Dr Pierre Busson

Dr Reini Fernandez De Luco

Marie-Dominique Galibert

Keywords:

In the three years that have lead up to the completion of my PhD project, many people have contributed towards it. This support came in many forms, as shared knowledge or technical skills, words of advice, or an open ear. As the popular saying goes, a problem shared is a problem halved, and I take this opportunity to thank all those who have helped me along the way.

Yann and Agnès both deserve the biggest of thank-yous. They welcomed me in and helped me every step of the way, from the first experiments to the final proof-readings. Their kindness, wisdom and openness to discussion is a true credit to them, and has made my time as a PhD student under their supervision that much more enjoyable. Of course, I also thank all the other members of the EGD team for their good spirits, making my time amongst them both pleasant and productive. Such a balance is not always easy to reach, and I would also like to thank Matthieu and Laure for lightening the mood with their banter during the otherwise stressful final months of my benchwork.

As well as inside the lab, science has a way of bringing people together outside of it. Even though Covid-19 tried its best to bring any form of social life to a standstill, I still have much to be grateful for in this regard. I would like to thank all those who have been involved in LUCA meetings and events, sharing our stories in a laid back setting was invaluable in putting my own experience into perspective. Special thanks to all those in the institute with whom I've shared the occasional joke and banter as our paths have crossed, it may not seem like much but sometimes it makes all the difference between a bad day and a good one. Although we haven't always been the best at catching up with each other, I would also like to thank Judikael, for inadvertently getting me into this project, and for his calmness and down-to-earth attitude whenever we have a chat.

A PhD can quite easily be likened to a roller-coaster ride, and my utmost gratitude goes to my girlfriend, Wendy, for being the rock in my life, making the most of the ups and helping me through the downs.

INTRODUCTION

A TP63 centric view of the TP53 family I. The TP53 family, an evolutionary toolbox for genome and tissue maintenance and integrity For its continued prosperity, any species must be able to create healthy offspring. In order to do so, it is crucial that its germline be protected from stress-induced genomic instabilities. In modern day species, this protection can be found from humans all the way back to choanoflagellates and sea anemones, attesting of its early emergence and billionyear old importance. This function is carried by the TP53 family genes. In humans, this ancestral gene family has three members: TP53, TP63 and TP73. The earliest and unique member of this family of genes, as present in choanoflagellates and sea anemones, is closest to the modern TP63/TP73 genes. Duplication of this gene led to the appearance of TP53. It is thought that TP53 filled the need for a guardian of somatic tissue integrity as multicellular organisms grew more complex and began to rely on the proliferation of stem cells for adult tissue homeostasis. Recent analysis of the elephant shark genomic sequences identified all three family members in its genome. This suggested that two consecutive duplication events led to two TP53 family genes in early vertebrate, rapidly followed by the appearance of a third member to compose the family as it is now know in all modern vertebrates. In all Gnathostomata, clearly distinct versions of TP63 and TP73 exist (Belyi et al. 2010;Lane et al. 2011). Recently published work on the family revealed that all three family members are already found in lamprey pushing back the origin of distinct TP73 and 63 genes before Gnathostomata radiation about 500 Million years ago [START_REF] Biscotti | The P53 Gene Family in Vertebrates: Evolutionary Considerations[END_REF], see Figure 1.

All three proteins share a common basic organisation which reflects their role as transcription factors: an N-terminal transactivation domain, a central DNA binding domain, and additional C-terminal domains required for oligomerisation and regulation (Figure 2).

The DNA binding domain of all three proteins is highly conserved but each family member diverges more significantly on the other functional domains. Globally TP63 is closer to TP73 than to TP53. Each family member has unique biological properties. The broad functions of these three genes in mammals have best been elucidated through a series of Loss of TP53 function in mice is associated with an increase in the early onset of multiple tumours [START_REF] Lavigueur | High Incidence of Lung, Bone, and Lymphoid Tumors in Transgenic Mice Overexpressing Mutant Alleles of the P53 Oncogene[END_REF][START_REF] Donehower | Mice Deficient for P53 Are Developmentally Normal but Susceptible to Spontaneous Tumours[END_REF]. Human mutations in TP53 are causal to the Li-Fraumeni syndrome, a rare disease characterized by an autosomal dominant transmission and by a dramatic increase in many different types of cancers [START_REF] Malkin | Germ Line P53 Mutations in a Familial Syndrome of Breast Cancer, Sarcomas, and Other Neoplasms[END_REF]).

TP73 knockouts are accompanied by dramatic developmental disorders:

hippocampal dysgenesis, hydrocephalus, chronic airway infections and sterility but do not develop spontaneous tumours as opposed to TP53 deficient mice (A. [START_REF] Yang | P73-Deficient Mice Have Neurological, Pheromonal and Inflammatory Defects but Lack Spontaneous Tumours[END_REF].

These effects are being mediated at least in part by TP73's control of the differentiation of multiciliated cells [START_REF] Napoli | Unifying the P73 Knockout Phenotypes: TAp73 Orchestrates Multiciliogenesis[END_REF]. Mutations in the human TP73 gene lead to reduced production of motile cilia that impact both respiratory and cognitive systems.

Patients have defective respiratory functions, associated with respiratory infections because of impaired mucociliary clearance, and impaired brain development [START_REF] Wallmeier | Mutations in TP73 Cause Impaired Mucociliary Clearance and Lissencephaly[END_REF].

TP63 was the last gene of the family to be cloned (A. [START_REF] Yang | P63, a P53 Homolog at 3q27-29, Encodes Multiple Products with Transactivating, Death-Inducing, and Dominant-Negative Activities[END_REF]. TP63

knockouts in mice lead to severe developmental defects in epidermis stratification, limb formation and fusion of craniofacial buds (Annie [START_REF] Yang | P63 Is Essential for Regenerative Proliferation in Limb, Craniofacial and Epithelial Development[END_REF].These data paint a clearer picture where TP63 and TP73 are crucial during differentiation and morphogenesis, whereas TP53 acts as a somatic tumour suppressor. This is further demonstrated by the high frequency of TP53 mutations in cancer, reaching around 50% [START_REF] Baugh | Why Are There Hotspot Mutations in the TP53 Gene in Human Cancers?[END_REF], whereas TP63 and TP73 mutations are rare in cancers. In Humans, heterozygous TP63 mutations are the cause of several autosomal dominant syndromes frequently characterised by ectrodactyly, cleft lip and palate and ectodermal dysplasia, which I will develop on in a following dedicated chapter.

II. Three genes, many more isoforms

The structure and the regulation of the three genes is complex (Figure 3). The TP53 family genes are generally under the control of two major alternative promoters producing mRNAs that differ in their 5' end. Combined with alternative splicing and sometimes alternative translation, this generates an impressive array of protein isoforms.

The TP53 gene's expression can lead to at least 12 different isoforms -p53, p53β, p53γ, proteins (Murray-Zmijewski, [START_REF] Murray-Zmijewski | P53/P63/P73 Isoforms: An Orchestra of Isoforms to Harmonise Cell Differentiation and Response to Stress[END_REF].

The p53 family proteins have a modular organisation with functional or regulatory domains sequentially distributed along the protein. I will take the structure of p63, the main focus of my thesis, as a paradigm (Figure 4). Three transcription initiation sites have been identified on the TP63 locus (A. [START_REF] Yang | P63, a P53 Homolog at 3q27-29, Encodes Multiple Products with Transactivating, Death-Inducing, and Dominant-Negative Activities[END_REF]Beyer et al. 2011). They are termed P0, P1 and P2. These promoters generate 3 different 5' terminal exons (exon U1, 1 and 4).

Four different translation initiation codons are described (AUG1, AUG2, AUG3, AUG4) and may produce 4 different N-termini that correspond to the isoforms GTAp63 (P0, ATG0), TA*p63 (P1, ATG1), TAp63 (P1, ATG2) and ΔNp63 (P2, ATG3). The 4 different encoded N terminal peptides are shown (Figure 4A) and are in the same reading frame. At the 3' end of the gene, two cassette exons (14 and 15) and 2 terminal exons (12 and 16) can produce multiple alternative C-terminal extremities. Because the alternative exons (15,[START_REF] Straub | The C-terminus of p63 contains multiple regulatory elements with different functions[END_REF] are not in the same reading frame, alternative splicing leads to frame shifting and a stop codon appears in the terminal exon [START_REF] Straub | The C-terminus of p63 contains multiple regulatory elements with different functions[END_REF] earlier than in the longest α isoform. Altogether The use of the proximal promoter (P1) leads to proteins which possess an Nterminal transactivation domain (TA) similar to the p53 transactivation domain, these are the "p53" isoforms and the "TAp63" or "TAp73" isoforms depending of the gene. The use of the distal promoters (P2) leads to proteins entirely or partially lacking a TA, these are the "Δp53", "ΔNp63" or "ΔNp73" isoforms.

From N to C-terminal the longest TAp63α isoform is composed of a transactivation domain, a DNA binding domain, an oligomerisation domain, a sterile alpha motif domain and a transcriptional inhibitory domain. Most of the alternative splicing events in the TP53/63/73 pre-mRNAs take place closer to the 3' end, favouring the production of protein isoforms with variable C-terminal domains. It is striking that no alternative splicing events affect the tetramerization domain. The minimal domains common to all of the p63 isoforms are the DNA binding domain and the tetramerization domain. This minimal configuration corresponds to the ΔNp63γ isoform. The active conformation of the p53 family transcription factors is a tetramer (a dimer of dimers, Figure 5B) [START_REF] Brandt | Conservation of DNA-Binding Specificity and Oligomerisation Properties within the P53 Family[END_REF]). The different p53/63/73 isoforms can bind to each other creating heterotetramers. The interaction is created via their oligomerisation domain. The homology between the oligomerisation domains of p63 and p73 enables them to bind to each other, however neither of them can bind to native p53 [START_REF] Davison | P73 and P63 Are Homotetramers Capable of Weak Heterotypic Interactions with Each Other but Not with P53[END_REF]. Some mutated forms of p53 are however capable of interacting with p63 or p73 isoforms (S. [START_REF] Strano | Physical and Functional Interaction between P53 Mutants and Different Isoforms of P73[END_REF]Sabrina Strano et al. 2002;[START_REF] Bergamaschi | P53 Polymorphism Influences Response in Cancer Chemotherapy via Modulation of P73-Dependent Apoptosis[END_REF][START_REF] Gaiddon | A Subset of Tumor-Derived Mutant Forms of P53 down-Regulate P63 and P73 through a Direct Interaction with the P53 Core Domain[END_REF][START_REF] Stindt | Functional Interplay between MDM2, P63/P73 and Mutant P53[END_REF]. The formation of heterotetramers opens the door to many possible functional consequences on the recognition and the activation or repression of their target genes. Indeed, despite a conservation rate of around 60% between p53, p63 and p73 DNA binding domains [START_REF] Dötsch | P63 and P73, the Ancestors of P53[END_REF]) and highly similar DNA binding specificities (see Figure 2 and 5), their respective target genes appear to be mostly non overlapping, especially between p63 and p53 [START_REF] Fontemaggi | Identification of Direct P73 Target Genes Combining DNA Microarray and Chromatin Immunoprecipitation Analyses[END_REF][START_REF] Gallant-Behm | ΔNp63α Represses Anti-Proliferative Genes via H2A.Z Deposition[END_REF][START_REF] Riege | Dissecting the DNA binding landscape and gene regulatory network of p63 and p53[END_REF].

III. Molecular functions and regulations of p63

The main focus of my thesis being the regulation and the functional consequences of the alternative splicing of TP63, I will therefore narrow down my field of study to the TP63 transcription factor. We should however keep in mind that its interactions with the other family members may complexify the picture even more.

III.1. Different functions among different isoforms

III.1.1. TAP63 vs ΔNP63

• differential expression

Based on GTEx data, there are 11 types of TP63 positive tissues in a human body: skeletal muscle, bladder, testis, endocervix, ectocervix, mammary gland, prostate, minor salivary gland, vagina, esophageal mucosa and skin (See Results). Out of these tissues, TATP63 is only expressed in testis, where it represents 96% of all TP63, and in skeletal muscle where it is the only N-terminal isoform detected [START_REF] Marshall | Tissue-Specific Expression of P73 and P63 Isoforms in Human Tissues[END_REF]. TATP63 is also expressed in the female germline where it participates in the maintenance of genome integrity. This concurs with evidence found in a prior study of human cell lines that concluded that ΔNTP63 isoforms are mainly expressed in cell lines of mostly ectodermal origin, and TAP63 is almost (with at least the exception of germ cells) only expressed in cell lines of mesodermal origin [START_REF] Sethi | A Global Analysis of the Complex Landscape of Isoforms and Regulatory Networks of P63 in Human Cells and Tissues[END_REF].

• differential functions

At the protein level, the differentiating factor between TAp63 and ΔNp63 isoforms is that the former possess an N-terminal transactivation domain similar to that of p53, whereas the latter does not. This TA domain is functional as a p53-like transcriptional activity is observed following transfection of the different TA isoforms. The different ΔN isoforms are unable to transactivate a p53 reporter vector (Petitjean et al. 2008).

ΔNp63 isoforms are nonetheless not devoid of transactivation activity thanks to a second transactivation domain located between the oligomerisation domain (OD) and the sterile alpha motif (SAM) (see Figure 2). This big difference between the two classes of isoforms is enough to give them many diverging functions. TAp63 is mainly associated with apoptosis, cell cycle and muscle development, while ΔNp63 is associated with stemness, survival, senescence, oncogenesis and metabolism [START_REF] Fisher | P63-Related Signaling at a Glance[END_REF]. These broad and diverging functions are a gross representation of the underlying interactions between p63 isoforms and the various cellular signalling pathways.

The current view is that TA isoform are mainly activated upon DNA damage to allow for a control of genome integrity while ΔNp63 isoforms control and interact with a vast array of downstream effector genes and signalling pathways that affect development and tissue homeostasis.

III.1.2. C-terminal isoforms

• differential expression

TP63α is the major isoform, representing 67 to 97% of overall TP63 in all tissues except skeletal muscle where TP63γ is most abundant, around 85%. In contrast, in tissues besides skeletal muscle, TP63γ only represents 0 to 24% of the expressed TP63. TP63β falls somewhere between the two, representing 5-21% of all TP63 in the epithelial tissues with highest TP63 expression. These percentages represent RNA levels, and were obtained from the analysis of GTEx RNAseq data [START_REF] Marshall | Tissue-Specific Expression of P73 and P63 Isoforms in Human Tissues[END_REF]). An RNAseq analysis of multiple human cell lines came to a similar conclusion in cells expressing ΔNTP63: TP63α is the main isoform, several log folds higher than both TP63β and TP63γ.

Again, TP63β was found to be more expressed than TP63γ, which was only detected at low levels in some head and neck squamous carcinoma cell lines [START_REF] Sethi | A Global Analysis of the Complex Landscape of Isoforms and Regulatory Networks of P63 in Human Cells and Tissues[END_REF]. One of the main caveats in the analysis of the expression of p63 C-terminal isoforms is the lack of protein data due to the absence of efficient and isoform-specific antibodies. • differential functions

The difference between C-terminal isoforms resides in the presence (or absence) of several functional protein domains. Yet, aside the TID that has a clearly demonstrated function in the repression of TA-domain dependent transcription upon DNA damage [START_REF] Gebel | P63 Uses a Switch-like Mechanism to Set the Threshold for Induction of Apoptosis[END_REF], the SAM and the TA2 domain present in the α isoform are poorly described.

ΔNp63α is the most expressed and the best described isoform. ΔNp63α is involved in epigenomic and transcriptional control of epidermal differentiation. It is clear that the different C-terminal isoforms carry different functions as two different models of transgenic mice expressing only γ-like forms of p63 are unable to develop properly and present phenotypes highly similar but not identical to TP63 knockout mice. This demonstrates that the α carboxy-terminal extremity is required for mice development and that the γ isoform is incapable of functionally substituting for it (Wolff et al. 2009;[START_REF] Suzuki | The Carboxy-Terminus of P63 Links Cell Cycle Control and the Proliferative Potential of Epidermal Progenitor Cells[END_REF]. From a mechanistic point of view, the difference in the γ and α terminal regions is best defined in the context of the full length TAp63 isoforms. In this context, the transcriptional activity of p63 on p53-reporter genes is dependent upon the presence of the α or γ isoform. TAp63γ

is the most potent transactivator of a p53-dependent CDKN1A reporter gene. This is coherent with the in vivo results obtained in the mice described above as in this context, when only γ-isoforms were produced, the cell cycle was compromised due to CDKN1A activation [START_REF] Suzuki | The Carboxy-Terminus of P63 Links Cell Cycle Control and the Proliferative Potential of Epidermal Progenitor Cells[END_REF]. However it is yet not clear if some biological functions are specific to the γ isoform in the context of ΔNp63.

III.2. Regulation of TP63 expression

As with many genes, different levels of regulation contribute to TP63's ultimate expression level. These regulations can be transcriptional, post-transcriptional or post translational.

III.2.1. Transcriptional regulation of TP63

Many different pathways control TP63 expression and dictate the usage of one promoter or the others. As stated previously, 4 different transcription initiation sites are reported for TP63, note P0, P1, P2. P0 is constituted by an endogenous retroviral long terminal repeat sequence and drives the expression of testis specific GTATP63 [START_REF] Pitzius | TA*p63 and GTAp63 achieve tighter transcriptional regulation in quality control by converting an inhibitory element into an additional transactivation domain[END_REF]. Little extra information is available as to its regulation. P1 historically drive the expression of TATP63 and TA*TP63 respectively (A. [START_REF] Yang | P63, a P53 Homolog at 3q27-29, Encodes Multiple Products with Transactivating, Death-Inducing, and Dominant-Negative Activities[END_REF]. It is however now thought that the isoform initially referred to as TATP63 could be artefactual. The canonical TATP63 isoform as we now know it, and as described as transcript variant 1 in the Uniprot database, is in fact the historical TA*TP63 (Pokorná et al. 2021). P2 drives the expression of ΔNTP63 (Figure 4).

The use of these different promoters must be tightly controlled in order to ensure the precise tissue specific expression of each class of isoforms. Table 1 resumes the described transcription factors which are said to positively or negatively regulate TP63 transcription. Of note, both p53 and p63 have been described as positive and negative regulators of ΔNTP63. Knowledge of TATP63 transcription regulators is far more limited, with only a handful of described regulators. Of all the described regulators, some act directly at the promoters, but several enhancer regions have also been described for the TP63 gene. In the region corresponding to the first ΔNTP63 exon there is an enhancer referred to as C38/C40 [START_REF] Antonini | An Autoregulatory Loop Directs the Tissue-Specific Expression of p63 through a Long-Range Evolutionarily Conserved Enhancer[END_REF]2015a). When bound by ΔNp63 itself or CEPBδ, it activates TP63 transcription, whereas it is inhibited by CEPBα, CEPBβ, or

Pou3f1 [START_REF] Leslie | Genome-Wide Meta-Analyses of Nonsyndromic Orofacial Clefts Identify Novel Associations between FOXE1 and All Orofacial Clefts, and TP63 and Cleft Lip with or without Cleft Palate[END_REF]Antonini et al. 2015a; [START_REF] Barbaro | C/EBPdelta Regulates Cell Cycle and Self-Renewal of Human Limbal Stem Cells[END_REF]). An enhancer for the ΔNTP63 promoter has also been described downstream of the TP63 gene. In mice, SOX2

and PITX1 promote ΔNTP63 expression via a distal enhancer [START_REF] Sastre-Perona | De Novo PITX1 Expression Controls Bi-Stable Transcriptional Circuits to Govern Self-Renewal and Differentiation in Squamous Cell Carcinoma[END_REF].

Chromatin modifications are also central in regulating TP63 gene activity. The pioneer factor TFAP2C, which has been described for its role in preparing chromatin for epithelial lineage differentiation genes, binds to TP63 in multiple locations and promotes its expression (L. Li et al. 2019).

Histone acetylation and methylation can influence ΔNTP63 expression. Indeed, the histone acetyl-transferase inhibitor NOCL2 stimulates ΔNTP63 expression in epidermal cells [START_REF] Duteil | The Histone Acetyltransferase Inhibitor Nir Regulates Epidermis Development[END_REF]. The histone demethylase KMD6A inhibits TP63 expression by repressing one of its enhancers [START_REF] Andricovich | Loss of KDM6A Activates Super-Enhancers to Induce Gender-Specific Squamous-like Pancreatic Cancer and Confers Sensitivity to BET Inhibitors[END_REF]) whereas the histone methyltransferase DNMT3B promotes TP63 expression by maintaining hydroxymethylation within its enhancers [START_REF] Rinaldi | Dnmt3a and Dnmt3b Associate with Enhancers to Regulate Human Epidermal Stem Cell Homeostasis[END_REF]. The switch between the TA and ΔN promoters is associated with a switch in their histone acetylation, which appears to be mutually exclusive. Analysis in cell lines has shown that in skeletal muscle myoblasts, histones surrounding the TATP63 promoter show H3K27 acetylation while the ΔNTP63 promoter showed the same marks in keratinocytes (Pokorná et al. 2021). Besides histone modifications, DNA methylation may also repress TP63 expression as hypomethylation of the ΔNTP63 promoter is associated with increased expression in lung squamous cell CEPBβ carcinoma, whereas high methylation levels are found in normal tissue [START_REF] Duteil | The Histone Acetyltransferase Inhibitor Nir Regulates Epidermis Development[END_REF][START_REF] Li | TFAP2C-and P63-Dependent Networks Sequentially Rearrange Chromatin Landscapes to Drive Human Epidermal Lineage Commitment[END_REF].

III.2.2. mRNA stability

The interaction between certain RNA binding proteins and TP63 mRNAs can have a drastic impact on their half lives, and subsequently on the downstream quantities of protein which can be produced. Rbm38 is capable of interacting with a AU/U rich sequence in the 3'UTR shared by TP63α, β and δ(J. [START_REF] Zhang | RNPC1, an RNA-binding protein and a target of the p53 family, regulates p63 expression through mRNA stability[END_REF]. This interaction destabilises the TP63 mRNA of these isoforms, but stabilises TP63γ mRNA by interacting with its specific 3'UTR [START_REF] Yan | TAp63γ and ΔNp63γ are regulated by RBM38 via mRNA stability and have an opposing function in growth suppression[END_REF]. Rbm38 is induced during keratinocyte differentiation, placing it as one of the actors enabling cells to break away from the stemness-promoting effects of p63. Furthermore, Rbm38 is itself a target of p63, which creates a feedback loop dictating their relative abundance. The phosphorylation of Rbm38 at Ser-195 abrogates its effect on TP63α/β/δ (Yanhong [START_REF] Zhang | Serine 195 phosphorylation in the RNA-binding protein Rbm38 increases p63 expression by modulating Rbm38's interaction with the Ago2-miR203 complex[END_REF].

III.2.3. Protein stability and degradation

p63 isoforms are degraded in a proteasome-dependent manner after apposition of specific post-translational marks. Many enzymes have already been described for their participation in these modifications, mostly E3-ligases and kinases.

The first description of a p63-targeting E3-ligase came about in 2005 when it was described that the C-terminal region of p63α contains target sequences for the E3-ligase

Nedd4 and for the SUMO-conjugating enzyme Ubc9 [START_REF] Bakkers | Destabilization of ΔNp63α by Nedd4-Mediated Ubiquitination Ubc9-Mediated Sumoylation, and Its Implications on Dorsoventral Patterning of the Zebrafish Embryo[END_REF]. The p63 SAM domain present in α isoforms contains a PPPY motif which the E3-ligase Itch recognises and binds to. Itch can also recognise an 11 amino acid sequence in the N-terminus of all p63 isoforms, meaning Itch can target any p63 isoform to degradation [START_REF] Rossi | The E3 ubiquitin ligase Itch controls the protein stability of p63[END_REF]).

The PPPY motif can also be recognised by WWP1, a homologue of Itch (Y. [START_REF] Li | WW Domain-Containing E3 Ubiquitin Protein Ligase 1 Targets P63 Transcription Factor for Ubiquitin-Mediated Proteasomal Degradation and Regulates Apoptosis[END_REF]. Some degradation pathways require the collaboration of multiple factors:

MDM2 can bind to the SAM domain of p63 in the nucleus, provoking its translocation to the cytosol where the E3-ligase Fbw7 can then target it, in combination with the GSK3 kinase. This process cannot degrade p63γ since it does not have a SAM domain (Galli et al. 2010).

In terms of kinases, I have already mentioned the regulation of TAP63 by c-Abl. This same kinase is reputed to be able to phosphorylate ΔNP63α, albeit at different residues:

Y55, Y137 and Y308 [START_REF] Yuan | c-Abl phosphorylation of ΔNp63α is critical for cell viability[END_REF]. Strikingly, the consequences are opposite, these modifications actually stabilise ΔNP63α. The ΔNP63α can however also be degraded following phosphorylation. Indeed, phosphorylation of residues S385, T397 and S466 mediated respectively by ATM, CDK2 and p70s6K all lead to its degradation [START_REF] Huang | ATM kinase is a master switch for the ΔNp63α phosphorylation/degradation in human head and neck squamous cell carcinoma cells upon DNA damage[END_REF]. HIPK2 can also phosphorylate ΔNP63α at the T397 residue [START_REF] Lazzari | HIPK2 Phosphorylates ΔNp63α and Promotes Its Degradation in Response to DNA Damage[END_REF].

This illustrates the general importance of the C-terminal end of p63 for its regulation.

Therefore the alternatively spliced isoforms of p63 may be differentially susceptible to those regulations which does not simplify the analysis of the functional consequences of these regulations.

The importance of the correct regulation of p63 stability was highlighted recently by the identification p63 as a major player in thalidomide teratogenicity. In the early sixties, many expectant mothers were prescribed thalidomide to help alleviate morning sickness.

The steady flow of severe congenital birth defects that followed quickly led to its withdrawal from the market. The precise mechanism by which thalidomide use led to these defects remained a mystery for several decades. Some of the thalidomide-linked birth defects were strikingly similar to those which are observed in p63-linked human syndromes and prompted further enquiry. Using HaCaT cells and zebrafish embryos, a mechanism linking thalidomide, p63 and the birth defects was identified. Thalidomide treatment increases the activity of the CRL4 CRBN ubiquitin-ligase complex, which in turn targets both TAp63 and ΔNp63 for degradation. The degradation of TAp63 and ΔNp63

during embryogenesis respectively leads to developmental defects in the ear and in the limbs, explaining the deformities observed in newborns [START_REF] Asatsuma-Okumura | P63 Is a Cereblon Substrate Involved in Thalidomide Teratogenicity[END_REF].

III.2.4. miRNAs targetting TP63

Micro RNAs (miRNAs), are short RNAs of around 22 nucleotides produced from intronic or intergenic regions which are capable of influencing gene expression by hindering translation and stability of their target RNAs. Their action relies on the base pairing of a "seed sequence", around 6-8 nucleotides long, to their targets, generally within the untranslated regions. Because of the short seed-sequence, any given miRNA can potentially control hundreds of different mRNA targets. TP63 isoforms can be regulated by miRNAs and are separated into two target groups based on the two different 3'UTRs which they can possess. On one side, TP63α, β and δ, on the other, TP63γ.

Figure. 6b. Location of binding sites for miRNA on the TP63 3'UTRs

Upper panel, genomic view of the Human TP63 locus and mRNA isoforms. Lower panels, location of potential miRNA binding sites on the 3'UTR of the gamma (left) and alpha beta delta (right) TP63 isoforms.

Studies of mouse keratinocyte differentiation in vitro have shown that as cells terminally differentiate, with increased levels of involucrin, there is an inverse relation between p63 and mir203 levels, mir203 levels increasing and p63 levels decreasing. In mice, transgenic overexpression of miR-203a-3p in the basal epithelial layer using a keratin 14 promoter construct [START_REF] Yi | A skin microRNA promotes differentiation by repressing 'stemness[END_REF]) led to a decreased proliferation and an early activation of the epithelial differentiation program with accumulation of Keratin 10 expressing cells and depletion of proliferating basal cells. These changes were accompanied by dowregulation of TP63 expression. The up-regulation of miR-203a-3p in epithelial differentiating cells could be recapitulated in vitro using CaCl2 induced differentiation of HaCaT cells. In this context differentiation induced upregulation of mir203 and downregulation of TP63 mRNA. Specifically blocking miR-203 using an anti-miR oligonucleotide abolished the downregulation of TP63 mRNA prompting to hypothesize a direct role of miR203 on the control of TP63 mRNA. Bioinformatic analysis identified two binding sites for miR203 in the 3'most terminal exon of TP63 at two positions of the human and mouse 3'UTR shared by ΔNTP63α, β and δ [START_REF] Lena | MiR-203 Represses "stemness" by Repressing DeltaNp63[END_REF][START_REF] Yi | A skin microRNA promotes differentiation by repressing 'stemness[END_REF]. A direct regulation of miR-203 on the level of TP63 mRNA was confirmed using reporter assays and mutations of the miR-203 binding sites [START_REF] Lena | MiR-203 Represses "stemness" by Repressing DeltaNp63[END_REF]. Together, all these data make the case for a regulatory role of mir-203 in epidermal differentiation via downregulation of TP63. Yet, the regulation of TP63 by this miRNA may be somehow tissue specific as in a bladder carcinoma cell line that robustly expresses p63, no impact of miR-203a-3p on TP63 expression could be observed in the course of a screen for miRNA controlling TP63.

The importance of miRNA modulation of TP63 expression is further revealed in the case of development of Idiopathic pulmonary fibrosis. Idiopathic pulmonary fibrosis (IPF) is a type of interstitial pneumonia which was triggered by neither infection nor cancer. In this disease, affected areas of the lung show an increase in both TAp63 and ΔNp63 isoforms.

In BEAS-2B and A549 cells, TP63 expression is required for TGF-β1 induced fibrosis, which is worsened by TP63 overexpression. During IPF, miR184 is robustly downregulated according to multiple publicly available datasets (GEO accession numbers: GSE13316, GSE32638, GSE27430, GSE24323), as well as in tested tissue samples when compared to healthy tissue. In the BEAS-2B and A549 cells, overexpression of mir184 inhibited TGF-β1-induced fibrosis, which could be partially reversed by concomitant TP63 overexpression. In mice treated with bleomycin (BLM) to develop IPF, p63 protein levels are increased and mir-184 levels are decreased. The experimental overexpression of mir-184 reduces the p63 increase. TP63 therefore seems convincingly involved in the development of IPF, secondary to outside stimuli (J. [START_REF] Li | MiR-184 Targets TP63 to Block Idiopathic Pulmonary Fibrosis by Inhibiting Proliferation and Epithelial-Mesenchymal Transition of Airway Epithelial Cells[END_REF]. Which TP63 isoforms are involved in this disease has not been investigated.

IV. p63 developmental functions

Many different TP63 mice models have been developed in order to address different aspects of p63 developmental functions [START_REF] Vanbokhoven | P63, a Story of Mice and Men[END_REF]. Studying their developmental defects was the starting point in understanding the biological importance of TP63. Initially, the lack of skin and limbs were the most obvious consequences of TP63 knockouts, but p63's developmental importance has now been broadened to most epithelial tissues, and some non epithelial tissues such as bone and muscle.

IV.1. p63 in the establishment of the epidermis

Protection against the harshness of the outside environment is a vital need for all living organisms. In many animals, this protection is provided by the largest organ of the body, the epidermis, and its many different types of specialisations. Depending on the environment and the stage of development, epidermis can be as varied as hairy skin with sebaceous glands, feather-displaying skin, or muco-ciliary epithelia.

Briefly after cloning of the TP63 cDNA, knockout experiments of TP63 in mice (Nota: TP63 is also called Trp63 in mice) showed a severe lack of epidermal stratification leading to dessication and death of the newborn pups (Annie [START_REF] Yang | P63 Is Essential for Regenerative Proliferation in Limb, Craniofacial and Epithelial Development[END_REF]. These first experiments underlined the essential role of TP63 in generating an epidermis.

Immunohistochemistry of the epidermis of perinatal pups revealed a strong expression of p63 localised in the basal layers of the epidermis. Analysis of the molecular markers of the simple epithelium present at E12 in both control and knockout mice, before any stratification happens, revealed that the knockout mice lacked keratins 5 and 14 compared to the control mice. These markers are normally expressed by the basal cells. At E17 the control mice embryos have a stratified epidermis while knockout mice only present dispersed clusters of cells. In both cases, differentiation markers loricrin, filaggrin and involucrin are expressed. These data indicate that in the absence of TP63, there is a lack of basal progenitor cells, a lack of proliferation and terminal differentiation happens with no stratification.

Fluorescent labelling of cells of the stratifying epidermis in post-larval zebra fish embryos has shed some light on the chronological importance of p63 in its establishment [START_REF] Rangel-Huerta | The Dynamics of Epidermal Stratification during Post-Larval Development in Zebrafish[END_REF]. Different populations of cells compose the stratified epidermis of the late zebrafish larvae. Epidermal Stem cells located closer to the basal membrane proliferate moderately and generate committed progenitor cells through asymmetric cell division. These epidermal stem cells express the highest levels of p63.

The committed progenitor cells are highly proliferative cells of the basal and suprabasal compartments and generate the cells that make up the upper layers of the epidermis. They express low levels of p63 and one of their hallmarks is the increased phosphorylation of ser66/68 of p63. This phosphorylation event is involved in the cytoplasmic relocalisation of p63 and increased degradation. As cells move upwards in the suprabasal compartment, p63 expression is completely lost. Furthermore, the rise in proliferative capacity is correlated to a rise in the levels of phosphorylated p63, which is mostly located in the cytoplasm. These findings are close to what has been described in human epidermis [START_REF] Suzuki | Increased P63 Phosphorylation Marks Early Transition of Epidermal Stem Cells to Progenitors[END_REF] as well as during wound healing of adult mouse skin (Suzuki et Senoo 2013) where p63 was detected both in basal and suprabasal layers, with an increase in phosphorylated p63 in the suprabasal layer as p63 levels decrease.

In vitro, Ca2+ induced differentiation of human keratinocytes leads to this same increase of phosphorylated p63 and decrease of total p63. In long term cultures of epidermal holoclones (cultures derived from the epithelial stem cells), the cells showing higher levels of p63 phosphorylation have a clearly lower proliferative capacity [START_REF] Suzuki | Increased P63 Phosphorylation Marks Early Transition of Epidermal Stem Cells to Progenitors[END_REF]. Together, all these data point in the same direction: p63 is required for the establishment and maintenance of a population of basal stem cells. Loss of p63, which can be secondary to its phosphorylation, guides cells towards the transiently amplifying stage, and its ultimate loss allows cells to terminally differentiate after a few rounds of divisions. Phosphorylated p63 can therefore be used as a marker of the initial stages of epidermal differentiation. This however raises the question of the initial activation of TP63 expression in the basal cells of the epidermis. Work realised in Xenopus laevis embryos provided some clues about the triggers of TP63 expression. The epidermis of the Xenopus larvae proved to be an excellent model of muco-ciliary epithelium development [START_REF] Walentek | Xenopus Epidermal and Endodermal Epithelia as Models for Mucociliary Epithelial Evolution, Disease, and Metaplasia[END_REF]. As shown on Figure 6, early TP63 expression is first detected in the non neural ectoderm and delineates the neural ectoderm as neurulation progresses (Yann Audic, unpublished data).

Haas and colleagues (Haas et al. 2019) identified the Wnt/β-catenin signalling pathway as promoter (Ruptier et al. 2011;[START_REF] Kjolby | Genome-Wide Identification of Wnt/β-Catenin Transcriptional Targets during Xenopus Gastrulation[END_REF]. The induced expression of ΔNTP63 led to increased proliferation and reduced differentiation of the multi-ciliary cells showing that p63 controls the proliferative potential of basal cells of the Xenopus epidermis.

IV.2. p63 regulates many other epithelia

Epithelia are not limited to skin and on the contrary are among the earliest evolved tissues in multicellular organisation. They can derive from all three embryonic layers. TP63 expression can be found in all types of epithelia from ectodermal, endodermal, or even mesodermal origin and delineates epithelial stem cells in these different contexts. A nice example (see Figure 7) from this epithelia-wide TP63 expression could be found in the early work of Annie Yang on TP63 cloning (A. [START_REF] Yang | P63, a P53 Homolog at 3q27-29, Encodes Multiple Products with Transactivating, Death-Inducing, and Dominant-Negative Activities[END_REF] where clear p63 labelling could mostly be observed in the basal cells of epithelia from ectodermal, mesodermal or endodermal origin.

A series of interesting xenograft experiments of rat epithelial stem cells onto mice have shown ΔNTP63 expression to be associated with a potential for multipotency. Indeed, ΔNTP63 expressing cells from squamous epithelia can be xenografted and lead to successful establishment of hairy epidermis with development of hair follicles and sebaceous glands. ΔNP63 expressing cells from epithelia of different embryonic origins can also give rise to hair epidermis and sebaceous glands following an intermediary culture step, with the exception of tracheal cells which never successfully produce sebaceous glands or hair follicles. Interestingly, despite differentiating into hair epidermis, the grafted cells kept expressing transcription factors associated with their tissue of origin [START_REF] Claudinot | Tp63-Expressing Adult Epithelial Stem Cells Cross Lineages Boundaries Revealing Latent Hairy Skin Competence[END_REF]. This work therefore highlights the pluripotency of ΔNTP63, which rather than allowing for trans-differentiation, seems to keep cells open to commit to a multitude of fates in a reversible manner. This is in line with the discovery that ΔNTP63 expression is a favourable factor in the reprogramming of differentiated cells using some of the Yamanaka transcription factors [START_REF] Alexandrova | ΔNp63 Regulates Select Routes of Reprogramming via Multiple Mechanisms[END_REF]. The ultimate fate of the differentiating cells being determined by the cellular environment and external clues in which it is developing. This suggests that p63 expression patterns are probably quite similar in all stratified epithelia, with expression in stem cells and loss towards terminal differentiation.

