Laura Mary Orcid

ORCID Sylvie Jaillard

Laura Mary
email: laura.mary@chu-rennes.fr

Delphine Leclerc

David Gilot

Marc-Antoine Belaud-Rotureau

The TALE never ends: a comprehensive overview of the role of PBX1, a TALE transcription factor, in human developmental defects

Keywords:

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Numerous classes of homeodomain (HD) transcription factors (TFs) have been described in the animal kingdom. One of these highly conserved super-classes of TFs is characterized by the insertion of a three aminoacid loop in the HD which has been named the TALE TF (for Three Amino acid Loop Extension, [START_REF] Bertolino | A novel homeobox protein which recognizes a TGT core and functionally interferes with a retinoidresponsive motif[END_REF]). Two TALE families, the MEINOX and PBC TFs, are able to interact with HOX and non-HOX proteins (reviews by [START_REF] Jozwik | Pioneer factors in hormone-dependent cancers[END_REF][START_REF] Laurent | PBX proteins: Much more than Hox cofactors[END_REF][START_REF] Merabet | To Be Specific or Not: The Critical Relationship Between Hox And TALE Proteins[END_REF][START_REF] Penkov | Analysis of the DNA-binding profile and function of TALE homeoproteins reveals their specialization and specific interactions with Hox genes/proteins[END_REF][START_REF] Pi | E2A-PBX1 functions as a coactivator for RUNX1 in acute lymphoblastic leukemia[END_REF]). They also have the ability to recruit regulators of chromatin accessibility and transcriptional co-repressors [START_REF] Asahara | Pbx-Hox heterodimers recruit coactivator-corepressor complexes in an isoform-specific manner[END_REF][START_REF] Laurent | PBX proteins: Much more than Hox cofactors[END_REF][START_REF] Longobardi | Biochemistry of the tale transcription factors PREP, MEIS, and PBX in vertebrates[END_REF]. These highly versatile functions make MEINOX and PBC proteins key factors in development, cell maintenance and cancer (see reviews by [START_REF] Blasi | A tale of TALE, PREP1, PBX1, and MEIS1: Interconnections and competition in cancer[END_REF] for cancer and (Licia [START_REF] Selleri | Building a perfect body': Control of vertebrate organogenesis by PBX-dependent regulatory networks[END_REF] for development).

The PBC family is composed of four proteins, namely PBX 1 to 4, with highly conserved sequences among vertebrates. As TALE TFs, their structure is composed of an HD and two protein-protein-interacting domains named PBC-A and B. The HD enables PBX proteins to interact with other TFs and DNA, but the PBC domains are .

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critical for interactions between PBX and MEINOX (PKNOX (also named PREP) and MEIS) proteins [START_REF] Longobardi | Biochemistry of the tale transcription factors PREP, MEIS, and PBX in vertebrates[END_REF]. Hetero-dimerization of these proteins is essential to allow PBX DNA-binding capacities, inducing: 1) the import of PBX proteins into the nucleus by hiding their nuclear export signals (NES) (Abu-Shaar, [START_REF] Abu-Shaar | Control of the nuclear localization of Extradenticle by competing nuclear import and export signals[END_REF][START_REF] Jaw | Direct interaction of two homeoproteins, homothorax and extradenticle, is essential for EXD nuclear localization and function[END_REF], 2) conformational changes of both partners stabilizing their interaction with DNA [START_REF] Mathiasen | The flexibility of a homeodomain transcription factor heterodimer and its allosteric regulation by DNA binding[END_REF], 3) the selection of targeted DNA with PBX protein binding properties relying on the DNA consensus sequence of their MEINOX partner [START_REF] Blasi | A tale of TALE, PREP1, PBX1, and MEIS1: Interconnections and competition in cancer[END_REF]. Since the phenomena of PKNOX or MEIS binding to PBX proteins are mutually exclusive [START_REF] Blasi | A tale of TALE, PREP1, PBX1, and MEIS1: Interconnections and competition in cancer[END_REF],

the properties of PBX are linked to its partner. Briefly, PKNOX and MEIS proteins have more or less opposite effects on cell fate. PKNOX proteins favour transcription initiation and preferentially bind to the promoter regions of housekeeping genes.

PKNOX1 has been extensively studied in the setting of both development and cancer [START_REF] Laurent | PBX proteins: Much more than Hox cofactors[END_REF][START_REF] Penkov | Analysis of the DNA-binding profile and function of TALE homeoproteins reveals their specialization and specific interactions with Hox genes/proteins[END_REF]; it is ubiquitously expressed during development and is also considered as a tumour-suppressing gene. PKNOX2 is less well known, and shares structure and functions that are close to PKNOX1. PKNOX2 is mainly expressed in the heart, the brain, muscles, limb buds and the ovaries, and regulates gene expression, with less effective binding properties to PBX proteins than PKNOX1 [START_REF] Fognani | Characterization of PREP2, a paralog of PREP1, which defines a novel subfamily of the MEINOX TALE homeodomain transcription factors[END_REF][START_REF] Haller | Prep2: Cloning and expression of a new prep family member[END_REF][START_REF] Zhou | Misexpression of Pknox2 in mouse limb bud mesenchyme perturbs zeugopod development and deltoid crest formation[END_REF]. MEIS proteins target promoter-remote regions of developmental genes and favour cellular survival and proliferation. MEIS1, expressed early and ubiquitously, is involved in cancer, hematopoiesis and embryonic development, especially for eye, limb, heart and vasculature formation (review by [START_REF] Paul | Homeobox gene Meis1 modulates cardiovascular regeneration[END_REF]). MEIS2 shares common functions with MEIS1 and is expressed during development mainly in neural structures, neural crest-derived cells, .

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the head and the gut, with overlapping patterns with MEIS1 [START_REF] Berenguer | Role of Retinoic Acid Signaling, FGF Signaling and Meis Genes in Control of Limb Development[END_REF][START_REF] Fujita | De novo MEIS2 mutation causes syndromic developmental delay with persistent gastro-esophageal reflux[END_REF]. Finally, MEIS3 has been far less studied in mammals. Its role in cancer varies with the tumour type [START_REF] Meng | Effect of myeloid ecotropic viral integration site (MEIS) family genes on tumor microenvironment remodeling and its potential therapeutic effect[END_REF]. Meis3 has been shown

to regulate neural crest cell migration, neural plate organization and pancreatic formation during mouse development (J. [START_REF] Liu | Three-amino-acid-loopextension homeodomain factor Meis3 regulates cell survival via PDK1[END_REF].

1.2) PBX1 expression and role

Most of the PBX proteins are expressed throughout the embryonic stages (as early as the blastula stage in vertebrates [START_REF] Choe | Meis cofactors control HDAC and CBP accessibility at Hox-regulated promoters during zebrafish embryogenesis[END_REF]) and maintained in mature tissues. During development, PBX1 is able to bind to promoters and promote histone modifications and chromatin accessibility, promoting gene transcription [START_REF] Choe | TALE factors poise promoters for activation by Hox proteins[END_REF][START_REF] Grebbin | Pbx1 is required for adult subventricular zone neurogenesis[END_REF]. This binding can precede efficient gene transcription by hours or days [START_REF] Choe | TALE factors poise promoters for activation by Hox proteins[END_REF]. In addition, PBX1 (and TALE TFs in general) can use distinct DNAbinding motifs and protein partners depending on the embryonic stage. This suggests key roles for PBC proteins, from early development and throughout life (for an extensive review on TALE TFs and development, see [START_REF] Selleri | Building a perfect body': Control of vertebrate organogenesis by PBX-dependent regulatory networks[END_REF]).

In the PBC family, PBX1 and 2 are the most widely expressed both during development and in mature organs [START_REF] Roberts | Localization of Pbx1 transcripts in developing rat embryos[END_REF]L. Selleri et al. 2001), with overlapping patterns [START_REF] Capellini | Pbx1/Pbx2 requirement for distal limb patterning is mediated by the hierarchical control of Hox gene spatial distribution and Shh expression[END_REF]. Intriguingly, only PBX1 seems to be mandatory for embryonic development, since Pbx2-KO mice are viable and healthy (Licia [START_REF] Selleri | The TALE Homeodomain Protein Pbx2 Is Not Essential for Development and Long-Term Survival[END_REF]. Multiple hypotheses may explain such a discrepancy, but a quantitative model, relying on global cell concentration of Pbx proteins, seemed the most accurate (see [START_REF] Selleri | The TALE Homeodomain Protein Pbx2 Is Not Essential for Development and Long-Term Survival[END_REF]) and paragraph 3.3 below). Numerous studies have investigated ubiquitous patterns of PBX1 expression .

