Asx Asxl

Philip Yuk Kwong

Alexandra Stuetzer

Anne-Marie Martinez
email: marie.martinez@igh.cnrs.fr

Wolfgang Fischle
email: wfischl@gwdg.de

Giacomo Cavalli
email: giacomo.cavalli@igh.cnrs.fr

p53-binding protein 1 Abdominal-B AE-1 Binding Protein 2 Antennapedia Gene Complex Antennapedia Antennapedia Nasobemia Anaphase Promoting Complex Absent Small or Homeotic 1 Additional Sexcomb

Keywords: Polycomb, Histone, Chromatin, Epigenetics

CP190 CPC CTCF CycB dac DAM-ID Dap DNMT

Table of Content

List of Figures

I. Overview In vitro histone methyltransferase assay suggested that, while H3K27me3S28A retained normal stimulatory effect on PRC2 activity assayed on nucleosomes reconstituted using XII wild type recombinant histones, recombinant H3S28A nucleosomes appeared to be a suboptimal substrate for PRC2. Hence the observed phenotypes in H3S28A mutant in vivo could be in part caused by intrinsic structural defects introduced by the alanine substitution, in addition to the lack of H3S28ph.

Collectively, our data indicate an essential role of H3S28 in the catalysis of H3K27 methylation and its potential modes of action are discussed.

La sérine 28 de l'histone H3 joue un rôle essentiel dans la répression des gènes dépendante des protéines PcG chez la drosophile. Les tests in vitro de l'activité histone-méthyltransférase montrent, d'une part, qu'un peptide mutant H3K27me3S28A conserve sa capacité à stimuler l'activité de PCR2 sur des nucléosomes reconstitués et, d'autre part, que des nucléosomes reconstitués comportant la mutation H3S28A sont un substrat que PRC2 méthyle moins efficacement que les nucléosomes non mutés. Ainsi, les phénotypes observés dans des mutants H3S28A in vivo pourraient s'expliquer en partie par des défauts intrinsèques de structure des histones introduits par la substitution de la sérine 28 en alanine, en plus de l'absence de la phosphorylation H3S28ph.

Résumé de la thèse

Dans leur ensemble, ces résultats révèlent un rôle essentiel de H3S28 dans la catalyse des méthylations de H3K27 et les potentiels mécanismes d'action sont discutés.

Overview of chromatin biology and epigenetics

The genetic materials presented as long threads of DNA are organized as chromatin within the confined space of eukaryote nuclei. Specifically, small basic proteins known as histones constitute the fundamental packaging materials of our genome. There are five canonical histones namely the linker histone H1, and the core histones H2A, H2B, H3 and H4 1 . Two H2A-H2B dimers assembled with a H3-H4 tetramer to form a histone octamer.

A nucleosome core particle is formed with a DNA thread spanning 147 bp wrapping around the histone octamer for 1.67 turns. DNA fragments connecting adjacent nucleosomes are referred as linker DNA on which linker histone H1 can bind and regulate chromatin compaction. As such nucleosome represents the basic packaging unit of eukaryotic genome, and together with other chromatin-bound factors, successive nucleosomal arrays can be folded into higher order structures. Nucleosome occupancy of the underlying DNA sequence represents a general accessibility barrier to trans-acting factors. And since the first characterization of reversible acetylation on histone tails, we now recognize that histone proteins can capacitate a great variety of chemical modifications to elicit specific functions [START_REF] Kouzarides | Chromatin modifications and their function[END_REF] . This constitutes the histone code hypothesis [START_REF] Strahl | The language of covalent histone modifications[END_REF] the broad implications of epigenetic regulators in differentiation and diseases, lessons learnt from the PcG system will be transferable to chromatin events and benefits both basic and clinical research.

Histone Modifications

Post-translational modifications of histones represent one of the most central epigenetic regulations of DNA-templated processes. In this chapter, the function of a selection of histone marks (Fig. I) will be discussed in a Drosophila-centric view. Examples from other model organisms will also be included when necessary.