Ectoderm

Mesoderm

Endoderm

Endoderm/mesoderm Mesoderm The striking epidermal defects and the early lethality associated with TP63 knockout initially masked the many diverse developmental functions of TP63. From the perspective of what we now know of TP63, it is striking that "almost" normal embryos could develop from animals defective in TP63 expression. One could have envisaged that early defects in TP63 could lead to much more drastic phenotypes.

IV.2.1. p63 in the eye: corneal regeneration

In mammals, the integrity of the cornea is ensured by a stock of corneal epithelial stem cells. These stem cells are preferentially located in the limbus, a ring-like structure that surrounds the cornea and delimits it from the surrounding sclera. Similarly to what has been described in the epidermis, in the ocular surface epithelia, ΔNP63α is the most dominant isoform, with lesser expression of ΔNP63β and ΔNP63γ, as determined by isoform specific RT-PCR. No TA isoforms were detected in the eye [START_REF] Kawasaki | Expression and Tissue Distribution of P63 Isoforms in Human Ocular Surface Epithelia[END_REF].

Immunofluorescence staining of p63 in normal human cornea indicates it is present in basal limbal cells but nearly absent from the cornea. ΔNp63 localizes both to the limbal stem cells and to the committed central cells of the cornea, ΔNp63 expression is lost in the most differentiated superficial cells [START_REF] Robertson | Characterization of ΔNp63 isoforms in normal cornea and telomerase-immortalized human corneal epithelial cells[END_REF]. Following corneal injury however, p63 is detected in the basal and suprabasal layers of both the limbus and the cornea. Isoform specific in situ hybridization analysis of the TP63 mRNA expressed in these tissues showed that in the injured corneas there is a significant increase in ΔNTP63β and ΔNTP63γ in the suprabasal layers, in opposition to the ΔNTP63α mRNA which was strictly confined to the basal layer. The p63 positive cells found in the cornea following injury are cells that have migrated from the limbal tissue to regenerate the cornea [START_REF] Iorio | Isoforms of DeltaNp63 and the Migration of Ocular Limbal Cells in Human Corneal Regeneration[END_REF]. It is not clear at this stage what role the shift in p63 isoforms from basal to suprabasal compartments plays in the physiological process of corneal wound healing. In the clinical context, p63 positivity is used as a positive predictor of success of regenerative therapies which relies on grafting of limbal holoclones to eyes which have suffered corneal loss or deterioration [START_REF] Rama | Limbal Stem-Cell Therapy and Long-Term Corneal Regeneration[END_REF].

IV.2.2. p63 in the uterus and vagina

Both the uterus and the vagina derive from the Müllerian ducts. Once mature, the uterus is lined by a simple columnar epithelium, while the vagina is lined by stratified squamous epithelia. The uterus and the proximal 1/3 part of the vagina are of mesodermal origin while the 2/3 distal part is of ectodermal origin. The basal cells of the vagina express ΔNp63α, while those of the uterus do not (Takeshi [START_REF] Kurita | Differential Expression of P63 Isoforms in Female Reproductive Organs[END_REF]. This is therefore an interesting model to study how the switch between commitment to stratified epithelium or simple epithelium is operated. The Müllerian ducts from TP63 -/-mice pups show normal uterine development, however their vagina lacks a stratified epithelium. Recombination grafting experiments where neonatal uterine mesenchyme is associated with vaginal epithelium fail to allow vaginal p63 expression and epithelial stratification. Reversely, when vaginal mesenchyme is associated to neonatal uterine tissue, epithelial uterine expression of p63 and subsequent stratification is triggered (T. [START_REF] Kurita | Roles of P63 in Differentiation of Müllerian Duct Epithelial Cells[END_REF]. Therefore it is likely that clues from the neighbouring tissues are necessary to instruct cells to express TP63 and favour epithelial stratification. In the context of female genital tract development, the expression of TP63 was shown to be under the control of the estrogenomimetic diethylstilbestrol (DES). Exposure of mice fetuses to diethylstilbestrol (DES) provokes cervico-vaginal adenosis, which is characterized by the presence of simple columnar epithelium instead of a stratified columnar epithelium. At least one aspect of this disease is based on the loss of p63 expression following DES exposure (Takeshi [START_REF] Kurita | Roles of P63 in the Diethylstilbestrol-Induced Cervicovaginal Adenosis[END_REF]. This places ΔNP63α as a major trigger for epithelium commitment towards the stratified squamous type, and acts in response to mesenchymal clues which have yet to be identified in this context.

IV.2.3. p63 in mammary tissue development and maintenance

TP63 -/-mice as well as ΔNTP63 specific knockout mice are devoid of mammary glands (Annie [START_REF] Yang | P63 Is Essential for Regenerative Proliferation in Limb, Craniofacial and Epithelial Development[END_REF]Mills et al. 1999;[START_REF] Chakrabarti | ΔNp63 Promotes Stem Cell Activity in Mammary Gland Development and Basal-like Breast Cancer by Enhancing Fzd7 Expression and Wnt Signalling[END_REF][START_REF] Koster | P63 Induces Key Target Genes Required for Epidermal Morphogenesis[END_REF]), indicating that one or several of the ΔNp63 isoforms are required for their development. When reintroduced into cleared mammary fatpads, epithelial stem cells expressing ΔNTP63 from lentiviral vectors are capable of partially regenerating the missing organ, contrary to epithelial stem cells expressing TATP63 [START_REF] Chakrabarti | ΔNp63 Promotes Stem Cell Activity in Mammary Gland Development and Basal-like Breast Cancer by Enhancing Fzd7 Expression and Wnt Signalling[END_REF]. Interestingly, the stem cell capacity of the ΔNTP63 transduced epithelial stem cells was critically dependant upon Wnt signalling and the expression of WNT5A and FZD7, a receptor and a ligand of the Wnt pathway, was upregulated upon overexpression of ΔNTP63 in a human mammary epithelial cell line. It is now thought that at the molecular level, ΔNp63 promotes stemness through activation of the Wnt/β-catenin pathway [START_REF] Lindley | The WNT-controlled transcriptional regulator LBH is required for mammary stem cell expansion and maintenance of the basal lineage[END_REF]. The stemness properties induced by the p63 expression are often associated with dedifferentiation as can be observed when loss of p63 in mammary basal cells shifts their expression of markers away from the basal and towards the more luminal phenotype, and oppositely when ectopic expression of p63 in luminal cells shifts them back towards a more basal phenotype [START_REF] Yalcin-Ozuysal | Antagonistic Roles of Notch and P63 in Controlling Mammary Epithelial Cell Fates[END_REF].

Epithelia are dynamic structures whose integrity is kept balanced by a moderate renewal of the cells composing the epithelium and a progressive shedding of the oldest differentiated cells. Stem cells are generally not highly proliferating cells but rather slow dividing cells that generate and eventually replenish (in the case of injury) a population of transient amplifying cells or progenitor cells with higher proliferative capacities. These expression rather than ΔNp63, as did GLI3 down-regulation. Altogether, this indicated that in mammary glands, a reciprocal interaction between p63 and IHH controls the production of progenitor cells from Mammary stem cells (Na Li et al. 2008). Interestingly, inhibition of Hedgehog signalling pathways by deletion of the SMO gene (Smoothened) in ectodermal tissues by conditional knockout led to the production of a glandular epithelium somehow capable of differentiation into mammary glands [START_REF] Gritli-Linde | Abnormal Hair Development and Apparent Follicular Transformation to Mammary Gland in the Absence of Hedgehog Signaling[END_REF]. This suggests that control of proliferation and differentiation in a p63 context is highly dependent of the hedgehog signalling pathway.

IV.3. p63 in non-epithelial tissue developmental

IV.3.1. Limbs/bones

Besides the lack of any stratified squamous epithelium, Trp63-/-mice also showed severe limb and craniofacial development issues. p63 is normally found in the apical ectodermal ridge (AER) of the limb buds. At the RNA level, ΔNTP63γ is the main isoform expressed during limb development from E10.5 to E12 in normal mice. ΔNTP63α but not ΔNTP63β is also expressed during this period, at lesser levels than ΔNTP63γ and there is a progressive increase in TATP63 expression. Fgfr8 and Fgfr4, positive regulators of the AER, are proposed transcriptional targets of ΔNp63γ. Jag2, a negative regulator of the AER, seems to be a target of TAp63γ (Kawata et al. 2017a).

Study of mouse chondrocytes in vitro has shown that the shift from proliferative cells, which express mostly TATP63 α and β, to hypertrophic cells is accompanied by a significant increase in TATP63γ and ΔNTP63β mRNA (Gu et al. 2013a). Depletion of ΔNTP63α in mouse chondrocytes has no effect on the ossification process of limbs, digits and tail bone. Depletion of TATP63α however provokes a delay in the ossification of digits and tailbone in mice [START_REF] Lu | Distinct function of P63 isoforms during embryonic skeletal development[END_REF]. Overexpression of TATP63α in mouse chondrocytes in vivo leads to accelerated ossification. In vitro, overexpression of TATP63γ in ATDC5 cells, a mouse cell line used for in vitro studying of skeletal development (Yao et Wang 2013a), led to an increase in col10a, a marker of hypertrophic differentiation.

The exact participation of different P63 isoforms in the formation and limbs and the ossification process that ensues is therefore unclear. There is an apparent importance of p63γ in the establishment of limbs as opposed to the dominant function of p63α in the establishment of epithelia.

IV.3.2. Muscle

shRNA mediated knockdown of TATP63 in spontaneously immortalised C2C7 mouse muscle cells increases their proliferation rate by removing CDKN1A and CDKN1C inhibition. Overexpression of TATP63γ in this same cell line led to a decrease in proliferation [START_REF] Ciuffoli | Myoblasts rely on TAp63 to control basal mitochondria respiration[END_REF]. In vitro differentiation of C2C7 cells leads to a vast increase of TATP63γ, while knockdown of TATP63 in these cells impairs their capacity to organise into myotubes (Cefalù et al. 2015). Immunohistochemical staining of tissue samples from various types of striated skeletal derived cancers has shown that p63 has a cytoplasmic expression in these cells. Furthermore, immunoelectron microscopy identified p63 presence at the Z striations. This is one of the rare indications of a non-transcription related function of p63, which could therefore be involved in myofirbil organisation or function [START_REF] Martin | Cytoplasmic P63 Immunohistochemistry Is a Useful Marker for Muscle Differentiation: An Immunohistochemical and Immunoelectron Microscopic Study[END_REF].

IV.3.3. Heart

The severity of the epidermal phenotype of TP63 -/-mice is such that it may have led to other potentially lethal phenotypes being overlooked. In work published in 2011, the impact of TP63 deficiency on cardiac development was identified [START_REF] Rouleau | TAp63 Is Important for Cardiac Differentiation of Embryonic Stem Cells and Heart Development[END_REF].

Without TP63, mouse pup hearts present with ventricular dilation, thin ventricular walls, poor trabeculation and impaired myofibrillogenesis. Besides this, the mitochondria also seemed to be affected, impaired mitochondrial function in muscle cells has since also been identified [START_REF] Ciuffoli | Myoblasts rely on TAp63 to control basal mitochondria respiration[END_REF]. The defects in heart development could explain in utero mouse mortality, as well as participate to the already well established peri-natal mortality of the pups. Strikingly though, p63 is not expressed in the E18.5 fully developed heart, or in any of the cells of the heart lineage in previous stages. Instead, p63 seems to be expressed by the associated endoderm. During the differentiation of mouse embryonic stem cells into embryonic bodies with beating cardiomyocytes, there is a switch in expression of TP63 mRNA. TATP63 is expressed during the first stages, and progressively ΔNTP63 isoforms become the most abundant. Depleting TP63 in these cells by shRNA leads to embryonic bodies devoid of beating cardiomyocytes. This phenotype is rescued by the transfection of GATA6 expression plasmids. GATA6 is a factor known to be involved in both endodermal formation and cardiogenesis. Furthermore, ChIP-seq analysis revealed activin-a as a target gene of p63. Activin-a is an endodermal stimulator of early cardiogenesis. Similarly as the expression of GATA6, addition of exogenous Activin-a to cultures of sh-TP63 embryonic stem cells restored their ability to form beating cardiomyocytes. Together, these data [START_REF] Rouleau | TAp63 Is Important for Cardiac Differentiation of Embryonic Stem Cells and Heart Development[END_REF] show that TP63 expression in the embryonic endoderm participates in its crosstalk with adjacent mesoderm, stimulating some aspects of cardiogenesis. Analysis of the TP63 isoforms in the END2 endodermal cell line revealed TATP63γ to be the dominant isoform. It is not clear at this point whether this is also the case in vivo, which would be interesting to confirm a non-epithelial role of the γ isoform.

IV.3.4. Cochlea

Some patients with TP63 mutation-related ectodermal dysplasia suffer from hearing loss. Analysis of the E18.5 TP63 -/-mouse embryos showed defects in the cochlear duct.

RT-PCR analysis of TP63 isoforms expressed in the cochlea of wild type E18.5 mouse embryos revealed that TATP63 is present rather than ΔNTP63. Furthermore, TATP63 -/mice show variable levels of sensorineural deafness. Microarray analysis of gene expression in SaOs-2 cells (human osteosarcoma) revealed that TAp63α regulates several members of the Notch pathway, including Hes5 and Atoh1 for which putative p63 response elements have been identified. Both genes are downregulated in E18.5 TAP63 -/-cochlear extracts. These data show a clear development role for TAp63α in the maturation of the cochlea [START_REF] Terrinoni | Role of P63 and the Notch Pathway in Cochlea Development and Sensorineural Deafness[END_REF]).

V. p63 in germline protection

So far, we have discussed the crucial role of ΔNP63 in epithelial homeostasis. The oldest function of the entire p53 family, however, lies in protecting the integrity of the germline. This function is still ensured today by a p63 homologue in Caenorhabditis elegans [START_REF] Schumacher | The C. Elegans Homolog of the P53 Tumor Suppressor Is Required for DNA Damage-Induced Apoptosis[END_REF][START_REF] Derry | Caenorhabditis elegans p53: Role in Apoptosis, Meiosis, and Stress Resistance[END_REF], Drosophila melanogaster [START_REF] Ollmann | Drosophila P53 Is a Structural and Functional Homolog of the Tumor Suppressor P53[END_REF], Ciona intestinalis [START_REF] Heering | Structural investigations of the p53/p73 homologs from the tunicate species Ciona intestinalis reveal the sequence requirements for the formation of a tetramerization domain[END_REF], and sea anemones (Belyi et al. 2010). In all these species, as well as in mammals, p63 or its orthologues are activated following DNA damage [START_REF] Gebel | P63 Uses a Switch-like Mechanism to Set the Threshold for Induction of Apoptosis[END_REF]. Just like the functions in stratified epithelia seem specific to ΔNp63 isoforms, germline protection seems to be the speciality of the TA isoforms and relies on the activity of the first transactivation domain. Indeed, ΔNTP63 knockout mice phenocopy the knockouts of all isoforms, and specific TAP63 knockout mice are not accompanied by developmental defects, indicating that TAp63 isoforms are dispensable for ΔNp63-like developmental functions. However, TAp63 is strongly expressed in oocytes, and in TAP63 -/-mice, primordial follicular oocytes survive exposure to UV radiation which is lethal to them in WT mice, indicating that TAp63 is involved in eliminating oocytes following DNA damage (Suh et al. 2006). The p53 status had no influence on oocyte survival, despite its role as a DNA damage sensor in somatic cells. The activation of TAp63 leads to activation of proapoptotic factors such as PUMA and NOXA [START_REF] Kerr | DNA damage-induced primordial follicle oocyte apoptosis and loss of fertility require TAp63-mediated induction of Puma and Noxa[END_REF]).

In the mammal female germline, oocytes go through an important step before meiotic division that ensures maximum genetic recombination. At the zygotene stage of prophase I, the chromosomes are aligned and subjected to many double stranded breaks by the type II topoisomerase Spo11 in order to promote crossovers [START_REF] Keeney | Meiosis-Specific DNA Double-Strand Breaks Are Catalyzed by Spo11, a Member of a Widely Conserved Protein Family[END_REF]. Having had the chance to crossover, most of these breaks are repaired by homologous recombination enzymes DMC1 and RAD51 [START_REF] Hinch | The Configuration of RPA, RAD51, and DMC1 Binding in Meiosis Reveals the Nature of Critical Recombination Intermediates[END_REF]. Five days after birth, mouse pup oocytes are strongly positive for TAp63 (Suh et al. 2006). By this point oocytes are arrested in prophase I of meiosis (dictyate arrest), and TAp63 may eliminate those with enduring DNA damage, ensuring only a healthy pool of oocytes remains. Following this, the oocytes will stay blocked until many years later before luteinising hormone increase jumpstarts their cell cycle to become mature for ovulation.

In oocytes which show no signs of DNA damage, TAp63 is maintained in an inactive dimer conformation. DNA damage triggers the phosphorylation of TAP63 by the tyrosine kinase c-Abl at residues 149, 171 and 289. This provokes the loss of interaction of the terminal inhibitory domain with the transactivation domain. The dimers can then regroup as active tetramers, with high affinity for DNA targets. The C-terminal region of ΔNp63α appears crucial in the inactivation of TAp63 by interacting with and masking the TA domain in the dimer conformation [START_REF] Gebel | P63 Uses a Switch-like Mechanism to Set the Threshold for Induction of Apoptosis[END_REF]. The downside, for TAp63, is that a tetrameric conformation exposes the consensus sites for binding to Itch and MDM2, the E3-ligases that target it for degradation [START_REF] Rossi | The E3 ubiquitin ligase Itch controls the protein stability of p63[END_REF][START_REF] Jiang | SUMO-specific protease 2 in Mdm2-mediated regulation of p53[END_REF].

There is therefore a direct relationship between the capacity of TAP63 to regulate its target genes, and its capacity to be degraded [START_REF] Ying | DNA-Binding and Transactivation Activities Are Essential for TAp63 Protein Degradation[END_REF]).

In the male germline, studies in mice knockouts for TP63 (-/-) reveal an increased number of gonocytes compared to wild type mice, and adult TP63 (+/-) male mice also show more gonocytes with abnormal morphologies [START_REF] Petre-Lazar | The Role of P63 in Germ Cell Apoptosis in the Developing Testis[END_REF]). Interestingly, it is the γ isoform that seems most expressed in the quiescent period following the fetal mitotic phase of gonocytes. More recent work (Beyer et al. 2011) has identified an Nterminal TATP63 isoform (GTATP63) with an extended 5' region which expresses itself specifically in the male germline. This extended region is encoded in one exon (U1) that is expressed from a third promoter located upstream of promoter P1 and P2. This isoform, called GTATP63, results from the integration of a retroviral LTR sequence upstream of the TP63 locus in hominid. In a way similar to the genomic integrity control provided by the TA isoform, cisplatin treatment of cultured cells leads to increased apoptosis when GTATP63 is expressed. This places GTAp63 as both the male equivalent of TAp63's oocyte protection role, and also as a possible testicular tumour suppressor. It is however not clear whether the activation of GTAp63 relies on the same model as the activation of TAp63 in oocyte.

VI. Pathological involvement of p63:

VI.1. p63 associated human syndromes

Just as the homozygous knockouts of TP63 in mice lead to severe, mainly ectodermal, phenotypes, heterozygous mutations in the TP63 gene have been associated with six different syndromes in humans that mainly present ectodermal defects in a general sense. These syndromes combine defects falling into one or several of the following categories: ectodermal dysplasia, orofacial clefting and limb malformation (Figure 8). Ectodermal dysplasia consists in the absence or malformation of ectoderm derived structures such as skin, hair, teeth and sweat glands, Ectrodactyly is a malformation of the hands or feet linked to the absence of one or several digits, and orofacial clefting is the incomplete closure of facial structures during development. bind to its target sequences, whereas those observed in AEC must be the result of defects in interaction between p63 and its partners. Mutations in LMS have been found to affect the TID, which could indicate that poor p63 regulation is involved. However, a regulatory effect of the TID on ΔNP63 isoforms has not been proven as it has been for the TAP63 isoforms [START_REF] Gebel | P63 Uses a Switch-like Mechanism to Set the Threshold for Induction of Apoptosis[END_REF]. As ectodermal p63 functions are dependant on ΔN isoforms, the effect of these mutations could therefore be linked to defects in the recruitment of p63 partners known to interact with the TID such as HDACs [START_REF] Ramsey | Physical association of HDAC1 and HDAC2 with p63 mediates transcriptional repression and tumor maintenance in squamous cell carcinoma[END_REF]. SHFM mutations are mostly found within the DBD, but also within the TID. Altogether, it is more than likely that there is overlap in target genes affected in each of these conditions, with specific differences likely stemming from modified interactions between p63 and its partners.

VI.2. Cancer

Since p63 is related to p53, the most infamous tumour suppressor, there has been a

vast amount of work performed in the last twenty years to characterise its participation in cancer. Unlike TP53, TP63 is very rarely mutated in cancers. However, based on the TCGA pan cancer atlas, TP63 amplification is the most common alteration and happens in 1/3 of lung squamous carcinoma cases, 1/5 of oesophageal adenocarcinomas and 1/6 of cervical squamous carcinomas, head and neck squamous cell carcinomas or ovarian serous cystadenocarcinomas (Figure 9).

VI.2.1. p63 as a biomarker

Many studies have been performed to assess p63 expression in a wide variety of cancers and normal tissues. Often these studies focus on one particular type of cancer, with one particular antibody, therefore overseeing any specific isoform involvement.

Recently, novel standardised work has been done in order to have a clear picture of p63 expression across all cancers [START_REF] Steurer | p63 expression in human tumors and normal tissues: a tissue microarray study on 10,200 tumors[END_REF]. Unsurprisingly, p63 expression is almost exclusively found in tumours derived from normal p63-expressing tissues. These are, for instance, squamous cell carcinomas of all locations, thymic tumours, urothelial carcinomas and basal cell carcinoma. p63 positive cells have however also been occasionally found in tumours deriving from p63 negative tissues of origin, such as gastric cancers, pancreatic cancers, and endometrial and ovarian cancers. In none of these cases was p63 expression associated with a worse outcome. p63 loss in cancers which typically express it, on the contrary, is frequently associated with poor outcome. This has been described in bladder cancer (X. Lin et al. 2014), oesophageal and laryngeal squamous cell carcinomas (Y. [START_REF] Takahashi | Reduced Expression of P63 Has Prognostic Implications for Patients with Esophageal Squamous Cell Carcinoma[END_REF][START_REF] Borba | Expression Profile of P63 in 127 Patients with Laryngeal Squamous Cell Carcinoma[END_REF], Head and neck squamous cell carcinoma (Lakshmanachetty et al. 2019a), and could also be the case in breast cancer (Xiaojuan [START_REF] Wang | P63 Expression in Normal, Hyperplastic and Malignant Breast Tissues[END_REF]. Besides the predictive potential of its loss, p63 is also very useful in differentiating different types of cancer within a tissue, or distinguishing metastatic from local tumour cells. In lungs, for instance, p63 is useful for distinguishing adenocarcinomas (p63 -) from squamous cell carcinomas (Bishop et al. 2012a).

VI.2.2. Tumour suppressor and oncogenic roles of p63

Despite being mainly expressed in cancer cells corresponding to normal p63 expressing cells, there is still a basis for evaluating how p63 can actively contribute to the disease. Indeed, if TP63 is rarely mutated in cancers, TP63 overexpression and/or amplification has been described in squamous cell carcinomas of various origins [START_REF] Sniezek | Dominant Negative P63 Isoform Expression in Head and Neck Squamous Cell Carcinoma[END_REF][START_REF] Massion | Significance of P63 Amplification and Overexpression in Lung Cancer Development and Prognosis[END_REF][START_REF] Deyoung | Tumor-Specific P73 Up-Regulation Mediates P63 Dependence in Squamous Cell Carcinoma[END_REF][START_REF] Hibi | AIS Is an Oncogene Amplified in Squamous Cell Carcinoma[END_REF][START_REF] Tanière | TP53 mutations, amplification of P63 and expression of cell cycle proteins in squamous cell carcinoma of the oesophagus from a low incidence area in Western Europe[END_REF][START_REF] Yamaguchi | Frequent Gain of the P40/P51/P63 Gene Locus in Primary Head and Neck Squamous Cell Carcinoma[END_REF]. In spite of its overexpression, loss of TP63 is often associated with disease progression and poor outcome [START_REF] Barbieri | Loss of P63 Leads to Increased Cell Migration and Up-Regulation of Genes Involved in Invasion and Metastasis[END_REF][START_REF] Tucci | Loss of p63 and its microRNA-205 target results in enhanced cell migration and metastasis in prostate cancer[END_REF]Lakshmanachetty et al. 2019a). Overall, investigated p63 functions in cancer can be broken down into three broad categories: metastasis and the Epithelial Mesenchymal transition (EMT), apoptosis, and proliferation.

• Metastasis and the EMT EMT is a differentiation process promoting cell motility and invasive properties.

These properties are the consequences of specific gene expression program that rely both on transcriptional and post-transcriptional regulators. EMT will be introduced in more in detail in the next chapter.

In Hs578T breast cancer cells which lack TP63 expression, forced expression of ΔNTP63α leads to the inhibition of cellular outgrowth in a matrigel matrix. A similar decrease in invasiveness was observed when ΔNTP63α was expressed in lung cancer cell lines H1299 (nscl carcinoma) and A549 (adenocarcinoma). In this particular study [START_REF] Miranti | Controlling Cell Surface Dynamics and Signaling: How CD82/KAI1 Suppresses Metastasis[END_REF], no effect was noted on EMT markers N-cadherin, E-cadherin and vimentin. Upon transfection with vectors encoding a mutated DBD version of TP63, these effects were not found. The inhibition of invasiveness by ΔNp63α is therefore mediated by its DNA binding capacity. Gene expression profiling identified subsets of genes whose expression is modified by expression of either wild type ΔNp63α, ΔNp63α-(C306R) with a mutated DBD or ΔNp63α-(C526W) with a mutated SAM domain. Some genes affected by wild type ΔNp63α expression overlapped with genes regulated by either one or the other of the mutants. There was no overlap between genes regulated by expression of ΔNp63α-(C306R) or by expression of ΔNp63α-(C526W). ΔNp63α's transcriptional impact can therefore be split three ways: genes that require the DBD and SAM domain to both be functional, genes that solely need the DBD, and genes that solely need the SAM domain.

This last category is likely comprised of genes targetted by other transcription factors which have affinity for p63. This work also identified CD82, a known suppressor of metastasis [START_REF] Miranti | Controlling Cell Surface Dynamics and Signaling: How CD82/KAI1 Suppresses Metastasis[END_REF], as a target of WT ΔNp63α in Hs578T cells as well as in HaCaT,

FaDu and MCF10a cells [START_REF] Wu | ΔNp63α activates CD82 metastasis suppressor to inhibit cancer cell invasion[END_REF].

In bladder cancer ΔNp63α transcriptionally promotes mir-205 which in turn inhibits pro-EMT transcription factors Zeb1 and Zeb2. Knockdown of ΔNP63α in bladder carcinoma cell lines leads to increased mesenchymal markers. SNAI2 (Slug), another pro-EMT transcription factor, is positively controlled in the tested cells, with levels decreasing upon knockdown and increasing upon overexpression of ΔNp63α (Tran et al. 2013c). In prostate cancer, loss of TP63 is accompanied by a decrease in mir-205 levels and an increase in cell migration and metastasis [START_REF] Tucci | Loss of p63 and its microRNA-205 target results in enhanced cell migration and metastasis in prostate cancer[END_REF]). The mir-205 is also a target of ΔNTP63β in highly metastatic oral squamous cell carcinoma (OSCC) SQUU-B cells, giving

ΔNTP63β anti-EMT properties [START_REF] Hashiguchi | Tumor-suppressive roles of ΔNp63β-miR-205 axis in epithelial-mesenchymal transition of oral squamous cell carcinoma via targeting ZEB1 and ZEB2[END_REF]. Indeed, ΔNTP63β expression shifts the phenotype of SQUU-B cells form spindle like to cobblestone-like, promoting their growth and decreasing their migratory capacity. ΔNTP63β expression is also accompanied by an increase in cytokeratins CK5 and CK14 expression and a drop in N-cadherin and Vimentin expression, with no effect on E-cadherin. Vimentin expression is associated with loss of p63 at the tumour front in HNSCC, and is of poor prognosis for the patients [START_REF] Goto | Possible Involvement of ΔNp63 Downregulation in the Invasion and Metastasis of Oral Squamous Cell Carcinoma via Induction of a Mesenchymal Phenotype[END_REF].Conversely, In HS-2 cells, another HNSCC cell line, depletion of ΔNTP63 by siRNA leads to a change in cell shape from polygonal to spindle-like, a decrease in epithelial markers CK5 and CK14 and an increase of mesenchymal markers Vimentin and N-cadherin. These ΔNP63 cells concomitantly gain migratory abilities and decrease their proliferation [START_REF] Goto | Possible Involvement of ΔNp63 Downregulation in the Invasion and Metastasis of Oral Squamous Cell Carcinoma via Induction of a Mesenchymal Phenotype[END_REF]. In transformed and non transformed keratinocytes, ΔNTP63α specific knockdown leads to loss of epithelial markers and a gain of mesenchymal markers. This is echoed by the gain of migratory ability upon ΔNTP63α knockdown in HaCaT-RG, SCC-6 and HEK cells [START_REF] Barbieri | Loss of P63 Leads to Increased Cell Migration and Up-Regulation of Genes Involved in Invasion and Metastasis[END_REF]. In EP156T prostate cancer cells, clones which have undergone EMT lose TP63 expression. Re-expressing TP63 changes their spindle-like morphology to a near-cobblestone appearance and limits their ability to migrate, without completely changing the cells back to an epithelial phenotype. Conversely, depletion of TP63 in epithelial EP156T cells does not provoke a loss of epithelial appearance [START_REF] Olsen | P63 Attenuates Epithelial to Mesenchymal Potential in an Experimental Prostate Cell Model[END_REF].

Mice with a conditional epithelial knockout of TP63 (Krt14 driven Cre, loxp sites around the DBD encoding region of TP63) show increased tumour burden following exposure to 4-Nitroquinoline- when they are depleted in TP63 compared to control shRNA cells. Therefore, there seems to be solid proof defending a protective role of ΔNP63α against metastasis [START_REF] Bergholz | ΔNp63α regulates Erk signaling via MKP3 to inhibit cancer metastasis[END_REF]. According to another study of MCF10a cells, knockdown of ΔNTP63α and β leads to an EMT like phenotypic change, whereas depletion of all isoforms did not effect their epithelial phenotype. Knockdown of TP63 isoforms decreased the cell's proliferation in both cases. Oppositely overexpression of ΔNTP63γ selectively induced EMT in the context of a depletion of all TP63 isoforms. ΔNp63γ therefore seems to have a pro-EMT role, which is counterbalanced by the abundant presence of ΔNp63α (Lindsay et al. 2011a).

ΔNp63γ has also been reported to promote EMT and invasiveness in an HPV E6/E7expressing human foreskin keratinocyte (HFK) cellular model [START_REF] Srivastava | P63 Drives Invasion in Keratinocytes Expressing HPV16 E6/E7 Genes through Regulation of Src-FAK Signalling[END_REF]2018a).

Altogether, most studies indicate that ΔNp63α acts against cellular migration, either by inhibiting the EMT or by specifically modulating actors of cellular adhesion. In certain circumstances however, ΔNp63α could also enhance metastasis. Indeed, when exposed to TGF-β, ΔNp63α promotes metastatic dissemination by inhibiting miR-527 and miR665

(Rodriguez [START_REF] Calleja | ΔNp63α Silences a MiRNA Program to Aberrantly Initiate a Wound-Healing Program That Promotes TGFβ-Induced Metastasis[END_REF]. This could have a big impact in the case of metastasis of ΔNp63α expressing cancer cells to the bone niche which is naturally rich in TGF-β.

ΔNp63β could have a similar role as ΔNp63α in protecting cells from EMT. On the other hand, ΔNp63γ could have pro-EMT properties, but considering this effect has not always been observed [START_REF] Bergholz | ΔNp63α regulates Erk signaling via MKP3 to inhibit cancer metastasis[END_REF], further work needs to be performed.

• Apoptosis p63 has been described as both an activator and a repressor of apoptosis.

In human Hep3B cells (hepatocellular carcinoma), transfection of TATP63α induces apoptosis. Gene expression analysis by microarray showed an induction of death receptor genes as well as genes involved in intrinsic apoptosis triggering. In Saos2 cells (osteosarcoma), TATP63α expression also triggers cell cycle arrest and apoptosis by activation of p21 and transcriptional activation of pro-apoptotic genes. TATP63α sensitizes the cells to CD95, TNF, TRAIL, and mitochondrial induced apoptosis, and thus sensitizes cells to chemotherapeutic agents [START_REF] Gressner | TAp63alpha Induces Apoptosis by Activating Signaling via Death Receptors and Mitochondria[END_REF].

Uveal melanoma cell lines have low TP63 expression and low PERP expression. In particular, OCM-1 cells, which do not express P63 following loss of chromosome 3q, enter apoptosis accompanied by TP53 and PERP upregulation after TP63 transfection. [START_REF] Awais | p63 is required beside p53 for PERP-mediated apoptosis in uveal melanoma[END_REF]. It has been previously described that PERP is a transcriptional target of p63, and that p63 could cooperate with p53 and p73 in its activation following DNA damage [START_REF] Flores | P63 and P73 Are Required for P53-Dependent Apoptosis in Response to DNA Damage[END_REF]. p63 interacts with HDAC1 and HDAC2 in SCC cells. p63 can therefore bind to its target genes and recruit HDACs to remodel the chromatin in a transcriptionpermissive manner. One such target is PUMA [START_REF] Ramsey | Physical association of HDAC1 and HDAC2 with p63 mediates transcriptional repression and tumor maintenance in squamous cell carcinoma[END_REF]. In zebrafish, ΔNp63α

is required for ER-stress induced apoptosis in the epidermis, via transcriptional activation of PUMA and subsequent caspase-3 activation, independently of p73 and p53. [START_REF] Pyati | p63 mediates an apoptotic response to pharmacological and disease-related ER stress in the developing epidermis[END_REF]). p63α could therefore promote apoptosis in ways both with and without cooperation of p73 and p53.

In FLIPs (also anti-apoptotic) and FLIPr increase when ΔNTP63α is overexpressed, including after UV exposure. TP63 null mice have lower caspase-8 levels but higher activated caspase levels and lower FLIP staining in K14 positive cells [START_REF] Borrelli | P63 Regulates the Caspase-8-FLIP Apoptotic Pathway in Epidermis[END_REF]).

In bladder carcinoma 5637 cells, ΔNTP63α knockdown by siRNA leads to an increase in apoptosis and sensitizes the cells to DNA damage induced apoptosis [START_REF] Lee | A dominant negative form of p63 inhibits apoptosis in a p53-independent manner[END_REF] Some HNSCC cells do not express ΔNp63α while still expressing TAp63β. Their survival is proposed to be linked to increased levels of Bcl-2, which has an anti-apoptotic function. In JHU-029 cells, overexpressing Bcl-2 prevents p63 depletion-mediated apoptosis (Rocco et al. 2006) The exact effect of p63 on apoptosis seems to be somewhat cell type-specific. At least some of the variability of effects likely comes from the presence or absence of other p53 family members, as well as potential mutations in p53. Indeed, many apoptotic genes possess response elements which can be targetted by several members of the family, which creates the perfect opportunity for competition. Apoptotic pathways are also notoriously sensitive to the cellular micro-environment, which is often neglected in cellular studies.

• Proliferation, survival and senescence

In oesophageal squamous carcinoma cells, TP63 knockdown leads to a drop in proliferation. In these cells, TP63 levels correlate with Akt activation [START_REF] Ye | p63 regulates growth of esophageal squamous carcinoma cells via the Akt signaling pathway[END_REF].

In murine keratinocytes, the overexpression of ΔNTP63α creates a c-Rel nuclear accumulation by its physical association to ΔNp63α's C-terminus. This protects the cells from growth arrest signals and promotes their proliferation in an NF-kB dependant manner [START_REF] King | The P53 Homologue ΔNp63α Interacts with the Nuclear Factor-ΚB Pathway to Modulate Epithelial Cell Growth[END_REF]. In mice keratinocytes, ΔNp63α is also said to repress p16 ink4a and p19 arf , counteracting cellular senescence [START_REF] Ha | Dysregulated ΔNp63α Inhibits Expression of Ink4a/Arf, Blocks Senescence, and Promotes Malignant Conversion of Keratinocytes[END_REF]. Conditional depletion of TP63 in mice leads to a cellular senescence increase and a reduction of their lifespan [START_REF] Keyes | p63 deficiency activates a program of cellular senescence and leads to accelerated aging[END_REF]. In HNSCC cell lines, the TCF3 transcription factor targets the TP63 gene, stimulating its expression. In turn, the increase in ΔNp63 promotes cellular proliferation. In oesophageal and lung squamous carcinoma patients, TCF3 and TP63 levels are correlated, and increased TCF3 expression has a negative impact on patient survival [START_REF] Xu | E47 Upregulates ΔNp63α to Promote Growth of Squamous Cell Carcinoma[END_REF]). Overall, these different studies place p63 as a signalling node which promotes proliferation and represses senescence, two properties which contribute to cancerous growth.