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during development, mainly in mouse embryos [START_REF] Capellini | Scapula development is governed by genetic interactions of Pbx1 with its family members and with Emx2 via their cooperative control of Alx1[END_REF](Capellini et al. , p. 20, 2008;;[START_REF] Dimartino | The Hox cofactor and proto-oncogene Pbx1 is required for maintenance of definitive hematopoiesis in the fetal liver[END_REF][START_REF] Golonzhka | Pbx regulates patterning of the cerebral cortex in progenitors and postmitotic neurons[END_REF][START_REF] Grebbin | Pbx1 is required for adult subventricular zone neurogenesis[END_REF][START_REF] Lampe | An ultraconserved Hox-Pbx responsive element resides in the coding sequence of Hoxa2 and is active in rhombomere 4[END_REF][START_REF] Lescroart | Hox and Tale transcription factors in heart development and disease[END_REF][START_REF] Manley | Abnormalities of caudal pharyngeal pouch development in Pbx1 knockout mice mimic loss of Hox3 paralogs[END_REF][START_REF] Schnabel | Pbx1 regulates nephrogenesis and ureteric branching in the developing kidney[END_REF]L. Selleri et al. 2001;[START_REF] Sonnet | Characterization of TALE genes expression during the first lineage segregation in mammalian embryos[END_REF][START_REF] Vitobello | Hox and Pbx factors control retinoic acid synthesis during hindbrain segmentation[END_REF]. In human adults, PBX1 is ubiquitously expressed, predominantly in glandular tissues, female tissues and the bladder [START_REF] Fagerberg | Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics[END_REF]. It remains difficult to define the role of PBX1 proteins because alternative splicing can produce up to seven isoforms for PBX1 in humans, only three with known functions (PBX1a, PBX1b and PBX1d [START_REF] Asahara | Pbx-Hox heterodimers recruit coactivator-corepressor complexes in an isoform-specific manner[END_REF][START_REF] Sengupta | The SLE-associated Pbx1-d isoform acts as a dominant-negative transcriptional regulator[END_REF]). All proteins exhibit a degree of specificity in their patterns of expression [START_REF] Laurent | PBX proteins: Much more than Hox cofactors[END_REF][START_REF] Schnabel | Expression of Pbx1b during mammalian organogenesis[END_REF][START_REF] Sengupta | The SLE-associated Pbx1-d isoform acts as a dominant-negative transcriptional regulator[END_REF]. PBX1a is mainly expressed in the brain and during adulthood. Conversely, PBX1b is expressed ubiquitously and preferentially during the embryonic period (Catherine A. [START_REF] Schnabel | Pbx1 regulates nephrogenesis and ureteric branching in the developing kidney[END_REF]. The PBX1d isoform seems specific to CD4+ T cells and is considered as a susceptibility factor for lupus [START_REF] Cuda | Pre-B cell leukemia homeobox 1 is associated with lupus susceptibility in mice and humans[END_REF][START_REF] Niu | The PBX1 lupus susceptibility gene regulates CD44 expression[END_REF]. PBX1 has numerous partners leading to hetero-dimerization or hetero-trimerization (PKNOX1 and 2 proteins, MEIS1 and 2, FOXC1, HOX proteins etc. [START_REF] Asahara | Pbx-Hox heterodimers recruit coactivator-corepressor complexes in an isoform-specific manner[END_REF][START_REF] Berry | FOXC1 Transcriptional Regulatory Activity Is Impaired by PBX1 in a Filamin A-Mediated Manner[END_REF][START_REF] Dard | The human HOXA9 protein uses paralog-specific residues of the homeodomain to interact with TALE-class cofactors[END_REF][START_REF] Laurent | PBX proteins: Much more than Hox cofactors[END_REF]). In these larger complexes, PBX1 is able to recruit chromatin-remodelling factors with either repressing or enhancing activity [START_REF] Asahara | Pbx-Hox heterodimers recruit coactivator-corepressor complexes in an isoform-specific manner[END_REF][START_REF] Laurent | PBX proteins: Much more than Hox cofactors[END_REF][START_REF] Penkov | Analysis of the DNA-binding profile and function of TALE homeoproteins reveals their specialization and specific interactions with Hox genes/proteins[END_REF]. PBX1 target genes include ubiquitous developmental genes (PAX1/PAX9, WNT9B/WNT3, PITX3, etc. [START_REF] Capellini | Pbx1/Pbx2 govern axial skeletal development by controlling . Accepted Article Polycomb and Hox in mesoderm and Pax1/Pax9 in sclerotome[END_REF][START_REF] Ma | Associations of Polymorphisms in WNT9B and PBX1 with Mayer-Rokitansky-Küster-Hauser Syndrome in Chinese Han[END_REF][START_REF] Villaescusa | A PBX1 transcriptional network controls dopaminergic neuron development and is impaired in Parkinson's disease[END_REF]) and organ-specific genes (HIF1A, .

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DCX, TH, GNRH genes, etc. (Britta M. [START_REF] Grebbin | PBX1 as Pioneer Factor: A Case Still Open[END_REF][START_REF] Kocabas | Hypoxic metabolism in human hematopoietic stem cells[END_REF][START_REF] Rave-Harel | TALE Homeodomain Proteins Regulate Gonadotropin-releasing Hormone Gene Expression Independently and via Interactions with Oct-1[END_REF])), driving cell proliferation, pluripotency and differentiation [START_REF] Fukuda | Survivin modulates genes with divergent molecular functions and regulates proliferation of hematopoietic stem cells through Evi-1[END_REF][START_REF] Gemel | Fibroblast growth factor-8 expression is regulated by intronic engrailed and Pbx1-binding sites[END_REF][START_REF] Kocabas | Hypoxic metabolism in human hematopoietic stem cells[END_REF][START_REF] Wei | PBX1 promotes the cell proliferation via JAK2/STAT3 signaling in clear cell renal carcinoma[END_REF]. with three functional domains: an HD and two protein-protein-interacting domains, PBC-A and B (Figure 1). The heptad 77-84 in PBC-A and two regions in PBC-B are required for PBX1-MEINOX interactions [START_REF] Bruckmann | Mapping the native interaction surfaces of PREP1 with PBX1 by cross-linking mass-spectrometry and mutagenesis[END_REF]. Between the PBC-A and PBC-B domains, a poly-alanine stretch (108-150) confers flexibility on PBX1 and thus enhances its DNA-binding ability by favouring a conformational change upon binding [START_REF] Blasi | A tale of TALE, PREP1, PBX1, and MEIS1: Interconnections and competition in cancer[END_REF]. DNA binding of PBX1 HD relies strongly on an alpha-helix (located in position 271-288) [START_REF] Farber | Concerted dynamics link allosteric sites in the PBX homeodomain[END_REF]. PBX1 HD is able to recognize a generic octameric motif (TGATNNAT), where N represents nucleotides that are specified by the HOX partner of PBX1 alone or PBX1-PKNOX/MEIS dimers [START_REF] Longobardi | Biochemistry of the tale transcription factors PREP, MEIS, and PBX in vertebrates[END_REF].

However, DNA motif recognition of PBX1 varies through development and depends on its partners [START_REF] Ladam | TALE factors use two distinct functional modes to control an essential zebrafish gene expression program[END_REF]. Initially, PBX1 was thought to interact only with HOX proteins carrying a specific hexapeptide motif in their homeodomain called a W motif [START_REF] Neuteboom | The hexapeptide LFPWMR in Hoxb-8 is required for cooperative DNA binding with Pbx1 and Pbx2 proteins[END_REF]L. Selleri et al. 2001).

However, recent studies have demonstrated that PBX1 is also able to interact with other amino acid motifs of HOX HD in place of, or in addition to, the W motif [START_REF] Dard | The human HOXA9 protein uses paralog-specific residues of the homeodomain to interact with TALE-class cofactors[END_REF][START_REF] Merabet | To Be Specific or Not: The Critical Relationship Between Hox And TALE Proteins[END_REF]. The use of these alternative motifs allows HOX .

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proteins to adopt different conformations, increasing the range of binding sites that are recognized. This increase in DNA recognition and binding gives the HOX-PBX1 complex the ability to recognize and bind low-affinity, non-consensus binding sites [START_REF] Merabet | To Be Specific or Not: The Critical Relationship Between Hox And TALE Proteins[END_REF] and thus increases the number of genes regulated by PBX1-HOX partners [START_REF] Dard | The human HOXA9 protein uses paralog-specific residues of the homeodomain to interact with TALE-class cofactors[END_REF].

Localization of PBX1 is under the control of two nuclear localization signals (NLS) located in the HD [START_REF] Berthelsen | The subcellular localization of PBX1 and EXD proteins depends on nuclear import and export signals and is modulated by association with PREP1 and HTH[END_REF], and two nuclear export signals (NES) located close to and in the PBC-A domain [START_REF] Saleh | A conformational change in PBX1A is necessary for its nuclear localization[END_REF]. As a monomer, PBX1 is folded in such a way that the NES (N-terminus) comes into close contact with the HD, hiding the NLS and thus retaining PBX1 in the cytosol [START_REF] Saleh | A conformational change in PBX1A is necessary for its nuclear localization[END_REF]. PBX1 heterodimerization changes the conformation of PBX1, exposing the NLSs [START_REF] Longobardi | Biochemistry of the tale transcription factors PREP, MEIS, and PBX in vertebrates[END_REF]. The nuclear import of PBX1 can also be based on serine phosphorylation, depending on the cell type (Kilstrup-Nielsen, [START_REF] Kilstrup-Nielsen | PBX1 nuclear export is regulated independently of PBX-MEINOX interaction by PKA phosphorylation of the PBC-B domain[END_REF].