1 H3K4me

H3K4me is one of the hallmarks of active genes [START_REF] Filion | Systematic protein location mapping reveals five principal chromatin types in Drosophila cells[END_REF][START_REF] Kharchenko | Comprehensive analysis of the chromatin landscape in Drosophila melanogaster[END_REF] . In Drosophila, H3K4me1 is enriched at enhancer regions disregarded of their activity, it also appears at the middle of active genes [START_REF] Bonn | Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development[END_REF] . H3K4me2/3 marks are enriched at active gene promoters and TSS [START_REF] Kharchenko | Comprehensive analysis of the chromatin landscape in Drosophila melanogaster[END_REF][START_REF] Yin | A high-resolution wholegenome map of key chromatin modifications in the adult Drosophila melanogaster[END_REF][START_REF] Schuettengruber | Cooperativity, specificity, and evolutionary stability of polycomb targeting in Drosophila[END_REF][START_REF] Schuettengruber | Functional anatomy of polycomb and trithorax chromatin landscapes in Drosophila embryos[END_REF] Drosophila Trithorax (Trx) and Trithorax-related (Trr) are the principle enzymes that deposit H3K4me1. Both of their SET domains display H3K4me1-specific HMT activity in vitro. And their respective SET domain specific mutants (trx z11 and trr [START_REF] Strahl | The language of covalent histone modifications[END_REF] ) individually lead to a partial drop of H3K4me1 level. Combination of both mutants lead to a complete lost of H3K4me1 while render H3K4me3 level normal in Drosophila embryo [START_REF] Tie | Trithorax monomethylates histone H3K4 and interacts directly with CBP to promote H3K27 acetylation and antagonize Polycomb silencing[END_REF] . Both Trx and

Trr interact with CBP (CREB-binding protein), and in vitro HAT assay suggests that specifically H3K4me1, but not H3K4me2 and H3K4me3, enhances CBP-mediated H3K27ac [START_REF] Tie | Trithorax monomethylates histone H3K4 and interacts directly with CBP to promote H3K27 acetylation and antagonize Polycomb silencing[END_REF] .

H3K4me1 is enriched at enhancers and PREs and since trxz [START_REF] Bonn | Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development[END_REF] mutant alleviated the transformation phenotype of Pc 3 , it is thought that H3K4me1 counteracts PcG silencing via CBP recruitment and H3K27ac [START_REF] Tie | Trithorax monomethylates histone H3K4 and interacts directly with CBP to promote H3K27 acetylation and antagonize Polycomb silencing[END_REF] . CBP also interacts directly with dUtx, whose H3K27me3 demethylase activity would counteract PcG silencing [START_REF] Gutierrez | The role of the histone H2A ubiquitinase Sce in Polycomb repression[END_REF] .

Drosophila Set1 protein was identified base on the search for sequence homology to yeast Set1 protein. Affinity purification of FLAG-HA-dSet1 interacting candidates identifies components similar to human and yeast COMPASS complex. And in vitro HMT assay confirmed dSet1 complex catalyzed all methylation states of at H3K4 [START_REF] Ardehali | Drosophila Set1 is the major histone H3 lysine 4 trimethyltransferase with role in transcription[END_REF] . RNAi approach and genetic mutants confirmed that dSet1 was responsible to catalyze H3K4me2 and H3K4me3 in vivo [START_REF] Mohan | The COMPASS family of H3K4 methylases in Drosophila[END_REF]19 . Also RNAi knock down of or mutation in COMPASS components including the TrxG protein Ash2 resulted in reduction of bulk H3K4 methylation states, most notably for H3K4me3 19 . Similar to yeast and mammalian models 20,21 , the putative Drosophila H2B ubiquitin ligase dBre1, is required for efficient catalysis of H3K4me3 [START_REF] Mohan | The COMPASS family of H3K4 methylases in Drosophila[END_REF]20,22 .

H3K4me3 interacts with chromatin remodelers Isw1/NURF 23,24 , Yng1-NuA3 HAT complex 25 as well as the basal transcription machinery TFIID 26 . It also binds Chd1 27,28 , through which spliceosome components are recruited 29 . And in response to DNA damage, H3K4me3 of cell proliferation genes recruits ING2 and its associated HDAC complex for gene repression 30 .

H3K4me1 and H3K4me2 are demethylation substrates of the amine oxidase dLsd1 / Su(var)3-3 31,32 . Besides, another demethylase known as Lid, specifically removes

H3K4me3 33,34 . Intriguingly, Lid is genetically classified as a TRX group protein which might appear contradictory to the accumulation of H3K4me3 upon loss of function of Lid 35 . One explanation is that the reader of H3K4me3 Chd1 is dilution upon overaccumulation this histone mark and hence some of the Chd1 target genes cannot be appropriately activated 35 . Another explanation lies in the fact that Lid inhibits Rpd3 HDAC, and lost of Lid might liberate Rpd3 and cause excessive deacetylation at H3K27

and favor ectopic PcG gene repressions 34 .

Since H3K4me decorates active genes across the genome, it is expected to play a vital role in active transcription. However, a recent histone replacement study of combined nonmethylatable H3K4R mutations on both H3.2 and H3.3 showed no deregulation on Hox genes and developmental regulators expression. This observation challenges the essential necessity of H3K4 methylation or it hints functional redundancy from other histone modifications 36 .

2 H3K9me

H3K9me decorates condensed heterochromatin in the fly genome, notably in the pericentromeric regions, the telomeres and the arms of chromosome 4. In a classical Drosophila model of position effect variegation [37][38][39] , the white gene required for red eye pigment synthesis is placed in proximity to the pericentromeric region as a result of chromosome rearrangement. The spreading of pericentromeric heterochromatin leads to varied degree of silencing of the white gene and hence a variegated eye color [37][38][39] .