In summary, there is little doubt that TP63 is important. The many different functions of TP63 can be assigned to 2 categories, control of the genome integrity, functions that are primarily taken care of by TAp63 isoforms and control of epithelial homeostasis and differentiation that are mainly the consequences of ΔNp63 function. Many articles refer to the importance of the different C-terminal isoform of TP63 as important for the function of either the TA or the ΔN but so far no references have described mechanisms that could control the accumulation of any of these C-terminal isoform. In this context my work was mainly aimed at the identification of regulators of the expression of those isoforms.

Maturation of pre-mRNA and its actors

I. Maturation of pre-mRNA

Although similar on many levels, gene organisation and expression varies greatly within the three great orders of life. Bacteria and Archaea generally opt for a streamlined approach with most of their mRNA being collinear to their genome. This allows for a smaller genome size per overall number of transcripts, thus optimising the speed of both replication and transcription. Since they are devoid of a nucleus, there is no physical barrier for access to the ribosomes, and translation into proteins can therefore happen straight after RNA synthesis. In eukaryotes, genes are usually discontinuous, alternating between sequences destined to be included in the mature mRNA, respectively named exons and introns. This was first discovered following the observation of adenovirus DNA-RNA hybrids. Indeed, depending on the maturation of the RNA, different DNA loops were formed to allow for base pairing of the corresponding sequences [START_REF] Chow | An Amazing Sequence Arrangement at the 5' Ends of Adenovirus 2 Messenger RNA[END_REF]. This groundbreaking discovery earned Phillip A. Sharp and Richard J. Roberts the Nobel prize in physiology and medicine in 1993.

The presence of a nucleus in eukaryotes means that translation can never happen immediately after transcription. This opens up an extra window during which the newly synthesised pre-mRNA can sustain a series of modifications to become a mature mRNA.

First, the newly synthesised RNA receives a 5' cap of 7-methyl-guanosine. Once capping is complete, the cap-binding complex signals for the beginning of splicing, the process by which introns are removed and exons are joined together. An endonucleolytic cleavage defines the 3' end of the RNA molecule which subsequently receives a poly-adenosine tail.

These steps happen in a co-transcriptional context [START_REF] Bird | Ribozyme Cleavage Reveals Connections between MRNA Release from the Site of Transcription and Pre-MRNA Processing[END_REF][START_REF] Rigo | The RNA Tether from the Poly(A) Signal to the Polymerase Mediates Coupling of Transcription to Cleavage and Polyadenylation[END_REF][START_REF] Mata | RNA Polymerase II C-Terminal Domain Mediates Regulation of Alternative Splicing by SRp20[END_REF][START_REF] Fong | Stimulatory Effect of Splicing Factors on Transcriptional Elongation[END_REF][START_REF] Furger | Promoter proximal splice sites enhance transcription[END_REF][START_REF] Kornblihtt | Chromatin, Transcript Elongation and Alternative Splicing[END_REF][START_REF] Laurencikiene | RNA editing and alternative splicing: the importance of co-transcriptional coordination[END_REF][START_REF] Listerman | Cotranscriptional Coupling of Splicing Factor Recruitment and Precursor Messenger RNA Splicing in Mammalian Cells[END_REF][START_REF] Mccracken | The C-Terminal Domain of RNA Polymerase II Couples MRNA Processing to Transcription[END_REF][START_REF] Rigo | The RNA Tether from the Poly(A) Signal to the Polymerase Mediates Coupling of Transcription to Cleavage and Polyadenylation[END_REF], and many of the factors involved can interact with the RNA polymerase II, resulting in mutual stimulation and regulation. By interacting with the c-terminal domain (CTD) of the polymerase, splicing factors can promote transcription elongation [START_REF] Fong | Stimulatory Effect of Splicing Factors on Transcriptional Elongation[END_REF]. Likewise, interaction with the RNA-pol II optimises the efficiency of the 3' processing of the pre-mRNA [START_REF] Rigo | The RNA Tether from the Poly(A) Signal to the Polymerase Mediates Coupling of Transcription to Cleavage and Polyadenylation[END_REF], and in turn the definition of the last exon with following cleavage and polyadenylation participates in transcription termination [START_REF] Dye | Terminal Exon Definition Occurs Cotranscriptionally and Promotes Termination of RNA Polymerase II[END_REF].

I.1. Correct recognition of exons and intron boundaries requires specific sequences in specific places

In order to correctly process pre-mRNA into mature mRNA, the cellular machinery must be capable of distinguishing intronic sequences from exonic sequences. At the most basic level, this is ensured by four elements that define the substrates of the biochemical splicing reaction. The first is the 5' splice site (5' SS), consisting of the following consensus sequence: AG/GURAGU, with the GU dinucleotide marking the 5' extremity of the intron.

I.2. The challenge of large introns

As mentioned, setting the boundaries between internal exons and introns requires paired 5' and 3' splice sites. With eukaryotic intron size sometimes reaching beyond 100 kilobasepairs (L. [START_REF] Fedorova | Puzzles of the Human Genome: Why Do We Need Our Introns?[END_REF], how does this machinery efficiently recognize and join two splice sites so far apart?

The first question that has been addressed, is whether the splicing machinery preferentially defines an exon, or an intron first? In vertebrates, exons are usually much smaller than introns, and it therefore seems likelier that splice site pairing happens through exons than through introns. This model of assembly is called "exon definition" [START_REF] Berget | Exon Recognition in Vertebrate Splicing ( ) *[END_REF][START_REF] Robberson | Exon Definition May Facilitate Splice Site Selection in RNAs with Multiple Exons[END_REF]) (Figure 11). Sequences presented in (Figure 10)

are sufficient in the case of internal exons, however the recognition of the first exon and the last exon require other elements. Indeed, the first exon is flanked by the 7-methyl guanosine cap on one side and a 5'SS on the other. The cap is recognised by the capbinding complex (CBC) which in turns stimulates the formation of the ribonucleoprotein particles required for splicing and stabilizes them at the 5'SS [START_REF] Lewis | A Nuclear Cap-Binding Complex Facilitates Association of U1 SnRNP with the Cap-Proximal 5' Splice Site[END_REF]. The last exon is flanked by a 3'SS and a poly(A) site. The presence of the terminal exon's 3'SS has a positive effect on cleavage and polyadenylation thanks to interactions across the exon [START_REF] Nesic | Upstream Introns Influence the Efficiency of Final Intron Removal and RNA 3'-End Formation[END_REF].

In cases where introns are relatively short, it is thought that splice sites are paired through the intron, referred to as "intron definition". Ancient eukaryotes such as yeast or drosophila have a majority of short introns (Goguel et al. 1993, Talerico andBerget 1994) and subsequently rely more on intron definition, whereas more recently evolved eukaryotes such as mammals have an abundance of long introns and thus heavily rely on exon definition. Both of these modes can however co-exist in any organism, and even within a single pre-mRNA. Regardless of intron size, initial exonic or intronic definition, the splicing reaction will always rely on the pairing of a 5'SS and a 3'SS through the processed intron. (Figure 11)

5'exon 3'exon

To deal with long introns, several processes have been described. Firstly, in drosophila, a ratchet-like mechanism has been described whereby large introns are excised in a multi-step process. This recursive splicing process requires the presence of special ratcheting points (RPs) which act as both acceptors and donors. [START_REF] Hatton | Generation of Alternative Ultrabithorax Isoforms and Stepwise Removal of a Large Intron by Resplicing at Exon-Exon Junctions[END_REF][START_REF] Burnette | Subdivision of Large Introns in Drosophila by Recursive Splicing at Nonexonic Elements[END_REF]. In mammals, RPs are very rare, but the recursive mechanism could still be important while relying on different sequences, notably dispersed 3'SS and BPS dispersed throughout the intron [START_REF] Hayashi | Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs[END_REF]X.-O. Zhang et al. 2018). The second proposed mechanism for dealing with large introns is based on the presence of complementary sequences which favour the creation of stemloop structures throughout the intron. In mammals, the frequency of such elements is much higher than in drosophila, with most of the sequences being SINE or LINE elements.

In primates, roughly 80% of the hairpin-forming structures were Alu-sequences [START_REF] Shepard | The Peculiarities of Large Intron Splicing in Animals[END_REF]. It is thus proposed that as retrotransposition contributed to expanding genomes, the repeated elements they introduced enabled a new and efficient means of regrouping 5' and 3' extremities of large introns, thus alleviating the selective pressure on RP sequences.

I.3. The spliceosome: where the magic happens

The splicing process requires the dynamic positioning and readjustment of 5 small nuclear RNAs (snRNAs) and roughly 100 proteins. These constitute the spliceosome. The five snRNAs are commonly named U1, U2, U4, U5 and U6. These interact with Sm, or L-Sm proteins to form the small ribonucleoprotein (snRNP) complexes of the same name [START_REF] Will | Spliceosome Structure and Function[END_REF]. In some rare cases, the U1 and U2 snRNPs are substituted by snRNPs U11 and U12 resulting in a complex called the "minor" spliceosome, which mainly recognises non-canonical intronic AT/AC dinucleotides [START_REF] Steitz | Where in the Cell Is the Minor Spliceosome?[END_REF]). Here, I will focus only on the description of the major spliceosome. Far from being a static entity, the spliceosome goes through many different states as the pre-mRNA splicing reaction progresses, through recruitment and release of partners and structural changes. These steps make up the splicing cycle as represented in Figure 12, which I will describe next.

Before any splicing reaction can occur, the different required sequences are brought together as the earliest form of the spliceosome: the E-complex. To form the E complex (E for early), the first step is the association of U1 snRNP with the 5'SS, which requires basepairing between the 5'end of U1snRNA and the exonic and intronic sequences of the 5' splice site. Besides binding of U1 snRNP to the 5' splice site, the E-complex also involves association of other proteins with the 3' splice site. Splicing Factor-1 (SF1)

recognises the BPS, while U2AF subunits U2AF 65 and 35 recognize the polypyrimidine tract (Py-tract) and the 3'SS AG respectively. The assembly of these factors allows the successful bridging of the intron. The next event is the association of U2 snRNP with the branch point which is mediated through displacement of U2AF and SF1 generating the stable pre-spliceosome complex also referred to as the A complex. The U2snRNA-premRNA interaction requires basepairing between conserved nucleotides of U2snRNA and the branch point sequence leading to the formation of a helix in which the branch point adenosine bulges out. To create a complete spliceosome, the pre-assembled U4/U6-U5

tri-snRNP adds itself to the structure, and the pre-B complex is thus formed. The destabilisation of U1 basepairing to the 5'SS and subsequent basepairing of U6 to an intronic subset of 5'SS nucleotides allows the complex to rearrange itself, gaining splicing competence. This process requires RNA helicase activity, both to separate U1 from the pre-mRNA and to dissociate U4 from its base-paired partner U6. Prp28 insures the first step, eliminating U1 while Brr2 insures the second. The extensive basepairing between U6snRNA and U4snRNA is thus destabilised allowing the 3'end of U6snRNA to basepair with the 5'end of U2snRNA, still associated with the branch point. Concomitantly, U5snRNA basepairing with terminal nucleotides of the 5' exon is enhanced. The newly rearranged, catalytically active, spliceosome is now the B act complex. ATP dependant changes in folding bring part of U2 closer to U6 and the bound pre-mRNA to form the catalytic site. The U1 and U4 snRNAs dissociate during the transition between the pre-B and the B act complex. Thus, U1snRNA is involved in the early recognition of the 5'SS whereas U4snRNA is involved in the delivery of U6snRNP to the spliceosome and prevents premature activation of U6snRNA. Once everything is set for the branching reaction, the C complex is formed. There, the 5' exon is cleaved by a nucleophilic attack of the BP adenosine's free 2' hydroxyl group on the 5'SS guanosine's phosphate group, forming a looped structure known as the lariat (Figure 12). The first step of the splicing

A B

reaction is then complete, with the spliceosome still attached to the 5' exon and the intermediate lariat. This intermediate complex is referred to as C*. Additional structural rearrangements are required to proceed to the second step. U5snRNA, already in interaction with the intronic part of the 5'SS, allows the second nucleophilic attack of the free hydroxyl of the 5'exon on the 3'SS guanosine's phosphate group, leading to the fusion of the two exons together (Figure 12B). This completes the formation of the P complex.

Following the second step of the splicing, the snRNPs remain associated with the lariat whereas the exon-exon product is released. Finally the snRNPs are released from the lariat, which is debranched and degraded. With two exons now joined together, and a released lariat, the actors of the spliceosome may then be recycled for reuse in subsequent splicing reactions.

I.4. Cleavage and polyadenylation

With the 5' end of the pre-mRNA having received a 7-methyl guanosine cap, and the sequence contents of the future mRNAs established through splicing, the 3' end also needs to be taken care off. With the exception of cell-cycle regulated histone mRNAs, which adopt a 3' terminal stem loop structure after cleavage [START_REF] Gallie | The Histone 3′-Terminal Stem-Loop is Necessary for Translation in Chinese Hamster Ovary Cells[END_REF], the 3' extremity of mRNAs is matured through two sequential reactions: an The sequences defining cleavage and polyadenylation sites are loosely defined.

Because of the great length of eukaryotic genes, this means that besides the regular sets of PAS a pre-mRNA possesses, it usually also has a number of cryptic PAS. In case of recognition, these would lead to premature cleavage and polyadenylation. To prevent this, an additional activity of U1 snRNP called telescripting is to bind to U-rich regions the RNA independently from the spliceosome, and acts as a protector against premature cleavage and polyadenylation [START_REF] Berg | U1 SnRNP Determines MRNA Length and Regulates Isoform Expression[END_REF][START_REF] So | A Complex of U1 SnRNP with Cleavage and Polyadenylation Factors Controls Telescripting, Regulating MRNA Transcription in Human Cells[END_REF].

The core machinery responsible for the cleavage and polyadenylation of pre-mRNA is exclusively constituted of proteins in mammals. Here, I will briefly describe the eight main actors presented in .

interactions with the other 3'end processing factors, with a notable interaction between its C-terminal domain and hFip1 and CPSF-160.

 Poly(A) binding protein (PABP): PABP are involved in the reaction while the addition of the poly(A) tail occurs and are essential for determining the length of the poly(A) tail. They also increase the efficiency of the polyadenylation reaction. PABP binds to adenine residues via strongly conserved residues in its two RRMs.

 Symplekin: Symplekin likely acts as the main scaffold protein of the 3' end processing complex. It stably binds to both CPSF and CstF.

The precise order in which the described actors of cleavage and polyadenylation come together as a stable cooperative complex on the pre-mRNA is not fully elucidated.

We do however have many clues to put the puzzle together. First of all, CF Im has a very high affinity for RNA and promotes the assembly of the complex [START_REF] Rüegsegger | Human Pre-MRNA Cleavage Factor Im Is Related to Spliceosomal SR Proteins and Can Be Reconstituted in Vitro from Recombinant Subunits[END_REF]. It is therefore thought that CF Im could be the first factor in place. Purification of splicing complexes led to the co-identification of both CF Im and CPSF. The two factors must therefore bind the RNA early on, which would make CPSF the second factor in place, bound to the hexanucleotide [START_REF] Rappsilber | Large-Scale Proteomic Analysis of the Human Spliceosome[END_REF]. Then comes CstF, fixing itself to the DSE, and CF IIm joins in too [START_REF] Neve | Cleavage and polyadenylation: Ending the message expands gene regulation[END_REF]. PAP recruitment is ensured by CPSF [START_REF] Kaufmann | Human Fip1 Is a Subunit of CPSF That Binds to U-Rich RNA Elements and Stimulates Poly(A) Polymerase[END_REF]). The whole complex stabilises itself, with extra help from symplekin.

Once the complex is in place, the catalysis of the endonucleotidic reaction can begin. This is likely performed by CPSF73 [START_REF] Ryan | Evidence That Polyadenylation Factor CPSF-73 Is the MRNA 3' Processing Endonuclease[END_REF]. The resulting 3'-OH of the upstream RNA can then be polyadenylated in a subsequent step ensured by CPSF, PAP and PABN1. Around 250 adenine residues are usually added and covered by Poly(A) binding protein nuclear-1 (PABPN1). When the poly(a) tail reaches its final length, the interaction between CPSF and the PAP ceases, marking the end of the reaction. On the other side of the cleavage, the downstream RNA fragment has a free 5' phosphate extremity and therefore gets degraded by Xrn2 (S. [START_REF] West | Human 5′ → 3′ Exonuclease Xrn2 Promotes Transcription Termination at Co-Transcriptional Cleavage Sites[END_REF].

This degradation participates in the recycling of of the RNA pol II.

II. Alternative splicing, alternative cleavage and polyadenylation, and their regulation II.1. Alternative splicing

Since the generalisation of high throughput RNA sequencing, it has been estimated that more than 95% of Human genes are expressed as two or more mRNA isoforms [START_REF] Ben-Dov | Genome-Wide Analysis of Alternative Pre-MRNA Splicing *[END_REF]. Alternative promoters, 5' and 3' splice sites and alternative cleavage and polyadenylation sites can be used in combination. The majority of described isoforms stem from cassette exon skipping, 3' or 5' alternative splice site selection, mutually exclusive exons or retained introns. (Figure 15). As mentioned previously, the likelihood for an exon to be included in the mature mRNA is firstly defined by the strength of its splice sites. An exon with weak 5' and 3' splice sites followed by an exon with stronger splice sites is likely to be skipped. In a similar way, two 5' or two 3' splice sites of differing strengths may be close together, generating alternative donor or acceptor sites. At the most basic level, alternative splicing is therefore a stochastic process. The functions of alternative mRNA isoforms frequently diverge, and evolution selected strategies to control splicing in a direction that is favourable to the cell and ultimately to the organism. The regulation of these splicing events can be responsive to outside stimuli, such as signalling molecules or biophysical and biochemical changes, they can also be tissue-specific or change throughout the development and differentiation of a given tissue. Alternative splicing patterns are therefore adaptive to physiological conditions, allowing an organism to respond to changes in the environment by determining how its genome is expressed. Of course, there are many more alternative splicing events than there are different splicing regulators. Most splicing regulators are also not strongly tissue-specific. This means that for the most part, there is no singular responsibility between one regulator and one alternative splicing event. Rather, the outcome of alternative splicing is the net result of all the different activators and repressors bound to the pre-mRNA, but also other contextual elements such as changes in the RNA structure, chemical modifications or mutations in its sequence. On top of this, since 80% of pre-mRNA splicing happens co-transcriptionally [START_REF] Girard | Post-Transcriptional Spliceosomes Are Retained in Nuclear Speckles until Splicing Completion[END_REF], the speed of transcription can have a direct impact on which splicing sites get used. Faster transcription can favour the use of stronger splice sites, whereas a slower transcription rate gives a chance to weaker splice sites to be used. Epigenetic modifications can also influence the outcome of splicing both directly through chromatinadaptor complexes, or indirectly by changing the local nucleosome distribution. Indeed, an .

increase in nucleosomes often leads to RNA polymerase II pauses, which create opportunities for splicing complexes to initiate [START_REF] Luco | Epigenetics in alternative pre-mRNA splicing[END_REF].

The three previously mentioned splicing sequences, namely the 5'SS, BPS and the 3'SS, to which we could add the polypyrimidine tract and the AGEZ, are required for correct spliceosome assembly, but they are not sufficient to determine the outcome of alternative splicing. These sequences are degenerate, meaning that sequence variations create strong splice sites and weak splice sites depending on their relative affinity to the splicing machinery. The strength of the splice sites determines how likely an exon is to be included in the mature mRNA. Some exons are surrounded by splice sites so strong that they are constitutively included, while others are often skipped. The strength of the splice site, however, is not the be-all and end-all of exon use as there are additional sequences which recruit proteins capable of stimulating or inhibiting spliceosome assembly. These sequences are classed as intronic or exonic splicing silencers or enhancers, depending on their location and function (Figure 16). The existence of splicing enhancers and silencers opens up a whole new possibility.

Beyond dictating where exons and introns start and finish, they also make up a splicing code [START_REF] Fu | Towards a Splicing Code[END_REF][START_REF] Baralle | The Splicing Code[END_REF] by which pre-mRNA molecules can be matured into several different mRNAs by including or omitting different exons. This models of splice site selection places proteins as the main regulators. However, RNA itself could be RNA-binding motif (RBM) proteins, arginie-serine-rich (SR) proteins (including SRSF2) and heterogeneous nuclear ribonucleoproteins (hnRNPs) bound to exonic or intronic regulatory elements can promote or prevent the recognition of the 5' splice site by the U1 small nuclear ribonucleoprotein (snRNP) or of the 3' splice site by SF1, U2AF2, U2AF1, or U2 snRNP, thus affecting splice site choices and therefore alternative splicing decisions. (from [START_REF] Bonnal | Roles and mechanisms of alternative splicing in cancer -implications for care[END_REF]. .

S R S F 1 / S R S F 2 U2AF1 U2AF2 SF1 GURAGU YNURAY YYY AG
involved in this regulation and there is emerging evidence of a larger role of non-coding RNA in exon selection (Y. [START_REF] Yu | Dynamic Regulation of Alternative Splicing by Silencers That Modulate 5′ Splice Site Competition[END_REF][START_REF] Khanna | Regulation of alternative splicing by short non-coding nuclear RNAs[END_REF].

II.2. Alternative cleavage and polyadenylation

A large proportion of pre-mRNAs actually possess several sets of cleavage and polyadenylation sites, which can be alternatively used or skipped. When several polyadenylation sites are present in an RNA, the most distal sequence is usually the strongest [START_REF] Moucadel | Beyond the 3′ end: experimental validation of extended transcript isoforms[END_REF]). In the case where a proximal CPA site is used, the matured RNA will be devoid of all downstream exons or sequences following cleavage.

There are conceptually four different forms of alternative cleavage and polyadenylation (Figure 17):

• Tandem 3'UTR APA, when the APA sites are located within the same terminal exon, generating 3'UTRs of different lengths but sharing some identical sequences.

• Alternative terminal exon APA: in this case the different APA sites are located on different terminal exons. This influences both the length of the coding sequence and the nature of the 3'UTR of the transcript.

• Intronic APA: this mode relies on the recognition of cryptic ASA sites within introns, resulting in an extension of the otherwise non-terminal exon which precedes them, and a novel 3'UTR.

• Internal exon APA: an APA site is used within an internal exon, making it the terminal exon. A shorter transcript is thus created with a novel 3'UTR. If the exon is a coding exon, this may lead to non-stop decay [START_REF] Vasudevan | Non-Stop Decay-a New MRNA Surveillance Pathway[END_REF].

The

II.3. Regulatory factors

The maturation of pre-mRNA by alternative splicing and alternative cleavage and polyadenylation offers the possibility for products from a single gene to have differing, context specific, functions or regulations. For this to work, however, the splicing machinery must receive specific instructions at the right place and time. In higher vertebrates, there are only a handful of tissue specific RNA binding proteins, and so it is proposed that the regulation is ensured by a combinatorial effect of varying levels of ubiquitous proteins.

These signals are what constitute the cellular "splicing code".

In a broad sense, any protein which possesses a domain capable of interacting with RNA, and therefore forming RNPs, can be classed as an RBP. The interaction with an RNA sequence or structure is generally provided by a globular protein domain of which there are three main kinds: the RRM, or RNA recognition motif, the KH or K homology domain, and the DEAD box helicase domains. Not all interactions between RNAs and RBPs are sequence or structure specific, a good example of these being the components of the exon junction complex. In this example, a complex of four subunits (eiF4A3, MAGOH, Y14 and BTZ) is recruited 20 to 24 nucleotides upstream of an exon-exon junction directly after splicing has occurred (Le [START_REF] Hir | The Spliceosome Deposits Multiple Proteins 20-24 Nucleotides Upstream of MRNA Exon-Exon Junctions[END_REF]. This recruitment is performed by CWC22, an abundant constituent of the active spliceosome, by interaction with the eiF4A3 subunit [START_REF] Steckelberg | CWC22 Connects Pre-MRNA Splicing and Exon Junction Complex Assembly[END_REF][START_REF] Boehm | Exon Junction Complexes Suppress Spurious Splice Sites to Safeguard Transcriptome Integrity[END_REF].

The combination of RBPs bound to an RNA is modified as the pre-mRNA matures to an mRNA and is exported to the cytoplasm. Therefore, the RNP compositions evolve throughout the mRNA's life, and can therefore impact many different functions: capping, pre-mRNA maturation, nuclear export and mRNA localisation, protection or degradation, and even modulating RNA function through covering of regulatory portions, be they in UTRs or in coding sequences, and aiding in the apposition of epitranscriptomic marks. We may also consider the functional impact of the interaction between an RNA and a protein in the opposite way: some classes of RNA, such as lncRNAs, could just as well be regulating as being regulated by their cognate binding proteins.

In the context of this introduction, I shall focus on the two main families of RNA binding proteins involved in pre-mRNA splicing regulation : SR and hnRNP proteins.

II.3.1. The SR protein family

SR proteins are usually activators of splicing, with a particular affinity for ESEs. The

II.3.2. The hnRNP protein family

Members of the hnRNP family (heterogeneous nuclear RiboNucleoProtein) owe their RNA binding capabilities to three sorts of domains: an RRM domain, in most cases, and sometimes either K Homology domains (KH) or Arginine-Glycine-Glycine domains (RGG). HnRNPs are present throughout the nucleus, ready to bind to nascent transcripts.

They are capable of binding in a non-specific manner to RNA, but in the case of splicing regulation, they usually bind with high affinity to splicing silencer sequences.

II.4. Functional and pathological results of aberrant splicing

There are several ways in which mutations can be pathological by modifying splicing. (Figure 18). First of all, mutations can happen within a cis acting region. This can effect either the core splicing sequences or the regulatory elements. Mutations in cis These mutations therefore result in a loss of function of the mutated allele. There can also be trans effects of a mutation if it directly disrupts a splicing actor, either by direct mutation of the gene enconding the factor or by mutation affecting its expression level. In this case, the resulting disease can be due to the accumulation of many aberrant splicing events. In

II.5. Therapeutic strategies to modulate pathological splicing

Of the known genetic causes of disease, 15% are attributed to pre-mRNA splicing defects [START_REF] Krawczak | The Mutational Spectrum of Single Base-Pair Substitutions in MRNA Splice Junctions of Human Genes: Causes and Consequences[END_REF]. In some cases of deleterious splicing, therapeutic action is possible. Antisense oligonucleotides targetting regulatory regions, or directed against aberrant splice sites, can lead to either the skipping of a particular splicing event or the degradation of a pathological mRNA. Therapeutic splicing modulation in pathological cells can be achieved by either fine-tuning the activity of splicing regulators or by precisely targeting a single spliced isoform. A good example of a splicing modulator is Spliceostatin A [START_REF] Corrionero | Reduced Fidelity of Branch Point Recognition and Alternative Splicing Induced by the Anti-Tumor Drug Spliceostatin A[END_REF]. This drug inhibits pre-mRNA splicing in vitro and in vivo by binding to SF3b, a protein component of U2 small nuclear ribonucleoprotein (snRNP), which is essential for recognition of the pre-mRNA branch point (Figure 12A). However, only a fraction of splicing events are affected. Differences in base-pairing strength with U2 snRNA in this region lead to different sensitivities of 3′ splice sites to the drug. Through splicing modulation, Spliceostatin A impacts the expression of key regulators of cell division, including cyclin A2 and Aurora A kinase, which can contribute to the anti-proliferative activity of the drug.

Considering the importance of phosphorylations in activating and repressing proteins involved in splicing regulations, several kinase-targetting drugs have been developed [START_REF] Ohe | Modulation of Alternative Splicing with Chemical Compounds in New Therapeutics for Human Diseases[END_REF]. NB-506, for example, inhibits the phosphorylation of SF2/ASF by topoisomerase I, preventing the splicing of pre-mRNAs containing SF2/ASF target sequences [START_REF] Pilch | Specific Inhibition of Serine-and Arginine-Rich Splicing Factors Phosphorylation, Spliceosome Assembly, and Splicing by the Antitumor Drug NB-506[END_REF]. Another example, TG003, inhibits the kinase activity of Clk kinases, potent regulators of the SR proteins [START_REF] Muraki | Manipulation of Alternative Splicing by a Newly Developed Inhibitor of Clks *[END_REF]. This drug has shown therapeutic potential in the treatment of cystic fibrosis by limiting the inclusion of a pseudo-exon [START_REF] Shibata | Mechanism-Based Personalized Medicine for Cystic Fibrosis by Suppressing Pseudo Exon Inclusion[END_REF], in Duchenne muscular dystrophy to promote the skipping of a mutated exon [START_REF] Nishida | Chemical Treatment Enhances Skipping of a Mutated Exon in the Dystrophin Gene[END_REF] and in anhidrotic ectodermal dysplasia with immunodeficiency, also by preventing the inclusion of a deleterious pseudoexon [START_REF] Boisson | Rescue of Recurrent Deep Intronic Mutation Underlying Cell Type-Dependent Quantitative NEMO Deficiency[END_REF]).

These small molecules often lack specificity, and their exact mechanisms of action are not always well understood. RNA-based therapeutics offer the potential to target any molecule [START_REF] Kole | RNA Therapeutics: Beyond RNA Interference and Antisense Oligonucleotides[END_REF][START_REF] Havens | Splice-switching antisense oligonucleotides as therapeutic drugs[END_REF]. Short antisense oligonucleotides targetting splicing are referred to as splice switching antisense oligonucleotides (SSOs). Depending on the chosen target location, they can either inhibit RNA:protein or RNA:RNA interactions. Thanks to their high specificity, SSOs can be used to negatively impact the inclusion of a sequence by binding locally to a splice site or a enhancer/silencer. This is useful to prevent the use of cryptic splice sites, for suppressing or introducing frameshifts and premature stop codons, or to direct splicing towards beneficial rather than detrimental isoforms. SSOs can also be directed against silencer sequences, which promotes the use of specific exons. This can be used as a compensatory mechanism in the case of mutations that weaken important splice sites [START_REF] Havens | Splice-switching antisense oligonucleotides as therapeutic drugs[END_REF]. Beyond splicing, antisense oligonucleotides are also potent in targetting pathological cleavage and polyadenylation events [START_REF] Vorlová | Induction of Antagonistic Soluble Decoy Receptor Tyrosine Kinases by Intronic PolyA Activation[END_REF]).

The most successful example of the power of sequence targetted therapy is in the treatment of spinal muscular atrophy (SMA). SMA is an autosomal-recessive monogenic disease caused by mutations or deletions in the SMN1 gene [START_REF] Lefebvre | Identification and Characterization of a Spinal Muscular Atrophy-Determining Gene[END_REF].

Patients present with motor neuron degeneration that leads to progressive muscle weakness and, in severe cases, respiratory failure and death. Disease severity is inversely correlated to the abundance of functional SMN [START_REF] Mailman | Molecular Analysis of Spinal Muscular Atrophy and Modification of the Phenotype by SMN2[END_REF][START_REF] Wirth | Mildly Affected Patients with Spinal Muscular Atrophy Are Partially Protected by an Increased SMN2 Copy Number[END_REF]), a ubiquitously expressed protein involved in pre-mRNA splicing, snRNP biogenesis, transcription, stress response, apoptosis and axonal transport. In theory, the lack of SMN1 gene expression can be compensated by expression of its paralogous gene SMN2.

SMN2's coding sequence diverges from SMN1 by a single translationally silent substitution in exon 7 (c.840C>T). Unfortunately, this substitution is sufficient to provoke skipping of exon 7 in most SMN2 transcripts (Figure 19) and leads to the production of a truncated mRNA, SMN2Δ7. SMNΔ7 codes for an SMN protein isoform that is unstable and does not .

function in the same manner as the full-length SMN isoform. Therapeutic efforts have therefore focused on correcting this splicing event.

An antisense oligonucleotide derivative, SPINRAZA (nusinersen), has been approved for use on pre-symptomatic newborns and young children. Its binding to SMN2 pre-mRNA removes hnRNPA1 from the cis-RNA silencer flanking the 5'SS of SMN2's exon 7. The exon 7 can therefore be included, leading to the production of functional SNM protein [START_REF] Hua | Enhancement of SMN2 Exon 7 Inclusion by Antisense Oligonucleotides Targeting the Exon[END_REF][START_REF] Beusch | Tandem hnRNP A1 RNA recognition motifs act in concert to repress the splicing of survival motor neuron exon 7[END_REF][START_REF] Haché | Intrathecal Injections in Children With Spinal Muscular Atrophy: Nusinersen Clinical Trial Experience[END_REF][START_REF] Foust | Rescue of the Spinal Muscular Atrophy Phenotype in a Mouse Model by Early Postnatal Delivery of SMN[END_REF][START_REF] Finkel | Nusinersen versus Sham Control in Infantile-Onset Spinal Muscular Atrophy[END_REF][START_REF] Mercuri | Nusinersen versus Sham Control in Later-Onset Spinal Muscular Atrophy[END_REF].

Though nusinersen provides an appreciable therapeutic gain, it requires frequent injections, and merely delays the onset of the disease. Other therapies are therefore being developed. RISDIPLAM (Evrysdi), a small molecule targetting SMN2 splicing has now also been authorised for clinical use. RISDIPLAM binds to the 5'SS of SNM2's exon 7 and promotes its inclusion. Studies performed on SNM-C5, a small molecule similar to RISDIPLAM, suggest that the stimulation of exon 7's inclusion is based on the stabilisation of a free adenine at its 5'SS, increasing the strength of the splice site by promoting binding to the U1 snRNP [START_REF] Campagne | Structural Basis of a Small Molecule Targeting RNA for a Specific Splicing Correction[END_REF].

III. Focus on the polypyrimidine tract binding protein (PTBP1) hnRNP

During my thesis work, one particular hnRNP has waltzed into the limelight: PTBP1.

The PTB family actually has three members sharing strong sequence conservation: the almost ubiquitous PTBP1, PTBP2, expressed primarily in neuronal tissue, and PTBP3, expressed in some immune cells. Binding of PTB proteins to RNA is made possible by the presence of four RRM domains, two of which are capable of interacting with each other, creating a loop like structure. As their name suggests, they share the capacity to bind to pyrimidine-rich sequences, with a proposed consensus of repeated UCUU or CUCUCU sequences (Oberstrass et al. 2005; Upadhyay et Mackereth 2020). Of course, the simplicity and in vivo abundance of these consensus sequences make it very difficult to accurately predict where PTB proteins will actually bind.

The abundance of PTBP1 in cells is determined through an elegant NMDdependant feedback loop. By exclusion of exon 11, the PTBP1 RNA suffers a frame shift which subsequently leads to its degradation by NMD. In Xenopus laevis, exon 11 inclusion is stimulated by ESRP1 [START_REF] Méreau | A Posttranscriptional Mechanism That Controls Ptbp1 Abundance in the Xenopus Epidermis[END_REF]. When PTBP1 levels reach a certain threshold, the PTBP1 protein binds to PTBP1 pre-mRNA and represses the exon 11, leading to a decrease in its own expression [START_REF] Wollerton | Autoregulation of Polypyrimidine Tract Binding Protein by Alternative Splicing Leading to Nonsense-Mediated Decay[END_REF]). In a similar manner, PTBP1 negatively controls PTBP2 expression by promoting the skipping of a cassette exon, leading to a frameshift and subsequent NMD (Boutz, Stoilov, et al. 2007;[START_REF] Makeyev | The MicroRNA MiR-124 Promotes Neuronal Differentiation by Triggering Brain-Specific Alternative Pre-MRNA Splicing[END_REF][START_REF] Spellman | Crossregulation and Functional Redundancy between the Splicing Regulator PTB and Its Paralogs nPTB and ROD1[END_REF].

PTBP1 levels are also subjected to regulation by miRNAs. During neuronal differentiation, mir-124 inhibits PTBP1 which allows PTBP2 expression [START_REF] Makeyev | The MicroRNA MiR-124 Promotes Neuronal Differentiation by Triggering Brain-Specific Alternative Pre-MRNA Splicing[END_REF]). During myoblast differentiation, muscle-specific microRNA miR-133 expression is associated with the reduced level of PTBP1 (Boutz, Chawla, et al. 2007). In human pancreatic islets responding to the presence of high levels of glucose, mir-133a is induced leading to lower PTBP1 levels and lower insulin biosynthesis rates by the effect of mRNA destabilization (C. [START_REF] Wang | Polypyrimidine Tract-binding Protein (PTB) Differentially Affects Malignancy in a Cell Line-dependent Manner[END_REF].

PTBP1 is involved in many steps during the life of its mRNA targets with described roles in their translation, splicing, stability and localisation [START_REF] Sawicka | Polypyrimidine-Tract-Binding Protein: A Multifunctional RNA-Binding Protein[END_REF]). These different functions are possible since PTB proteins possess both a nuclear localisation signal (NLS) and a nuclear export signal (NES) and can therefore shuttle between the nucleus and the cytoplasm [START_REF] Kamath | Nucleocytoplasmic Shuttling of Polypyrimidine Tract-binding Protein Is Uncoupled from RNA Export[END_REF]. PTBP1 protects transcripts from NMD degradation by displacing the RNA helicase UPF1 from its target sites. This inhibition is dependant on the enrichment of high affinity PTBP1 binding sites within a mRNA's 3'UTR, which enables protection even in the case of multiple UTF1 binding events [START_REF] Fritz | The RNA-Binding Protein PTBP1 Promotes ATPase-Dependent Dissociation of the RNA Helicase UPF1 to Protect Transcripts from Nonsense-Mediated MRNA Decay[END_REF].