Even if the target genes of PBXs are not fully discovered yet, these proteins, and especially PBX1, have long been linked to cell fate, embryonic development and cancer in humans. The first human phenotype linked to PBX1 dysfunction was acute leukaemia by way of the formation of an E2A-PBX1 fusion transcript [START_REF] Kamps | The human t(1;19) translocation in pre-B ALL produces multiple nuclear E2A-Pbx1 fusion proteins with differing transforming potentials[END_REF][START_REF] Lin | Oligomeric self-association contributes to E2A-PBX1-mediated oncogenesis[END_REF]. Further studies have also highlighted the role of PBX1 in promoting the proliferation of various solid tumours [START_REF] Blasi | A tale of TALE, PREP1, PBX1, and MEIS1: Interconnections and competition in cancer[END_REF][START_REF] He | A hydrophobic residue in the TALE homeodomain of PBX1 promotes epithelial-to-mesenchymal transition of gastric carcinoma[END_REF][START_REF] Jung | Ovarian Cancer Chemoresistance Relies on the Stem Cell Reprogramming Factor PBX1[END_REF][START_REF] Magnani | The pioneer factor PBX1 is a novel driver of metastatic progression in ERα-positive breast cancer[END_REF][START_REF] Wei | PBX1 promotes the cell proliferation via JAK2/STAT3 signaling in clear cell renal carcinoma[END_REF].

2) PBX1 variations in humans

The PBX1a transcript (NM_002585.4) will be used as a reference in the present manuscript. Point variations and structural variants with an allele count of 1 were omitted, since we could not exclude the presence of very mildly affected individuals in the databases used (gnomADv2, DGV database and [START_REF] Alsmadi | Genome at Juncture of Early Human Migration: A Systematic Analysis of Two Whole Genomes and Thirteen Exomes from Kuwaiti Population Subgroup of Inferred Saudi Arabian Tribe Ancestry[END_REF][START_REF] Audano | Characterizing the Major Structural Variant Alleles of the Human Genome[END_REF][START_REF] Thareja | Sequence and analysis of a whole genome from Kuwaiti population subgroup of Persian ancestry[END_REF][START_REF] Uddin | A High-Resolution Copy Number Variation Resource for Clinical and . Accepted Article Population Genetics[END_REF][START_REF] Wong | Deep Whole-Genome Sequencing of 100 Southeast Asian Malays[END_REF]. Two hundred and fifty-eight point variations or small indels and 24 structural variations (15 deletions, 6 duplications and 3 insertions) have been reported to date in PBX1. Only 2 of these late deletions have an impact on the PBX1 coding region (exon 1, [START_REF] Alsmadi | Genome at Juncture of Early Human Migration: A Systematic Analysis of Two Whole Genomes and Thirteen Exomes from Kuwaiti Population Subgroup of Inferred Saudi Arabian Tribe Ancestry[END_REF][START_REF] Thareja | Sequence and analysis of a whole genome from Kuwaiti population subgroup of Persian ancestry[END_REF]). The exact count for each variation type is reported in Supp. Table S1.

Thirty patients with heterozygous pathogenic mutations, and partial or complete heterozygous deletions of PBX1 have been reported in the literature [START_REF] Alankarage | Functional characterization of a novel PBX1 de novo missense variant identified in a patient with syndromic congenital heart disease[END_REF][START_REF] Bertoli-Avella | Successful application of genome sequencing in a diagnostic setting: 1007 index cases from a clinically heterogeneous cohort[END_REF][START_REF] Eozenou | The TALE homeodomain of PBX1 is involved in human primary testis-determination[END_REF][START_REF] Fitzgerald | Inherited intragenic PBX1 deletion: Expanding the phenotype[END_REF][START_REF] Fromer | De novo mutations in schizophrenia implicate synaptic networks[END_REF][START_REF] Heidet | Targeted Exome Sequencing Identifies PBX1 as Involved in Monogenic Congenital Anomalies of the Kidney and Urinary Tract[END_REF][START_REF] Tanno | PBX1 haploinsufficiency leads to syndromic congenital anomalies of the kidney and urinary tract (CAKUTHED) in humans[END_REF][START_REF] Riedhammer | Identification of a Novel Heterozygous De Novo 7-bp Frameshift Deletion in PBX1 by Whole-Exome Sequencing Causing a Multi-Organ Syndrome Including Bilateral Dysplastic Kidneys and Hypoplastic Clavicles[END_REF][START_REF] Slavotinek | De novo, deleterious sequence variants that alter the transcriptional activity of the homeoprotein PBX1 are associated with intellectual disability and pleiotropic developmental defects[END_REF], and 10 additional patients are reported in the ClinVar, DECIPHER and HGMD databases. Their clinical data is summarized in Table 2. The PBX1 structure and its related variants are summarized in Figure 1 and Supp. Figure S1. None of these variations was reported in gnomAD, except one (NM_001204961.1:c.862C>T, p.(Arg288Ter) in a heterozygous state and at a frequency of 0.3.10 -4 ).

2.1) PBX1 loss-of-function (LOF)

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Whole or partial gene deletions account for 43% of all the PBX1-mutated patients, and nonsense variations (stop gains) or frame-shift mutations (including indels) amount to 23% of the patients. Nonsense or frame-shift variations mainly affect the polyalanine stretch between PBC-A and PBC-B (56%). Only one nonsense variation was located outside a functional domain (Figure 1).

To date, seventeen patients have been reported with whole or partial deletions of PBX1 [START_REF] Fitzgerald | Inherited intragenic PBX1 deletion: Expanding the phenotype[END_REF][START_REF] Heidet | Targeted Exome Sequencing Identifies PBX1 as Involved in Monogenic Congenital Anomalies of the Kidney and Urinary Tract[END_REF][START_REF] Tanno | PBX1 haploinsufficiency leads to syndromic congenital anomalies of the kidney and urinary tract (CAKUTHED) in humans[END_REF]) of whom 3

were siblings [START_REF] Fitzgerald | Inherited intragenic PBX1 deletion: Expanding the phenotype[END_REF]. The size of these deletions ranged from 37 kb [START_REF] Fitzgerald | Inherited intragenic PBX1 deletion: Expanding the phenotype[END_REF] to 18 Mb (patient 398083 from DECIPHER). Nine patients were described as presenting LOF variations (nonsense, splice or frame-shift variations, including indels) and 6 had documented phenotypes. The clinical data of these patients is summarized in Figure 2 panel A and extensively reported in Supp.

Table S2. Phenotypes are highly similar between variants resulting in LOF and PBX1

gene deletions; we therefore considered them as the same entity. Most of these patients (83%) displayed various combinations of renal and/or urinary tract anomalies, the most common defect being renal hypoplasia. The only genital anomaly reported was cryptorchidism. Dysplastic ears were frequently reported (43%), sometimes associated with deafness. Cardiac malformations were mainly conotruncal defects and patent ductus arteriosus. Rare cases of arrhythmia were also documented.

Interestingly, these cardiac anomalies were only reported in patients with PBX1

deletions. The hypothesis of the contiguous gene syndrome to explain certain extrarenal phenotypes has been proposed (Le [START_REF] Tanno | PBX1 haploinsufficiency leads to syndromic congenital anomalies of the kidney and urinary tract (CAKUTHED) in humans[END_REF] but the cardiac phenotype could not be linked to another gene in close proximity to PBX1. The implication of PBX1 in the cardiac phenotype is discussed below.

2.2) PBX1 missense variations

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Missense variations were diagnosed for thirteen (33%) of the PBX1-mutated patients. Most of these missense variations identified in the HD of PBX1 lie in the NLS (Figure 1), with two recurrent positions affected (Arg234 and Arg235), which were studied using functional analyses [63,64](see below).

Of these 13 patients, 9 had detailed clinical features [START_REF] Arts | Paternal mosaicism for a novel PBX1 mutation associated with recurrent perinatal death: Phenotypic expansion of the PBX1-related syndrome[END_REF] In contrast with PBX1 LOF patients, genital anomalies were frequent and severe. Only one 46,XX individual was described in this cohort as having a bicornuate uterus. Six of the 8 46,XY individuals had various severe phenotypes ranging from cryptorchidism and micropenis to complete sex reversal. These last phenotypes included a nearly normal female phenotype for both internal and external genitalia and gonads (1 patient), various gonadal anomalies (ovotestis, streak gonads) and internal genitalia malformations. Lung hypoplasia was observed in 4 patients. For 3 patients, this lung hypoplasia was associated with diaphragmatic thinning or defect (different from diaphragmatic hernias).

Functional assessment of PBX1 HD variations

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cytoskeleton, namely NMHCB or FLNA, retains PBX1 in the cytosol [START_REF] Berry | FOXC1 Transcriptional Regulatory Activity Is Impaired by PBX1 in a Filamin A-Mediated Manner[END_REF][START_REF] Huang | Nonmuscle Myosin Promotes Cytoplasmic Localization of PBX[END_REF]. Both proteins compete with MEIS1 to bind PBX1, but it is the interaction between MEIS1 (or PKNOX1) and PBX1 that enables PBX1 conformational change and exposure of its NLS [START_REF] Longobardi | Biochemistry of the tale transcription factors PREP, MEIS, and PBX in vertebrates[END_REF][START_REF] Saleh | A conformational change in PBX1A is necessary for its nuclear localization[END_REF]. It is tempting to postulate that PKNOX1 overexpression could partially overcome mutant PBX1 cytoplasmic retention. On the contrary, a myc-tag may worsen an abnormal conformational change induced by a morbid variation in the PBX1 NLS domain.