III.1. Role in translation

III.2. PTBP1's role in pre-mRNA splicing

During pre-mRNA splicing, PTBP1 often enters into competition with other regulatory proteins. In alpha-tropomyosin, PTBP1 competes with RBM4 for the binding to a CU-rich element. PTBP1 binding to this regulatory element inhibits the inclusion of muscle-specific exons in alpha-tropomyosin mRNA, whereas RBM4 stimulates them(J.-C. Lin et Tarn 2005, 4). PTBP1 competes with TIA to promote the skipping of exon 12 of Myosin Phosphatase Target Subunit 1 (MYPT1) by binding to a U-rich element downstream of the exon's 5'SS. Skipping exon 12 is one of the events necessary for smooth muscles to shift from a short-phasic to a fast-phasic contractile phenotype [START_REF] Shukla | Competition of PTB with TIA proteins for binding to a Urich cis-element determines tissue-specific splicing of the myosin phosphatase targeting subunit 1[END_REF]. Competition between PTBP1 and TIA1 has also been described for the maturation of Fas (Apo-1/CD95) pre-mRNA. Fas can exist as a membrane bound receptor which triggers apoptosis upon binding of its ligand, or as a soluble protein which inhibits apoptosis by neutralizing Fas-ligand. The soluble form is produced following skipping of exon 6. PTBP1 inhibits the inclusion of this exon by binding to an exonic silencer and inhibiting the fixation of U2AF to the upstream polypyrimidine tract [START_REF] Izquierdo | Regulation of Fas Alternative Splicing by Antagonistic Effects of TIA-1 and PTB on Exon Definition[END_REF]. PTBP1 also represses the splicing of c-src's exon N1 onto exon 4 by preventing the binding of U2AF to the exon 4, which stalls the assembly of the prespliceosomal E complex [START_REF] Sharma | Polypyrimidine Tract Binding Protein Blocks the 5′ Splice Site Dependent Assembly of U2AF and the Prespliceosomal E Complex[END_REF].

So far, there have been three described modes by which PTBP1 can repress the inclusion of an exon:

• PTBP1 can bind its target pre-mRNA near the branch point, in the polypyrimidine tract, thus hindering its recognition by U2AF65 and the subsequent assembly of the splicing machinery. Good examples of this mode of action are the fibronectin EIIIB exon [START_REF] Norton | Polypyrimidine tract sequences direct selection of alternative branch sites and influence protein binding[END_REF]) and exons 2 and 3 of the α-tropomyosin [START_REF] Gooding | MBNL1 and PTB Cooperate to Repress Splicing of Tpm1 Exon 3[END_REF][START_REF] Mullen | Alpha-Tropomyosin Mutually Exclusive Exon Selection: Competition between Branchpoint/Polypyrimidine Tracts Determines Default Exon Choice[END_REF]. Additional factors other than U2AF65 such as Celf1 could also antagonize the PTBP1 repressive interaction (R. [START_REF] Spellman | Regulation of Alternative Splicing by PTB and Associated Factors[END_REF]. (Figure 20. The action of PTBP1 in blocking exon and intron definition is a way to control many splicing fates. By binding to intronic or exonic elements, it can prevent the assembly of a productive spliceosome at the targetted exon, and can also prevent the exon definition by disrupting SR protein-dependent interactions across it.

A-B)

AGGURAGU

As illustrated by the above examples, PTBP1 is one of the best described hnRNPs responsible for negative regulation of splicing by causing exon skipping. In some cases however, it can also act as an activator. In hnRNPA1 pre-mRNA, PTBP1 counteracts SRp30c's repressive function. SRp30c binds to a conserved intronic element (CE9) in hnRNPA1 pre-mRNA, resulting in the repression of its downstream 3'SS. PTBP1 inhibits this mechanism by fixing itself to a CU rich region two nucleotides upstream of SRp30c's binding site [START_REF] Paradis | hnRNP I/PTB can antagonize the splicing repressor activity of SRp30c[END_REF].

III.3. PTBP1's role in RNA polyadenylation

During pre-mRNA maturation, besides its influence on splicing, PTBP1 is also capable of recognizing polyadenylation signals. Indeed, PTBP1 shares an affinity with CsTF63 for polypyrimidine rich sequences. Several studies demonstrate that PTBP1 competes with CstF64 for binding to the pyrimidine-rich DSE. As a result of this competition, PTB inhibits mRNA 3′ end cleavage, leading to a significant down-regulation of polyadenylated transcripts and a corresponding increase in unprocessed "read-through" transcripts (Castelo-Branco et al. 2004).

Sequences surrounding polyadenylation signals often include auxiliary cis-acting sequences, including U-rich upstream motifs and G-rich sequences (GRSs) positioned both upstream and downstream of the pA signal (Figure 14). Both cis-elements have an enhancing function in 3′ end formation through the binding of trans-acting proteins, including PTBP1 [START_REF] Danckwardt | Splicing Factors Stimulate Polyadenylation via USEs at Non-Canonical 3' End Formation Signals[END_REF][START_REF] Neve | Cleavage and polyadenylation: Ending the message expands gene regulation[END_REF]. PTBP1 can therefore affect the choice of polyadenylation sites leading to mRNAs with differing 3' ends, or proteins with different C-termini (Figure 17). Cis-acting elements promote the use of suboptimal polyadenylation signals and in the case of PTB, this regulation appears to be linked to its function as a splicing repressor. A well documented example of the involvement of PTBP1 in directing the choice of terminal exon is the calcitnonin/calcitonin gene-related peptide (CT/CGRP) pre-mRNA. By binding to an intronic pseudoexon in CT/CGRP pre-mRNA, PTBP1 favours the inclusion of an alternative 3' terminal exon containing an alternative polyadenylation signal (Figure 21).

The calcitnonin/calcitonin gene-related peptide (CT/CGRP) pre-mRNA presents two cell type-specific 3' terminal exons. Thyroid C cells use exon 4 as CT/CGRP's 3' terminal exon to produce the CT peptide. In neuronal cells, exon 4 is excluded, and inclusion of exons 3, 5 and 6 allows the production of a longer mRNA which codes for the CGRP peptide. The exon 4 is defined by a weak 3'SS and a weak polyadenylation signal. In order to be included, it therefore requires an intronic enhancer (ISE). This ISE is located downstream of intron 4 and contains a pseudo-5'SS, a pyrimidine rich sequence and U repeats (H. [START_REF] Lou | An Intron Enhancer Containing a 5' Splice Site Sequence in the Human Calcitonin/Calcitonin Gene-Related Peptide Gene[END_REF]. In vitro cleavage experiments have shown that the pseudo-5'SS and the pyrimidine rich sequence are both required for efficient cleavage and polyadenylation of exon 4. Moreover, the U rich sequence is said to contribute to exon 4's 3'SS recognition (H. [START_REF] Lou | An Intron Enhancer Recognized by Splicing Factors Activates Polyadenylation[END_REF][START_REF] Zhu | U1 snRNP-Dependent Function of TIAR in the Regulation of Alternative RNA Processing of the Human Calcitonin/CGRP Pre-mRNA[END_REF]. Another regulatory sequence present within exon 4 enables the use of its 3'SS. These activator sequences are recognised by many factors including SR proteins [START_REF] Lou | Regulation of Alternative Polyadenylation by U1 snRNPs and SRp20[END_REF]. In vitro, PTBP1 interacts both with the sequence located upstream of the polyadenylation signal and with the polypyrimidine rich sequence (Figure 21.C). In cells, overexpression of PTBP1 favours exon 4 usage by activating its cleavage step (H. Lou et Gagel 1999;[START_REF] Lou | An Intron Enhancer Recognized by Splicing Factors Activates Polyadenylation[END_REF].

PTBP1 is therefore a very potent regulator of alternative splicing and polyadenylation. Despite having been categorised for many years as ubiquitous, variation in PTBP1 levels are responsible for the regulation of many different types of posttranscriptional regulation and more specifically of alternative splicing and cleavage and polyadenylation events.

C.

B.

A.

III.4. PTBP1 and Cancer

PTBP1 expression may affect cancer initiation and progression by inducing aberrant splicing events in transcripts involved in many aspects of tumour progression. Cancer specific splicing events have been reported at the mRNA level in colon, bladder, and prostate cancer [START_REF] Thorsen | Alternative Splicing in Colon, Bladder, and Prostate Cancer Identified by Exon Array Analysis[END_REF]) Cancer specific alternative splicing events are also promising markers for cancer diagnosis [START_REF] Venables | Unbalanced Alternative Splicing and Its Significance in Cancer[END_REF]as well as genetic diseases in a broader sense [START_REF] Xiong | The human splicing code reveals new insights into the genetic determinants of disease[END_REF].

For reasons still unknown, cancer cells usually shift their metabolism from oxidative phosphorylation to aerobic glycolysis, a process termed the Warburg effect. This shift is dependent on the splicing of two exons that allow the production of two isoforms of pyruvate kinase: PKM1 and PKM2 in patients presenting with Her-2 positive breast cancer therefore shows potential as a novel therapeutic approach [START_REF] Wang | PTBP1 promotes the growth of breast cancer cells through the PTEN/Akt pathway and autophagy[END_REF]. In a similar way, PTBP1 is overexpressed in colorectal cancers, based on immunohistochemical staining of patient tumour samples. Again, PTBP1 depletion in a multitude of colorectal cancer cell lines increased their proliferation, but also inhibited their colony forming ability which translates to a limited capacity of anchorage-free growth. In mice, xenografts of HCT116 (colorectal carcinoma) cells have decreased tumorigenicity when depleted in PTBP1 (H. [START_REF] Takahashi | Significance of Polypyrimidine Tract-Binding Protein 1 Expression in Colorectal Cancer[END_REF].

In glioblastoma multiform tumours, PTBP1 could influence growth by promoting the production of the fibroblast growth factor receptor 1 (FGFR-1) with increased affinity for FGF. In this model, PTBP1 binds to intronic splicing silencers upstream and downstream of the α exon, which then gets skipped, leading to the production of high-affinity FGFR-1.

Furthermore, PTBP1 is dramatically increased during the malignant transformation of the glial cells [START_REF] Jin | Fibroblast Growth Factor Receptor-1 α-Exon Exclusion and Polypyrimidine Tract-Binding Protein in Glioblastoma Multiforme Tumors[END_REF]. Analysis of migration rates of glioma cell lines U251

(glioblastoma) and LN229 (glioblastoma) cells has shown that knocking down both PTBP1 and PTBP2 impedes their motility (Cheung et al. 2009, 2).

In ovarian cancer, PTBP1, as well as SRp20, are overexpressed. They could promote exon skipping and partial intron retention in the ABC transporter "multidrug resistance protein 1" (MRP1) mRNA. Some of these splicing modifications in MRP1 promote resistance to doxorubicin, a drug frequently used in the treatment of recurrent or refractory ovarian cancer(Xiaolong [START_REF] He | Alternative Splicing of the Multidrug Resistance Protein 1/ATP Binding Cassette Transporter Subfamily Gene in Ovarian Cancer Creates Functional Splice Variants and Is Associated with Increased Expression of the Splicing Factors PTB and SRp20[END_REF]). During immortalisation of ovarian epithelial cell lines, PTBP1 levels increase. Its knockdown in A2780 cells impairs their proliferation, invasion and anchorage-independant growth, reminiscent of its effect on colorectal cancer cells(X. [START_REF] He | Knockdown of Polypyrimidine Tract-Binding Protein Suppresses Ovarian Tumor Cell Growth and Invasiveness in Vitro[END_REF]).

Despite the convincing derease in proliferation of many cell types, both normal and cancerous, PTBP1 overexpression has not been associated with a gain in proliferative or malignant abilities. Furthermore, in contrast with the previously cited examples, In PC-3M

(prostate carcinoma) and T84 (colon adenocarcinoma) cells, depleting PTBP1 leads to a decrease in migratory abilities. (C. [START_REF] Wang | Polypyrimidine Tract-binding Protein (PTB) Differentially Affects Malignancy in a Cell Line-dependent Manner[END_REF]. Considering the broad ranging targets of PTBP1, its likely that its participation to cancer is strongly context and cell-type dependant. PTBP1's described involvement in different cancer types is summarised in

Cancer, splicing and the EMT

In cancers of epithelial origin, which constitute the vast majority of cancers [START_REF] Christofori | New Signals from the Invasive Front[END_REF], the oncogenic process starts out as excessive cellular proliferation accompanied by angiogenesis in order to sustain the newly established tumour mass [START_REF] Hanahan | Hallmarks of Cancer: The Next Generation[END_REF]. Cancers which are diagnosed in these early stages have the best clinical outcome [START_REF] Whitaker | Earlier Diagnosis: The Importance of Cancer Symptoms[END_REF]. The break between early stages and advanced stages of cancer is secondary to the invasion-metastatic cascade. The first step, invasion, sees epithelial cells from the tumour front acquire mesenchymal traits in order to delaminate, break the basement membrane and enter the surrounding conjunctive tissue.

There, the second step can take place: intravasation. In this step, the cancer cells which had broken away from the initial tumour now find their way into the microvasculature of the vascular and lymphatic systems. These cells can then circulate around the body and are as such referred to as circulating tumour cells (CTCs). In order to establish a new tumour, these cells have to subsequently leave the circulatory system, in a step called extravasation, and colonise a new location. The CTCs then revert to an epithelial phenotype which can sustain rapid proliferation, and subsequently form a new tumour. In some cases, the cells which broke free from the initial tumour may never gain entry into the circulatory system, and may form localised secondary tumours [START_REF] Valastyan | Tumor Metastasis: Molecular Insights and Evolving Paradigms[END_REF][START_REF] Thiery | Epithelial-Mesenchymal Transitions in Tumour Progression[END_REF].(Figure 22). Considering all this, we realise that understanding and gaining control of the first step of the metastatic cascade would be an incredible leap forward in preventing aggressive forms of cancer. So how exactly do cells change their phenotype, from epithelial to mesenchymal, and back again?

The epithelial to mesenchymal transition (EMT) in its broadest terms can be characterised by a loss of adherence between an epithelial cell and its partners, with a concomitant loss of its apico-basal polarity and instead gain of a front-back polarity (Figure 23). GSC, TCF4, FOXC2, PRR1 and TWIST are indirect repressors of CDH1 [START_REF] Peinado | Snail, Zeb and BHLH Factors in Tumour Progression: An Alliance against the Epithelial Phenotype?[END_REF]J. Yang et Weinberg 2008). Changes in expression and activity of these classical EMT transcription factors of course lead to more consequences than simply E-cadherin loss, and there are therefore many molecular events which have been used as an EMT readout, the most frequently used being expression of Vimentin, Fibroenctin and N-Cadherin [START_REF] Zeisberg | Biomarkers for epithelial-mesenchymal transitions[END_REF]. Furthermore, these transcription factors are capable of regulating each other both transcriptionally and posttranscriptionally.

During embryogenesis, the EMT happens first during gastrulation when the epiblast cells of the primitive ectoderm differentiate into primary mesenchymal cells. Later on during development, EMT is also crucial for the correct formation of the secondary palate, which ensures the separation of the oral an nasal cavities of mammals [START_REF] Nawshad | Transforming Growth Factor Beta (TGFbeta) Signalling in Palatal Growth, Apoptosis and Epithelial Mesenchymal Transformation (EMT)[END_REF][START_REF] Martínez-Alvarez | Snail Family Members and Cell Survival in Physiological and Pathological Cleft Palates[END_REF][START_REF] Takigawa | Terminal Differentiation of Palatal Medial Edge Epithelial Cells in Vitro Is Not Necessarily Dependent on Palatal Shelf Contact and Midline Epithelial Seam Formation[END_REF]. In adults, the EMT also occurs physiologically during wound healing, and pathologically during cancer as described above [START_REF] Kalluri | EMT: When Epithelial Cells Decide to Become Mesenchymal-like Cells[END_REF]. There is however quite some contrast between the EMT which occurs during development and that which occurs during the metastatic cascade. In the former, cells differentiate fully from epithelial to mesenchymal states, whereas in the latter, epithelial cells tend to gain some mesenchymal traits while retaining most of their original properties. This is referred to as partial-EMT (P-EMT), and is thought to be the main mechanism involved during metastasis, perhaps even allowing migration of whole groups of cells (Pal et al. 2021a;[START_REF] Grigore | Tumor Budding: The Name Is EMT. Partial EMT[END_REF][START_REF] Saxena | Hypoxia, Partial EMT and Collective Migration: Emerging Culprits in Metastasis[END_REF]. The co-expression of both mesenchymal and epithelial markers is said to give cells an advantage in surviving in and exiting the vascular system (M. [START_REF] Yu | Circulating Breast Tumor Cells Exhibit Dynamic Changes in Epithelial and Mesenchymal Composition[END_REF]. With the advent of single cell sequencing, markers expressed only in subsets of cells undergoing partial EMT, which would otherwise have been lost in a bulk assessment, are coming to light, and provide researchers with a spectrum of markers corresponding to the spectrum of cellular states (Pal et al. 2021a). In HNSCC, and by using single cell analysis of intratumoral heterogeneity, P-EMT genes have been identified which correlate with poor patient prognosis, despite no overall expression of EMT transcription factors (Puram et al. 2017a).

Canonical EMT transcription have also been associated with poor prognosis in HNSCC [START_REF] Wan | Prognostic Value of Epithelial-Mesenchymal Transition-Inducing Transcription Factors in Head and Neck Squamous Cell Carcinoma: A Meta-Analysis[END_REF]) which highlights both the heterogeneity of the disease and the necessity to take both EMT and P-EMT into consideration when assessing the potential aggressivity of a tumour.

In such a plastic cellular landscape, besides transcription factor activity, posttranscriptional regulation is thought to be a strong component of the regulation of EMT [START_REF] Nieto | EMT: 2016[END_REF]. This potentially places the fine tuning potential of alternative splicing at the forefront of P-EMT regulation. In line with this, RNA biding proteins are frequently up or down regulated in cancer, and mutations in RBPs associate with abnormal splicing events of which many are cancer drivers or other cancer hallmarks [START_REF] Sebestyén | Large-scale analysis of genome and transcriptome alterations in multiple tumors unveils novel cancer-relevant splicing networks[END_REF]. In one study of ZEB1-induced EMT in human non-small-cell lung cancer cells, ESRPs and RBM47 were reported to work in cooperation towards an epithelial splicing pattern, while QKI promoted a mesenchymal pattern(Y. [START_REF] Yang | Determination of a Comprehensive Alternative Splicing Regulatory Network and Combinatorial Regulation by Key Factors during the Epithelial-to-Mesenchymal Transition[END_REF]. In another study, this time in breast cancer cells induced into EMT by TWIST activation, ESRP1 was once again found associated with EMT relevant splicing events, as well as PTBP1 and RBFOX2 (Shapiro et al. 2011a).

AIMS OF THE STUDY

The many examples of the involvement of p63's isoforms during development, in the maintenance of adult tissues, and in oncogenic mechanisms highlight the pleiotropic functions of this transcription factor. The sometimes overlapping, sometimes mutually exclusive, and sometimes opposing effects of its isoforms underscore their requirement for tight regulation. A large part of the balance of p63 isoforms in a cell comes from posttranscriptional regulation and more precisely from control of its alternative splicing. If some examples of control of TP63 mRNA stability have been described,so far absolutely no information exists regarding the mechanisms controlling TP63 isoforms production. My thesis project therefore aimed to explore the regulation of TP63's isoforms at the RNA level, and investigate the functional impacts of modified isoform ratios in a human epithelial context.

The first axis of the project revolves around the discovery of a novel regulatory factor controlling the choice of alternative splice sites which operate the switch between producing TP63 γ or the α, β and δ isoforms. We identified the RNA binding protein PTBP1 as a negative regulator of the production of the TP63γ isoform and demonstrated its direct repression of TP63γ isoform production. The work that went into this aspect of the project has been summarised as the article "The splicing factor PTBP1 represses TP63γ isoform production in squamous cell carcinoma" and is deposited as a preprint on www.bioRxiv.org (https://biorxiv.org/cgi/content/short/2021.10.01.462767v1). This work is currently being submitted for publication.

Considering the published evidence towards differential roles of p63α and p63γ in epithelial cell biology, the second axis of my work was an attempt at identifying broad phenotypic shifts or more specific molecular events associated with a modification of the balance of TP63 isoforms in HaCaT keratinocytes or in HNSCC cell lines. To this end, we looked for modifications in the expression of epithelial or mesenchymal markers in cells with modified ratios of TP63 isoforms. We also investigated effects on cellular migration and proliferation, and we measured the expression of previously described TP63 target genes after overexpression or depletion of specific isoforms. Considering the exploratory nature of this work, most presented data for this part was not as thoroughly replicated as it was for the first part.

At the protein level, detection of p63' minoritary isoforms γ and β is complex due to low expression levels and lack of specific antibodies. It is further complexified by proteolysis of p63α that leads to accumulation of lighter proteins that are in the same molecular weight range as TP63γ. This means that immunoprecipitation and identification of the native proteins is tricky, if not outright impossible without isoform-specific antibodies.

To try and solve this problem, we collaborated with the Etablissement Français du Sang (EFS) to try and produce novel monoclonal mouse antibodies which could either recognise all TP63 isoforms, or specifically recognise the Pp3 α or p63 γ isoforms.

Finally, since TP63 circular RNAs have been identified as part of an oncogenic axis in lung squamous cell carcinoma, we sought out to determine whether they are also expressed in our P63 positive HNSCC cell lines. Furthermore, these circRNAs include regions which we have described as regulated by PTBP1. We therefore enquired upon the possibility that PTBP1 also regulates TP63 circRNA biogenesis.

RESULTS

Chapter 1. Functional characterisation of the involvement of the RNABP PTBP1 in the regulation of TP63 isoform production.

INTRODUCTION

The TP63 gene encodes a conserved transcription factor, p63, controlling epithelial development and homeostasis (1,2). In Humans, heterozygous mutations in TP63 lead to developmental syndromes affecting ectodermal tissue derivatives (3). Overexpression or amplification of TP63 is observed in multiple cancer types generally from epithelial origin (4) and p63 staining therefore serves as a classification biomarker in some malignancies including skin cancer [START_REF] Smirnov | p63 Is a Promising Marker in the Diagnosis of Unusual Skin Cancer[END_REF]. It discriminates carcinoma from non carcinoma breast cancer types, and lung carcinoma from lung adenocarcinoma in combination with other markers [START_REF] Travis | International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society International Multidisciplinary Classification of Lung Adenocarcinoma[END_REF][START_REF] Amin | Best practices recommendations in the application of immunohistochemistry in urologic pathology: report from the International Society of Urological Pathology consensus conference[END_REF]. It is also a prognostic marker as TP63 loss is associated with metastatic progression in head and neck squamous cell carcinoma [START_REF] Lakshmanachetty | Loss of TP63 promotes the metastasis of head and neck squamous cell carcinoma by activating MAPK and STAT3 signaling[END_REF].

TP63 encodes multiple protein isoforms owing to alternative promoters and alternative splicing or polyadenylation. The TP63 gene structure is evolutionary conserved and beside exons, several intronic regions also present similarities among vertebrates. The complexity of the TP63 gene structure allows for the production of multiple protein isoforms (Fig. 1A). This conserved complexity suggests some evolutionary pressure presumably associated with the function of the different isoforms [START_REF] Belyi | The origins and evolution of the p53 family of genes[END_REF][START_REF] Lane | Conservation of all three p53 family members and Mdm2 and Mdm4 in the cartilaginous fish[END_REF]. accumulating DNA damage in a way similar to p53 in somatic cells [START_REF] Suh | p63 protects the female germ line during meiotic arrest[END_REF]. A tissue-specific 5' TA variant coined GTA TP63 has been described in Hominid testis where it is proposed to similarly act as a guardian of the male germ line [START_REF] Beyer | Endogenous retrovirus drives hitherto unknown proapoptotic p63 isoforms in the male germ line of humans and great apes[END_REF].

Several C-terminal ends are produced from combinations of cassette and alternative terminal exons. These isoforms encode p63 proteins harboring different combinations of the C-terminal domains. The longest isoform p63α contains a phosphodegron (PD) ( 13), a sterile-alpha-motif (SAM) [START_REF] Mcgrath | Hay-Wells syndrome is caused by heterozygous missense mutations in the SAM domain of p63[END_REF], a transcriptional inhibitory domain (15) and a regulatory sumoylation region [START_REF] Straub | The C-terminus of p63 contains multiple regulatory elements with different functions[END_REF]. The shortest mRNA isoform TP63γ encodes only the oligomerisation and DNA binding domains. It is generated by the use of an internal terminal exon and thus has a 3' untranslated region (3'UTR)

different from the other C-terminal isoforms. Since all p63 isoforms contain the oligomerisation domain, they may all interfere with each other's functions. Most described functions of the α isoform concern its involvement in epithelia, and the p63γ isoform is unable to support proper epithelial development [START_REF] Wolff | The α / β carboxy-terminal domains of p63 are required for skin and limb development. New insights from the Brdm2 mouse which is not a complete p63 knockout but expresses p63 γ -like proteins[END_REF]. Instead, p63γ promotes the onset of an epithelial to mesenchymal (EMT) transition in keratinocytes [START_REF] Srivastava | ΔNp63 is np63γ/Src/Slug Signaling Axis Promotes Epithelial-to-Mesenchymal Transition in Squamous Cancers[END_REF], promotes osteoblastic differentiation [START_REF] Curtis | TAp63γ and ΔNp63 is Np63β promote osteoblastic differentiation of human mesenchymal stem cells: regulation by vitamin D3 Metabolites[END_REF] and favors terminal myogenesis [START_REF] Cefalù | TAp63gamma is required for the late stages of myogenesis[END_REF]. The functions of p63 are therefore strongly dependent on the regulation of the production of the different 5' and 3' isoforms in a tissue-specific manner. Despite the acknowledged importance of the differential role of the p63 C-terminal isoforms, the regulation of the multiple alternative splicing events involved in p63 pre-mRNA maturation remains unaddressed [START_REF] Pokorná | The foggy world(s) of p63 isoform regulation in normal cells and cancer[END_REF].

Regulation of alternative splicing events requires the assembly of the spliceosome machinery onto splice sites (noted 5'SS and 3'SS with respect to the introns) and the definition of the cleavage and polyadenylation sites (CPA) [START_REF] Herzel | Splicing and transcription touch base: cotranscriptional spliceosome assembly and function[END_REF]. The tissue-specific choices among different alternative splicing and polyadenylation events are generally controlled by a combination of regulatory sequence elements, silencers or enhancers and their cognate trans-acting factors, generally RNA binding proteins (RBPs). The differential tissue distribution of these RBPs will often account for the tissue-specific regulation of alternative splicing [START_REF] Fu | Context-dependent control of alternative splicing by RNA-binding proteins[END_REF]. We reasoned that by analyzing relative abundance of RBPs and TP63 alternative splicing events in RNASeq data from many tissues we could prioritize the RBPs most likely to intervene in TP63 splicing.

Here, we show by combining exploration of TCGA and GTEX Data, RNA/protein biochemistry and reverse genetics experiments that higher inclusion of the γ exon is associated with poorer survival of Head and Neck squamous Cell Carcinoma (HNSCC) patients and we identify the RBP PTBP1 as a direct inhibitor of the inclusion of the γ exon in HNSC cell lines. This regulation of TP63 alternative splicing by PTBP1 appears to be essential as it is evolutionarily conserved, being present in Xenopus laevis, a model amphibian that diverged from amniotes about 360 Million years ago. Targeting the PTBP1-mediated regulation of TP63 expression may therefore be a means to modify TP63 isoform ratio.

MATERIAL AND METHODS

Cell lines

HNSC cell lines (TCP-1012) were obtained from ATCC, HaCaT cells were obtained from CLS Cell Lines Service. HeLa, HaCaT, FaDu and Detroit 562 cells were cultured in DMEM medium (Gibco), A253 cells were cultured in Mc Coy's 5A (Gibco), SCC-9 and SCC-25 cells were cultured in DMEM/F12 (Gibco) supplemented with 400 ng/mL of hydroxycortisone (H0888, Sigma-Aldrich). For siRNA depletion, cells were seeded on 6-well plates at 150000 cells per well.

After seeding, a jetPRIME transfection mix (Polyplus) was added, containing 100 pmol of target siRNA or negative controls.

Xenopus embryos microinjection

Xenopus laevis eggs were obtained from WT females and fertilized using standard procedures [START_REF] Paris | Changes in the polyadenylation of specific stable RNA during the early development of Xenopus laevis[END_REF]. When indicated 30 ng of MoPtbp1 ( 25) or 30 ng of control morpholino (GeneTools) was injected into each blastomere of two-cell embryos in a volume of 13.8 nl, using a

Nanoject II (Drummond). For rescue experiments, 1 fmol of mRNA-encoding morpholino-resistant PTBP1-V5R was co-injected. Embryos were allowed to develop at 22 °C and collected at stages 26 according to Nieuwkoop and Faber stages [START_REF] Nieuwkoop | Normal Table of Xenopus Laevis (Daudin): A Systematical and Chronological Survey of the Development from the Fertilized Egg Till the End of Metamorphosis[END_REF]. Total RNA was extracted using RNeasy columns (Qiagen) from a pool of 3 embryos at stage 26. RNA were analysed by RT-PCR for 25 cycle using radiolabeled PCR primer.

Minigene construction and analysis

The TP63 exon 12 minigene was constucted by gibson assembly of Beta-globin and TP63

gene fragments obtained by PCR amplification from HaCaT genomic DNA using oligonucleotides pairs hGbE1_E2, hTP63_gamma, hGb_E3 and cloned into ApaI, NheI digested pmirglo (Promega).

Construction was controled by sequencing. HaCaT cells were transfected with plasmid minigene and selected using G418 (Gibco). Expression and splicing of the minigene was assessed by RT-qPCR using primers presented in the reagent table.

RNA extraction, reverse transcription and qPCR

Total RNA was extracted from cells using nucleospin RNA kits (Macherey Nagel, #740955.50) following the manufacturer's instructions. One µg of total RNA was reverse transcribed with superscript II enzyme (Invitrogen) and random primers (Invitrogen #58875).

Dilutions of cDNA (1:20) were amplified with PowerSybr Green Master mix (Appliedbiosystems, #4367659) using a Quantstudio (TM) 7 flex real-time PCR 384 well system.

Western-blot

Cells were washed with PBS 1X and lysed in 2X Laemmli sample buffer (4% SDS, 20% glycerol, 0,125M tris-Hcl pH 6.8, 2.5% β-mercaptoethanol, 0.2% bromophenol blue ). Denatured protein samples (10', 95°C) were loaded onto 8-12% SDS-PAGE gels. Transfer onto nitrocellulose membranes ( Amersham Protran 0.45µm NC) was performed using the trans-blot turbo transfer system (Biorad). Membranes were blocked for one hour with a 5% fat-free milk TBST solution (1X TBS, 0.1% Tween 20), incubated with primary antibody solutions (16h, 4°C). After three washes in TBST (5'), the membranes were incubated with anti-mouse or anti-rabbit secondary antibodies coupled to HRP (1h at 20°C), washed three times in TBST (5'). Revelation was performed on an Amersham AI680 imager with either ECL-select (Amersham) or West pico PLUS (Thermo-fisher scientific) chemiluminescent substrate, depending on anticipated signal strength.

TCGA data analysis.

TP63 isoform and splicing in HNSC patient samples were quantified using clinical and expression data from firebrowse.org and splicing quantification from TCGA splice SEQ [START_REF] Ryan | TCGASpliceSeq a compendium of alternative mRNA splicing in cancer[END_REF]. All expression, splicing and survival analysis were conducted in R. For kaplan-meier estimation, the package survival was used and a log rank test ( 28) applied. Scripts are available from Gitlab (project ID:29285922).

GTEX data analysis

To identify RBPs with tissue expression correlated to TP63 tissue specific junction uage, we analysed RNASeq data from GTEx Analysis 8.0. The GTEx data are composed of 17 382 samples from 54 different tissues obtained from 980 donors. For each sample s, the total number of reads Ts is calculated. The median read depth Mrd is calculated form each sample and a read depth normalisation factor (Ns) for each sample as:

N s = T s Mrd
Normalized gene expression Egs for gene g and sample s is computed as:

Egs= ∑ i=1 n junction gsi ×N s
Tissue and samples expressing TP63 are selected and used for correlation analysis. RBPDB [START_REF] Cook | RBPDB: a database of RNA-binding specificities[END_REF] provided a list of RBPs for which we calculated the RNA expression in the TP63 expressing samples. For each of the 21 TP63 junctions the junction usage is computed as:

junctionusage is = junction is ∑ i=1 21 junction is
The pearson correlation coefficient and associated p-value is calculated for each RBP with the junction usage of the TP63 junction. Top correlated RBPs are then selected for further analysis. R scripts are available on gitlab (project ID:29285922).

Fluorescent RNA

Transcription templates were synthesized by PCR (GOTAQ G2, Promega) using a hybrid T7promoter/sequence-specific forward primer and a sequence-specific reverse primer. PCR products were checked on native agarose gel and purified on columns (DC5, Zymoresearch).

Fluorescent RNAs were transcribed (37°C, 3h) in (Transc. buffer (1X), 5mM dTT, rNTPs 0. polyacrylamide gels [START_REF] Clerte | Characterization of multimeric complexes formed by the human PTB1 protein on RNA[END_REF]. Bound and free RNA were quantified on a Typhoon FLA 9500. To estimate the Kd, a non linear model of the form (bound/total RNA) ~ 1/(1 + Kd/[PTBP1]) was fitted to the data for each RNA using R script [START_REF] Ryder | Quantitative analysis of protein-RNA interactions by gel mobility shift[END_REF].

PTBP1 γcomplex γimmunoprecipitation

Ten millions cells were lysed in (50mM TrisHCl, 0.1M NaCl, 1 % NP40, 0.1 % SDS, 0. RNAs were analyzed by RT-PCR or RT-qPCR .

RESULTS

Higher γTP63 γγ γexon γinclusion γis γassociated γwith γpoor γoutcome γin γHNSC γpatients.

While many HNSC tumors overexpress TP63 (Fig. 1B and [START_REF] Campbell | Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas[END_REF])), the expression and impact of its C-terminal isoforms on patient survival is unclear. We used TCGASpliceSeq data [START_REF] Ryan | TCGASpliceSeq a compendium of alternative mRNA splicing in cancer[END_REF] to evaluate inclusion of exons composing the splice variants of TP63 in HNSC patients. The ΔNTP63 NTP63 isoforms are characterized by transcription initiation at exon 4 (Fig. 1A and SFig. 1A) and account for more than 99 % of total TP63 (Fig. 1C) in both normal and tumor samples. The TATP63 isoforms are barely detectable. Among the two terminal exons 12 and 16, the inclusion of exon 16 accounts for about 98 % of the TP63 RNA (isoforms α, β or δ, see SFig.1B for isoform structure). Conversely, usage of exon 12, found exclusively in TP63γ, is weak (around 2 % in average). A small but significant increase in the usage of exon 12 is observed in tumors samples compared to normal tissues (p=0.010, Wilcoxon test) with a parallel decrease in exon 16 (Fig. 1C). The exclusion of exon 15 measures TP63β, while the exclusion of the pair of exons 14 and 15 measures TP63δ. No difference could be observed between tumors and normal sample for these splicing events. We sought to determine whether differential exon usage in tumors could be associated with difference in survival probability for patients (Fig. 1D). The measure of inclusion of exon 12 or of both exon 14+15 in tumor samples could discriminate patient survival rates. Higher inclusion (psi >= 4.07%) of the exon 12 was associated with a lower survival of patients (median half-life = 862 days compared to 2319 days, p = 0.00363). Lower inclusion of exon 14+15 was also associated with a more moderate decreased survival (psi < 98.5%, median half-life = 1466 days compared to 2319 days, p = 0.0177). A higher inclusion of TP63 γ exon appears therefore detrimental for patient survival.

Candidate γRBPs γcontrolling γTP63 γγ γexon γinclusion. Because tumors samples may be highly heterogeneous due to tumor type, location or mingled tissues, we sought to identify potential regulators of TP63 splicing using normal tissues data. We searched for RNA-binding proteins (RBPs) whose expression correlated to TP63 junctions usages. RNASeq junction quantification were obtained from the GTEX consortium [START_REF] Lonsdale | The Genotype-Tissue Expression (GTEx) project[END_REF]. They represent quantifications from 54 different tissues or conditions. The correspondence between GTEX and TCGA nomenclatures for TP63 is shown in SF1A. We calculated the normalized expression for TP63 in each sample and all tissues. Among the 54 tissues, ten (10/54, 18.5 %) were selected as expressing TP63 (Fig. 2A). These tissues where mainly of epithelial origin, as expected for TP63, but EBV-transformed lymphocytes and skeletal muscles also showed a notable TP63 expression.

For each tissues and sample we quantified the usage of the 21 individual TP63 junctions (SFig.2A). The junction usage is strikingly different between tissues (SFig.2B). We can distinguish three groups of tissues: Muscle, Epithelia, and EBV-transformed lymphocytes. Eleven junctions are used at similar levels across tissues. Among those, four are very weakly used (J4-6, J5-5', J5'-6 and J13-16) and seven are constitutive splicing events included at high levels in all tissues. Ten junctions are differentially used across tissues. Junctions (J1-2, J2-3, J3-5, J4-5) are representative of differential promoter usage between epithelial tissue (ΔNp63 is N isoforms favored) and muscle and EBV-transformed lymphocytes (TA isoforms favored). Muscle specific differences (J11-12 and J11-13, red boxes) are visible. Theses differences are not observed in EBV-transformed lymphocytes.

The competition between these two mutually exclusive splicing reactions (J11-12, J11-13) sharing an identical 5' splice site allows for the production of the γ or [α, β, δ] terminal isoforms. The quantification of these two reactions (Fig. 2B) clearly shows that they are oppositely regulated, with epithelial tissues favoring the exclusion of the exon γ and the inclusion of the γ terminal exon being promoted in skeletal muscle.