Thus, these studies suggest that variations in the NLS lead to a partial loss of PBX1 nuclear localization, but that they also prevent, at least partly, PBX1 interaction with some of its cofactors, two defects that could explain the phenotype induced in humans which recalls, but does not fully phenocopy, the Pbx1-KO mouse phenotype.

Two other variations in the HD have been reported to date but not extensively

Functional assessment of PBX1 non-HD variations

Only one variation in the PBC domains was analysed in a functional study by [START_REF] Slavotinek | De novo, deleterious sequence variants that alter the transcriptional activity of the homeoprotein PBX1 are associated with intellectual disability and pleiotropic developmental defects[END_REF], with the protocol previously mentioned. The authors demonstrated that, as for variations in the HD, expression levels of the mutant .

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PBX1 Arg227Pro were comparable to the WT protein. Mutant proteins also reduced transactivation properties in HEK293T cells but not in a Pbx1-KO murine cell line.

The authors observed no change in nuclear levels in cells expressing the mutant protein compared to WT transfected cells.

The four other variations have not been extensively studied. One patient from a cohort of schizophrenic individuals was described with a substitution of the threonine in position 88 by an isoleucine [START_REF] Fromer | De novo mutations in schizophrenia implicate synaptic networks[END_REF]). This variation is located in PBX1 NES. PBX1 NES prevents PBX1 nuclear localization by interacting with its NLS [START_REF] Saleh | A conformational change in PBX1A is necessary for its nuclear localization[END_REF] and it is masked after PBX1 dimerization with PKNOX1 [START_REF] Berthelsen | The subcellular localization of PBX1 and EXD proteins depends on nuclear import and export signals and is modulated by association with PREP1 and HTH[END_REF]. The substitution of a threonine by a more hydrophobic amino acid could disrupt NES/NLS interactions. In fact, the NLS has an arginine-rich structure that could be destabilized by hydrogen bond modifications, but this hypothesis remains to be explored.

Two siblings were described with a variation substituting the arginine in position 107 for a tryptophan [START_REF] Arts | Paternal mosaicism for a novel PBX1 mutation associated with recurrent perinatal death: Phenotypic expansion of the PBX1-related syndrome[END_REF]). This Arg107 is located just before one poly-alanine segment (Leu108-Tyr150) that plays an important role in PBX1 (Supp. Figure S1).

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One patient was reported with a substitution of the arginine in position 184 by a proline [START_REF] Alankarage | Functional characterization of a novel PBX1 de novo missense variant identified in a patient with syndromic congenital heart disease[END_REF][START_REF] Slavotinek | De novo, deleterious sequence variants that alter the transcriptional activity of the homeoprotein PBX1 are associated with intellectual disability and pleiotropic developmental defects[END_REF]. The Arg184 was conserved up to Drosophila. This substitution could disrupt the alpha helix structure implicated in interaction with MEINOX proteins ( [START_REF] Bruckmann | Mapping the native interaction surfaces of PREP1 with PBX1 by cross-linking mass-spectrometry and mutagenesis[END_REF], HOPE results).

Moreover, this arginine is located in a MAP-kinase recognition motif [START_REF] Calvo | inhibitory switch derepressed by pbx, hox, and Meis/Prep1 partners regulates DNA-binding by pbx1 and E2a-pbx1 and is dispensable for myeloid immortalization by E2a-pbx1[END_REF] and phosphorylation is a way for PBX1 to reach the nucleus [START_REF] Kilstrup-Nielsen | PBX1 nuclear export is regulated independently of PBX-MEINOX interaction by PKA phosphorylation of the PBC-B domain[END_REF], see above).

Finally, another patient was reported with a substitution of the methionine in position 224 by a lysine [START_REF] Slavotinek | De novo, deleterious sequence variants that alter the transcriptional activity of the homeoprotein PBX1 are associated with intellectual disability and pleiotropic developmental defects[END_REF]. Met224 is conserved in all species (including C. elegans). This methionine is located in a potential "minimal inhibitory helix", a domain that could play a role in preventing PBX1 monomers from binding DNA, an effect suppressed when PBX1 dimerizes [START_REF] Calvo | inhibitory switch derepressed by pbx, hox, and Meis/Prep1 partners regulates DNA-binding by pbx1 and E2a-pbx1 and is dispensable for myeloid immortalization by E2a-pbx1[END_REF]. However, the function of this domain has not been assessed in more recent studies.

Thus, missense variations in the PBC domains can lead to various deleterious effects: absence of interaction with MEINOX proteins, modification of DNA binding stringency or alterations in nuclear import. However, more robust functional studies are needed to assess the role of these variants.

3) Shedding light on human PBX1-induced phenotypes using animal models

Animal models of heterozygous PBX1 LOF are not helpful in explaining these developmental anomalies, since heterozygous mice for a Pbx1 null allele merely exhibit smaller size than their wild-type littermates, even if they express only half the normal level of the protein (L. [START_REF] Selleri | Requirement for Pbx1 in skeletal patterning and programming chondrocyte proliferation and differentiation[END_REF]. One notable exception concerns mice heterozygous for the p.(Arg184Pro) mutation that sometimes present with a phenotype close to that of their KO littermates, although it tends to be less severe .

Accepted Article [START_REF] Alankarage | Functional characterization of a novel PBX1 de novo missense variant identified in a patient with syndromic congenital heart disease[END_REF]). However, PBX1-null mouse malformations clearly recall the developmental phenotype reported in humans (Figure 2 panel C)(L. [START_REF] Selleri | Requirement for Pbx1 in skeletal patterning and programming chondrocyte proliferation and differentiation[END_REF].

3.1) Head, face and neck morphogenesis anomalies

The main defect observed in Pbx1-KO mice is a homeotic transformation of structures derived from the second branchial arch and the first pharyngeal groove into first branchial arch derivatives (L. [START_REF] Selleri | Requirement for Pbx1 in skeletal patterning and programming chondrocyte proliferation and differentiation[END_REF]. PBX proteins are regulators of HOXA2 expression in rhombomere 4 [START_REF] Lampe | An ultraconserved Hox-Pbx responsive element resides in the coding sequence of Hoxa2 and is active in rhombomere 4[END_REF], which gives rise to the ectomesenchyme of the second branchial arch [START_REF] Couly | The regeneration of the cephalic neural crest, a problem revisited: The regenerating cells originate from the contralateral or from the anterior and posterior neural fold[END_REF]. However, the mouse phenotype is not completely reminiscent of Hoxa2-KO mice and Pbx1-deficient animals also present with delayed third pouch development, including thymus and parathyroid [START_REF] Manley | Abnormalities of caudal pharyngeal pouch development in Pbx1 knockout mice mimic loss of Hox3 paralogs[END_REF]). Pbx1-KO mice also display external and inner ear anomalies that are not fully explained by Hox gene dysregulation. Hmx1, a homeodomain transcription factor, plays a role in lateral facial morphogenesis, notably in pinna formation, and possesses a downstream regulatory sequence that can be bound by the Hox-PBX-MEIS complex [START_REF] Rosin | A distal 594 bp ECR specifies Hmx1 expression in pinna and lateral facial morphogenesis and is regulated by the Hox-Pbx-Meis complex[END_REF].

Deletions of this highly conserved regulatory region are responsible for the dumbo phenotype in mice and rats [START_REF] Quina | Deletion of a conserved regulatory element required for Hmx1 expression in craniofacial mesenchyme in the dumbo rat: A newly identified cause of congenital ear malformation[END_REF]. Mutations in another homeobox gene,

Emx2

Accepted Article

cleft lip and palate [START_REF] Ferretti | A conserved Pbx-Wnt-p63-Irf6 regulatory module controls face morphogenesis by promoting epithelial apoptosis[END_REF]. Interestingly, none of the PBX1-mutated patients previously reported presented oral clefts, but SNP in PBX1, WNT9B and TP63 was significantly associated with non-syndromic cleft lip and palate in humans [START_REF] Maili | PBX-WNT-P63-IRF6 pathway in nonsyndromic cleft lip and palate[END_REF].

3.2) Skeletal and limb deformities or hypoplasia

Mice KO for Pbx1 develop constant and severe skeletal anomalies compared to what is observed in humans. These anomalies mainly involve the cervical and thoracic vertebrae, the ribs and sternum, and proximal structures of the limbs (L. [START_REF] Selleri | Requirement for Pbx1 in skeletal patterning and programming chondrocyte proliferation and differentiation[END_REF]. The scapula, clavicle and humerus in particular are hypoplastic and misshapen, resembling some malformations described in patients. In mice, Pbx1 and Pbx2 control both Polycomb and Hox TFs (namely Bmi, Eed, Hoxa4, Hoxb5, Hoxb8, Hoxc6, and Hoxd3) to establish hind limb positioning, [START_REF] Capellini | Pbx1/Pbx2 govern axial skeletal development by controlling . Accepted Article Polycomb and Hox in mesoderm and Pax1/Pax9 in sclerotome[END_REF]. Pbx1, in association with Emx2, also controls the expression of scapula-specific genes such as Alx1

Accepted Article

humans, but most of the patients reported to date were children and we cannot fully assess the impact of their PBX1 variant on their adult stature.