We hypothesized that correlations between RBP expression and junction usage could allow us to pinpoint RBPs acting as splicing enhancers or splicing silencers affecting the tissue specific inclusion of the γ exon. Using the RBPs represented in the RNA Binding Protein Database ( 29), we first determined their expression in TP63 expressing tissues. We could analyse the expresssion levels of 386 different RBPs in the different tissues (Fig. 2C). Based on individual samples, we computed the correlations between RBP expression and junction usage relevant to the γ exon (J11-12 and J11-13). Among the 10 most correlated RNA binding proteins (Fig. 2D), eight were positively correlated and two were negatively correlated to γ exon inclusion (J11-12). As expected the correlation was inverted when considering the alternative junction (J11-13). These 10 RBPs are strongly expressed in muscle (FXR1, GRSF1, RBM24, CSDE1/UNR, HTATSF1, PABPC4, A2BP1/ RBFOX1, CSDA/YBX3) or in epithelial tissues (PTBP1, SRSF9).

A TP63 EXPRESSION IN TISSUES • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • 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• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 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• • • • • • • • • • • • • • • • • • • 0
B SPLICE JUNCTION USAGE • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 5
To determine whether expression of the selected RBPs was coherent with higher γ exon inclusion in HNSC patients with poor survival, we compared the differential expression of the RBPs in tumors selected for having high or low levels of γ exon that discriminate among patient survival (Fig. 1C). Of the 10 RBPs tested, only three (SFRS9, PTBP1, GRSF1) are differentially (Wilcoxon, p<0.05) expressed at the RNA level between tumors from patients with high or low γ exon inclusion (Fig. 2E). SFRS9 is more highly expressed in samples with high γ exon inclusion (Wilcoxon, p= 0.0385), which is not in accordance with the GTEX data where SRSF9 is negatively correlated to γ exon inclusion (Fig. 2D). PTBP1 and GRSF1 are low and high, respectively, in patients with high gamma (Wilcoxon, p= 0.0094 and p= 0.0389, Fig. 2E). The same RBPs are negatively and positively correlated, respectively, with γ in GTEX data (Fig. 2D). Therefore PTBP1 and GRSF1 can coherently be involved in γ exon regulation. However, because GRSF1 is mainly a mitochondrial RBP and is weakly expressed in epithelial cells [START_REF] Jourdain | GRSF1 regulates RNA processing in mitochondrial RNA granules[END_REF] while PTBP1 is a well defined alternative splicing regulator expressed in epithelial tissues, we chose to test whether experimental depletion of PTBP1 could enhance TP63 γ exon inclusion in a panel of HNSCC cell lines (FaDu, SCC9, SCC25, Detroit562 and A253) from different tissues of origin.

PTBP1 γrepresses γendogenous γTP63 γγ γexon γinclusion γin γsquamous γcarcinoma γcell γlines.

Using two different antibodies we analyzed p63 and p63α-specific expression by westernblot (Fig. 3A) in a panel of HNSC cell lines. Both antibodies detected a major isoform around 70 kDa, coherent with the major ΔNp63 is NP63α isoform (MW 66 kDa). In addition to the 70 KDa protein the pan-p63 antibody (4A4) detected lower molecular weight bands undetected using the α-specific antibody (D2K8X). This suggested that isoforms other than ΔNp63 is NP63α are expressed at lower levels in the tested cells. The identity of these isoforms can not be inferred from these experiments. We used RT-qPCR to evaluate the abundance at the RNA level of total TP63 and of its three predominant isoforms (α, β, γ). We estimated that global TP63 expression levels were higher in SCC25, then A253 and SCC9, then FaDu, Detroit 562 and non transformed keratinocytes HaCaT.

As expected, it is barely detectable in HeLa cells used as a negative control (Fig. 3B). We used isoform-specific RT-qPCR to quantify C-terminal isoforms (Fig. 3C). In all cell types tested, the α isoform is the major one ranging from 87 to 95 % of TP63 mRNA. Detroit 562 cells have the lowest α proportion. Accordingly, β and γ isoforms are in higher proportions in Detroit 562 cells with 8 % and 2.7 % of each respectively. The expression of the β and γ isoforms are variable among the cell lines with the Detroit 562 and the SCC25 cells having the highest relative proportion of A) p63 expression was evaluated in HaCaT, HeLa and HNSC cells by western blot and immunodetection using a pan-p63 antibody (4A4) or a p63α-specific antibody (D2K8X) (n=3). Even loading was controlled using PCNA or β-tubulin antibodies as indicated. B) TP63 RNA expression was measured by RT-QPCR with one pair of primer detecting all isoforms and quantification was normalised to a calibration curve obtained from TP63 plasmid DNA dilutions (n=3). C) Quantification of TP63α, β or γ percent spliced-in using isoform-specific primer pairs (n=3). D) upper panel, quantification of TP63γ isoform and PTBP1 RNA in control and PTBP1 depleted HNSC cell lines (n=3). Lower panel, evaluation of PTBP1 depletion by western blot (n=3), PCNA serves as a loading control.

• • • • • • • • • • • • • • • SCC9 • NT • SiCTRL • SiLUC • SiPTBP1a • SiPTBP1b • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •
D • • • • • • •• • • • • • • • • • • TP63α • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • TP63β TP63γ A253 Detroit FaDu HaCaT SCC25 SCC9 A253 Detroit FaDu HaCaT SCC25 SCC9
TP63γ (2.75 and 1.5% respectively) while other cell lines had all less than 1 % of TP63γ mRNA (Fig. 3C).

We efficiently depleted PTBP1 in the five HNSC cell lines using two different siRNAs (Fig. 3D, upper panels). In all five cell lines, depletion of PTBP1 led to an increase in TP63γ abundance as shown by the lower TP63γ ΔNp63 is CT in PTBP1 depleted cells (Fig. 3D, lower panels). The TP63γ levels were therefore inversely related to PTBP1 levels. This demonstrates that PTBP1

represses the accumulation of the TP63γ isoform in HNSC cell lines.

PTBP1 γbinds γto γTP63 γpre-mRNAs γsequences γin γvivo γon γthe γγ exon 3'Splice γsite γand γCPAs.

Direct regulation of TP63 splicing would require binding of PTBP1 to regulatory elements located in the pre-mRNA of interest. To determine whether PTBP1 was bound to TP63 pre-mRNAs in vivo we immuno-precipitated specifically PTBP1/RNA complexes from HaCaT extracts and analyzed the pre-mRNA content of the complexes by RT-PCR and RT-qPCR (Fig. 4A,4B). Presence of TP63 pre-mRNA was specifically detected in input and PTBP1 immunoprecipitates but not using control Ig or beads only. To determine whether PTBP1 was preferentially associated to TP63 pre-mRNA or mRNA, we used primers specific to TP63 mRNAs (ex8-ex9, ex11-12 ) or pre-mRNA (int10-ex11, int11-ex12, int12-ex13). As shown in Fig. 4B, the most significant and robust enrichment was observed with the primer pair specific to the pre-mRNA region closest to the γ exon (int11-ex12). Control RT-qPCR performed in absence of reverse transcriptase did not show PCR amplification (data not shown). Therefore, on TP63 pre-mRNA, PTBP1 is preferentially bound to the region that is up-regulated following PTBP1 depletion.

The RBPmap database details known binding data for hundred of RBPs [START_REF] Paz | RBPmap: a web server for mapping binding sites of RNA-binding proteins[END_REF]. Binding motifs for PTBP1 represent predicted binding sites (PBS) and were plotted along the TP63 γ exonic region (Fig. 4C). Examination of the genomic sequences surrounding TP63 γ exon identifies 4 blocks of conservation among vertebrates : an upstream intronic region (a), a region overlapping the cleavage and polyadenylation site, remain unbound to PTBP1. FRAGT3, FRAGT4 and FRAGT6 and CPA2 associate strongly with human PTBP1 protein with binding affinities better than (CPA2, FRAGT6, ~ 60 nM) or comparable to (FRAGT3, FRAGT4, ~ 150 nM) the positive PTBP1binding control RNA (150PYWT, ~ 104 nM) [START_REF] Hamon | Polypyrimidine tract-binding protein is involved in vivo in repression of a composite internal/3' -terminal exon of the Xenopus alpha-tropomyosin Pre-mRNA[END_REF].

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 1
Altogether, this demonstrated the direct association of PTBP1 on regulatory regions surrounding the γ exon 3'Splice site and CPAs on the TP63 pre-mRNA in vivo in an epithelial cell lines.

PTBP1 γrepresses γTP63 γγ γexon γinclusion γin γa γminigene γin γepithelial γcells.

If PTBP1 can control TP63γ exon inclusion by directly binding to these cis-elements, then the splicing of a minigene composed of the TP63 genomic sequence should be under the control of PTBP1. To test this, we stably transfected HaCaT cells with minigenes containing TP63 genomic sequences within an efficient β-globin globin splicing context. The structure of the minigene is shown in Fig. 5A. We depleted PTBP1 in HaCaT keratinocytes using either of two different siRNAs (siP1A and siP1B, Fig. 5B). As previously shown in HNSC cell lines (Fig. 3D), PTBP1 depletion strongly increases the abundance of TP63 γ in HaCaT cells (Fig. 5C). This demonstrates that, like in transformed cells, the accumulation of the endogenous TP63γ isoform is at least partly repressed by PTBP1 in non-transformed HaCaT keratinocytes.

The TP63 minigene splicing is also dependent on PTBP1 abundance (Fig. 5D). Upon PTBP1 depletion, the splicing between E2 and the γ terminal exon was increased 2 times (paired t.test, pvalue = 0.0016, n=3) while the alternative possibility, splicing of E2 to E3, was decreased about 2 times (paired t.test, pvalue = 0.004, n=3). This demonstrates that the genomic region surrounding the γ terminal exon is sufficient to allow for its PTBP1-dependent regulation.

PTBP1 γrepresses γTP63 γγ γinclusion γsince γat γleast γ350 γMillions γyears.

Clusters of PTBP1 PBS are located in-between evolutionarily conserved regions a and b and in regions b, c and d (Fig. 4C). Because of this interesting conservation of non coding regions, we addressed the regulation of the PTBP1-dependent regulation of the γ exon in an evolutionary distant organism. It is estimated that amphibians and mammals diverged from their common tetrapod ancestor roughly 360 millions years ago (37). Within amphibians, Xenopus laevis is a wellestablished model of vertebrate development. The exon/intron structure of the TP63 locus is well conserved between Xenopus and human with conserved coding exons ( in blue on Fig. 6A) serving as milestones along the sequence. In Xenopus, two annotated additional exons (11' and *, in green) are present (Fig. 6A). Exon * is very weakly used and exon 11' encode an alternative peptide in frame with the rest of the coding sequence. A terminal exon corresponding to the human gamma exon is present in both species.

Previously, we used antisense morpholino oligonucleotides to deplete Ptbp1 from developing embryos, and we analyzed RNA expression by RNAseq (25). Here, we re-investigated these data to focus on Xenopus tp63. Notably, the depletion of Ptbp1 led to an increase in the γ exon [START_REF] Beyer | Endogenous retrovirus drives hitherto unknown proapoptotic p63 isoforms in the male germ line of humans and great apes[END_REF] inclusion in embryos as judged by the Sashimi plot on Fig. 6B. On this example, reads spanning junction 11-12 or junction 11'-12 increased from 0 to 35, and from 5 to 56 respectively. This was accompanied by an accumulation of sequencing reads on the γ exon. To independently confirm these data, we analyzed Xenopus embryos by RT-PCR (Fig. 6C). The analyzed embryos were previously injected with a control antisense morpholino oligonucleotide (lane 2), a morpholino against PTBP1 (lane 3), the same morpholino co-injected with an mRNA encoding PTBP1 made immune to the morpholino by a few nucleotidic substitutions (lane 4), or the rescue RNA alone (lane 5). As shown on Fig. 6C, depletion of PTBP1 leads to the specific accumulation of RT-PCR products 11-12 and 11-11'-12, revealing an increased inclusion of the γ exon. That this increase is due to a specific change in RNA splicing in this region is shown by the unchanged signal when another region of tp63 mRNA is amplified (primers in exons 14 and 15). Upon restoration of PTBP1 expression from an injected rPTBP1 mRNA which is resistant to morpholino inhibition, the γ exon inclusion was restored to control levels.

These results demonstrate that the repression of γ exon usage by the RNA binding protein PTBP1 is a mechanism that emerged at least in tetrapods and has been conserved across 360 Millions years of evolution. Together, these data reveal that PTBP1 controls TP63γ exon inclusion by binding to discrete and conserved regulatory elements located in this pre-mRNA region. The γ exon inclusion is hence restricted by the presence of PTBP1, decreasing the abundance of the TP63 γ isoform in normal and cancerous epithelial cell lines where PTBP1 is abundantly expressed.

DISCUSSION

Alternative splicing misregulation in cancer cells is emerging as a major theme in the alteration of gene expression observed during the cancerous process. This offer new routes of tumor classification or treatment [START_REF] Bonnal | Roles and mechanisms of alternative splicing in cancer -implications for care[END_REF]. Using a heuristic approach we identified PTBP1 as a major repressor of TP63γ production in normal and malignant epithelial cells. This is the first identification of a trans-acting factors controlling the splicing pattern of TP63 in a tissue-specific manner.

Predicted PTBP1 binding sites were located in evolutionarily conserved regions framing exon 12 in the TP63 pre-mRNA and we demonstrated that PTBP1 binds with high affinity to discrete elements of the TP63 pre-mRNAs in vitro and in cells. These elements coincide with the 3' SS and the CPA of the γ exon.

Model of γ terminal exon regulation by PTBP1.

PTBP1 is generally observed as an inhibitor of the splicing or polyadenylation reaction.

However some cases of exon activation by PTBP1 have been reported [START_REF] Lou | Polypyrimidine Tract-Binding Protein Positively Regulates Inclusion of an Alternative 3′-Terminal Exon[END_REF]. This inhibition is generally the consequence of the inhibition of the recognition of the 3' splice site by U2AF [START_REF] Saulière | The polypyrimidine tract binding protein (PTB) represses splicing of exon 6B from the beta-tropomyosin pre-mRNA by directly interfering with the binding of the U2AF65 subunit[END_REF] or of the CPA site by CSTF (Castelo-Branco et al. 2004). On Xenopus tropomyosin mRNA, PTBP1 inhibits the function of an intronic silencer element located upstream of a terminal exon [START_REF] Sommer | PTB regulates the processing of a 3'-terminal exon by repressing both splicing and polyadenylation[END_REF]. The multiple RNA-recognition motifs of PTBP1 and its possible multimerisation on its targets offer multiple modes of interaction [START_REF] Oberstrass | Structure of PTB Bound to RNA: Specific Binding and Implications for Splicing Regulation[END_REF]. PTBP1 binding to a pre-mRNA at several sites can either hinder the regulated exon or exclude it by creating a loop through interaction of PTBP1 in the upstream 5' and downstream 3' region surrounding the regulated exon [START_REF] Spellman | Novel modes of splicing repression by PTB[END_REF]. Gruber and colleagues [START_REF] Gruber | Discovery of physiological and cancer-related regulators of 3′ UTR processing with KAPAC[END_REF] identified repeats of CU dinucleotides, reminiscent of PTBP1 binding sites, as preferentially associated with differentially used CPA in tumors versus normal samples of gliobastomas. PTBP1 levels were strongly anti-correlated with the length of these RNAs suggesting that PTBP1 acts as an inhibitor of CPA on tandem UTRs harboring sequential polyadenylation sites. Binding of PTBP1 on the CU-rich regions around the CPA would hinder its recognition by the CPA-bound machinery favoring the alternative unmasked CPA. The context of the TP63 γ exon is different as it is an internal terminal exon in competition with the splicing of a classic exon defined by 5' and 3'SS. As interactions between splicing factors involved in the recognition of the 3'SS and the CPA bound factors are central in the efficient processing of terminal exon [START_REF] Millevoi | An interaction between U2AF 65 and CF Im links the splicing and 3′ end processing machineries[END_REF], we can propose that by its dual binding to the 3'SS and the CPA, PTBP1 is likely interfering with the ability of U2AF and CPA factors to interact with 3'SS and CPA respectively and therefore impede their ability to define the γ exon.

In HNSCC cells depleted in PTBP1, the inclusion of the γ exon is still modest (see Fig. 3D).

It is therefore possible that additional inhibitory factors are at play in theses cell lines. We can exclude that this limited effect is because of a redundant role of the orthologues of PTBP1 as codepletion experiments of PTBP1 and PTBP2 barely increase the γ exon inclusion (data not shown).

One alternative is that the limited γ exon inclusion in HNSC cells is due to intrinsic properties of this γ exon, with sub-efficient 3'SS and CPA that would only drive limited inclusion. In this case efficient terminal exon γ inclusion observed in other tissues would require specific factors to enhance exon γ inclusion. Such factors should be looked for in tissues where the exon γ is predominantly used and we can assume that germ cell or muscle cells may prove useful to identify such factors.

Which role for the γ terminal exon ?

The importance of TP63 in development is clearly demonstrated from works in numerous models. In Xenopus , tp63 is required for the proper muco-ciliary epithelial development [START_REF] Haas | ΔNp63 is N-Tp63 Mediates Wnt/β-Catenin-Induced Inhibition of Differentiation in Basal Stem Cells of Mucociliary Epithelia[END_REF]. The evolutionarily conserved regulation of the γ exon by PTBP1 suggests that the p63γ harbors specific and required functions in certain cellular type or tissues. These functions may come from its impact on the p63 coding sequence, from a difference in the sequences of the 3'UTRs between γ and the α or β isoforms, or from more complex phenomenon such as regulation of circular RNA production as already described for the TP63 gene [START_REF] Cheng | c ircTP63 functions as a ceRNA to promote lung squamous cell carcinoma progression by upregulating FOXM1[END_REF]. At the RNA level, because of its different 3'UTR, and the γ isoforms have a different susceptibility to miRNAs controlling TP63 expression than the α, β isoforms [START_REF] Lin | The microRNA feedback regulation of p63 in cancer progression[END_REF]. It is therefore possible that the production of the γ isoform provides the cell with a way to escape some miRNA-mediated tissue-specific regulation of TP63. However this change in 3'UTR would also result in a change in the encoded p63 isoforms.

It is unlikely that the γ-specific peptide encoded in the terminal exon has any conserved functions by itself because of its important divergence between species. Indeed, only 15/37 (40.5%) aminoacids are conserved between Xenopus and Human γ peptides while the overall identity of ΔNp63 is Np63α proteins is 85 %. Both in Human and in Xenopus, the γ isoform is strikingly different from the α isoform. In Human, it is deprived of 231 amino acids forming the SAM domain, a transcription inhibitory domain, a transcription activation domain and a phosphodegron. These domains are of paramount importance for the correct function of TP63 as demonstrated by the Ankyloblepharon-Ectodermal defects Cleft lip/palate (AEC) and Rapp-Hodgkin (RHS) syndromes associated with mutations altering these domains [START_REF] Rinne | p63-Associated Disorders[END_REF]. Yet p63γ retains the DNA binding and oligomerisation domains allowing the protein to form hetero-complexes that have altered transcriptional functionality [START_REF] Petitjean | Properties of the six isoforms of p63: p53-like regulation in response to genotoxic stress and cross talk with ΔNp63 is Np73[END_REF].

In general, the ability of an alternative isoform to affect protein complex functions increases with the number of monomers composing the complex [START_REF] Bergendahl | The role of protein complexes in human genetic disease[END_REF]. p63 forms homo-or hetero-tetramers composed of various p63 or p73 proteins. It is therefore possible that even low amounts of the γ isoform affect the functions of p63 as a transcription factor. It is unclear yet if the low amount of TP63γ at the RNA levels are strictly correlated to similarly low protein level. Indeed, expression experiments showed that ΔNp63 is Np63γ was the most stable p63 isoform in Hep3B cells (Petitjean et al. 2008). This may be due to ΔNp63 is Np63γ being deprived of the Fbw7 E3 ligase phosphodegron and of of the lysine residues (K494,/505) involved in ubiquitinylation and destabilisation of p63α [START_REF] Galli | MDM2 and Fbw7 cooperate to induce p63 protein degradation following DNA damage and cell differentiation[END_REF][START_REF] Prieto-Garcia | Maintaining protein stability of ∆Np63 via USP 28 is required by squamous cancer cells[END_REF]. Quantification of the γ isoform at the protein level is complex as no γ -specific antibodies are currently available. We may expect that state of the art mass-spectroscopy will allow for isoform specific quantification of protein in the near future [START_REF] Lau | Splice-Junction-Based Mapping of Alternative Isoforms in the Human Proteome[END_REF].

The overexpression of ΔNp63 is Np63γ in the context of global p63 depletion leads to increased EMT in both a breast cancer cell line [START_REF] Lindsay | Role of DeltaNp63gamma in epithelial to mesenchymal transition[END_REF] and in a 3D organotypic model of invasion ((18). This p63γ-associated EMT is promoted through a TP63/SRC/SNAIL2 transcriptional axis [START_REF] Srivastava | ΔNp63 is np63γ/Src/Slug Signaling Axis Promotes Epithelial-to-Mesenchymal Transition in Squamous Cancers[END_REF]. However in HNSC patients, we failed to observe any increased accumulation of SRC in TCGA tumor samples with higher expression of the gamma isoform (data not shown). Another possibility arises from the existing feed-back loop where Wnt/B-catenin signaling promotes TP63 expression [START_REF] Haas | ΔNp63 is N-Tp63 Mediates Wnt/β-Catenin-Induced Inhibition of Differentiation in Basal Stem Cells of Mucociliary Epithelia[END_REF][START_REF] Ruptier | TP63 P2 promoter functional analysis identifies β-catenin as a key regulator of ΔNp63 is Np63 expression[END_REF] and p63 represses Wnt/B-catenin signaling [START_REF] Katoh | Repression of Wnt/βcatenin response elements by p63 (TP63)[END_REF][START_REF] Lakshmanachetty | TRP63/TP63 loss accelerates skin tumorigenesis through activation of Wnt/β-catenin signaling[END_REF]. This feedback loop could be essential for the maintenance of proper balance between differentiation and proliferation. One can speculate that this balance could be tilted by the modification of the activity of the p63 transcription factor associated with an altered ratio of the p63 C-terminal isoforms.

We observed that increased expression of TP63γ in tumors is associated with HNSC patients having a lower survival. While we can not conclude that this lower survival is a direct consequence of TP63γ expression, we identified PTBP1 as being more lowly in these tumor samples. It is therefore possible that the health status of these patients is associated with a PTBP1-specific altered splicing pattern that is reflected in the splicing of TP63. In this context measuring TP63 isoforms in tumor biopsies could serve as a biomarker to identify patients with altered splicing patterns and potentially altered survival.

"PTBP1 controls TP63 alternative splicing in HNSCC tumour cells": Discussion and perspectives

This work led to the first identification of a transacting factor, the RNA binding protein PTBP1, in the regulation of the decisive splicing event of TP63's pre-mRNA that determines whether it will become a γ isoform, or any one of the [α, β, δ] isoforms. The depletion of PTBP1 in human keratinocytes, in human HNSCC cells or in Xenopus laevis embryos invariably leads to the increase of the TP63γ RNA. This dates this regulatory process to at least 350 million years ago, prior to the divergence of Lissamphibians.

Interestingly, a recent publication highlighted the importance of pre-mRNA splicing regulation by SRSF3 in the evolutionarily conserved control of the biophysical properties of transcription factors. This therefore emphasizes that regulation of transcription factor activity may be efficiently mediated by alternative splicing regulation [START_REF] Kawachi | Regulated Splicing of Large Exons Is Linked to Phase-Separation of Vertebrate Transcription Factors[END_REF].

The conservation of the TP63 gene structure and of its regulation is suggestive of their strong importance in development. Interestingly, the p63γ specific peptide does not show strong conservation between species. This suggests that it is the fact that this isoform is devoid of C-terminal functional domains, rather than its novel peptide, that requires tight control. In adults, TP63γ is only significantly expressed in skeletal muscles [START_REF] Marshall | Tissue-Specific Expression of P73 and P63 Isoforms in Human Tissues[END_REF]. During embryogenesis, it is expressed during limb bud development (Kawata et al. 2017b), and is associated with the maturation of chondrocytes from proliferative cells to hypertrophic cells (Gu et al. 2013b;Yao et Wang 2013b). This isoform could therefore be specifically required at certain key points during development, in mesoderm derivatives rather than in ectoderm derivatives as TP63α is. p63γ can be integrated into heterotetramers with p63α thanks to their oligomerisation domain, and shares the same DNA binding domain without any of the C-terminal domains. It is therefore likely that p63γ can alter the effect of p63α on its target sites in a dominantnegative-like manner, effectively serving as a damper to p63α mediated effects.

At the most basic level, cancer can be described as a pathological loss of control of cellular homeostasis. This dysregulation classically involves gain of oncogenic functions and loss of tumour suppressor functions. These errors are often attributed to genetic abnormalities such as mutations, deletions or duplications. Once the oncogenic process has been initiated, activation and repression of numerous signalling pathways give cancer its trademark heterogeneity. A majority of proteins exist as several different isoforms dictated by alternative splicing. In some of these cases, shifting between two isoforms can significantly impact cellular proliferation, migration, invasion or cell death. Shifting between isoforms to a more cancer-favourable proteome requires cancer cells to adapt the expression of regulatory factors. Alteration of alternative splicing programs during tumourigenesis can be illustrated by the recently described roles of multiple RNA binding proteins (SRSF3, RBM22, PTBP1 and RBM3) in the development of glioblastomas. The accumulation of these post-transcriptional regulators was associated with development of glioblastomas, modification of alternative splicing and defavorable cellular properties such as proliferation and cell migration [START_REF] Fuentes-Fayos | Splicing Machinery Dysregulation Drives Glioblastoma Development/Aggressiveness: Oncogenic Role of SRSF3[END_REF]). Among the many different processes involved in tumour initiation and progression, the epithelial to mesenchymal transition is particularly prone to regulation by alternative splicing. Many effectors of the EMT can be expressed as either epithelial or mesenchymal isoforms. Shapiro and colleagues (Shapiro et al. 2011b), identified an EMT-associated alternative splicing signature that was dependent on a small number of RNA BP. Reversely, modification of transcription factor activity can alter expression of RBP. This may be illustrated more specifically by the association of individual RBPs with tumourigenesis. In this context, increased levels of c-myc promote an increase in polypyrimidine tract binding protein 1 (PTBP1) levels in gliomas. In turn, PTBP1 helps cancer cells proliferate by shifting the splicing of the pyruvate kinase PKM premRNA from PKM1 to PKM2 which switches the cellular energy production from oxidative phosphorylation to aerobic glycolysis and favour production of the metabolites necessary for transcription and replication [START_REF] Cj | HnRNP Proteins Controlled by C-Myc Deregulate Pyruvate Kinase MRNA Splicing in Cancer[END_REF]. In breast, lung, colon and bladder tumours, the SR protein SRSF1 is upregulated and enhances proliferation and migration while repressing apoptosis. In oral squamous cell carcinomas showing advanced dysplasia or lymphatic metastasis, SRSF3 is upregulated and promotes the expression of EMT markers [START_REF] Ghigna | Cell Motility Is Controlled by SF2/ASF through Alternative Splicing of the Ron Protooncogene[END_REF][START_REF] Karni | The gene encoding the splicing factor SF2/ASF is a proto-oncogene[END_REF][START_REF] Ezponda | The Oncoprotein SF2/ASF Promotes Non-Small Cell Lung Cancer Survival by Enhancing Survivin Expression[END_REF][START_REF] Anczuków | THE SPLICING FACTOR SRSF1 REGULATES APOPTOSIS AND PROLIFERATION TO PROMOTE MAMMARY EPITHELIAL CELL TRANSFORMATION[END_REF]. p63α has been shown to repress the EMT in various types of cancer [START_REF] Bergholz | Role of p63 in Development, Tumorigenesis and Cancer Progression[END_REF][START_REF] Wu | ΔNp63α activates CD82 metastasis suppressor to inhibit cancer cell invasion[END_REF]Hu et al. 2017b;Lakshmanachetty et al. 2019a;[START_REF] Barbieri | Loss of P63 Leads to Increased Cell Migration and Up-Regulation of Genes Involved in Invasion and Metastasis[END_REF][START_REF] Goto | Possible Involvement of ΔNp63 Downregulation in the Invasion and Metastasis of Oral Squamous Cell Carcinoma via Induction of a Mesenchymal Phenotype[END_REF][START_REF] Tucci | Loss of p63 and its microRNA-205 target results in enhanced cell migration and metastasis in prostate cancer[END_REF]Tran et al. 2013a), and p63γ has been shown to have a pro-EMT effect in breast cancer cells and in HPV E6/E7-expressing keratinocytes.

Considering our identification of an association between the high proportion of TP63γ and poor patient survival in HNSCC, it seems quite plausible that PTBP1 could be used by cancer cells as a regulator of EMT. An increase in p63γ could perhaps have a similar effect as the loss of p63α which is frequently observed in advanced cases of HNSCC (Lakshmanachetty et al. 2019a).

More than half of human genes possess alternative promoters [START_REF] Kimura | Diversification of transcriptional modulation: Large-scale identification and characterization of putative alternative promoters of human genes[END_REF]). These act in combination with downstream alternative splicing events to generate multiple isoforms. In tissues, the occurrence of p63γ seemed more often associated with TA (=P1) promoter usage. Global bioinformatic analysis of transcripts has shown that genes with multiple promoters tend to have more alternative splicing events, and the outcome of these splicing events is correlated to promoter use [START_REF] Xin | Alternative Promoters Influence Alternative Splicing at the Genomic Level[END_REF]. At the functional level, the promoter's architecture influences splicing outcome [START_REF] Cramer | Functional Association between Promoter Structure and Transcript Alternative Splicing[END_REF]1999).

So what are the different possibilities that may explain such correlation? One can envisage that common factors are acting both on promoter usage and on alternative splicing to favour regulatory events that would be coupled. These events could be upstream events, signalling pathways for example, that are simultaneously targeting transcription factor and regulatory RNA binding proteins, therefore promoting a correlated regulation. These pathways could also act by promoting specific modifications of the chromatin or of transacting factors that would impact transcription and splicing.

Alternatively modifications carried by transcription factors or RNApol II could act on the recruitment of regulatory RNA binding proteins during transcription. Changes in the biophysical properties of the RNA polymerase itself, for example transcription speed or pausing, could impact alternative splicing. These modification of RNA polymerase characteristics could be general in a cell and still cause specific impact on different promoters or, they could be the consequence of alternative promoters usage favoring the loading of specific cofactors or by the distinct recruitment of factors involved in both transcription and splicing [START_REF] Kornblihtt | Multiple links between transcription and splicing[END_REF][START_REF] Braunschweig | Dynamic integration of splicing within gene regulatory pathways[END_REF][START_REF] Merkhofer | Introduction to Cotranscriptional RNA Splicing[END_REF]. In prostate cancer for example, c-Myc binds to the promoter of Sam68, and by controlling transcription speed, favours the inclusion of an exon required for the functional full length mRNA [START_REF] Caggiano | c-MYC empowers transcription and productive splicing of the oncogenic splicing factor Sam68 in cancer[END_REF]. Alternative promoters are often used differentially between tissues or developmental time-points [START_REF] Pankratova | Alternative Promoters and Tissue-Specific Regulation of Mouse Oct-1 Gene Transcription[END_REF][START_REF] Edupuganti | Alternative SET/TAFI Promoters Regulate Embryonic Stem Cell Differentiation[END_REF]S.-H. Liang, Hassett, et Omiecinski 2005). In the case of TP63, the TA isoforms are mostly expressed in the germline cells, whereas the ΔN isoforms are expressed in the basal layers of many epithelia. In our work, we have shown that TP63γ is hardly expressed in most tissues, but is robustly expressed in skeletal muscle cells.

Furthermore, the majority of muscular TP63 is of the TATP63 isoform, suggesting that it is TATP63γ which is expressed in this tissue, as reported recently by Marshall and colleagues in an independent analysis of GTEX dataset [START_REF] Marshall | Tissue-Specific Expression of P73 and P63 Isoforms in Human Tissues[END_REF]). Through our experiments in human epithelial cell lines, we have shown that PTBP1 represses the use of the TP63γ-specific terminal exon. However, the induction of TP63γ upon PTBP1 depletion in these cells remains modest. In these cells, TP63 is almost exclusively expressed as ΔNTP63 isoforms. This dichotomy of promoter use between skeletal muscle cells and the epithelial cells we have used could provide an additional explanation to the limited effect of PTBP1 depletion on TP63γ levels, on top of the possible existence of TP63γ splicing activators. It would be very interesting to compare PTBP1's impact on the maturation of TATP63 pre-mRNAs to that which we have documented for ΔNTP63 transcripts. This last approach could be undertaken in muscular cells where PTBP1 expression is low. One could use differentiable muscular cell lines or in vivo models of development such as Xenopus to address simultaneously the regulation and the functional consequences of isoform switching.

Chapter 2. Investigation of TP63 isoform function in human epithelial cells

The different experiments presented as part of the first chapter highlighted the direct role of PTBP1 in the repression of TP63's γ isoform. The analysis of patient RNA seq data from the HNSC TCGA database also revealed a correlative link between the amount of TP63 γ present in a tumour sample and the duration of the patient's survival. This could mean that more TP63γ is present in tumours of worse prognosis as part of broader modifications to the RNA processing landscape, and could therefore potentially serve as a readout of these events. It could also mean that TP63γ somehow exerts a negative effect on survival, and could therefore warrant investigation as a causative event. The survival of HNSCC patients is directly related to whether they have developed metastases or not. The breaking point between a primary tumour and full on metastatic disease is thought to be dependant on the epithelial to mesenchymal transition (EMT) (Yun [START_REF] Zhang | Epithelial-to-mesenchymal transition in cancer: complexity and opportunities[END_REF]. This is a process by which the adhesive apico-basal polarity of an epithelial cell is lost as it changes into a mesenchymal cell. EMT is promoted by many changes in the components and architecture of the cytoskeleton and associated adhesion molecules.

Complex transcriptional [START_REF] Cook | Context Specificity of the EMT Transcriptional Response[END_REF] and post-transcriptional programs (Shapiro et al. 2011b) are the driving forces in EMT. These changes provide affected cells with extra migratory capacities and, if coupled with the expression of matrix-remodelling enzymes, can also lead to increased invasive potential. The whole process of EMT is gradual and reversible, and there is therefore an EMT-MET continuum whereby cells can exist in any state from fully epithelial to fully mesenchymal [START_REF] Pastushenko | Identification of the Tumour Transition States Occurring during EMT[END_REF]). In the case of metastasis, it is thought that a partial EMT allows some epithelial cells from the primary tumour to break loose and migrate to a distant site where they go through a MET phase to anchor themselves down and proliferate into a new tumour, acting as "cancer stem cells" [START_REF] Saitoh | Involvement of Partial EMT in Cancer Progression[END_REF].

Associating p63 with EMT can at first seem surprising given its well described role in the differentiation and maintenance of epithelia. However, if we think in terms of isoforms, it begins to make more sense. p63α has been described as an inhibitor of EMT, both in breast cancer and bladder cancer cells (Tran et al. 2013b;[START_REF] Yoh | Repression of p63 and induction of EMT by mutant Ras in mammary epithelial cells[END_REF]. In agreement with this, HNSCC tumour progression is associated with a loss of TP63 expression (Lakshmanachetty et al. 2019b). p63γ has been described to promote the EMT process in both breast cancer cells (Lindsay et al. 2011b) and HPV transformed human keratinocytes [START_REF] Srivastava | p63 drives invasion in keratinocytes expressing HPV16 E6/E7 genes through regulation of Src-FAK signalling[END_REF]2018b). p63γ is the shortest of the p63 isoforms, and has the theoretical possibility to act as a dominant negative to p63α. In physiological conditions, the α and γ isoforms show non overlapping expression in tissues: TP63α is expressed in several epithelial tissues, whereas TP63γ is expressed in muscles. We therefore hypothesized that the TP63γ isoform could be involved in promoting certain mesenchymal properties in cells.

Comparison of EMT-related phenotypes between PTBP1 depleted and TGF-β treated HaCaT cells

Thanks to our identification of the repression of TP63γ by PTBP1, we had the possibility to use siRNAs directed against PTBP1 as a means to modestly increase the abundance of TP63γ in our cell lines. This had the advantage of simplicity since it did not require the labour intensive process of generating cell lines overexpressing TP63γ, and would be a good model of physiologically possible levels of TP63γ. We chose to proceed with HaCaT cells, as these have already been thoroughly used for the study of EMT [START_REF] Räsänen | TGF-Beta1 Causes Epithelial-Mesenchymal Transition in HaCaT Derivatives, but Induces Expression of COX-2 and Migration Only in Benign, Not in Malignant Keratinocytes[END_REF][START_REF] Hatta | Keratin 13 Gene Is Epigenetically Suppressed during Transforming Growth Factor-Β1-Induced Epithelial-Mesenchymal Transition in a Human Keratinocyte Cell Line[END_REF]. They have the advantage of being sensitive to EMT induction by TGF-β, which we included as a positive control for EMT onset. The use of TGF-β to trigger EMT in HaCaT cells requires sensitization by serum-depletion. We therefore settled on comparing the effect of depleting PTBP1 with the effect of the addition of 2ng/mL of TGF-β on HaCaT cells cultured in 0.5% serum media. To appreciate the advent of an EMT, we combined western blotting with immunofluorescence imaging to look at modifications in abundance and/or localisation of commonly used markers. We chose to look at E-cadherin, Fibronectin, SRC and Slug as these markers have been previously associated with TP63γ mediated EMT in keratinocytes (Srivastava et al. 2018b). After 96 hours of treatment, TGF-β treatment clearly resulted in Fibronectin and Slug accumulation as observed by Western-blot, while no effect was observed on either E-cadherin or SRC levels (Figure 24 The targets we chose were however markers that are known to be affected when cells transition strongly towards the mesenchymal phenotype [START_REF] Scanlon | Biomarkers of Epithelial-Mesenchymal Transition in Squamous Cell Carcinoma[END_REF]. It is possible that we would have missed a more subtle, partial-EMT triggered by PTBP1 depletion by limiting ourselves to these. The fact that we did not reproduce the EMT phenotype described following TP63γ overexpression in (Srivastava et al. 2018b) is likely down to the vast difference in TP63γ levels between our respective experiments. Indeed, by overexpressing TP63γ using a pENTR11 based vector, the authors achieved p63γ levels which were dramatically stronger than the endogenous p63α. Following PTBP1 depletion, TP63γ levels in our cells still make up less than 10% of the overall expressed TP63 isoforms, as illustrated in the first chapter. respond to TGF-β treatment (Puram et al. 2017b). One of the limiting factors in identifying an effect of the TP63γ isoform is its low expression, even following PTBP1 depletion.