3.3) Abnormal cardiac outflow tract development

Pbx-deficient mice present heart defects that mimic the malformations observed in humans, arising from abnormal cardiac outflow tract development [START_REF] Stankunas | Pbx/Meis deficiencies demonstrate multigenetic origins of congenital heart disease[END_REF]). The anomalies observed in mice partly depend on which Pbx protein is deficient, but Pbx1 seems to be the main gene implicated in these malformations, with relatively minor roles for Pbx2 and Pbx3. These effects on the cardiac outflow tract are at least partially explained by the PBX1 requirement for correct expression of PAX3 in pre-migratory neural crest cells, a cell population highly implicated in heart development (review by [START_REF] Lescroart | Hox and Tale transcription factors in heart development and disease[END_REF]). We can also postulate a role of MEIS2 in this expression, since MEIS2 mutations in humans lead to similar defects in neural crest-derived structures [START_REF] Douglas | De novo missense variants in MEIS2 recapitulate the microdeletion phenotype of cardiac and palate abnormalities, developmental delay, intellectual disability and dysmorphic features[END_REF][START_REF] Fujita | De novo MEIS2 mutation causes syndromic developmental delay with persistent gastro-esophageal reflux[END_REF]). At cellular level, PBX1 controls both cardiac precursor proliferation in synergy with GATA4, and cell cycle arrest in mature cardiomyocytes by transcriptional activation of CDKN2a, with a suspected role of MEIS1 as a cofactor [START_REF] Hatzistergos | Tumor Suppressors RB1 and CDKN2a Cooperatively Regulate Cell-Cycle Progression and Differentiation During Cardiomyocyte Development and Repair[END_REF].

Thus, PBX1 is a key factor in heart formation, and it is worth noting that cardiac anomalies are not very frequent in patients with PBX1 LOF, unlike patients carrying missense variants (26% vs. 55%). In fact, some authors [START_REF] Stankunas | Pbx/Meis deficiencies demonstrate multigenetic origins of congenital heart disease[END_REF]) [START_REF] Selleri | Requirement for Pbx1 in skeletal patterning and programming chondrocyte proliferation and differentiation[END_REF]. This finding highlights the redundancy of PBX proteins [START_REF] Capellini | Pbx1/Pbx2 requirement for distal limb patterning is mediated by the hierarchical control of Hox gene spatial distribution and Shh expression[END_REF]. In contrast, PBX1 missense variants can lead to defective PBX1 proteins retaining PBX1 cofactors (dominant-negative effect, see paragraph 4 and Figure 3), preventing partial rescue by other PBX proteins and leading to severe cardiac phenotypes.

demonstrated

3.4) Lung hypoplasia and delayed maturation

PBX1 (and especially the PBX1b isoform) is expressed early in the pulmonary mesenchyme during the initial stages of budding and outgrowth of the bronchial tree.

3.5) Diaphragmatic thinning and defects

.

Accepted Article

A few patients carrying PBX1 missense variations presented diaphragmatic thinning and defects [START_REF] Arts | Paternal mosaicism for a novel PBX1 mutation associated with recurrent perinatal death: Phenotypic expansion of the PBX1-related syndrome[END_REF][START_REF] Slavotinek | De novo, deleterious sequence variants that alter the transcriptional activity of the homeoprotein PBX1 are associated with intellectual disability and pleiotropic developmental defects[END_REF]. By studying the embryonic transcriptome in the search for genes implicated in diaphragmatic hernia,

3.6) Pancreas hypoplasia and splenic anomalies

.

Accepted Article

Pbx1-KO mice are also characterized by hypoplastic pancreas and asplenia (L. [START_REF] Selleri | Requirement for Pbx1 in skeletal patterning and programming chondrocyte proliferation and differentiation[END_REF]. Intriguingly, only two patients (siblings) presented asplenia or small spleen associated with accessory spleens. No pancreatic phenotype (pancreas hypoplasia, maldigestion, diabetes…) was reported among PBX1 patients. In Pbx1 mouse mutants, Tlx1 and Nkx2.5, early markers for splenic progenitor cells, are not expressed [START_REF] Brendolan | A Pbx1-dependent genetic and transcriptional network regulates spleen ontogeny[END_REF]. PBX1 is able to bind to TLX1 promoter, alone and in cooperation with TLX1 itself, underlining the fact that PBX1 is a key element in spleen development. During embryogenesis, both PBX1 and PBX2 are expressed in the developing pancreas. PBX1 is mainly expressed in the pancreas mesenchyme, unlike PBX2, which is more broadly expressed, including in the pancreatic epithelium [START_REF] Zhang | Pax6 is regulated by Meis and Pbx homeoproteins during pancreatic development[END_REF]. Both PBX proteins, in association with PKNOX1, bind to the pancreatic PAX6 enhancer [START_REF] Zhang | Pax6 is regulated by Meis and Pbx homeoproteins during pancreatic development[END_REF]. Thus, in Pbx1-deficient mouse embryos, Pax6 expression is strongly reduced in the pancreas, which is not the case for Pdx1, although this TF is also a cofactor of Pbx1 [START_REF] Asahara | Pbx-Hox heterodimers recruit coactivator-corepressor complexes in an isoform-specific manner[END_REF]. Finally, as demonstrated in the limbs [START_REF] Capellini | Pbx1/Pbx2 requirement for distal limb patterning is mediated by the hierarchical control of Hox gene spatial distribution and Shh expression[END_REF], the pancreatic defects observed in Pbx1-KO mouse embryos are more severe when a second Pbx2 hit is present [START_REF] Zhang | Pax6 is regulated by Meis and Pbx homeoproteins during pancreatic development[END_REF]). In the adult pancreas, the expression of Pbx1 isoforms differs in exocrine and endocrine tissues, Pbx1b being expressed in acinar cells and Pbx1a preferentially in Langherans islets [START_REF] Asahara | Pbx-Hox heterodimers recruit coactivator-corepressor complexes in an isoform-specific manner[END_REF]. The absence of pancreatic phenotypes among patients could be explained by a partial rescue of PBX1 function by PBX2, as suggested for heart development (see above). Finally, mild pancreatic features could be underreported by patients or clinicians, or appear later in life (since most of the PBX1-mutated patients described were young children).

3.7) CAKUTHED

.

Accepted Article

In KO mice, renal anomalies are similar to the human CAKUTHED phenotype, with hypoplastic caudally positioned and ventrally rotated kidneys. Renal hypoplasia is linked to oligonephronia, due to a reduced nephrogenic zone and nephronic differentiation (Catherine A. [START_REF] Schnabel | Pbx1 regulates nephrogenesis and ureteric branching in the developing kidney[END_REF]. [START_REF] Schnabel | Pbx1 regulates nephrogenesis and ureteric branching in the developing kidney[END_REF]) demonstrated that Pbx1 was expressed in the nephrogenic mesenchyme, mainly in the stroma. Renal defects in KO mice rely on both deficient ureteric branching and an abnormal extension of the induced nephrogenic mesenchyme. These anomalies could be the consequence of abnormal interactions between Pbx1 and other TFs, such as Hoxd genes [START_REF] Patterson | Atlas of Hox gene expression in the developing kidney[END_REF]. The Hoxd gene cluster has been shown to be implicated in the regulation of ureteric branching response and the maintenance of the structural integrity of the tubular epithelia (Di-Poï, Zákány, & Duboule, 2007). However, the Hoxd genes are known to interact with Pbx1 and Meis1 during limb bud pattern development [START_REF] Capdevila | Control of vertebrate limb outgrowth by the proximal factor Meis2 and distal antagonism of BMPs by Gremlin[END_REF].

Similar interactions could explain the renal phenotype observed in Pbx1-KO mice and PBX1-mutated patients, but this hypothesis remains to be explored. Moreover, PBX1 also has a prominent role in the establishment of renal vascular patterns: conditional Pbx1-KO in mouse renal vascular mural cells (VMC) leads to a loss of Pdgfrb downregulation, premature VMC differentiation, vascular pattern defects and thus early kidney dysfunction [START_REF] Hurtado | Pbx1-dependent control of VMC differentiation kinetics underlies gross renal vascular patterning[END_REF]. These findings suggest a prominent role of PBX1 in the induction and differentiation of the renal mesenchyme and especially of vasculature progenitors, essential for organ development and function.

3.8) Genital organs and genitalia anomalies

Analyses of mice KO for Pbx1 showed that the anomalies observed for kidneys were part of a larger spectrum of defects of the urogenital ridge mesodermal .

Accepted Article

derivatives (Catherine A. [START_REF] Schnabel | Pbx1 regulates nephrogenesis and ureteric branching in the developing kidney[END_REF]. PBX1 is expressed early in the coelomic epithelium, nephrogenic cord and adrenogenital precursors, it is maintained in the somatic cells of the undifferentiated gonad and finally in the interstitium of the mature gonad (Catherine A. [START_REF] Schnabel | Pbx1 regulates nephrogenesis and ureteric branching in the developing kidney[END_REF]. Gonads of Pbx1-KO animals initiate sexual differentiation but remain rudimentary due to reduced cell proliferation rate, and the expression of the steroid hydroxylase is barely detectable in gonads of male mutant mice. Interestingly, the authors also demonstrated that, in addition to gonadal anomalies, the mice completely lacked adrenal formations, due to the absence of Nr5a1 expression (SF-1, a key transcription factor implicated in steroidogenesis and sexual differentiation, review by [START_REF] Tremblay | A mutated form of steroidogenic factor 1 (SF-1 G35E) that causes sex reversal in humans fails to synergize with transcription factor GATA-4[END_REF]).

However, SF-1 expression appeared to be retained in gonadal cells, suggesting a differential regulation of Nr5a1 expression depending on the tissue (Catherine A. [START_REF] Schnabel | Pbx1 is essential for adrenal development and urogenital differentiation[END_REF]. These results demonstrate that Pbx1 does not interfere in the establishment of the urogenital ridge intermediate mesoderm but plays a prominent role in the establishment of the bipotential gonad (Catherine A. [START_REF] Schnabel | Pbx1 is essential for adrenal development and urogenital differentiation[END_REF] and is active upstream from Nr5a1 only in adrenal development. WNT9B, HOXA10 and EMX2. These last two genes are known to interact with or be recruited by PBX1 during limb formation and osteoblast activation [START_REF] Capellini | Scapula development is governed by genetic interactions of Pbx1 with its family members and with Emx2 via their cooperative control of Alx1[END_REF][START_REF] Gordon | Pbx1 represses osteoblastogenesis by blocking Hoxa10-mediated recruitment of chromatin remodeling factors[END_REF]. It is also worth noting that Hoxa10-KO male mice present bilateral cryptorchidism [START_REF] Satokata | Sexually dimorphic sterility phenotypes in HoxalO-deficient mice[END_REF], a phenotype also observed in some 46,XY patient carriers of PBX1 truncating variants. Thus, we can hypothesize that a partial disruption of PBX1 interaction with these effectors, especially HOXA10,

could

Accepted Article

diaphragmatic thinning. This discrepancy has already been discussed by [START_REF] Slavotinek | De novo, deleterious sequence variants that alter the transcriptional activity of the homeoprotein PBX1 are associated with intellectual disability and pleiotropic developmental defects[END_REF]. Genotype-phenotype correlations are of importance to further assess protein function but above all to provide appropriate genetic counselling and suggest the most suitable management for the patients.

Phenotypic variation depending on the mutation type is a mechanism previously described for other TFs. For example, GLI3 mutations cause Greig syndrome (MIM# 175700) in case of inactivating variations or haplo-insufficiency (through gene deletion), unlike truncating variants in the middle third of the gene, which are responsible for Pallister-Hall syndrome (MIM# 146510). Similarly, we can postulate different mechanisms to explain the phenotypic variability observed in PBX1.

4.1) Nonsense-mediated decay (NMD)

Stop or frame-shift mutations in PBX1 are thought to lead to mRNA NMD, as described for other TFs such as PAX6 (Lima Cunha, Arno, Corton, & Moosajee, 2019).

The NMD of PBX1 mRNA could lead to a decrease in PBX1 concentration, but the WT allele and other PBX proteins (Figure 3) could counterbalance this loss, at least partially. There is partial functional redundancy between PBX proteins [START_REF] Stankunas | Pbx/Meis deficiencies demonstrate multigenetic origins of congenital heart disease[END_REF]. However, total concentration levels of all PBX proteins are also of importance for normal cellular functions, as explained above for cardiac development.

Dose-dependency has been previously reported for other TALE TFs, such as MEIS1 [START_REF] Marcos | Meis1 coordinates a network of genes implicated in eye development and microphthalmia[END_REF]. Haplo-insufficiency has been described in numerous diseases caused by mutations in TFs [START_REF] Seidman | Transcription factor haploinsufficiency: When half a loaf is not enough[END_REF]. Haplo-insufficiency could be the only pathogenic mechanism reported for the gene (for example, MYRF, HAND2 or NFIB, [START_REF] Cohen | Haploinsufficiency of the basic helix-loop-helix transcription factor HAND2 causes congenital heart defects[END_REF][START_REF] Rossetti | Review of the phenotypic spectrum associated with haploinsufficiency of MYRF[END_REF], or it could be associated with various gene function disruptions caused by missense mutations (for example PAX6).

However, these later mutations are often responsible for phenotypes that are milder .

Accepted Article

4.3) Dominant-negative or gain-of-function (GoF) effects

Missense variations can also lead to dominant-negative or GoF effects, especially when proteins are engaged in multimeric complexes. This type of mechanism is well known in TP53-linked pathologies for example, where TP53 tetramers formed by both normal and mutant proteins (Gencel-Augusto 2020) affect the stability and localization of the complex. These mechanisms were also observed in complexes composed of different subunits, such as the chromatin-remodelling SWI/SNF complex. Mutations in genes encoding subunits of this complex are known to cause Coffin-Siris syndrome (MIM# 135900): depending on the protein affected, both dominant-negative effects and GoF have been described (Kosho, Okamoto, & Coffin-Siris Syndrome International Collaborators, 2014). Since PBX1 forms heterodimers or trimers with various cofactors, we can postulate that PBX1 missense variations can generate the expression of abnormal PBX1 proteins that can compete with normal PBX1 TF, either for cofactor dimerization or DNA binding (Figure 3).

4.4) Second genetic hit and epigenetic alterations

However, since PBX1 can interact with various cofactors with pleiotropic modalities, a second genetic hit (such as a rare SNP) in one of these cofactors, in another TALE protein, or in one of PBX1 DNA-binding sites, could play a role in this phenotypic variability. The potential role of a second variation in another TALE protein has been partially discussed above: Pbx1 KO mouse models have a more severe phenotype when they carry a heterozygous variant in another Pbx protein [START_REF] Capellini | Pbx1/Pbx2 govern axial skeletal development by controlling . Accepted Article Polycomb and Hox in mesoderm and Pax1/Pax9 in sclerotome[END_REF]. We can postulate that partial disruption of a MEIS or PKNOX partner could worsen the phenotype. The hypothesis of a second genetic hit has .

Accepted Article

already been reported for other diseases linked to TF mutations, such as SHH for example: some of the intra-familial variability observed for SHH-induced holoprosencephaly has been linked to a second pathogenic variation in another causative gene [START_REF] Nanni | The mutational spectrum of the sonic hedgehog gene in holoprosencephaly: SHH mutations cause a significant proportion of autosomal dominant holoprosencephaly[END_REF]. Animal models support this hypothesis of a second hit, with a more severe phenotype when there are additive variants in partner or target genes (for example, otocephaly and otx2 in zebrafish, [START_REF] Chassaing | OTX2 mutations to the otocephaly-dysgnathia complex[END_REF] or double mutations in MECP2 and CDKL5 in Rett-like syndrome [START_REF] Jdila | Unusual double mutation in MECP2 and CDKL5 genes in Rett-like syndrome: Correlation with phenotype and genes expression[END_REF]).

In contrast, TF binding variations induced by rare SNPs in TFs binding sites have been recently described, especially in common diseases such as thalassemia [START_REF] Cheng | Novel compound variants of the AR and MAP3K1 genes are related to the clinical heterogeneity of androgen insensitivity syndrome[END_REF][START_REF] Savinkova | An Experimental Verification of the Predicted Effects of Promoter TATA-Box Polymorphisms Associated with Human Diseases on Interactions between the TATA Boxes and TATA-Binding Protein[END_REF]. Despite growing knowledge in this field, in silico predictions of variations in TF binding sites remain challenging and require experimental validation [START_REF] Moradifard | A preliminary computational outputs versus experimental results: Application of sTRAP, a biophysical tool for the analysis of SNPs of transcription factor-binding sites[END_REF]. Finally, variations in sequence or level of expression of miRNAs targeting PBX1 could partly explain both intra-and inter-individual variability by modulating the impact of PBX1 variations from one tissue (or individual) to another. Recent studies have linked the roles of miRNAs to PBX1 function and degradation in both development processes and cancer [START_REF] Cao | MicroRNA-141-3p inhibits the progression of oral squamous cell carcinoma via targeting PBX1 through the JAK2/STAT3 pathway[END_REF][START_REF] Liu | MicroRNA-181 regulates the development of Ossification of Posterior longitudinal ligament via Epigenetic Modulation by targeting PBX1[END_REF].

Shedding light on these mechanisms would be of help to better understand PBX1 function and regulation, and also to refine genetic counselling.

5) Concluding remarks

First discovered in cancer, PBX proteins, and especially PBX1, have proved to be essential in organism development, from the early gastrulation stages to adulthood. .