PTBP1's ortholog PTBP2 is known to be induced following PTBP1 depletion (Boutz, Stoilov, et al. 2007;[START_REF] Makeyev | The MicroRNA MiR-124 Promotes Neuronal Differentiation by Triggering Brain-Specific Alternative Pre-MRNA Splicing[END_REF][START_REF] Spellman | Crossregulation and Functional Redundancy between the Splicing Regulator PTB and Its Paralogs nPTB and ROD1[END_REF] and shares a partial overlap of its functions with PTBP1 [START_REF] Vuong | PTBP1 and PTBP2 Serve Both Specific and Redundant Functions in Neuronal Pre-MRNA Splicing[END_REF]. Therefore phenotypes associated with depletion of PTBP1 could be partially masked by the redundant functions of PTBP2. We therefore sought to co-deplete PTBP1 and PTBP2 to favour accumulation of TP63γ. For this, SCC-9 cells were transfected with siRNAs against PTBP1 (SIP1 is SiPTBP1a), PTBP2 (SIP2), TP63γ (SIG) or combinations of each (Figure 26). While siP1 depleted specifically PTBP1 and concomitantly increased PTBP2 levels compared to non-transfected cells, siP2 could deplete both PTBP2 and most remarkably PTBP1. Depletions of PTBP1 and PTBP2 was confirmed by RT-qPCR when compared to non-transfected SCC-9 cells. Co depletion experiments with siP1 and siP2 efficiently depleted PTBP1 and reduced levels of PTBP2 to the levels observed in control cells.

TP63γ levels seemed marginally increased when both siRNAs against PTBP1 and PTBP2

were combined and were specifically reduced upon treatment with SIG alone or in combination with siP1P2. Therefore, we could rule out a strong effect of PTBP2 reexpression following PTBP1 depletion on the overall levels of TP63γ. SiG led to some downregulation of overall TP63 expression and of TP63α expression. It would probably be worth investigating whether the effect of TP63γ depletion on TP63 is the result of an indirect effect of the siRNA on the stability of TP63 pre-mRNA or if down-regulation of p63γ protein itself is responsible for this effect. This would set TP63γ as a factor promoting TP63 alpha expression.

In our continued search for potential effects of PTBP1 knockdown on EMT and similar to what we did on HaCaT cells, we treated SCC-9 cells with 2ng/mL TGF-β for 48, 96 or 144h. We then compared the impact of the addition of TGF-β with the impact of siRNA mediated depletions of PTBP1 and PTBP2 on the expression of various EMT and partial EMT genes using RT-qPCR (Figure 27). Partial-EMT (P-EMT) genes are expressed by cells which have started to differentiate from an epithelial state, undergoing the transition towards the mesenchymal state without necessarily fully committing to it

, TP63γ (SiG) or combinations of each. Gene expression of PTBP1, PTBP2, and TP63 isoforms was measured by RT-qPCR and plotted as -ddCT, using non transfected cells as a control.

Figure.27. SCC-9 cells were cultured in 10% serum containing medium and either treated with 2ng/mL TGF-β1 or transfected with siPTBP1 and siPTBP2, siTP63γ or a combination of the three. Gene expression was measured by RT-qPCR and analysed by the ddCT method. The reference gene is RPLP0. siRNA depleted samples were normalised to untransfected cells, and each TGF-β treated sample was normalised according to its associated untreated control.

SERP1 SNAI2 SNAI1 TGFBI VIM KRT16 KRT5 LAMB3 CDH2 PDPN CDH1 EPCAM FN1 ITGA6 KRT14 T G F 4 8 h T G F 9 6 h T G F 1 4 4 h s iP 1 P 2 s iG s iP 1 P 2 G T G F 4 8 h T G F 9 6 h T G F 1 4 4 h s iP 1 P 2 s iG s iP 1 P 2 G T G F 4 8 h T G F 9 6 h T G F 1 4 4 h s iP 1 P 2 s iG s iP 1 P 2 G T G F 4 8 h T G F 9 6 h T G F 1 4 4 h s iP 1 P 2 s iG s iP 1 P 2 G T G F 4 8 h T G F 9 6 h T G F 1 4 4 h s iP 1 P 2 s iG s iP 1 P 2 G -ddCT Epithelial Mesenchymal P-EMT -2 0 2 4 -2 0 2 4 -2 0 2 4
(Figure 23). The epithelial phenotype of cells is characterised by an apico-basal polarity, and low mobility through tight cell-cell and cell-basement membrane junctions (Figure 23).

The mesenchymal phenotype is in complete opposition: cells are motile and invasive, they have a front-back polarity (Etienne-Manneville 2008), and do not adhere strongly to cells or the matrix. P-EMT cells can share any of these properties, and in HNSCC, cells in between the two states tend to retain epithelial markers including E-cadherin as they acquire certain mesenchymal markers (Pal et al. 2021b). We chose to look at three groups of markers: CDH1 (E-cadherin), EPCAM, KRT5, KRT14, KRT16 were used as markers of the epithelial state; TGFBI, SERP1, LAMB3, PDPN and ITGA6 were used as P-EMT markers; and SNAI2, SNAI1, FN1, CDH2 and VIM were used as mesenchymal markers (Puram et al. 2017b).

We first established the differential expression of the three sets of markers upon TGF-β treatment as a way to induce EMT. Amongst the epithelial markers, only KRT16

was decreased in the days following TGF-β treatment. This is in corroboration with work indicating that KRT16 levels are decreased in a subgroup of HNSCC patients with poor metastasis-free survival [START_REF] Jung | A Poor Prognosis Subtype of HNSCC Is Consistently Observed across Methylome, Transcriptome, and MiRNome Analysis[END_REF]). Among P-EMT markers, TGFBI and SERP1

were increased to their maximum observed level as soon as 48h after TGF-β treatment,

LAMB3 and PDPN showed a more gradual increase, with their highest levels reached at 144h after TGF-β treatment. These genes are therefore probably part of a second differentiation step in the context of a TGF-β-induced EMT and are likely expressed in response to the activation of earlier targets. Despite being associated with increased migration and invasion in HNSCC [START_REF] Kinoshita | Tumour-Suppressive MicroRNA-29s Inhibit Cancer Cell Migration and Invasion by Targeting Laminin-Integrin Signalling in Head and Neck Squamous Cell Carcinoma[END_REF], as well as hepatocellular carcinomas and metastatic breast cancer [START_REF] Lv | RNA Interference Targeting Human Integrin Α6 Suppresses the Metastasis Potential of Hepatocellular Carcinoma Cells[END_REF][START_REF] Brooks | ITGA6 Is Directly Regulated by Hypoxia-Inducible Factors and Enriches for Cancer Stem Cell Activity and Invasion in Metastatic Breast Cancer Models[END_REF]) ITGA6 levels showed no changes in any of the tested conditions. The mesenchymal markers FN1 (Fibronectin) and CDH2 (N-Cadherin) were surprisingly expressed as soon as 48h after TGF-β treatment as evidenced in Figure 27, which highlights the fact that the transition from an epithelial to a mesenchymal phenotype does not necessarily require the cells to progressively move through intermediary states. Considering SCC-9 cells are naturally heterogeneous, with a subset expressing P-EMT genes in normal growth conditions (Puram et al. 2017b), it is also possible that it is these cells that were rapidly pushed by the TGF-β treatment towards a more mesenchymal state revealed by FN1 and CDH2 increased expression. SNAI2 and VIM showed a more progressive induction, and SNAI1

was not affected at all. Therefore TGF-β treatment induces an EMT in SCC9 cells with rapid induction of P-EMT markers and some EMT markers.

Depleting PTBP1 and PTBP2 had no impact on expression of the epithelial markers, and only a moderate impact on the EMT or P-EMT markers tested. Only an increase in VIM expression could be observed. Depleting TP63γ had no effect on epithelial markers, no effect on P-EMT markers and led to some induction of SNAI2 and VIM expression suggesting, counter-intuitively, that TP63γ could be pro-epithelial. Altogether, downregulation of PTBP1 and/or TP63γ showed no particular pattern in EMT marker expression.

In colorectal cancer cells, PTBP1 stimulates EMT and migration by repressing the expression of ATG10 [START_REF] Jo | Polypyrimidine Tract-Binding Protein 1-Mediated down-Regulation of ATG10 Facilitates Metastasis of Colorectal Cancer Cells[END_REF]. In lung carcinoma cells, PTBP1 provokes the skipping of exon11a in Mena pre-mRNA to produce a Mena isoform that promotes migration and invasion. PTBP1 levels are significantly increased in lung adenocarcinoma compared to normal tissue, and contributes to its metastatic ability (S. Li et al. 2019).

Adenocarcinoma cells typically do not express ΔNTP63 (Bishop et al. 2012b). Considering these results, it is likely than any pro-EMT effects resulting from an increase of p63γ would be lost within the many consequences of PTBP1 depletion, some of which could be opposed to the EMT onset. From these experiments, we can therefore conclude that depleting PTBP1 and 2 does not provoke an epithelial to mesenchymal transition, but we cannot say whether an increased expression of TP63γ outside of this context would impact the EMT.

Identifying p63 targets in HNSCC cells

Considering we have so far not been able to identify a role of p63's γ isoform in the EMT process, we wished to identify some of its target genes in a broader context and test for any overlap with known P63α target genes.

Our first approach was to use PTBP1 and PTBP2 depletion to induce TP63γ expression and combine it with with TP63γ depletion to try to rescue the changes in expression that would therefore identify differentially expressed genes in SCC-9 and FaDu cell lines (Figure 28), as we had done previously for the study of the EMT. For this, we chose a short list of genes reputed to be targetted by p63 in the literature: CDKN1A 28.A). Upon depletion of PTBP1, PTBP2 or PTBP1+ PTBP2, an increase in TP63γ was observed in FaDu and SCC9 cells as visualized by RT-PCR (Figure 28.B). This increase was abolished when cells were treated with siRNA against TP63γ. In FaDu cells, depletion of TP63γ led to a significant increase in CDKN1A and a significant decrease in PERP when compared to siP1P2, the most extreme conditions with respectively the least and the most TP63γ (Figure 28.C). In SCC9 cells, only SOX4 was significantly increased upon depletion of TP63γ when compared to siP1P2. Co-depletion of PTBP1/2 and TP63γ restored SOX4 levels to the levels in PTBP1/2 depleted cells. P63γ could therefore be a negative regulator of SOX4 in SCC-9 cells, and of CDKN1A in FaDu cells. This corresponds with the inhibitory role of P63α on these two targets as stated in the literature, and would suggest that p63γ does not have an antagonistic function in targetting these genes.

We tried to alter TP63γ levels by modifying availability of the regulatory RNABPs PTBP1 and PTBP2. One of the potential caveats of this approach is to hinder their potential impact on TP63 by altering other PTBP targets. Therefore, to more directly assess the potential function of P63α and P63γ we designed siRNAs targeting each of these isoforms specifically. Using these siRNAs, we sought to determine whether p63 target genes were influenced by p63α, p63γ, or perhaps both. For this, we chose to use SCC-9 cells which express low levels of p63 and A253 cells which express high levels of p63 (see chapter 1). Efficient knockdown of p63 was controlled at the protein (Figure Depleting TP63 α or γ had no impact on the expression of these EMT markers, suggesting that though p63α may protect against EMT, inhibiting its expression is not sufficient to induce one. As was previously observed in Figure 26, transfection of any of the siRNA targeting TP63γ led to partial but efficient depletion of p63 α as can be seen in (Figure 29.A). .

When measuring expression of potential p63 target genes, in SCC-9 cells, we had previously shown an increase of SOX4 expression following TP63γ knockdown using siRNA siG1 (Figure 28). Here, only siG1 showed this effect. Considering both TP63γ siRNAs efficiently deplete its RNA levels, it's likely that he increase in SOX4 was merely an off-target effect of siTP63γ n°1. There is no effect of TP63 depletion on SOX4 in the A253 cells. In SCC9 cells, TP63γ knockdown but not TP63α knockdown leads to a progressive decrease of MDM2 levels from 24 to 72h after treatment. Again, this was not observed in A253 cells. The increase in CDKN1A observed previously in FaDu cells following TP63γ depletion seems conserved in A253 cells until 48h, but not at 72h, and not in SCC-9 cells.

Overall, the limited fluctuation in expression levels of the tested targets coupled with the single experiment make it impossible to robustly conclude to an effect on any of these targets, and taking the matter further would require repeating the experiment.

The most striking conclusion of these experiments is actually not on p63's targets, but on p63 itself. Upon depleting TP63γ RNA, TP63α RNA is also depleted (Figure 29.B),

as well as the p63α protein (Figure 29.A). This effect is reproduced in both A253 and SCC-9 cells, and with two different siRNAs. Several mechanisms could potentially explain this effect.

p63 activates its own transcription through binding to C38 and C40 enhancer elements as shown in mouse primary keratinocytes [START_REF] Antonini | A composite enhancer regulates p63 gene expression in epidermal morphogenesis and in keratinocyte differentiation by multiple mechanisms[END_REF]. p63 selfactivation is also mediated by its binding to its own promoter. Both ΔNp63α and γ, as well as their TA counterparts are capable of activating a ΔNTP63 minimal promoter in Ptk2 cells (Potorous tridactylus kidney epithelial cells) [START_REF] Romano | Defining the Regulatory Elements in the Proximal Promoter of ΔNp63 in Keratinocytes: Potential Roles for Sp1/Sp3, NF-Y, and P63[END_REF].

TAP63γ also stimulates ΔNTP63 expression in non-small cell lung carcinoma cells and in immortalised mammary epithelial cells (N. [START_REF] Li | TA-P63-γ Regulates Expression of ΔN-P63 in a Manner That Is Sensitive to P53[END_REF]. A potential argument against this theory is the fact that in SCC-9 cells overexpressing TP63γ, TP63α levels are not increased at the RNA or protein levels (Figure 30. A and B). However, expressing ectopically ΔNP63γ in HeLa cells activates the transcription of the endogenous ΔNTP63 in a DBD-dependant manner [START_REF] Antonini | An Autoregulatory Loop Directs the Tissue-Specific Expression of p63 through a Long-Range Evolutionarily Conserved Enhancer[END_REF]. In HeLa cells TP63 is not expressed under normal circumstances, but it is in SCC-9 cells (Figure 30

C

H3K27, and DNA methylation. This effect seems restricted to siRNAs targetting promoter regions [START_REF] Hawkins | Promoter Targeted Small RNAs Induce Long-Term Transcriptional Gene Silencing in Human Cells[END_REF]). The interaction between Ago2 and the RNA pol II suggests that siRNAs targetting sequences downstream of the promoter could also have an impact on the dynamics of their transcription. These mechanisms have been reviewed by [START_REF] Weinberg | Transcriptional gene silencing in humans[END_REF][START_REF] Castel | RNA interference (RNAi) in the Nucleus: roles for small RNA in transcription, epigenetics and beyond[END_REF]. If TP63γ siRNA made its way into the nucleus, there is therefore a possibility that it could impact the transcription of the TP63 gene. This hypothesis would have deep consequences on the regular use of siRNA for isoform specific depletion, not only of TP63, but also in general.

Since TP63α is downregulated at both the RNA and protein levels, we can rule out any stabilizing action of γ on α at the protein levels without transcriptional consequences. It does remain possible that p63γ stabilizes p63α proteins, which in turn activate TP63

transcription. Removing p63γ in this context would therefore lead to a drop in p63α proteins, followed by a drop in overall RNA, which fits with our observations.

Using TP63α and TP63γ overexpressing SCC-9 cells to identify differential targets and functions

Whatever the mechanism by which ΔNTP63α is downregulated following TP63γ siRNA, the conclusion is the same: it is impossible to confidently identify isoform-specific targets of TP63 with this knock-down method. We therefore set out to test a gain of function approach by creating cell lines stably overexpressing ΔNTP63α or ΔNTP63γ. We initially chose to work with A253 and SCC-9 cells, as their initial levels of TP63α are respectively high and low, and SCC-9 cells already express some mesenchymal markers whereas A253 cells do not. However, we could not establish cell lines from A253 cells, and were forced to limit our study to SCC-9 cells. ΔNTP63 isoform expression was driven by a PGK promoter in order to limit the overexpression to near-physiological levels (Figure 30.A). isoforms. An increase in p63α strengthens the epithelial identity of SCC-9 cells, as shown by the increase in CDH1 and KRT5, while having no effect on mesenchymal markers (FN1, SNAI2, SNAI1, VIM, ZEB1) or P-EMT markers (SERP1, TGFBI). Overexpressing

TP63γ had no effect on any of these markers (Figure 30.C), and is therefore not sufficient to induce an EMT in these cells.

Since we had limited success in identifying robust changes in the expression of the p63 targets that we had previously selected, we sought to test some others (Figure 31.E).

HERC3 is directly transactivated by ΔNp63α in MCF10A and FaDu cells (Y. [START_REF] Chen | ΔNp63α down-regulates c-Myc modulator MM1 via E3 ligase HERC3 in the regulation of cell senescence[END_REF]. SERPINB13 is downregulated in HNSCC and is associated with a poor clinical outcome (de [START_REF] Koning | Downregulation of SERPINB13 Expression in Head and Neck Squamous Cell Carcinomas Associates with Poor Clinical Outcome[END_REF]. LAD1 is increased in HNSCC and promotes cellular growth and invasion (Abe et al. 2019). Both genes have been identified as p63 targets (Riege et al., s. d.). The absence of antibodies specific to the different C-terminal isoforms is a severe limitation in the study of both their functions and their pathological relevance. Isoform specific antibodies would allow the delineation of the expression domains of the different isoforms at the tissular, cellular or sub-celllular levels. From this, we could gain knowledge of the p63 containing complexes, which isoforms preferentially compose them, and where they are located.

The ability for health care practitioners to classify patients based on the expression of specific proteins in pathological samples allows them to adapt treatments to specific sub-populations of patients [START_REF] Duraiyan | Applications of immunohistochemistry[END_REF][START_REF] Lin | Standardization of Diagnostic Immunohistochemistry: Literature Review and Geisinger Experience[END_REF]. Furthermore, in the case of proteins existing as multiple isoforms, immunological assessment of patient samples must be adapted to take into consideration the divergence in biological and clinical significance between these isoforms. As an example, isoform specific recognition of the 14-3-3 protein is used to distinguish sporadic Creutzfeldt-Jacob disease from other types of dementia [START_REF] Everbroeck | 14-3-3 γ-Isoform Detection Distinguishes Sporadic Creutzfeldt-Jakob Disease from Other Dementias[END_REF][START_REF] Kuo | A Novel Antibody-Based Approach to Detect the Functional ERCC1-202 Isoform[END_REF]. Isoform specific antibodies have also been useful for furthering our basic understanding of some diseases.

In the case of Amyotrophic Lateral Sclerosis (ALS), C9orf72 isoform specific antibodies have identified differences in their subcellular localisations, which are modified in diseased cells [START_REF] Smeyers | C9ORF72: What It Is, What It Does, and Why It Matters[END_REF]S. Xiao et al. 2015). In Alzheimer's disease and other Tauopathies, antibodies specific to the 0N, 1N and 2N Tau isoforms have enabled the identification of their respective partners, pointing towards a role of the 2N isoform in disease (C. [START_REF] Liu | Co-Immunoprecipitation with Tau Isoform-Specific Antibodies Reveals Distinct Protein Interactions and Highlights a Putative Role for 2N Tau in Disease*[END_REF]).

As we have shown that the specific overexpression of the TP63γ isoform at the RNA level may be of prognostic value, it is important to determine whether this holds true at the protein level.

Isoform-specific antibodies are typically generated by using isoform-specific immunogenic peptides or recombinant proteins to promote antibody production (C. M. [START_REF] West | Generating Isoform-Specific Antibodies: Lessons from Nucleocytoplasmic Glycoprotein Skp1[END_REF]. As stated earlier, multiple protein isoforms are encoded in the TP63 gene (UniProtKB -Q9H3D4 (p63_HUMAN)) thanks to alternative splicing and alternative promoter usage. While some polyclonal or monoclonal antibodies are available to detect some p63 isoforms (Table 3), their capacities to potentially cross-react with the other family members (p53 or p73) in addition to their capacity to detect multiple isoforms render their usage tricky in histology (Nekulova et al. 2013b). Indeed, the initial use of antibodies that could not distinguish between the different N-terminal isoforms of p63 made the analysis of p63 functions and expression quite complex in the early years. While numerous p63 specific antibodies are available, only few are isoform specific. Many target the DNA binding domain or the oligomerisation domain and are therefore able to detect all p63 protein isoforms and sometimes cross-react with the other family members (p53 or p73).

Some are specific to the two N-terminal isoforms TA or ΔN and one, the D2K8X (Cell Signalling Technology) is specific to the C-terminal p63α isoform. None are specific to the p63γ or β isoforms. Therefore, we decided to try to develop novel p63 antibodies. Through a collaboration with the EFS (Etablissement Francais du Sang), we attempted to develop a panel of p63 antibodies that could prove useful in the study of p63 function or expression.

Developing an antibody panel free of licensing rights could prove useful if we want to adopt a commercial strategy further down the line. To develop such p63 antibodies, we selected specific peptides located in precise domains of the p63 protein sequence and for which we could predict the protein isoforms that would be detected. Four peptides were designed to detect a) all ΔN p63 isoforms, b) all the p63 protein isoforms, c) the p63 gamma isoforms (ΔN or TA) and d) the p63α isoforms (ΔN or TA) (Figure 33.A). We chose to avoid the p63β or δ isoforms as only respectively 5 (RIWQV) or 7 (RSGKSENP) aminoacids are specific to those isoforms (see sequence Figure 32).

Mice immunisation and control

Peptides were ordered as biotin-labeled and Keyhole limpet hemocyanin (KLH)labeled peptides from Protéogenix (SCHILTIGHEIM, France). Mice were immunized at the EFS using combinations of the chosen KLH-labeled peptides to increase the probability of a positive immune response. Because the absence of a p63γ specific antibody is the most problematic, we chose to immunize mice using either a single gamma_p63_1 peptide These extracts used so far came from cells overexpressing p63 isoforms at high levels, driven by a CMV promoter. We therefore sought to test if the KO85 antibodies could detect p63 γ specifically and with enough sensitivity on cells with lower overexpression of p63. For this we used a second set of p63 expressing vectors derived from pmirGLO, with a PGK promoter driving their expression.

SP|Q9H3D4-2|P63_HUMAN MGANIPMMGTHMPMAGDMNGLSPTQALPPPLSMPSTSHCTPPPPYPTDCSIVSFLARLGC 464 SP|Q9H3D4-4|P63_HUMAN MGANIPMMGTHMPMAGDMNGLSPTQALPPPLSMPSTSHCTPPPPYPTDCSIVRIWQV---461 SP|Q9H3D4-6|P63_HUMAN ------------------------------------------------------------ SP|Q9H3D4-8|P63_HUMAN MGANRSGKSENP------------------------------------------------416 SP|Q9H3D4-2|P63_HUMAN SSCLDYFTTQGLTTIYQIEHYSMDDLASLKIPEQFRHAIWKGILDHRQLHEFSSPSHLLR 524 SP|Q9H3D4-4|P63_HUMAN ------------------------------------------------------------ SP|Q9H3D4-6|P63_HUMAN ------------------------------------------------------------ SP|Q9H3D4-8|P63_HUMAN ------------------------------------------------------------ SP|Q9H3D4-2|P63_HUMAN TPSSASTVSVGSSETRGERVIDAVRFTLRQTISFPPRDEWNDFNFDMDARRNKQQRIKEE 584 SP|Q9H3D4-4|P63_HUMAN ------------------------------------------------------------ SP|Q9H3D4-6|P63_HUMAN ------------------------------------------------------------ SP|Q9H3D4-8|P63_HUMAN ------------------------------------------------------------ SP|Q9H3D4-2|P63_HUMAN GE 586 SP|Q9H3D4-4|P63_HUMAN -- SP|Q9H3D4-6|P63_HUMAN -- SP|Q9H3D4
Transfection of the pmirGLO TP63 γ expressing plasmid into SCC-9 or A253 cells that normally express p63 was chosen to allow for an estimation of the background detection in a more relevant cell line than HeLa (Figure 38). Multiclonal SCC-9 and A253 cell lines were obtained following these transfections and used for analysis. Extract from HeLa cells expressing p63γ was used as a positive control. In SCC-9 extracts (Figure both detectable signal levels, and an experimentally pertinent context for appreciation of background noise. The same samples were deposited in duplicate series to compare the 4A4 and KO85 antibodies (Figure 38). On SCC9 extracts, the 4A4 antibody recognised a protein just below 72 KDa, coherent with the detection of the endogenous p63α protein from SCC9 cells. In SCC9 cells mixed with a serial dilution of HeLa cell extract expressing p63γ, the 4A4 antibody detected a p63γ band and its likely degradation products specifically even at the strongest dilution (1/128). Comparatively, the KO85 only provided signal superior to background noise at the highest concentration of HeLa extract (1/16 dilution) and with an exposure 74 times longer. Several non-specific species were also recognised by this antibody with such a long exposure. From this experiment, we could estimate that the KO85 purified antibody is about 600 times less sensitive than the 4A4 antibody. Part of the background noise problem is likely down to the poorer sensitivity of the KO85 antibody compared to the 4A4 antibody, which forces the use of longer exposure times.

SCC-25 cells express high levels of p63α, and were therefore chosen to test the recognition of endogenous p63 by the KO1393/pan antibody and compare it to the commercial D2K8X and 4A4 antibodies (Figure 39). To further assess the specificity of the antibodies for p63 proteins, we depleted the SCC-25 cells by siRNA directed against all TP63 isoforms, or against TP63α specifically. A strong signal at 72 KDa is observed with the 4A4 and D2K8X antibodies and correspond to the p63 α isoform as confirmed by the vast decrease in this signal intensity upon treatment with siPan and siA1 or siA2. The KO1393/pan antibody was capable of recognising the same endogenous p63α band with a lesser intensity than the commercial antibodies. The KO1393/pan antibody detected significantly more non-specific bands than both the D2K8X and the 4A4 antibodies.

Furthermore, the p63α band recognised by the KO1393/pan antibody is much weaker than several of its non-specific targets. This raises serious concern over its potential use in immunofluorescence based applications where specificity is crucial.

Conclusion

Despite having developed novel monoclonal antibodies capable of detecting either all p63 isoforms or specifically the p63γ isoform when present at high concentrations, we have fallen short of achieving usable results when dealing with the endogenously low p63

Chapter 4. Identification of circRNAs derived from the TP63 gene in head and neck squamous cell carcinoma (HNSCC)

Since the initial discovery of circular RNA from viroids in the late 70s and the description of some singular cases of eukaryotic circular RNA in the 90s, the development of high throughput sequencing techniques has generalised the existence of such circular RNA [START_REF] Patop | Past, Present, and Future of CircRNAs[END_REF].

By an alternative pathway, pre-mRNAs can produce circulars RNA (circRNA). This process termed back-splicing is a splicing reaction where a donor site (SD) at the 3'end of an exon is spliced to an an upstream acceptor site (SA), which can be in the same exon or in a previous exon. The resulting phosphodiester bond creates a looped molecule: the circRNA (Figure 40). Over 25 000 circular RNAs, derived from around 15% of actively transcribed genes, were identified from a single cell type [START_REF] Jeck | Circular RNAs Are Abundant, Conserved, and Associated with ALU Repeats[END_REF]. Backsplicing of exons for circRNA formation can be considered as an unusual type of alternative splicing.

Back-splicing, however, represents less than 1% of canonical splicing in most endogenous human loci [START_REF] Zhang | The Biogenesis of Nascent Circular RNAs[END_REF]. The usage of splice sites is competitive between back-splicing and canonical splicing. Increasing back-splicing can therefore increase the proportion of linear RNAs lacking the circularized exons. When abundance of core spliceosomal factors becomes limited, it has been observed that back-splicing appears to be more efficient than the production of linear mRNA. This likely results from remodeling of cross-intron exon definition complexes (D. [START_REF] Liang | The Output of Protein-Coding Genes Shifts to Circular RNAs When the Pre-MRNA Processing Machinery Is Limiting[END_REF]. Indeed, exon definition complexes can be deposited on single exons, which become catalytically competent for back-splicing reactions (Xueni Li et al. 2019). This back-splicing event requires the donor and acceptor sites to be in close proximity to each other. This is made possible either by the presence of Inverted Complementary Sequences (ICS), such as Alu sequences, in the introns flanking circRNA-exons . Repetitive elements are enriched in the human genome, but in other organisms, ICS appear to be less crucial. As an illustration, production of circRNA in D. melanogaster is generated from exons with long flanking introns lacking ICS [START_REF] Zhang | The Biogenesis of Nascent Circular RNAs[END_REF]. Besides repeated elements, a number of RBPs that bind the flanking introns can be involved in circRNA biogenesis. By directly bridging distal splice sites, or by binding ICS to modulate back-splicing, RBPs can either stabilize the transiently formed intronic RNA pairs to facilitate back-splicing as illustrated by the RBP Muscleblind (Mbl) which promotes production of circMbl from its own pre-mRNA in D.melanogaster [START_REF] Ashwal-Fluss | CircRNA Biogenesis Competes with Pre-MRNA Splicing[END_REF], or destabilize RNA base-pairing through their helicase activity as illustrated by the loss of DHX9 which increases the levels of circRNAs (Aktaş et al. 2017, 9). The proximity of the dowstream donor to upstream acceptor sites can also arise from the presence of multiple RBPs which can interact with each other (D. [START_REF] Liang | Short Intronic Repeat Sequences Facilitate Circular RNA Production[END_REF]. For example, multiple hnRNPs and SR proteins regulate back-splicing of the laccase2 gene in D. melanogaster [START_REF] Kramer | Combinatorial Control of Drosophila Circular RNA Expression by Intronic Repeats, HnRNPs, and SR Proteins[END_REF].As a result of this process, circRNAs can either be constituted solely of RNA elements that would otherwise constitute exons, or include both exons and retained introns. (Figure 41)

The fact that these RNAs are looped implies they do not have any free extremities, and therefore are well protected from either 5'-or 3'-exonuclease degradation. They may indeed be very stable entities in a cell [START_REF] Enuka | Circular RNAs Are Long-Lived and Display Only Minimal Early Alterations in Response to a Growth Factor[END_REF]). These highly stable transcripts contains almost exclusively exonic sequences and localize mainly to the cytoplasm. For some genes, the abundance of the circular RNA may exceed that of the associated linear RNA. The steady state abundance of circRNA is thus a balanced result of the poor efficiency of circRNA production, nuclear transport and low turnover. The expression of these molecules is therefore probably not very dynamic.

Since they share the same sequence as their related mRNAs, circRNAs likely have a common share of miRNA binding sites with them. Increased stability coupled with the usual absence of translating ribosomes on circRNAs makes them efficient to trap miRNAs and RBPs in a sponge-like manner. For this purpose, several miRNA binding sites are required to achieve a measurable effect in a given cell [START_REF] Bosson | Endogenous MiRNA and Target Concentrations Determine Susceptibility to Potential CeRNA Competition[END_REF]).

The most striking example is the circular RNA CDR1as (also known as CiRS-7) which contains more than 70 conserved binding sites for miR-7 [START_REF] Piwecka | Loss of a Mammalian Circular RNA Locus Causes MiRNA Deregulation and Affects Brain Function[END_REF] [START_REF] Piwecka | Loss of a Mammalian Circular RNA Locus Causes MiRNA Deregulation and Affects Brain Function[END_REF]. This circRNA's mainly expressed in the brain, but is also found in pancreatic islet cells where its overexpression can improve insulin expression by inhibiting the mir7mediated insulin repression This modification of miRNA availability can therefore affect their impact on gene expression [START_REF] Hansen | Natural RNA Circles Function as Efficient MicroRNA Sponges[END_REF][START_REF] Ashwal-Fluss | CircRNA Biogenesis Competes with Pre-MRNA Splicing[END_REF]. It is noticeable that binding sites for miRNAs in circRNAs are only partially complementary to miRNAs, allowing miRNA intracellular transport, but unproductive for Argonaute 2 targeting and further degradation. However circRNA can be degraded by different cytoplasmic endonucleases such as RNase L which is activated during viral infection (C.-X. [START_REF] Liu | Structure and Degradation of Circular RNAs Regulate PKR Activation in Innate Immunity[END_REF] or the ribonuclease complex RNase P/MRP known to be responsible for tRNA + maturation and cleavage of ribosomal RNAs, long non-coding RNAs and mRNAs [START_REF] Park | Endoribonucleolytic Cleavage of M6A-Containing RNAs by RNase P/MRP Complex[END_REF]. CircRNAs are also proposed to interact with RNA binding proteins in a scaffolding manner and further influence their cellular availability (Xiang [START_REF] Li | Coordinated CircRNA Biogenesis and Function with NF90/NF110 in Viral Infection[END_REF][START_REF] Du | Foxo3 Circular RNA Retards Cell Cycle Progression via Forming Ternary Complexes with P21 and CDK2[END_REF]2017;[START_REF] Schneider | CircRNA-Protein Complexes: IMP3 Protein Component Defines Subfamily of CircRNPs[END_REF]. They may also encode peptides, provided they possess some internal ribosome entry sites [START_REF] Lei | Translation and functional roles of circular RNAs in human cancer[END_REF]. However circRNA-derived peptides are often truncated versions of the canonical proteins and lack essential functional domains. They may act as dominant-negative protein variants, decoys, or modulators of alternative protein complexes [START_REF] Legnini | Circ-ZNF609 Is a Circular RNA That Can Be Translated and Functions in Myogenesis[END_REF].

CircRNAs have been implicated in numerous diseases including cancer, cardiovascular disease, autoimmune diseases and neurological disorders. [START_REF] Zhou | Circular RNA: metabolism, functions and interactions with proteins[END_REF][START_REF] Kristensen | The Biogenesis, Biology and Characterization of Circular RNAs[END_REF]L.-L. Chen et Yang 2015). An additional interest arises from the important stability of circular RNAs. They may indeed be sufficiently stable in blood to serve as circulating biomarkers that could be sampled without invasive procedures.

Recently, in 2019, a TP63 circRNA was identified as potentially involved in lung squamous cell carcinoma (LUSC) development. This TP63 circRNA (hsa-circ-0068515) is elevated in advanced stages of this disease. By binding to mir-873-3p, the TP63 circRNA prevents this miRNA from decreasing FOXM1 levels, which is an inhibitor of cell cycle progression. This mechanism explains the increase of cellular proliferation and survival which follows the cytoplasmic increase of circTP63 in LUSC cells (Cheng et al. 2019). A number of other circRNAs have been identified in HNSCC, with a potential use as biomarkers (see Table 5) [START_REF] Guo | Circular RNAs and their roles in head and neck cancers[END_REF]. Functions for circRNAs have also been described in head and neck cancer cells, controlling proliferation and the cell cycle, migration, apoptotis and response to chemotherapy either positively or negatively (Guo et al. 2019). Considering the apparent importance of TP63 in HNSCC biology, we sought to establish whether its circRNAs are expressed in HNSCC cell lines.

Identifying TP63 circRNAs in HNSCC cell lines

From the TP63 locus, Julia Salzman and colleagues identified three TP63 circRNAs [START_REF] Salzman | Cell-Type Specific Features of Circular RNA Expression[END_REF]). The first one originates from the TA promoter, hence I shall not detail it further. The second one, hsa-circ-0068515, contains exons 11 and 13 which surround the γ terminal exon. The third one, hsa-circ-0068516, contains exons 14 and 15 (Figure 42.A). I shall therefore refer to them as circ13-11 and circ15-14 for the sake of simplicity.

Does PTBP1 influence TP63's back-splicing into circRNAs?

Of the many exons composing the TP63 pre-mRNA, it is interesting that the two described circRNAs fall within regions subjected to alternative splicing. Since we previously identified PTBP1 as a regulator of TP63 splicing, we sought to determine whether PTBP1 was also modulating the production of these circRNAs. By RT-qPCR, and using circ13-11 or circ15-14 back-splicing specific primers, we compared the abundance of circRNAs in PTBP1-depleted cells and control cells (Figure 44 A,B). For both cirRNAs, depleting PTBP1 had no impact on their expression levels. Abundance of TP63γ mRNA was increased in these same samples following PTBP1 depletion, serving as a positive control (Figure 44 C). One possible caveat is the fact that this analysis only detects presence or absence of the back-splicing junction, without knowing whether the RNA was in fact circular. Furthermore, this analysis was carried out on samples obtained after 48h of PTBP1 depletion. Since circRNAs are thought to be very stable, and their biogenesis infrequent, there may not have been enough time for an effect of PTBP1 depletion to be RNA was obtained from cells depleted in PTBP1 for 48h. Results were normalised to untreated cells and total TP63 was used as a reference gene following the ddCT method. siP1: siPTBP1. . visible on the pool of cellular TP63 circRNAs. Analysis should therefore be carried out at further time points, and should also include other methods of detection such as northern blotting, which could allow us to conclude on the levels of circular RNA rather than on the sole presence of a back-splicing event.