Accepted Article

The

  TALE never ends: a comprehensive overview of the role of PBX1, a TALE transcription factor, in human developmental defects Laura Mary, Delphine Leclerc, David Gilot, Marc-Antoine Belaud-Rotureau, Sylvie Jaillard To cite this version: Laura Mary, Delphine Leclerc, David Gilot, Marc-Antoine Belaud-Rotureau, Sylvie Jaillard. The TALE never ends: a comprehensive overview of the role of PBX1, a TALE transcription factor, in human developmental defects. Human Mutation, 2022, 43 (9), pp.1125-1148. 10.1002/humu.24388

  More recently, PBX1 constitutional heterozygous deletions or point variations have been linked to developmental defects, especially to congenital anomalies of the kidney and urinary tract (CAKUTHED; MIM# 617641)(Le Tanno et al. 2017). Despite growing knowledge on the role of PBX1, human developmental phenotypes induced by PBX1 . Accepted Article variations are not yet fully understood. The increasing number of cases and the pleiotropy of the symptoms are presented in this review focused on the human developmental phenotype induced by PBX1 variations.

Slavotinek

  et al. (Slavotinek et al. 2017) studied the functional roles of PBX1 mutations, notably generating the p.(Arg234Pro) and the p.(Arg235Gln) mutants in HEK293T transfected cells with plasmids encoding these variants. They demonstrated that these variations affected neither levels of protein expression nor nuclear localizations in their conditions. Both mutant proteins showed reduced transactivation properties of a luciferase gene preceded by a PBX1 promoter, but in a Pbx1-KO murine cell line, only p.(Arg234Pro) had reduced transactivation properties. The authors suggested that the interactions between endogenous PBX1 protein in HEK293T cells and mutated transfected PBX1 could adversely affect these results. Eozenou et al. (Eozenou et al. 2019) studied the p.(Arg235Gln) mutant in HEK293T cells and demonstrated that, despite a few mutant proteins localized in the nucleus, the variation favoured a cytoplasmic retention of mutated PBX1. Moreover, they demonstrated that the mutant protein was unable to form heterodimers with HOXB8. Although the binding of the mutant protein to PKNOX1 was maintained, the binding of mutant PBX1 to EMX2 was reduced and abolished with CBX2. The discrepancy in nuclear localization between these two publications that studied the same variation in the same cellular model raises interesting questions, such as the impact of in vitro approaches on PBX1 function. Eozenou et al. used myc-tagged PBX1 expression vectors, unlike Slavotinek et al., who transfected HEK293T cells with the cDNA of interest (plus PKNOX1 cDNA) and analysed the protein content of the cell compartments by Western-blotting experiments. Variations might also result to the expression levels of PBX1 reached after plasmid transfection. However, mechanisms other than the use of NLS enable PBX1 to reach the nucleus: phosphorylation of serine residues in the PBC-B domain also has a role in PBX1 nuclear import (Figure 1, (Kilstrup-Nielsen et al. 2003)). In contrast, interaction with proteins of the .

  studied: p.(Cys273Tyr) and p.(Gln279Arg)(ClinVar VCV000598775 and (Bertoli- Avella et al. 2021)). Both native amino acids are highly conserved across species (Supp. FigureS1). Both residues are positioned on a segment undergoing conformational changes (Ser271-Arg288,[START_REF] Blasi | A tale of TALE, PREP1, PBX1, and MEIS1: Interconnections and competition in cancer[END_REF])), which are important in increasing DNA binding ability. We can suggest that these variants alter DNA binding rather than PBX1 localization (as demonstrated with other variants by[START_REF] Slavotinek | De novo, deleterious sequence variants that alter the transcriptional activity of the homeoprotein PBX1 are associated with intellectual disability and pleiotropic developmental defects[END_REF])) or they disrupt interaction with non-HOX partners. Functional studies are required to explore these hypotheses.

  flexibility and DNA binding[START_REF] Blasi | A tale of TALE, PREP1, PBX1, and MEIS1: Interconnections and competition in cancer[END_REF]. Deletion of this helix region (positions 105-113) mildly altered the ability of PBX1 to dimerize with PKNOX1, but did not influence PBX1 dimerization with MEIS1 (Bruckmann et al. 2020). However, in silico prediction tools (HOPE tool (Dunlavy, O'Leary, Klimov, & Thirumalai, 2005; Venselaar, Te Beek, Kuipers, Hekkelman, & Vriend, 2010)) suggest that this amino acid substitution would be damaging for the correct folding of the alpha-helical region (positions 103-116, (Bruckmann et al., 2020)). Arg107 is conserved in all species

  , which is known to be regulated by PBX1 in limb development (see below), also have a role in middle and inner ear formation. Mice mutated on this gene present ossicular chain malformations and abnormal hair cell development[START_REF] Holley | Emx2 and early hair cell development in the mouse inner ear[END_REF]. Abnormal patterns or expression of these two genes, combined with HOXA2 dysregulation, could be involved in the cranial and hearing phenotypes observed in patients, but this remains to be further explored. Finally, it has been demonstrated that PBX proteins control facial morphogenesis by promoting epithelial apoptosis through the Pbx-Wnt-p63-Irf6 network, and a compound loss of Pbx proteins in mice leads to .

(

  [START_REF] Capellini | Scapula development is governed by genetic interactions of Pbx1 with its family members and with Emx2 via their cooperative control of Alx1[END_REF]. More recent studies have also suggested that PBX1 and PBX2 work together in regulating the pattern of the proximal limbs in a dose-dependent manner[START_REF] Eyal | Bone morphology is regulated modularly by global and regional genetic programs[END_REF]. PBX1/PBX2 also interact with GLI3, one of the main controllers of the global limb pattern regulatory network[START_REF] Eyal | Bone morphology is regulated modularly by global and regional genetic programs[END_REF]) and an organizer of neural tube patterns. At cellular level, mice lacking Pbx1 display diminished chondrocyte proliferation with precocious ossification (L.[START_REF] Selleri | Requirement for Pbx1 in skeletal patterning and programming chondrocyte proliferation and differentiation[END_REF]. Pbx1 normally repress the ability of Hoxa10 to activate osteoblast-related gene programs[START_REF] Gordon | Pbx1 represses osteoblastogenesis by blocking Hoxa10-mediated recruitment of chromatin remodeling factors[END_REF]. This property could explain why Pbx1-KO mice display early abnormal osseous formation. The fact that heterozygous mice for a Pbx1-null allele are smaller than WT animals also suggests early halting of growth as a result of premature ossification. This late phenotype has not been reported in .

  that the severity of heart defects in various Pbx-deficient mice correlated with the total concentration level of PBX proteins. We can postulate that a decrease in PBX1 concentrations through nonsense-mediated decay can be counterbalanced, at least partially, by other PBX proteins (Stankunas et al. 2008) in patients carrying PBX1 LOF variants. In fact, PBX proteins share the same HD sequence and are able to target the same binding sites (Longobardi et al. 2014; Penkov . Accepted Article et al. 2013). Pbx1-KO mice have a more severe phenotype while they also carry a second hit in Pbx2, despite the fact that Pbx2-KO mice are viable and healthy (L.

  Pbx1b immuno-reactivity persists in the pulmonary interstitium until adulthood in mice (C. A.[START_REF] Schnabel | Expression of Pbx1b during mammalian organogenesis[END_REF]. Pbx1-KO mouse embryos show severe lung hypoplasia (L.[START_REF] Selleri | Requirement for Pbx1 in skeletal patterning and programming chondrocyte proliferation and differentiation[END_REF]) and conditional KO in mouse lungs has demonstrated abnormal lung maturation with compact terminal saccules and severely reduced expression of surfactant genes[START_REF] Li | Pbx1 activates Fgf10 in the mesenchyme of developing lungs[END_REF]. The loss of Pbx1 in the lung mesenchyme also results in continuous vascular constriction and elastin matrix anomalies[START_REF] Mcculley | PBX transcription factors drive pulmonary vascular adaptation to birth[END_REF]. The role of Pbx1 in the developing lung resides, at least partly, in its ability to control the expression of Fgf10, a key factor for alveolar type-II cells and differentiation of lung epithelial progenitors, in collaboration with HoxB4 and Meis1[START_REF] Li | Pbx1 activates Fgf10 in the mesenchyme of developing lungs[END_REF]). PBX1 interactions with HOXA5, HOXB5 and, more recently, the repression of TBX2, are also of importance in tracheal budding, mesenchyme induction and lung maturation[START_REF] Jeannotte | Hoxa5: A Key Player in Development and Disease[END_REF][START_REF] Lüdtke | Combined genomic and proteomic approaches reveal DNA binding sites and interaction partners of TBX2 in the developing lung[END_REF].

  Russel et al.[START_REF] Russell | Congenital diaphragmatic hernia candidate genes derived from embryonic transcriptomes[END_REF] identified PBX1 as a candidate gene. In the Pbx1-KO mice they generated, the authors demonstrated that the homozygous mutant mice displayed diaphragmatic and muscle-pattern defects, with regions of the diaphragm reduced to a thin membrane with poor or absent musculature and abnormal muscle marker expression. The authors postulated that these defects originated from a dysregulation of Hoxa5 and Hoxb5 through dysregulation of retinoic acid (RA) synthesis, since previous studies had demonstrated that PBX1 was able to control RA synthesis[START_REF] Vitobello | Hox and Pbx factors control retinoic acid synthesis during hindbrain segmentation[END_REF]. Other signalling pathways, such as SHH (PBX1 interacts with GLI3 during limb formation, see above) could be implicated in the diaphragmatic defects highlighted in Pbx1-deficient mice and PBX1-mutated patients.The absence of PBX1 could lead to defects in the mesenchymal proliferation of the pleuroperitoneal folds, a structure from which the diaphragm originates. A recent study reported a decrease in Pbx1, Meis1, and Runx1 expression in rats with diaphragmatic defects and lung hypoplasia, suggesting reduced mesenchymal cell proliferation as a common mechanism for both diaphragmatic defects and abnormal bronchial budding in Pbx1-deficient rodents[START_REF] Takahashi | Pbx1, Meis1, and Runx1 Expression Is Decreased in the Diaphragmatic and Pulmonary Mesenchyme of Rats with Nitrofen-Induced Congenital Diaphragmatic Hernia[END_REF]. Impaired mesenchymal cell proliferation has been previously demonstrated as a mechanism leading to renal anomalies (see below). Finally, PBX1 is also a cofactor for proteins implicated in myoblast differentiation, such as Mcadherin, (Y.-J.[START_REF] Lin | The cooperation of cis-elements during M-cadherin promoter activation[END_REF]).