Conclusion

TP63 circRNAs are indeed expressed in HNSCC cells, with preliminary evidence suggesting that their relative levels are just as variable as their mRNA counterparts.

Interestingly, there does not seem to be a direct link between TP63 abundance and TP63 circRNA levels. A253 cells for example express high levels of TP63, yet they are almost the weakest in terms of circRNA expression. In view of this, it seems unlikely that circulating TP63 circRNAs could directly reflect the exact expression of TP63 within tumour cells. Since many normal epithelial tissues express TP63, there is likely a baseline level of circulating TP63 circRNA in the body. However, it would be interesting to see whether variations of this baseline can be measured during tumour development, reflecting the overall number of TP63 positive cells in the body. If this is the case, TP63 circRNAs could become a useful tool for early detection of many epithelial derived tumours.

The apparent dissociation between TP63 mRNA and TP63 circRNA levels highlights the existence of some kind of regulation independent of TP63's transcription rate. The circRNAs map to regions of the pre-mRNA which are subjected to alternative splicing. One possibility was therefore that the RNA binding protein PTBP1, which we have identified as a regulator of TP63 alternative splicing, could also exert an effect on the biogenesis of TP63 circRNAs. We tested this hypothesis by measuring the abundance of the respective back-splicing junctions or the TP63 circRNAs with or without PTBP1 depletion and found no difference. From this experiment, it is however impossible to conclude to an absence of influence from PTBP1, as stated previously, and this will therefore be the object of further enquiries. It is yet unclear whether the production of TP63 mRNA and TP63 circRNA should be correlated or on the contrary inversely correlated as both biosynthetic pathways are exclusive.

The work of Cheng and colleagues has highlighted a function of TP63 circRNA in a cancer context. It is however apparent that these circRNAs are not specific to cancer, we have notably detected them in HaCaT immortalised keratinocytes. It would be interesting to see whether they are conserved across multiple TP63-expressing species which could reflect the evolutionary degree of importance of their regulatory pathways. Likewise, we have so far only identified these circRNAs in an epithelial context, and it would be interesting to assess their expression in non-epithelial TP63-positive cells such as muscle, bone or germline cells. In the context of HNSCC, this work will be carried on in the lab with overexpression experiments of circRNAs to try and identify phenotypic changes and their associated regulatory pathways, which will likely involve miRNAs.

GENERAL DISCUSSION

From a global point of view, the most striking finding of my Ph.D work is the identification of a roughly 400 million year old regulatory mechanism that mediates the inhibition of the TP63γ isoform production. I have identified a conserved and functional regulatory interaction between the RNA binding protein PTBP1 and TP63 alternative splicing. In tissues with high PTBP1 expression, splicing of TP63 towards the longer C-terminal isoforms is favoured while in situations of lower PTBP1 expression the accumulation of an isoform deprived of most of the C-terminal domains is favoured. Obviously, the existence of this conserved and functional regulatory interaction between PTBP1 and TP63 alternative splicing raises multiple fundamental questions that may not have any answer yet.

• What is/are the function(s) of this shorter isoform that warranted its evolutionary conservation? And the corollary question: when did this alternatively spliced isoform emerge in the course of the TP53 gene family evolution?

Answering these two questions will require tackling problems linked to developmental biology on one side, and to evolutionary biology on the other side. The increased accumulation of genomic and transcriptomic datasets from many different species across the tree of life should allow to identify the turning point when TP63 gamma emerged. Was it before the divergence of TP63 and TP73 functions or was it later on, once the family was already composed of the three members (TP73/TP63/TP53)? The latter hypothesis would indicate that p63γ specific functions are likely linked to the expression of the other family members. TP73's expression pattern has been shown to overlap with TP63's in some situations [START_REF] Marshall | Tissue-Specific Expression of P73 and P63 Isoforms in Human Tissues[END_REF]. p73 participates in developmental processes that also require p63 activity such as the development of mucociliary epithelium. In this context, p63γ could be implicated in the fine tuning of certain developmental steps, outside of which its expression would be of limited use to cells. This would be in line with its overall absence in adult tissues, skeletal muscles aside. It would also explain the lack of any striking modifications in cellular phenotype following its overexpression as shown in the second results chapter. Furthermore, p63γ expression in striated skeletal muscle cells has been shown to be cytoplasmic, which contrasts with the general assumption that p63 always acts as a transcription factor. In the few instances where it is is expressed, p63γ is present as a TA isoform. This raises two possibilities: γ's use could be restricted to the TA isoform with no real requirement for the ΔN isoform, or γ splicing could be more efficient in the context of transcription from the TA promoter.

Tackling developmental functions of the γ isoform will require specific alteration of its expression in a complex organism. TP63 isoform specific knockouts mediated by CRISPR CAS could probably allow us to perform such gene editing, and promote only • Does the gamma isoform have a regulatory role on TP63α?

In the course of the experiments described in this thesis, we showed that specific downregulation of TP63γ led to a decrease in TP63α. While ruling out a potential artefact would require additional experiments, the fact that two different siRNAs targeting TP63γ

were capable of directing decreased TP63α expression points to a specific effect event though its mechanism is unclear. In particular it is obscure whether this mechanism is mediated by a drop in p63γ protein expression or whether it's mediated directly by the siRNA used. Previous work showed that HeLa cells overexpressing TP63γ started to express TP63α [START_REF] Antonini | An Autoregulatory Loop Directs the Tissue-Specific Expression of p63 through a Long-Range Evolutionarily Conserved Enhancer[END_REF]. We should reiterate these experiments, forcing

TP63γ expression in HeLa cells, with concomitant knockdown of TP63γ by siRNA to ascertain that p63γ is indeed involved in a self amplifying loop for TP63α activation.

• Does p63's c-terminal region affect its subcellular localisation?

In human adult tissues, the sole robust expression of TP63γ is observed in muscle tissues. Interestingly, rhabdomysosarcomas that are cancer derived from muscle cells are characterized by accumulation of p63 in a somehow unusual localisation as it appears to be cytoplasmic [START_REF] Martin | Cytoplasmic P63 Immunohistochemistry Is a Useful Marker for Muscle Differentiation: An Immunohistochemical and Immunoelectron Microscopic Study[END_REF]. Abnormal cytoplasmic localisation of TP63 as been associated with poor outcome in prostate cancer patient [START_REF] Dhillon | Aberrant Cytoplasmic Expression of P63 and Prostate Cancer Mortality[END_REF]) and in lung adenocarcinoma [START_REF] Narahashi | Cytoplasmic Localization of P63 Is Associated with Poor Patient Survival in Lung Adenocarcinoma[END_REF]). However it is unclear the nucleo-cytoplasmic relocalisation of p63 is mediated. Actually, it was unclear until recently how proper nuclear localisation of p63 is achieved [START_REF] Hazawa | ROCK -dependent Phosphorylation of NUP 62 Regulates P63 Nuclear Transport and Squamous Cell Carcinoma Proliferation[END_REF]. In rhadomyosarcomas, is the cytoplasmic localisation linked to the nature of the C-terminal region of p63 isoforms, or to altered regulation of the import pathway under the control of NUP62 and Rho Kinase [START_REF] Hazawa | ROCK -dependent Phosphorylation of NUP 62 Regulates P63 Nuclear Transport and Squamous Cell Carcinoma Proliferation[END_REF])? The specific localisation of p63 at the Z-striations in muscle cells suggests it is not merely present in the cytoplasm through lack of nuclear import; it would rather seem that p63 is purposefully addressed to this location to ensure a yet unknown function. It would be interesting to express different p63 in a muscle cell line, and follow their subcellular localisation following differentiation.

• Considering our demonstration of TP63γ repression by PTBP1 in epithelial cells, what consequence would PTBP1 expression have on TP63 isoforms produced in muscle cells?

We have mainly addressed the control of PTBP1 on TP63 alternative splicing using • Does regulation of TP63 alternative splicing have potential for diagnostic or therapeutic use?

While overexpression of TP63 is associated to specific types of cancer, most notably in squamous carcinomas (lung, head and neck, cervical) and oesophageal adenocarcinomas, disease progression is usually correlated to loss of TP63 expression.

We showed that in HNSCC patients, a high inclusion of TP63γ is associated with a shorter overall survival of the patients. While this does not demonstrate a causal link between the two phenomenon, this does however suggest that analysis of TP63 expression in tumours samples could allow refinement of tumour description and grading. However, no antibodies are so far available to address this question. The alternative to immunostaining would be to analyse the expression of TP63 isoforms at the RNA level in FFPE samples using RT-qPCR. Of course, it has to be noted that due to the correlative nature of the relationship between TP63γ expression and patient survival, TP63γ could have no causal affect at all.

Instead, it could be the result of an overall modified splicing landscape in cancer cells. The expression of the correct set of RNA BPs is an important element in cellular differentiation.

In cancer, disease progression is usually associated with a de-differentiation of cells, or trans-differentiation into aberrant cell types for the tissue of origin. In this context, measuring TP63γ levels could still be used as an interesting marker of aberrant splicing in epithelial cancer cells.

• TP63 alternative splicing: an entry point for disease producing mutations?

The regulatory mechanism we have identified likely controls some TP63 functions.

Apart from carcinogenesis, mutations in TP63 are causal to many different developmental syndromes affecting ectodermal derivatives. Among the repertoire of different syndromes, the location of the mutation relative to the functional domains of the encoded protein defines the spectrum of individual defects. One can envisage that mutations located in intronic regulatory domains affecting PTBP1 binding to TP63 could promote expression of the γ isoform. This could define TP63 dependent pathological consequences which should resemble those stemming from mutations producing truncated TP63 proteins. In a broader sense, mutations which increase the strength of the TP63γ exon's splice sites in cis or in trans could explain cases of patients showing a p63-attributed disease without having any mutations in regions corresponding to functional domains.

• Is TP63 functionally important besides its protein products?

There are two striking differences between TP63α and TP63γ. The first, and most obvious, is the lack of c-terminal regulatory domains in p63γ. This has long stood as an argument for a dominant-negative like role of γ on α. The experimental proof of this has yet to emerge. Furthermore, in an adult body, TP63γ is expressed in muscle cells where other isoforms are absent. This obviously circumvents any possible dominant-negative functions. The second difference lies in the difference in 3'UTR between TP63α and γ. In conclusion, we have established that the production of the TP63γ isoform has been tightly regulated for hundreds of millions of years by the RNA binding PTBP1. The strong conservation of its regulation contrasts with its overall extremely low expression.

This suggests that whatever the function of TP63γ, it is generally dispensable, but must be fundamentally important in one or several contexts to warrant its continued existence.

Despite previous descriptions of its involvement in the EMT, we have been incapable of reproducing such effects. In fact, we have been incapable overall of identifying a TP63γ

IV. Conclusion et perspectives ouvertes par ce travail

En résumé, au cours de ce projet de thèse nous avons réussi à identifier le premier régulateur de l'épissage alternatif de TP63 : la PTBP1. Cette régulation conditionne la production de l'isoforme γ vis-à-vis des autres isoformes plus longues. In cells, TP63 is involved in stem cell renewal, differentiation, proliferation, control of epithelial to mesenchymal transition, and in the control of the genomic integrity of germ cells. Alternative promoters and alternative splicing events allow for the production of at least eight different p63 protein isoforms. At the C-terminal end, α, β, γ and δ isoforms differ by the occurence of functional protein domains involved in the control of the activity or lifespan of the protein. The shorter γ isoform is produced by the use of a distinct alternative 3 'UTR. The existence of multiple isoforms is often used to explain the many and sometimes conflicting roles of TP63 in cellular phenotypes.

TP63 is often amplified or overexpressed in squamous cell carcinoma and we found a negative correlation between the γ isoform and Head and Neck Squamous Cell Carcinoma patient survival. By combining bioinformatic analysis of alternative splicing data and mechanistic analysis of TP63 alternative splicing, I identified a direct binding between the splicing regulator PTBP1 and TP63 pre-mRNA that controls the outcome of TP63 gene expression by repressing the production of the γ terminal exon in squamous cell carcinoma cell lines. This is the first identification of a transacting factor controlling TP63 splicing. The functional importance of this regulation is further supported by its conservation from Amphibians to Humans. However, questions concerning the functional consequences of changes in the abundance of the γ isoform remain open.

Figure. 1 .Figure. 2 .

 12 Figure.1. Schematic evolution of the TP63/73/53 gene family

Fig. 3 :

 3 Fig.3: Alternative promoters and alternative splicing events produce many RNA and protein isoforms. Schematic representation of the complex exon/intron architecture of TP53/63/73 genes.

  this leads to the four different C-terminal peptides shown on the figure. Not only are the Cand N-terminal peptides different but full peptidic domains are omitted or diverged inbetween the different isoforms. This is detailed in Figure4B-C

Figure 4 :

 4 Figure 4 : TP63 gene, RNA and proteins. A. Structure and genomic location of 5' and 3' splice sites of the TP63 human gene. "pA" denotes polyadenylation sites. ATGn indicates the start codon. N and C-terminal peptides of the indicated isoforms are shown. Exon composition of the 4 main different C-terminal isoforms is shown under the gene structure. B. Exon structure and color encoded peptidic domain of TP63. D. Domain composition of the main P63 protein isoforms.

Figure. 5 .

 5 Figure. 5. DNA Binding element and organisation of TP63 as tetramer. A. The DNA elements bound by the p63/73/53 proteins are presented as a logo motif based on the JASPAR 2016 database (http://jaspar.genereg.net/). B. Structure of the DNA binding domain of p63 associated with two response element (PDB : 3QYN)

Figure. 6 .

 6 Figure. 6. TP63 mRNA expression in developing Xenopus laevis by whole mount in situ hybridization. (A) positive and (B-C) negative control probe for ISH. (D-F) pools of embryos at the indicated stage were used to detect TP63 expression. (G, J, M, P) Anterior view of TP63 expression in embryos at the indicated stages, (HKNQ) dorsal views, (I,L,O,R) posterior views. The scale bar is 0.5 mm. (from Yann Audic, unplublished data) 23

Figure 7 .

 7 Figure 7. Immunolocalization of p63 in Human Epithelial Tissues from Yang et al. 1998 Paraffin sections of normal human epithelial tissues probed with monoclonal antibodies to p63 using an alkaline phosphatase reporter system. A. p63 staining in foreskin showing nuclear localisation of p63 in basal epithelial cells. B. p63 localisation to basal cells of ectocervical epithelium. C. p63 localisation in basal cells of vaginal epithelium. D. p63 staining of basal urothelium cells. E. p63 staining of the epithelial cell layer below luminal cells in prostate tissue.

  transient amplifying cells then differentiate. While most of the basal cells of epithelia express high levels of ΔNp63, the balance between TAp63 and ΔNp63 seems to sometimes play a regulatory role in balancing stemness with proliferation. Using a complex cell sorting procedure, Li and colleagues identified mammary cell populations that expressed either TA or ΔNp63. The enriched stem cell fraction express ΔNp63 in agreement with previous evaluation of ΔNp63 expression in stem cell while a progenitor cell population exclusively expressed TAp63 along with the Indian hedgehog (IHH) ligand. The strict exclusion of IHH expression and ΔNTP63 expression raised the possibilities that IHH is controlled by p63 or controls TP63 promoter selection. Overexpression of the TAp63γ isoform or downregulation of ΔNp63α activated IHH expression in immortalized mammary epithelial cells, demonstrating that TAp63 enhances hedgehog signalling. Both TA and ΔNp63 associate to the IHH locus in ChIPseq experiments. Furthermore, IHH activation depleted the hedgehog effectors GLI3/GLI3R, increased mitotic activity in the progenitor population and favoured TAp63

•

  EEC: The main syndrome associated with TP63 mutations which combine all three types of developmental defects. Ectrodactyly, Ectodermal dysplasia and Cleft lip/palate • AEC: Ankyloblepharon is characterised by the adhesions between upper and lower eye-lids, Ectodermal defects and cleft lip/palate syndrome. It is also referred to as Hay-Wells syndrome. • RHS: Rapp Hodgkin syndrome encompasses ectodermal dysplasia and cleft lip/palate syndrome. It is therefore quite close to the AEC. • LMS: Limb mammary syndrom is mainly represented by Ectrodactyly and defects in the development of the mammary glands and nipples. The ectodermal defects can be present but are generally mild. • ADULT: Acro-dermato Ungual-lacrymal-tooth syndrome. ADULT defects are mainly Ectrodactyly, Ectodermal dysplasia • SHFM4: Non syndromic Hand Foot Malformations are mainly associated with Ectrodactyly Mutations specific to each of these syndromes almost always fall within p63 functional domains (van Bokhoven et McKeon 2002). EEC mutations typically fall within the DNA binding domain whereas AEC mutations are usually located in the SAM domain.This suggests that symptoms observed in EEC are provoked by defects in p63's ability to

Figure 8 .

 8 Figure 8. TP63 mutations and overlapping human syndromes.The association of orofacial clefting with Ectodermal Dysplasia (ED) and limb malformations are the three hallmarks of TP63 dependent developmental defects that are differently combined in different TP63 dependent syndromes.. Ectrodactyly, Ectodermal Dysplasia, and Cleft lip/palate syndrome (EEC), Limb Mammary Syndrome (LMS), Ankyloblepharon-Ectodermal defects-Cleft lip/ palate (AEC), Acro-Dermato-Ungual-Lacrimal-Tooth syndrome (ADULT), Rapp-Hodgkin Syndrome (RHS) and non-syndromic Split Hand/Foot Malformation (SHFM4). (adapted fromRinne et al. 2006)

Figure. 9 :

 9 Figure. 9 : Alterations of the TP63 gene in Pan Cancer data (https:// www.cbioportal.org/) Distribution of amplifications, mutations, structural variants and deletions in different cancer types.

Next, the 3 '

 3 splice site (3'SS) is characterised by YAG/xx, the AG dinucleotide marking the 3' end of the intron. The 3'SS is preceded by a 10-20 pyrimidine stretch called the "polypyrimidine tract". The final element is the branch point sequence, a degenerate heptamer -YNYURAY-which must contain an adenine, located 20 to 40 nucleotides upstream of the 3'SS. (Figure. 10)

Fig. 10 .

 10 Fig.10. Schematic representation of splicing regulatory sequences within the Human pre-mRNA. Introns are minimaly characterised by three short conserved sequences: a 5'splice site (5SS), a branchpoint sequence (BPS) containing the branch point (BP) and a 3'splice site (3'SS). A polypyrimidine tract is also present between the BPS and the 3'SS. The branchpoint adenosine is indicated in purple. (Adapted from Wilkinson et al. 2020) .

Fig. 11 .

 11 Fig.11. Exon and intron definition models. The left panel depicts the intron definition model according to which pairing between the splice sites intially takes place across an intron when long exons are separated by introns shorter than 250bp. The right panel depicts the exon definition model which happens with introns longer than 250bp, the splice sites initially communicate across the exons. (from De Conti, Baralle and Buratti. 2012) .

Fig. 12 :

 12 Fig.12 :The splicing cycle A. The spliceosome is assembled in a highly ordered manner, activated to form the active site and remodeled extensively to perform the branching and exon ligation reactions, releae the mRNA and disassemble. ATPases from the DEADbox, DEAH-box and Ski-2 families depicted in red play crucial roles in this remodeling. B. Introns are removed following two trans-esterification reactions, branching and exon ligation, which are catalyzed at a single active site. Abbreviations: BP branch point, ILS intron-lariat spliceosome, mRNA messenger RNA, NTC Prp-19 associated complex, NTR Prp-19 associated complex, snRNP small nucleolar ribonucleoprotein, SS splice site.(from Wilkinson et al. 2020) .

  Figure 13)

Figure 14 .

 14 Cleavage and Polyadenylation Specificity Factor (CPSF): CPSF is constituted of five different subunits(CPSF-160, CPSF-73, CPSF-100, CPSF-30 and HFip1). The main subunit, CPSF-160, directly binds the PAS. CPSF-73 binds to the C/A cleavage site in a hexanucleotide dependant manner, and could be responsible for the endonucleotidic cleavage. CPSF-100 interacts with itself and with other proteins in the processing complex. CPSF-30 binds to the U-rich sequence and coordinates the assembly of other factors; its zinc finger motifs make it another possible candidate for the endonuclease activity. HFip1 interacts with both PAP and other CPSF subunits, it contacts the RNA on the U-rich upstream auxiliary sequence.  Cleavage stimulation factor (CstF): CstF-64 binds to the RNA on the G/U rich DSE by its conserved RRM motif, placing it physically downstream of the CPSF complex. It also interacts with CstF-77 and symplekin via a hinge domain, and with CF-1A via its carboxy-terminal domain. CstF-77 likely acts as a dimer, and can interact with the carboxy-terminal domain of the RNA-polII along with CstF-50.  Mammalian cleavage factor I (CF Im): CF Im is a heterodimer made up of a small subunit of 25kDa, and a choice of three large subunits of either 59, 68 or 72 Kda. Together, they interact with the RNA in a sequence-dependant manner, aiding in the definition of the polyadenylation site. Mammalian cleavage factor II (CF IIm): CF IIm is also composed of two subunits. HPcf11 can bind both the RNA-pol II and the RNA and subsequently influences transcription termination and the release of the 3'end processing factors from the polII HCip1 interacts with CF Im and CPSF.  Poly(A) polymerase (Pap1p/PAP): Pap1p is a required enzyme for cleavage and polyadenylation. The length of the poly(A) tail is dictated by the nature of its

Fig. 13 :

 13 Fig.13 : multiple factors affect definition of Cleavage and polyadenylation sites Multiple cues are factored in during alternative polyadenylation site choice. The gene's promoter, recruitment of regulatory proteins to the polyadenylation complex, the RNA pol II and its interaction with the RNA pol II associated factor PAF, various RNA binding proteins bound to the nascent transcript, presence of N 6methyladenosine (m 6 A) and inhibition by U1 snRNP all influence the final outcome of alternative polyadenylation.. (from Tian and Manley. 2017) .

Fig. 14 .

 14 Fig.14. Mammal cleavage and polyadenylation complex. The cis-regulatory elements, GU rich sequence (G/U), the cleavage site (CA) and the hexanucleotide (AAUAAA) are presented with the trans-acting factors of the cleavage and polyadenylation machinery. The protein composing the CPSF complex are shown in red, the Cstf complex in blue, CFIm and CF IIm in orange and green respectively.(from Mandel et al. 2008). .

Fig. 15 .

 15 Fig.15. Schematic depiction of the most common alternative splicing patterns. P1,P2 promoters, AAAA... cleavage and polyadenylation sites. pre-mRNA are shown on the left and matured mRNA products on the right. Exons are represented as boxes and splcing events as broken lines joining two 5' and 3' splcie sites (from Kai-qin Le et al. 2015). .

  Exonic splicing enhancers, or ESEs, typically stimulate the formation of the E complex by binding activator proteins. Intronic splicing enhancers work in much the same way, and tend to be localised close to the 5' and 3' splice sites where they help with the recruitment of splicing machinery. (S.[START_REF] Lin | SR Proteins and Related Factors in Alternative Splicing[END_REF]. Negative splicing regulation is provided by exon and intron splicing silencers, usually by changes in conformation of the RNA or by steric hindrance that decrease interaction of activating factors with enhancer sequences. The proteins involved belong to the hnRNP family and SR proteins[START_REF] Martinez-Contreras | HnRNP Proteins and Splicing Control[END_REF]). These factors recognize specific intronic or exonic sequences which facilitate or inhibit, in a position-dependent manner, the recognition of neighbouring splice sites by the spliceosome machinery. Most proteins bind to single stranded RNA elements and changing local RNA structure can inhibit such binding. The availability of active splicing regulatory proteins in the cell can be influenced through modulation of expression levels, post-translational modifications or by control of their nucleo-cytoplasmic localisation.

Fig. 16 .

 16 Fig.16. Cis-acting elements and trans-acting factors controling alternative splicing.RNA-binding motif (RBM) proteins, arginie-serine-rich (SR) proteins (including SRSF2) and heterogeneous nuclear ribonucleoproteins (hnRNPs) bound to exonic or intronic regulatory elements can promote or prevent the recognition of the 5' splice site by the U1 small nuclear ribonucleoprotein (snRNP) or of the 3' splice site by SF1, U2AF2, U2AF1, or U2 snRNP, thus affecting splice site choices and therefore alternative splicing decisions. (from[START_REF] Bonnal | Roles and mechanisms of alternative splicing in cancer -implications for care[END_REF]. .

  choice of polyadenylation site (PAS) during alternative polyadenylation can be influenced by various factors, including the gene promoter at the transcription start site (TSS); recruitment of factors that influence PAS choice; nucleosome density in the region around the PAS; RNA polymerase II (Pol II)-mediated transcription elongation by the Pol IIassociated factor (PAF) complex; the function of various RNA-binding proteins (RBPs) associated with the nascent transcript; the presence of N 6 -methyladenosine (m 6 A); and inhibition of polyadenylation by the splicing factor U1 small nuclear ribonucleoprotein (U1 snRNP). See the main text on cleavage and polyadenylation for more details.

Fig. 17 .

 17 Fig.17. The four modes of alternative polyadenylation. The combination of alternative splice sites and alternative cleavage and polyadenylation sites allows for mulitple flavors of terminal exon definitions. (from Elkon et al. 2013). .

  members of the family are related by the presence of one or two amino-terminal RRM domains, and a carboxy-terminal RS domain. By phosphorylation of the Serine residues of the RS domain, SR proteins can be moved from storage in nuclear speckles to active transcription sites. Most of these factors are ubiquitously expressed but their relative abundance or activity may differ in different tissues and can be modulated by induction of specific cellular signalling pathways SR proteins are mainly located in the nucleus but some (SRSF1, 3 and 7) are also exported to the cytoplasm where they aid with mRNA export. This process couples the nuclear export of an mRNA with its proper splicing and 3′ end processing[START_REF] Müller-Mcnicoll | SR Proteins Are NXF1 Adaptors That Link Alternative RNA Processing to MRNA Export[END_REF], as well as with mRNA decay and translation[START_REF] Sun | SF2/ASF Autoregulation Involves Multiple Layers of Post-Transcriptional and Translational Control[END_REF][START_REF] Sanford | A Novel Role for Shuttling SR Proteins in MRNA Translation[END_REF]).There are three proposed modes through which SR proteins could positively influence splicing. The first mode consists in stimulating the recruitment of U1 or U2AF when bound to an ESE. This could provide an extra positive signal for otherwise weak 5' and 3' splice sites. The second mode relies on the competition between the effects of SR and hnRNP proteins: by binding to the ESEs, SR proteins could inhibit the binding of hnRNPs to them, therefore promoting exon usage. The third and final mode by which SR proteins promote splicing is by creating a web of protein interactions which bridges the intron and contributes to the physical grouping of 5' and 3' splice sites.

FIG. 18 :

 18 FIG. 18: Different entry points and outcomes of altered splicing decisions.Alternative RNA splicing enables the production of distinct RNA isoforms from a single gene, providing transcriptional plasticity by controlling which RNA isoforms are expressed at a given time point in a given cell type. Defects in alternative splicing are frequently found in human tumours and result either from mutations in splicing-regulatory elements of specific cancer genes or from changes in the regulatory splicing machinery. Alterations of the splicing regulation may benefit cell proliferation or migration, or unable escape from cell death. Green: ESE or ISE, red: ESS or ISS

  both trans and cis cases, the mutations can lead to the expression of either dysfunctional proteins, or functional proteins at abnormal levels. In a more indirect manner, mutations can happen in non coding genomic regions, resulting in either cis effects by alteration of the regulatory role of 5' and 3' UTRs or by altering the function in trans of lncRNAs and other non coding RNAs, which could involve mechanisms such as sponging of splicing factors or scaffolding roles during the establishment of splicing complexes.

Fig. 19 .

 19 Fig.19. Modulation of splicing by SSOs in spinal muscular atrophy. Schematic representation of disease associated splicing in Spinal Muscular Atrophy (SMA) within the SMN2 pre-mRNA, and the antisense oligonucleotide targetting strategy used to therapeutically influence splicing. (from Havens and Hastings. 2016). .

  PTBP1 is capable of nucleo-cytoplasmic shuttling, and its function in internal ribosomal entry sites (IRES) mediated translation initiation of both cellular and viral mRNAs is extensively documented. Most translation initiation is mediated by the cap structure at the 5' end of the mRNA, which is recognized by the initiation factor complex eIF4F. The 40S ribosomal subunit interacts with eIF4F and scans along the RNA until it reaches the AUG start codon. In contrast, viral RNA from the picornoviridae family lack a cap structure and ribosomal recruitment occurs internally at highly structured elements known as IRES[START_REF] Balvay | Translational Control of Retroviruses[END_REF]. Interaction with the 40S ribosomal subunit is mediated by additional cellular factors, known as initiation of translation accessory factors (ITAFs) such as PTBP1. PTBP1 has been shown to be required in internal ribosome entry by a number of viral IRES(Martínez-Salas et al., s. d.). It has been proposed that PTBP1 may act as a chaperone to stabilize IRES structure to enable the recruitment of the ribosome and to position it at the start codon[START_REF] Song | Evidence for an RNA Chaperone Function of Polypyrimidine Tract-Binding Protein in Picornavirus Translation[END_REF]. Outside of the viral context, PTBP1 also plays an ITAF role in the translation of several cellular factors. For example PTBP1 positively regulates the IRES activities of p53 isoforms, relocating them from the nucleus to the cytoplasm during stress conditions[START_REF] Grover | Polypyrimidine Tract Binding Protein Regulates IRES-Mediated Translation of P53 Isoforms[END_REF].

Fig. 20 .

 20 Fig.20. Mode of actions of PTBP1 on alternative splicing and cleavage and polyadenylation. A. Alternative splicing. In the absence of PTBP1, the two exons are spliced together (a). PTBP1 masking of a splice site (b) or covering the targetted exon (c) prevents its inclusion into the mature mRNA. PTBP1 multimerisation can exclude an exon from being spliced by creating a loop in the pre-mRNA (d) B. Cleavage and polyadenylation. (a) Normal assembly of the cleavage and polyadenylation factors onto the pre-mRNA. (b) PTBP1 masks the 3'SS and the CPA signals of a terminal exon, preventing its recognition by both splicing and CPA factors. .

•

  When PTBP1 binding sites are dispersed around a regulated exon, PTBP1 proteins can act cooperatively to cover this exon, making it physically inaccessible to the splicing machinery. (Figure 20. A-C) • If PTBP1 sites are present before and after a regulated exon, PTBP1 proteins can regroup and bind to each other, effectively creating a loop which excludes the exon from the remainder of the pre-mRNA and potentially bringing the surrounding 5' and 3' splice sites in a closer conformation. This mechanism was first described for the N1 exon of c-src. (Figure 20. A-D)

Figure 21 :

 21 Figure 21 : PTBP1 controls the inclusion of a terminal exon in CT/CGRP RNA A. CT/CGRP regulated internal terminal exon. Low quantities of PTBP1 in neuronal cells and higher levels in thyroid C cells. B. Polyadenylation enhancer element located downstream of the terminal exon. The location of the PTBP1 binding motif is shown. C. Model for PTBP1 mediated activation of CT/CGRP polyadenylation.(fromLou et al. 1999)

  2020). PTBP1 has been identified as a potent regulator of PKM2 (David et al. 2010). Numerous reports highlight the involvement of PTBP1 in cancers of different origins and by modulating different cellular pathways. The following is a brief summary of some of their highlights. PTBP1 is upregulated in breast cancer tumours and cell lines, and correlates with her2 expression, lymph node metastasis and disease stage. PTBP1 knockdown depletes the strong invasive capacity of MDA-MB-231 cells and in mice, tumours derived from PTBP1 depleted MDA-MB-231 show slower growth than control cells. Depletion of PTBP1

Figure. 22 .

 22 Figure. 22. The sequential steps of the metastatic cascadeEpithelial sheets undergo initial abnormal proliferation and primary tumour formation. The epithelial to mesenchymal transition promotes the initial invasion of the underlining tissues and of the microvasculature. Cells can then disseminate in the body before before exiting the circulation and forming secondary tumours while reverting to a more epithelial phenotype. (from[START_REF] Thiery | Epithelial-Mesenchymal Transitions in Tumour Progression[END_REF] .

Figure. 23 :

 23 Figure. 23:The EMT as defined by the EMT international association (fromYang et al. 2020)

  The first promoter generates mRNAs encoding TAp63 isoforms comprising the N-terminal transactivation (TA) domain. The mRNAs produced from the second promoter encode only a partial TA domain and the encoded proteins are the ΔNp63 is Np63 isoforms. The ΔNp63 is Np63 isoforms are the most abundant ones in epithelial tissues, where they are present mainly in basal cells. They carry most of the p63 functions required for epidermal proliferation and differentiation. The TA isoforms are preferentially expressed during female germline differentiation where they protect the genome from

  5 mM each, Cy3-UTP 0.1mM (Jena Bioscience), RNAsin (1U/µl, Promega), T7 RNA polymerase (1,25 U/ µl), DNA template (12,5 ng/µl)). RNA were purified on G50 sephadex (GE-healthcare). and controlled by denaturing electrophoresis and fluorescent detection (Typhoon FLA 9500). Cy3 fluorescence and RNA absorbance were quantified on a De Novix DS-11 spectro/fluorimeter.Protein production and EMSAThe sequence encoding hsPTBP1 (NM_002819.5) was obtained from sourcebiosciences (IRAUp969B052D). The PTBP1 ORF was PCR amplified and subcloned by gibson cloning into pET21A+ (Novagen) digested by XhoI and NheI. The 6xHis-tagged hsPTBP1 was expressed in E.Coli (BL21) after induction by IPTG (1mM), and purified on nickel column using standard procedures. Protein was eluted with 250 mM Imidazole and concentrated on vivaspin (30KDa cutoff). Purifed protein was resuspended in (Sodium Cacodylate pH7.0 20mM, NaCl 100mM, EDTA 0.5 mM, DTT 1mM).The EMSA experiment were performed by mixing dilutions of hsPTBP1 in RNA Binding buffer (RBB: Sodium Cacodylate pH7.0 10mM, BSA 0.1µg/µl, yeast tRNA 0.1µg/µl, NaCl 100mM, MgCl2 1mM, DTT 1mM, Rnasin (Promega) 0.4U/µl, Heparin (Sigma) 1U/µl) with an equal volume of labeled RNA in RBB. RNA protein complexes were analyzed on native

5 %

 5 Deoxycholate , protease inhibitor cocktail P8340 0.1 %, RNAsin 400U, TurboDNAse 10U, DTT 1mM) and incubated 10 min at 37°C. Prior to immunoprecipitation, lysates were partially digested with RNAseT1 (Ambion) (0.25U per mg of lysate 5 min, 37°C). Lysates were pre-cleared with 50µl of dynabeads Protein-G magnetic for 1 hr at 4°C. The cleared supernatants were incubated with 50 µl of beads pre-coated with 40 µg of anti-PTBP1 antibodies (clone BB7) or mouse IgG Isotype Control (Invitrogen). Immunoprecipitations were conducted overnight at 4°C with constant shaking, then washed successively with IP300 (50 mM HEPES-K pH 7.5, 300 mM KCl, 0.05% NP-40, 0.5 mM DTT), IP500 (50 mM HEPES-K pH 7.5, 500 mM KCl, 0.05% NP-40, 0.5 mM DTT), IP750 (50 mM HEPES-K pH 7.5, 750 mM KCl, 0.05% NP-40, 0.5 mM DTT) and finally once the immunoprecipited samples were used for analysis of immunoprecipitated proteins by SDS-PAGE and western blotting. Co-immunoprecipitated RNAs were isolated by proteinase K treatment(30 min, 37°C in 150 µl of PK buffer (50 mM Tris pH 7.4, 1M NaCl, 0.5% NP40, 0.5mM EDTA, 0.1% SDS, 2M Urea, 1% Deoxycholate) containing 50 µg tRNA and 12 U proteinase K (ThermoFisher) followed by phenol-chloroform extraction and ethanol precipitation.

Figure 1 :

 1 FIGURE 1

  FIGURE 2

Figure 2 :

 2 Figure 2: Identification of PTBP1 as a RBP correlated to TP63 γ terminal exon splicing. A) TP63 gene expression in samples from TP63 expressing tissues (normalized reads count) (se , nse, (non-) sun exposed). B) Quantification of the usage of junctions 11-12 and 11-13 pertinent to γ terminal exon inclusion. C) RBP expression heatmap and hierarchical clustering of tissues based on RBP expression from GTEX data. D) Top 10 RBPs most correlated to the junctions involved in γ exon inclusion. E) Expression of SFRS9, PTBP1 and GRSF1 in two classes of patients with high or low γ exon 12 inclusion (threshold =4.07%) in tumors.

  FIGURE 3

Figure 3 :

 3 Figure 3: PTBP1 controls TP63 splicing in HNSCC cell lines.A) p63 expression was evaluated in HaCaT, HeLa and HNSC cells by western blot and immunodetection using a pan-p63 antibody (4A4) or a p63α-specific antibody (D2K8X) (n=3). Even loading was controlled using PCNA or β-tubulin antibodies as indicated. B) TP63 RNA expression was measured by RT-QPCR with one pair of primer detecting all isoforms and quantification was normalised to a calibration curve obtained from TP63 plasmid DNA dilutions (n=3). C) Quantification of TP63α, β or γ percent spliced-in using isoform-specific primer pairs (n=3). D) upper panel, quantification of TP63γ isoform and PTBP1 RNA in control and PTBP1 depleted HNSC cell lines (n=3). Lower panel, evaluation of PTBP1 depletion by western blot (n=3), PCNA serves as a loading control.