  Moreover, Pbx1-KO mice lack Müllerian ducts, but not Wollfian ducts which are normally formed and joined. These findings have led some authors to explore the role of PBX1 in Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. Burel et al. (Burel et al. 2006) did not find morbid variations in the HOX genes or PBX1 in MRKH patients, but Ma et al. (Ma et al. 2015) found an increased risk of MRKH syndrome in association with rare SNP in PBX1 and WNT9B, which are known to interact together in facial morphogenesis (see above and (Ferretti et al. 2011; Maili et al. 2020)). More recently, Chen et al. (Chen et al. 2021) also found MRKH candidate variants in

  explain certain mild phenotypes observed in PBX1-mutated patients. However, the discrepancies between mouse models and PBX1-mutated patients, especially for adrenal agenesis and sex-dependent gonadal/genital malformations, are not well known. These differences recall the variations observed between NR5A1-mutated patients and Nr5a1-KO mice: XY mice are sex-reversed and present adrenal agenesis, unlike patients who can either have isolated adrenal deficiency or 46,XY DSD(Biason-Lauber 2000). The phenotypes of Nr5a1-KO mice are known to vary depending on the strain studied, suggesting a prominent role of other genetic modifiers. This phenomenon could also play a partial role in the phenotypic variability observed in PBX1-mutated patients, where DSDs are not fully penetrant. These results demonstrate that the precise role of PBX1 in human gonadal and genital developmenthas not yet been fully understood.4) Genotype-phenotype variations in PBX1-mutated patientsTwo distinct phenotypes can be identified for PBX1-mutated patients (as suggested by[START_REF] Slavotinek | De novo, deleterious sequence variants that alter the transcriptional activity of the homeoprotein PBX1 are associated with intellectual disability and pleiotropic developmental defects[END_REF], Figure2 A and B). First, a phenotype of syndromic CAKUTHED with skeletal anomalies, deafness, ear dysplasia, neurodevelopmental delay and cryptorchidism is observed for patients carrying a LOF mutation or a gene deletion. A more severe phenotype is observed for patients with missense variations, with gonadal and genital anomalies that can lead to complete sex reversal in 46,XY individuals and lung hypoplasia sometimes associated with .

  than haplo-insufficiency[START_REF] Lima Cunha | The Spectrum of PAX6 Mutations and Genotype-Phenotype Correlations in the Eye[END_REF], unlike what is observed in PBX1mutated patients.4.2) mRNA instability and post-translational protein modificationsMechanisms other than NMD can alter mRNA stability or translation, leading to a drastic decrease in the total protein levels. mRNA secondary structures are known to alter protein expression through changes in mRNA half-life[START_REF] Mauger | MRNA structure regulates protein expression through changes in functional half-life[END_REF]. Any nucleotide alteration of the secondary structure of mRNA can lead to premature mRNA decay and haplo-insufficiency-like phenotypes in patients carrying PBX1 missense variations. Micro-ARNs (miRNA) are also of importance in regulating mRNA levels: 3'UTR variations affecting miRNA binding sites are implicated in human diseases such as Tourette's syndrome (review by[START_REF] Kawahara | Human diseases caused by germline and somatic abnormalities in microRNA and microRNA-related genes[END_REF]). Very recently, a SNP in the 3'UTR sequence of PBX1 was linked to poor prognosis in breast and gastric cancers, by alteration of the regulatory affinity of miR-522-3p[START_REF] Mohammadi | Rs6426881 in the 3'-UTR of PBX1 is involved in breast and gastric cancers via altering the binding potential of miR-522-3p[END_REF]. Thus, the occurrence of aberrant miRNA binding sites in PBX1 could also play a role in the milder phenotypes caused by missense variants. It is worth noting that PBX1 3'UTR variants are rare in the general population (64 variants reported in the gnomAD database, of which only 11 had an allele count > 10). However, 3'UTR variants are not fully explored by exome sequencing, a routine first-tier technique, and they may be under-diagnosed in CAKUTHED patients. Post-translational changes such as ubiquitinylation or phosphorylation are of importance for correct protein function or degradation. At least 14 phosophorylation sites are reported in PBX1 (https://www.phosphosite.org/). Only the sites located in the PBC-B domain have been studied in vivo, with proven effect on PBX1 nuclear . Accepted Article localization (Kilstrup-Nielsen et al. 2003), and no germline variation affecting, or creating, a phosphorylation site has been reported to date.

  As a transcription cofactor, PBX1 interacts with numerous proteins, including those of the HOX family, and reaches dozens of regulating sequences, even in the heterochromatin state, suggesting that PBX proteins could act as pioneer factors. This .Accepted Articlepleiotropy explains the polymalformative phenotypes observed in both Pbx1-deficient mice and patients carrying heterozygous PBX1 variations. By altering PBX1 cellular localization and its interaction with its partners or DNA binding, these variations impact the wide network under control of PBX1. However, despite a growing number of studies about this particular family of PBC/MEINOX proteins, questions are still emerging: the mechanisms behind the phenotypic variability among PBX1-mutated patients, the discrepancies between human phenotypes and animal models (such as sexual development anomalies), the whole landscape of PBX protein redundancy, or the key elements developed by organisms to compensate for PBX1 deficiency. The end of the TALE has not yet been written.

Figure 1 :

 1 Figure 1: Diagram of the linear structure of PBX1 longest isoform

Figure 2 :

 2 Figure 2: Comparison of the human phenotypes generated by PBX1

  Table 1 sums-up the most relevant PBX1 cofactors and target genes with implication in development demonstrated in functional studies. For extensive gene modifications induced by PBX1 alone or by the chimeric E2A-PBX1 protein, see (Diakos et al. 2014; Thiaville et al. 2012).

	PBX1 structure is similar to what is observed in other PBC family members,

  ; Eozenou et al. 2019; Kia, Sarafoglou, Mooganayakanakote Siddappa, & Roberts, 2019; Slavotinek et al. 2017)(Figure 2 panel B and Supp. TableS2). Renal or urinary anomalies were less prevalent in this group than in the PBX1 LOF group and were

milder (hypoplastic or horseshoe kidneys, ureteral dilatation or hyperechogenic kidneys). Dysplastic ears were rarer. One third of the patients had mild skeletal anomalies (brachydactyly, clinodactyly, radioulnar synostosis). Unlike PBX1 LOF patients, no central nervous system malformations were reported. Cardiac anomalies were frequent (55%) and similar to the cardiac defects found among patients carrying PBX1 deletions.

Table 2 : Molecular and clinical summary of previously published PBX1-mutated patients.

 2

						F	R
						a	e
						c	n
						e	al
						a	a	Ex
						n	n	ter
						d	d	na
		Prot				h	ur	l				Pul
		ein		Fu		e	in	ge		S		mo		N
		cha		nct		a	ar	nit	Gon	ke	Car	nar		C
		nge		ion	Chr	d	y	ali	adal/	let	diac	y		S		O
	Heterozygou	(NP		al	omo	a	a	a	uteri	al	malf	hy	De	a		t
	s variant	_00		stu	som	nl	nl	an	ne	an	orm	po	af	nl		h
	(NM_002585.	257	Expected	die	al	ie	ie	lie	anlie	lie	atio	pla	ne	ie	I	e	Article or accession
	4)	6.1)	effect	s	sex	s	s	s	s	s	ns	sia	ss	s	D	r	number
		p.(G
	c.145C>T

Acknowledgements

Figures 2 and 3 were created with the help of BioRender.com tool.

This research did not receive any specific grant from commercial, or not-for-profit sectors.

Authors would like to thank AVIESAN (Plan Cancer).

Data availability Statement

The data that support the findings of this study is openly available on ClinVar at https://www.ncbi.nlm.nih.gov/clinvar/ or DECIPHER at .

Accepted Article

https://www.deciphergenomics.org/ (reference numbers are listed in Table 2) or can be found in the publications listed in the Reference section.

Competing interests

There are no competing interests for any author.

Ethics approval statement

Ethics approval was not sought for the present study because it only includes previously published data from human and animal subjects.

Authorship

Laura Mary: Investigation, Writing -Original Draft, Delphine Leclerc: Review & Editing, David Gilot, Marc-Antoine Belaud-Rotureau, Sylvie Jaillard: Supervision, Writing -Review & Editing.

Accepted Article