3 '

 3 splice site (b), a broader area in the γ 3'UTR (c) and a smaller region overlapping the cleavage and polyadenylation (CPA) site (d).We performed electrophoretic mobility shift assay (EMSA) using recombinant PTBP1 and fluorescently labelled RNA molecules to determine the affinity of PTBP1 for RNA sequences representing the conserved sequence elements (Fig.4Dand SFig.3). We quantified the fluorescent RNAs in bound and unbound PTBP1 complexes and calculated a Kd based on PTBP1 concentration[START_REF] Ryder | Quantitative analysis of protein-RNA interactions by gel mobility shift[END_REF]. As presented in Fig.4D, RNA fragment 1 (FRAGT 1) composed solely of β-globin RNA sequences, FRAGT2 that is composed in part of β-globin RNA sequences and in part of the TP63 intronic region, FRAGT 5, FRAGT7 located totally in the γ exon, and CPA1 located close to the

FIGURE 4 Figure 4 :

 44 FIGURE 4

12 Figure 5 :

 125 FIGURE 5.

Figure 6 :

 6 FIGURE 6

Supplementary Figure 1 : 1

 11 TP63 gene structure A) Correspondence of the TCGA (upper lane) and GTEX (lower lane) annotations for the TP63 locus. Hg38 chromosomal locations of 5' and 3' splice sites are shown in green and black respectively. The main difference between both annotations stands in the presence of an additional exon, noted 5' here, in the GTEX gene model. The exon numbering used throughout the manuscript is from the TCGA annotation and presented underneath the GTEX gene model. B) Exon composition of the C-terminal isoforms γ, α, β, δ. Correspondance between the TP63 gene model from TCGA and GTEX Exon composition of the different C-terminal isoforms

3 :

 3 Electrophoretic mobility shift assays A) Validation of the purification of the recombinant human PTBP1 protein by gel electrophoresis and coomassie staing. B) Representative EMSA experiments conducted with fluorescently labeled RNA and recombinant PTBP1 proteins in increasing concentration (0 to 667 nM) (n >= 4). In each panel the lower bands in 0 nM PTBP1 correspond to free RNA probes.

Figure. 24 .

 24 Figure.24. Human keratinocytes (HaCat cells) were cultured in 10% or 0,5% serum containing medium and either treated with 2ng/mL TGF-β1 or transfected with PTBP1 siRNA. A. Total protein extracted from cells were analysed by western blot for E-cadherin, Fibronectin, Src, PTBP1, Slug and PCNA used as a loading control.(a) Control cells, 10% FCS. (b) Control cells, 0,5% FCS. (c) Transfection control cells, 0,5% serum. (d) TGF-β1 2ng/mL, 0,5% serum. (e) siPTBP1 transfected cells, 0,5% serum B. Immunofluorescence staining of fixed and permeabilised cells for E-cadherin (Red) and Fibronectin (Green) expression, counterstained with DAPI.

Figure. 25 .

 25 Figure.25. Human keratinocytes (HaCat cells) were cultured in 10% serum media and transfected with either PTBP1 siRNAs a or b or with control siRNAs. Cells were harvested after 96 hours of treatment. SCC9 cell extracts are loaded as control. A. Total protein was analysed by western blot for E-cadherin, Fibronectin and PTBP1, with PCNA used as a loading control. B. Immunofluroescence images were taken following E-cadherin, phalloïdin and DAPI staining. Images are the result of stacked Z-slices. C.Immunofluorescence staining of E-cadherin was performed, followed by measurement of E-cadherin staining intensity at the cell membrane using imageJ software. For each tested condition, 15 fields containing an average of 10 cells were imaged and analysed. Statistical significance of the difference of means was calculated by student's T test.

Figure. 26 .

 26 Figure.26. SCC-9 cells were transfected with siRNA against PTBP1 (SiP1), PTBP2 (SiP2), TP63γ (SiG) or combinations of each. Gene expression of PTBP1, PTBP2, and TP63 isoforms was measured by RT-qPCR and plotted as -ddCT, using non transfected cells as a control.94

(Figure. 28 .

 28 Figure.28. FaDu or SCC-9 cells were transfected with siPTBP1 and siPTBP2, siTP63γ or a combination of the three. Protein levels were assessed by western blot for the indicated proteins (A), TP63 isoforms expression was analysed by RT-PCR and gene expression was measured by RT-qPCR for the indicated target and plotted as the ddCT method. Reference gene is RPLP0, reference sample is untransfected cells.

  29.A) and RNA levels (Figure 29.B). As a readout of p63 transcriptional activity we measured the RNA levels of CDKN1A, MDM2, PERP and SOX4 (Figure 29.B), which hadshown somewhat promising results previously. As p63α has been linked with and anti-EMT role, we also looked at Fibronectin, E-Cadherin and Vimentin levels by western blot (Figure29.A). SCC-9 cells typically express more mesenchymal markers as illustrated here by their higher expression of Fibronectin than A253 cells in the same conditions.

Figure. 29 .

 29 Figure.29. SCC9 and A253 cells were transfected with the indicated SiRNA targeting either the alpha or the gamma TP63 isoforms. A. Total protein was analysed by western blot at two different time points after transfection to control for P63 and mesenchymal markers expression as indicated on the side of the blots. B. Expression of potential TP63 target genes was assessed by RT-qPCR and compared to untransfected cells. RPLP0 serves as reference gene. .

Figure. 30 .

 30 Figure.30. SCC9 cells were transfected with pmirGLO GFP vectors containing the TP63α or TP63γ coding sequences. Stable multiclonal cell lines were subsequently derived and used for experimental purposes. A. Schematic representation of pmirGLO derived TP63α or TP63γ expression vectors. B. Total protein was analysed by western blot to control P63 isoforms expression in the newly established SCC-9 cell lines C. Expression of epithelial or mesenchymal related markers was assessed in SCC9 cell lines by RT-qPCR and compared to the empty vector cell line. .

Figure 30 .

 30 B presents a western blot analysis of p63 expression in the 3 situations, empty vector, ΔNTP63α, or ΔNTP63γ. As shown using the 4A4 antibody, ΔNp63α is already expressed in empty vector cells. The levels of ΔNp63α increases in pmirGLO ΔNTP63α cells. An immunoreactive protein of low molecular weight is also detected in these cells. Considering the location of the 4A4 epitope in the N -terminal region of ΔNp63(Nekulova et al. 2013a), this band of about 20kDa is most probably the result of a cleavage occurring in the DNA binding domain of ΔNp63. pmirGLO ΔNTP63γ specifically drives the accumulation of a protein with a size and an immunoreactivity coherent with ΔNp63γ. (Figure 30.B). RT-qPCR quantification of ΔNTP63α, or ΔNTP63γ expression indicate that TP63γ was increased 32 fold, and TP63α, which is already reasonably well expressed, by 4 fold (Figure 30.C). Western blot analysis is also coherent with this estimate (Figure 30.C). Due to time constraints, we chose to evaluate proliferation and migration capacities with multi-clonal cell lines rather than isolate individual clones. First of all, the generated cell lines did not display any strong morphological changes compared to wild type cells (Figure 31.A). Proliferation of the cells was analysed by nucleus counting for 96 hours. Naive, control, ΔNTP63α, or ΔNTP63γ cells all have a similar proliferation rate (n=4, Figure 31B). Migration properties of the cells were evaluated using Ibidi cell culture inserts. Cells seeded at equivalent density were videorecorded after wounding and the percentage of wound closure plotted against time (Figure 31.C). SCC-9 control and naive cells were the slower migrating cells. Both ΔNTP63α, or ΔNTP63γ were faster to close the wound. However these experiments suffer from a quite high variability more specifically on the ΔNTP63α overexpressing cells that preclude drawing any conclusion.Albeit non significant, the tendency was towards a higher migration rate for TP63 overexpressing cells, a conclusion that would be rather counter intuitive (Figure31.D). It is possible that clonal analysis of the cells would allow for a more statistically robust analysis to be conducted. Finally we assessed using RT-qPCR some markers or known targets of p63. (Figure31.E). Having at hand cells that overexpress ΔNTP63α, or ΔNTP63γ, we chose to compare those overexpressing cells with SCC-9 cells following depletion of TP63

Figure. 31 .

 31 Figure.31. Phenotypic and molecular characterisation of SCC-9 multiclonal cell lines. A. Phase contrast microscopy images of subconfluent SCC-9 cells. WT: wild type, CTRL: cells expressing an empty pmirGLO GFP vector, TP63α and TP63γ: cells expressing TP63α and γ from a pmirGLO GFP vector (see fig. 30) B. Cellular proliferation measured by automatic counting of DAPI stained nuclei on fixed cells at the indicated time after plating. C. Wound closure assays. Migration assessment was performed by time lapse microscopy on Ibidi cell culture inserts. Images were taken every hour. D. Comparison of time to 95% closure. E. Expression of TP63 isoforms and selected previously described p63-target genes in overexpression cell lines or following 72h knock down of specific isoforms as indicated. .

(

  mice #112-115) or dual combinations of gamma specific peptide and of each of the other peptides: gamma + alpha (mice # 98-100 and # 102), gamma + ΔN ( mice #101,#116-118), gamma + pan (mice #108-111). For each condition, four mice were challenged by 3 peptide injections. ****************************************************************************************************************************************************************************************************************************** SP|Q9H3D4-2|P63_HUMAN LGRRCFEARICACPGRDRKADEDSIRKQQVSDSTKNGDGTKRPFRQNTHGIQMTSIKKRR 300 SP|Q9H3D4-4|P63_HUMAN LGRRCFEARICACPGRDRKADEDSIRKQQVSDSTKNGDGTKRPFRQNTHGIQMTSIKKRR 300 SP|Q9H3D4-6|P63_HUMAN LGRRCFEARICACPGRDRKADEDSIRKQQVSDSTKNGDGTKRPFRQNTHGIQMTSIKKRR 300 SP|Q9H3D4-8|P63_HUMAN LGRRCFEARICACPGRDRKADEDSIRKQQVSDSTKNGDGTKRPFRQNTHGIQMTSIKKRR 300 ************************************************************************************************************

Figure 32 :Figure 33 :

 3233 Figure 32: Protein sequence and peptides for the different p63 isoforms. Only ΔN isoforms are shown . Q9H3D4-2: ΔNp63 alpha, Q9H3D4-4: ΔNp63 beta, Q9H3D4-6: ΔNp63 gamma, Q9H3D4-8: ΔNp63 delta. The protein domains are identified using PFAM and the sequences are highlighted accordingly. The peptides used in the immunisation process are in bold. The epitope for the 4A4 monoclonal antibody is underlined 109

Figure 34 :

 34 Figure 34: Second round of immunisation and selection. The selected mice were challenged two more times with the indicated peptides. Elisa analysis of mice sera after 3 rounds of immunization with the indicated peptide combinations (on top of the graph). 4 (or 6 upper panel) sera dilutions were tested for each mouse on an irrelevant peptide or on the indicated p63 peptides. Mice IDs are indicated on the right of each graph. Signal is represented as arbitrary units (AU).

Table 4 :

 4 Hybridoma development and selection Fusion hybridomas were selected, amplified and tested for immunoreactivity to Pan or γ peptides. . similar to WB signals obtained with the 4A4 monoclonal antibody (Figure 37.A, lower panel)We compared the sensitivity of the KO1393 and KO85 antibodies to the 4A4 commercial pan-p63 antibody, by performing serial dilutions of HeLa cell extracts expressing p63α or γ on duplicate membranes (Figure37.B). The KO1393 was only slightly less sensitive than the 4A4 antibody, with similar detection time and roughly similar signal detection (compare the two upper panels). However the sensitivity of the KO85 monoclonal purified antibody was clearly lower than that of the 4A4 with longer required exposure time and absence of detection in the 1/64 dilution sample. Nonetheless, in the context of the detection of recombinant p63 protein, the specificity of these antibodies seemed excellent, especially the γ-specific KO85.

Figure 38 :

 38 Figure 38: Detection of endogenous and ectopic p63 isoforms using commercial pan-p63 (4A4), p63α specific (D2K8X) antibodies, or using pan-p63 (KO1393) and γ-specific (KO85) antibodies we developped. A. SCC-9 or A253 cells were transfected with control (pmirGLO GFP ctrl) or p63γ expressing plasmids (pmirGLO TP63 γ). Stable multiclonal lines were obtained by antibiotic selection. HeLa cells expressing TP63 γ under the control of a strong pCMV promoter serve as a positive control. PCNA is used as a loading control. B. SCC9 extract was mixed with a serial dilution of HeLa cell extract expressing TP63 γ. The size fractionated proteins were detected using either the 4A4 or the KO85 antibodies. Total cellular extract was used for western blotting.

Figure 39 :

 39 Figure 39: Detection of endogenous p63 comparing commercial pan-p63 (4A4) commercial p63α specific (D2K8X) antibodies, or pan-p63 antibodies (KO1393). SCC-25 cells were depleted in all p63 isoforms (siPan), specifically depleted in p63α (siA1 and siA2) or treated with control siRNAs (siCtrl and siLuc). NT cells received only transfectant. Total cellular extract was used for western blotting with the indicated antibodies .

Figure 40 .

 40 Figure 40. Schematic depiction of canonical (bottom) and backsplicing (top) of a pre-mRNA molecule. SD: splice donor. SA: splice acceptor. (From Yu and Kuo, 2019) .

Figure 41 .

 41 Figure41. Circular RNA biogenesis. Donor and acceptor splice sites can be brought into close proximity by pairing of repeated sequences within their flanking introns and/or by RBP interactions. CircRNA can also be formed by the lariat generated by exon skipping during alternative splicing as it can bring the splice sites of the exon it contains together. RBP: RNA Binding Protein; BP: Branch Point; SD: Splice site Donor; SA: Splice site Acceptor; BSJ: Back Splice Junction; EIcircRNA: exon-intron circRNA (Adapted from[START_REF] Kristensen | The Biogenesis, Biology and Characterization of Circular RNAs[END_REF]

Figure 42 .Table 5

 425 Figure 42. A. Schematic depiction of cirRNAs 13-11 and 15-14 production from the TP63 pre-mRNA B. Analysis of the abundance of TP63 mRNA (exons 10-11) and circs 13-11 and 15-14 by RT-PCR before and after poly(A) extension and RNAse R digestion. .

Figure 43 .BFigure 44 .

 4344 Figure 43. Alignments of sanger sequences obtained from the PCR products of A. 13Fwd/11Rv or B. 15-14Fwd/14Rv reactions. .

TP63α

  expression. Xenopus could be a model of choice for this work as embryos allow rapid access to developmental stages pertinent for TP63 biology and efficient F0 genome edition. This would be all the more interesting since TP63 isoform specific knockouts and re-expressions have already been performed in mice, which did not highlight any strong TP63γ related phenotypes. Direct access to the developing embryos could facilitate the observation and characterisation of potentially subtle effects, which may have been missed in a mouse model if embryo analysis was performed at the wrong stage. Analysis of Xenopus embryos could allow us to determine in which tissue or under which circumstances the gamma isoforms are required. If p63γ's function is indeed limited to a small number of cells, single cell RNAseq analysis could allow us to identify the cells of interest within an affected tissue. While SC-RNAseq is usually not indicated for analysis of alternatively spliced isoforms, alternative terminal exon usage can be discriminated by the classical sequencing techniques used in single cell analysis.

  loss of function methods. The model we propose of repression of the TP63γ isoform splicing by PTBP1 would gain to be challenged using gain of function experiments. It could be particularly interesting to symmetrically address the question using gain of function of PTBP1 in a muscular context to determine if accumulation of TP63 alpha consequentially occurs. A muscular cell model would also allow for the investigation of the participation of other RNA BPs on TP63 splicing, most notably potential activators of TP63γ splicing.

3 '

 3 UTRs are notoriously involved in post-transcriptionnal control of RNAs by binding to complimentary sequences or by recruiting protein factors. Their wide ranging regulatory action comes from influencing processes such as mRNA localisation, stability and translation. Part of their function is mediated through recruitment of miRNAs. Besides 3'UTRs, we have also shown that circRNAs derived from the TP63 pre-mRNA are expressed in HNSCC cell lines. These circRNAs share their sequence with some TP63 exons. In this circularised form, regulatory sequences which they may contain are protected from degradation. The existence of this stabilised forms of TP63 RNA could indicate that they possess regulatory functions which are totally independent from the protein.

Figure 1 .

 1 Figure 1. Au niveau protéique, les membres de la famille p53 partagent une organisation en domaines fonctionnels tout à fait comparable avec un domaine N-terminal de transactivation, un domaine central de liaison à l'ADN, un domaine impliqué dans leur oligomérisation et plusieurs domaines C-terminaux impliqués dans leurs régulations. En particulier, le domaine de liaison à l'ADN est très conservé entre les trois membres. Leurs spécificités fonctionnelles ont été largement appréciées grâce à des expériences de délétion de gènes chez la souris, et sont mises en relation avec des observations faites chez l'homme lors de la présence de mutations dans les différents membres de la famille.Les souris i n'exprimant plus de gène fonctionnel TP53 n'ont pas de problèmes de développement mais accusent une nette hausse d'apparition précoce de tumeurs de multiples origines comparé à leurs congénères sauvages. Chez l'humain, la mutation de TP53 est la cause du syndrome de Li-Fraumeni, une maladie autosomique récessive rare qui se caractérise par une augmentation importante du risque de cancer. D'une façon plus large, 50 % de tous les cas de cancers portent des mutations sur le gène TP53. La

  Il s'agit d'un mécanisme de régulation qui est au moins conservé du Xénope jusqu'à l'homme, dont la divergence remonte à près de 400 millions d'années. L'isoforme γ, ainsi que la régulation de sa production, sont donc fortement conservés, ce qui suggère une importance fonctionnelle suffisante à assurer une pression de sélection. Ce mécanisme est également conservé dans les cellules de carcinomes épidermoïdes de la tête et du cou, où TP63 est fréquemment amplifié et/ou surexprimé. Nous avons identifié une corrélation négative entre l'abondance de TP63 γ et la survie de patients atteints de carcinomes épidermoïdes de la tête et du cou. Dans des données d'expression tissulaires, l'isoforme γ est généralement très peu exprimée dans les différents tissus en comparaison à l'isoforme α, ceci à l'exception du muscle où elle est l'isoforme dominante. P63 est connu pour son rôle en tant que facteur de transcription, pour autant son expression musculaire semble être uniquement cytoplasmique ce qui suggère une fonction autre que transcriptionnelle. L'isoforme γ pourrait donc avoir une fonction tissu-spécifique restreinte aux muscles. Compte tenu de l'importance de p63 au cours du développement, il est également possible qu'elle ait une fenêtre temporelle d'utilité dans ce contexte. Dans les cellules épithéliales, il a été suggéré qu'elle pouvait promouvoir la transition épithélio-mésenchymateuse. Nous n'avons pas été capables d'étayer cette piste. Des anticorps spécifiques de p63γ seraient d'une aide cruciale dans l'analyse de ses fonctions protéiques, y compris sa localisation subcellulaire et au sein de complexes multimériques de p63. Bien que nous ayons réussi à développer des anticorps avec une bonne sensibilité et spécificité pour la protéine p63 surexprimée, ils restent à améliorer avant d'être utilisables dans l'analyse de p63 endogène. L'existence d'ARN circulaires de TP63 dans les cellules HNSCC offre également une nouvelle piste d'investigation de ses fonctions non-protéiques dans ce type de cancer. Le gène TP63 permet la production de multiples espèces moléculaires, ARNm, isoformes protéiques et ARN circulaires qui peuvent potentiellement toutes moduler la fonction cellulaire. La régulation de la production de ces différents acteurs semble importante puisque la structure génique de TP63 est fortement conservée. Déterminer quelle part prend chacun de ces acteurs dans le maintien d'un fonctionnement cellulaire normal s'avère une gageure qu'il conviendra d'aborder dans l'objectif d'élucider les fonctions de TP63 dans des contextes pathologiques pouvant être aussi bien associés à la carcinogenèse qu'aux problèmes de développement. Titre : Régulation post-transcriptionnelle des isoformes de TP63 par la PTBP1 dans les carcinomes épidermoïdes de la tête et du cou Mots clés : famille p53, épissage alternatif, biomarqueurs, RNABP, EMT. Résumé : La protéine tumorale p63 (p63) codée par le gène TP63 est un facteur de transcription de la famille de p53. TP63 est essentiel au développement embryonnaire, son absence empêche la mise en place de la peau et s'accompagne de malformations voir d'absence totale des membres supérieurs et de défauts de morphogenèse craniofaciale. Au niveau cellulaire, TP63 est impliqué dans le renouvellement des cellules souches, la différentiation et la prolifération cellulaire, la promotion de la transition épithéliomésenchymateuse ou bien encore le maintien de l'intégrité de la lignée germinale. Des promoteurs alternatifs et plusieurs événements d'épissage alternatif conduisent à la production d'au moins huit isoformes de p63. Côté C-terminal, on distingue quatre principales isoformes : α, β, γ et δ. L'existence de multiples isoformes est souvent utilisée pour expliquer la complexité des évènements cellulaires dépendant de TP63. TP63 est fréquemment amplifié et/ou surexprimé dans les carcinomes épidermoïdes et nous avons mis en évidence une corrélation négative entre le niveau d'expression des isoformes minoritaires γ et la survie des patients atteints de carcinomes épidermoïdes de la tête et du cou. Par une combinaison d'approches bioinformatique et d'analyses mécanistiques, j'ai pu mettre en évidence qu'une interaction directe entre la protéine de liaison aux ARN PTBP1 et l'ARN pré-messager de TP63 réprime l'utilisation de l'exon terminal γ dans un contexte de lignées cellulaires issues de carcinomes épidermoïdes. L'importance fonctionnelle de cette régulation est supportée par sa conservation des amphibiens à l'homme. Toutefois, les conséquences fonctionnelles d'une modification de l'abondance des isoformes minoritaires de p63 demeure une question en suspens. Title : Post-transcriptional control of TP63 isoforms by PTBP1 in Head and Neck Squamous Cell Carcinoma. Keywords : p53 family, alternative splicing, biomarkers, RNABP, EMT Abstract : The tumour protein p63 (p63), encoded by the TP63 gene, is a transcription factor belonging to the p53 family. TP63 is essential for embryonic development, its inactivation leading to defects in skin, limb and cranio-facial development.

Table 1 : Transcription factors targeting the TP63 gene

 1 .(Adapted from pokorna et al. 2021). The last column indicates the affected isoform (orange ΔNp63, blue TAp63), the second column the impact of the promoter (green:

	upregulation, red: down regulation)

. In FaDu and HaCaT cells, chemotherapeutic agents decrease ΔNTP63α transcription levels. Depleting ΔNTP63α in these cells by RNAi leads to sensitisation to apoptosis whereas overexpression of ΔNTP63α confers resistance to Dox and CDDP

	co-immunoprecipitation experiments. Activation of BCC3's promoter by TAp63β is
	inhibited by the presence of ΔNp63α. The inhibition could either be through competition for
	binding to the promoter, sequestration by formation of p63/p73 heterotetramers, or both.
	induced apoptosis (Xiaorong Li et al. 2012).
	In HNSCC JHU-029 (WT p53) and JHU-011 (p53 truncated) cells TP63 (mostly
	ΔNTP63α) is more expressed than in primary human keratinocytes. ShRNA against TP63
	induces apoptosis in both cell lines, which is rescued by concomitant expression of shRNA
	resistant ΔNTP63α. The p63 knockdown triggers PMAIP1 (NOXA) and BCC3 (PUMA)
	expression. Co-depletion of TP73 abrogates the onset of TP63-knockdown-triggered
	apoptosis. The function of p53 has no incidence on apoptosis induction in these cells. p73
	has been previously described as an activator of PUMA (Melino et al. 2004). Furthermore,
	endogenous ΔNp63α and TAp63β physically interact in JHU-029 cells as demonstrated by

  . PKM M2 is essential for glycolysis, as opposed to PKM

M1 which serves in oxidative phosphorylation. In cells, PKM M1 and M2 are rate limiting enzymes and considered to be cancer hallmarks. Outside of its metabolic roles, it is suggested that PKM2 could promote cancer progression through its kinase activity, by activating the EMT program, proliferation, angiogenesis and drug resistance

(Zahra et al.

Table 2 .

 2

	Type of cancer	Mechanism	Function/effect	Reference
	Melanoma	PTBP1/CD44v6	Migration of melanoma brain metastasis	Marzese et al., 2015
	Hepatoma	miR137 and miR206/↓PTBP1/PKM1	Negative effects related to tissue carcinogenesis characteristics and	Taniguchi et al., 2018
	Rhabdomyosarcoma	↓miR-133b/PAX3-FOXO1/ PTBP1 and ↓miR-1 and ↓miR-133b/PTBP1	Positive regulation of cancer-specific energy metabolism	Sugito et al., 2017
	Cervical cancer	HPV infection/PTBP1	Promotion of DNA replication and cancer cell proliferation	Xu et al., 2019
	Acute myeloid leukemia with mutated nucleophosmin	PTBP1/PKM2/phosphorylation of Beclin-1	Autophagic activation and cancer cell survival	Wang et al., 2019
	Anaplastic large cell lymphoma	PTBP1/PKM2/STAT3	Transcription of genes involved in oncogenesis cell survival and proliferation,	Hwang et al., 2017
	Chronic myeloid leukemia	AIC-47 and imatinib/↓PBTP1/↓PKM2	Perturbation of energy metabolism	Shinohara et al., 2016
	Gastric cancer	miR-133b/↓PTBP1	Negative regulation of the Warburg effect	Sugiyama et al., 2016
	Oral squamous cell carcinoma	PTBP1/SRSF3	Overexpression of SRSF3, oncogenesis	Guo et al., 2015
	Ovarian cancer	(1) PTBP1/CDC42 (2) ↓OVAAL/PTBP1/p27	(1) Filopodia formation, proliferation, invasiveness (2) Cellular senescence	He et al., 2015; Sang et al., 2018
			(1) Perturbation of the Warburg
	Bladder cancer	(1) miR-145/↓KLF4/↓PTBP1/↓PKMs (2) PTBP1/MEIS2/PKM2	effect and inhibition of carcinogenesis (2) Lymph node metastasis, tumor stage, histological grade, and poor	Minami et al., 2017; Takai et al., 2017; Xie et al., 2019
			prognosis
		(1) Gemcitabine/PTBP1/PKM2
	Pancreatic ductal	(2)	(1) Resistance to gemcitabine (2)	Calabretta et al.,
	adenocarcinoma	Linc-ROR/↓miR-124/PTBP1/PK	Resistance to gemcitabine	2016; Li et al., 2016
		M2
	Breast cancer	(1) PTBP1/PKM2 (2) PTBP1/PTEN-PI3K/Akt (3) PTBP1/FGFR1α/FGFR1β splicing	(1) Warburg effect, tumorigenesis, and transformation properties (2) Proliferation, invasion, and migration metastasis (3) Proliferation and	He et al., 2014; Ouyang et al., 2018; Zhao et al., 2019
	Renal cell cancer	(1) PTBP1/PKM2 (2) ↓PTBP1/HIF-1α pathway	(1) Proliferation, migration, and invasion (2) Inhibit cell migration, invasion, and angiogenesis	Jiang et al., 2017; Shan et al., 2018
	Glioblastoma	(1) ↓miR-124/PTBP1/ANXA7/EGFR (2) BAF45d/PTBP1 (3) PTBP1/USP5 (4) ↓CircSMARCA5/SRSF1/SRSF3/ PTBP1	(1) Glioblastoma progression (2) Malignant phenotype and undifferentiated cellular state (3) Oncogenesis (4) Invasion and migration	Izaguirre et al., 2012; Ferrarese et al., 2014; Aldave et al., 2018; Barbagallo et al., 2018
	Neuroblastoma	(1) MYCN/PTBP1/PKM2 (2) Hydrogen peroxide/PTBP1/sGC	(1) Proliferation, metastasis, and poor overall survival (2) Cellular adaptation to oxidative stress	Cote et al., 2012; Zhang SL et al., 2016
	Glioma	(1) PTBP1/RTN4 (2) PTBP1/IRES-like element/ADAR1 p110	(1) Proliferation, invasion, and migration (2) Gliomagenesis and proliferation	Cheung et al., 2009; Yang et al., 2015
	Colorectal cancer	(1) ↓miR-1 and ↓miR-133b/PTBP1/PKM2 (2) DDX6/c-Myc/PTBP1 (3) PTBP1/ATG10/EMT-associated proteins (4) PTBP1/cortactin	(1) Warburg effect (2) Warburg effect (3) EMT, migration and invasion (4) Migration and invasion	Taniguchi et al., 2015, 2016; Jo et al., 2017; Wang et al., 2017
		(1) ELK1/MYC/PTBP1/RAS-	(1) Tumorigenesis (2) Apoptosis	Hollander et al.,
	Colon cancer	MAPK (2) ↓TPBP1/TRAIL and	and perturbation of the Warburg	2016; Kumazaki et
		↓PTBP1/PKM2→PKM1 (3)	effect (3) Warburg effect and	al., 2016; Shan et al.,

Table 2 .

 2 Roles of PTBP1 in various cancers (from[START_REF] Wan | Prognostic Value of Epithelial-Mesenchymal Transition-Inducing Transcription Factors in Head and Neck Squamous Cell Carcinoma: A Meta-Analysis[END_REF]

1.1. Article: "PTBP1 controls TP63 alternative splicing in HNSCC tumour cells" TITLE PAGE THE SPLICING FACTOR PTBP1 REPRESSES TP63 γγ ISOFORM PRODUCTION IN SQUAMOUS CELL CARCINOMA

	Authors: William Taylor, David Reboutier, Luc Paillard, Agnès Méreau, Yann Audic
	Affiliation: Univ. Rennes, CNRS, Institut de Genetique et Developpement de Rennes (IGDR)
	UMR6290, Rennes, France.
	Corresponding author: Yann Audic, IGDR-UMR6290, Faculté de médecine 2 avenue du Pr Léon
	Bernard 35043 Rennes Cedex France , yann.audic@univ-rennes1.fr, +33 (0)2 23234475
	Running title: PTBP1 controls TP63 alternative splicing in HNSC tumor cells

keywords : Alternative splicing, RNA binding protein, RNA targets, EMT, biomarkers, p53 family Conflict of interest : "The authors declare no potential conflicts of interest." Financial support : Contrat Doctoral, University of Rennes 1 to William Taylor. This project was supported by the departmental committees of the Ligue contre le Cancer du Grand Ouest: 22, 29, 35.

Table 3 :

 3 most common antibodies used in TP63 studies

Apigenin/↓PTBP1/↓PKM2 oncogenesis 2017

Acknowledgements

Acknowledgment :

This work was funded by grants from Ligue Régionale contre le cancer (comité 22, 29, 35). WT was financed by a Contrat Doctoral from the Université de Rennes I. The authors wish to thank Christophe Tascon (IGDR-protein purification and analysis) for purification of the hsPTBP1 protein and Tiffany Yen Kway for her help with the correlation script.

ABSTRACT

The TP63 gene encodes the transcription factor p63. It is frequently amplified or overexpressed in squamous cell carcinomas. Owing to alternative splicing, p63 has multiple isoforms called α, β, γ and δ. The regulatory functions of p63 may be isoform-specific. The α isoform inhibits the epithelial to mesenchymal transition (EMT) and controls apoptosis, while the γ isoform promotes EMT. Here, we observed in TCGA data that a high ratio of the TP63γ isoform to the other isoforms is a pejorative factor for the survival of patients with head and neck squamous cell carcinoma (HNSCC). We therefore addressed the regulation of the γ isoform. In several tissues (GTEX data), the expression of the RNA-binding protein PTBP1 (polypyrimidine tract binding protein 1) is negatively correlated with the abundance of TP63γ. Accordingly, we demonstrated that PTBP1 depletion in HNSCC cell lines leads to an increase in abundance of the γ isoform. By RNA immunoprecipitation and in vitro interaction assays, we showed that PTBP1 directly binds to TP63 pre-mRNA in close proximity to the TP63γ-specific exon. The region around the TP63γ-specific exon was sufficient to elicit a PTBP1-dependent regulation of alternative splicing in a splice reporter minigene assay. Finally, we demonstrated that the regulation of TP63γ production by PTBP1 is conserved in amphibians, revealing that it encounters a strong evolutionary pressure.

Together, these results identify TP63γ as a prognostic marker in HNSCC, and identify PTBP1 as a direct negative regulator of its production. , s. d.). Again, no effect of TP63γ modulation could be seen.

Supplementary

Overall, we have been unable to reproduce previously described effects of p63 on the EMT despite being able to drive EMT using TGF-β treatment. Surprisingly, we also struggled to find any significant changes in the expression of previously described p63 targets following isoform-specific depletion or up-regulation. To move forwards in the identification of p63γ functions in HNSCC, clonal cell lines should be established overexpressing TP63α and γ. Rather than looking for individual targets, RNA sequencing of the different clones should be performed in order to identify their differentially expressed genes. Since there are currently no p63γ-specific antibodies, expression of tagged p63γ followed by Chip-seq could also be a good way of identifying its specific targets.

After the third injection, small aliquots of blood were taken from mice for analysis.

Elisa was conducted with serum dilutions against baits (p63 peptides) and control peptides to assess the development of an immune response against the peptides and determine the nature of the peptide targeted when combinations were used (Figure 33.B). Mice 114 and 115 developed a response against peptide γ. Mice 98 and 99 developed a response against peptide γ but not to peptide α. Mice 117 developed a response to peptide γ and not to peptide ΔN. Mice 109 and 110 developed responses to peptides γ and to peptide Pan-p63; and mice 108 and 111 developed responses to peptide Pan-p63 only. Therefore the immune responses appeared to be limited to peptides γ and Pan-p63. No response to peptides ΔN or α could be observed.

To further assess the ability of the sera to specifically detect the p63 isoforms of interest, we tested them by western-blot analysis using recombinant proteins. HeLa cells do not express p63 or only at very low levels. As such they are a suitable model to assess the capacity of the newly generated antibodies to detect recombinant p63 protein, with low endogenous targets which could confuse the results. As shown on To try to improve the sera's antibody titer and try to develop antibodies to α and ΔN peptides, the same mice were further challenged by two sets of injections with γ peptide, α peptide or ΔN peptide. Sera titer was controlled by Elisa (Figure 34). Elisa results confirmed the absence of immunisation against the ΔN and the α peptides and highlighted an increase in the titer of γ specific antibodies.

Based on these results we proceeded to select mice that would be used for fusion of B cells with myeloma cells to generate antibody producing hybridomas.

Hybridoma generation

To choose which mice were going to serve for hybridoma productions, the main criteria 4). We then tested these 8 clones by western-blot on recombinant protein as previously done for the sera (Figure 35). Among the γ specific clones, only the clone KO85 rendered a strong positive and specific signal to ΔNp63γ in western-blot. Among the Pan-specific clones, the results were somewhat unexpected. Only clone KO1393 gave a strong signal with both the ΔNP63α and ΔNP63γ recombinant proteins. The KO1393 pan-p63 clone also recognises a protein of similar molecular weight to p63γ in the p63α lane. This band is not recognised by the clone KO85 specific to p63γ, suggesting that this is most likely a degradation product of p63α. Among the three other pan-p63 clones, only signals from the ΔNP63γ recombinant protein could be detected at a somewhat low intensity level. No detection of the ΔNP63α could be observed.

Assessing the sensitivity and specificity of the purified antibodies

For further analysis we chose to focus on the KO1393/pan TP63 and KO85/γspecific clones. We wanted to determine the sensitivity and specificity of both clones.

Immunoglobulins from the hybridoma clone supernatants were purified on A/G proteins and concentrated. For the following experiments we exclusively used the purified antibodies.

To determine if the antibodies could be used to detect endogenous or recombinant protein by immunofluorescence, we used two distinct cell lines transfected with plasmids expressing p63 α, β or γ isoforms. HeLa cells were once again used, taking advantage of their lack of endogenous p63, and SCC-9 cells were used as an easy to transfect p63 positive cell line. In practice, neither cell line showed endogenous staining of p63 by IF, even using the 4A4 commercial antibody. Transfection of recombinant p63 proteins allowed us to achieve sufficient p63 levels to observe IF signals. In both cell lines, antibodies from the KO1393/pan clone specifically stained nuclei expressing p63 isoforms.

Likewise, antibodies from the KO85/ γ-specific clone only stained nuclei from cells transfected with ΔNTP63γ vectors. (see Figure 36). This indicated that both the pan and γ-specific clones could detect overexpressed p63 in immunofluorescence experiments, and the γ-specific clone could do this in an isoform-specific manner.

Assessing the capacity of the novel antibodies to recognise endogenous p63 in the immunofluorescence context will require the use of cell lines expressing higher p63 levels, or increasing the titer of the antibodies.

In parallel, we tested the specificity and sensitivity of the new p63 antibodies for the recognition of recombinant or endogenous p63 protein using western-blot. First, we tested the KO1393/pan and KO85/γ-specific antibodies on HeLa cell extracts overexpressing the three different p63 isoforms α β and γ as previously described (Figure 37.A). The KO85 antibody (Figure 37.A, upper panel) recognised recombinant p63γ with very little nonspecific signal, however the exposure time required for signal detection is quite high (31,4 s). The KO1393 purified antibody (Figure 37