Enhancer and suppressor genetic screens based on this PEV model identify many players regulating heterochromatin biogenesis. Among them are the Suppressor of variegation 3-9, Su(var)3-9, the first characterized H3K9-specific histone methyltransferase 40 and Su(var)2-5/HP1, the chromodomain-containing reader of H3K9me2/3 41,42 .

In addition to Su(var)3-9, dG9a is another H3K9 HMT [43][44][45] . In Drosophila germarium, H3K9me3 is catalyzed by both Su(var)3-9 and dSETDB1/Eggless, with the latter HMT specifically required for germ cell pericentromeric H3K9me3 deposition 46 . Notably, lost of germ cells H3K9me3 in eggless mutant resulted in sterility while double mutant of Su(var)3-9 and dG9a was fertile, despite a drastic reduction of all H3K9 methylation states 43 . While in somatic tissue, dSETDB1 is primarily responsible for H3K9me3 on the arm of chromosome 4.

H3K9me3 level also depends on the RNAi machinery 47 . In Drosophila, mutant of RNAi machinery showed a reduction of H3K9me3 and a diffused localization of HP1 48 . It is demonstrated that pericentromeric H3K9me3 recruits HP1 via its chromodomain to establish heterochromatin 41,42 , which is essential for kinetochore assembly and sister chromatids cohesion 49,50 . Consistent with this idea, compromised pericentromeric heterochromatin leads to chromosome segregation defects 51,52 . HP1 also helps to recruit and activate dKDM4A activity to regulate H3K36me3 level on heterochromatic genes 53,54 .

Recently, it is reported that Su(var)3-9 interacts with linker histone H1 to induce heterochromatin silencing on Drosophila transposable elements 55 .

Finally, H3K9me3 can be removed by the dual-specific demethylases dKDM4A and dKDM4B, both of them also target H3K36me3 56,57 .

3 H3K36me

Drosophila H3K36me is associated with active transcription and decorates along the gene body 58 , with H3K36me2 peaks located in proximity to the promoter and higher level of H3K36me3 concentrated at 3' end of the gene. Both methylation marks are enriched on the histone variant H3.3 [START_REF] Bonn | Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development[END_REF]59 .

Ash1, dSet2 and dMes4 are the primary enzymes for H3K36 methylations in Drosophila 59- 61 . dSet2 interacts with elongating RNA polymerase II (RNAPII) 60 and deposits H3K36me3 co-transcriptionally 58 . RNAi against dSet2 in cultured cells and larva suggested that dSet2 primarily acted on premethylated histone to catalyze H3K36me3. It is believed that methylation of H3K36 by dMes4 provides a substrate for dSet2 58,62 .

As a TrxG protein, Ash1 was once considered to deposit H3K4me3 in counteracting PcG silencing 63,64 . However, in vitro HMT assay with the use of histone lysine scanning mutations and a larger purified SET domain clarified Ash1 catalytic activity as an H3K36specific HMT 61 .

Lessons from yeast show that H3K36me inhibits ectopic transcription initiation from cryptic promoters within the gene body via various mechanisms. H3K36me recruits Rpd3S

HDAC via the chromo-domain of Eaf3 to deacetylate H3 and H4 along the 3' end of the transcribing ORF to avoid cryptic transcription initiation the gene body 65,66 . H3K36me also blocks the association of Asf1, to avoid exchange of preacetylated histones into the 3' end of the transcribing gene body 67 . Besides, H3K36me recruits chromatin remodelers Isw1b, which acts together with Chd1 to prevent trans-histone exchange and inhibits incorporation of acetylated histones into the gene body 68,69 .

H3K36me3 might also have a repressive function. In mammalian cells, the PWWP domain of DNMTA3 is recruited to H3K36me3 for DNA methylation 70 . Besides, Tudor domain of mammalian Pcl homologs specifically recognizes H3K36me3 and is implicated in chromatin targeting of PRC2 repressive complex [71][72][73][74] .

H3K36me is a substrate of the demethylases Kdm2A/B [75][76][77] and Kdm4A/B 53,54,56 .

Intriguingly both Drosophila 77 and mammalian Kdm2B is a component of a non-canonical PRC1 complex. Mammalian Kdm2 harbors a CXXC domain, which binds unmethylated CpG islands and is implicated in targeting PRC1/2 complex 78-81 (See Chapter I, p.37). In addition, H3K36me3 was shown to inhibit PRC2 activity, as discussed in Chapter I, p.70.

4 H3K79me

Drosophila Grappa is the yeast Dot1 homolog that is responsible for H3K79 methylations 82 . Grappa was recovered in a genetic screen as a suppressor of PcG silencing.

A mini-white transgene is normally repressed under the control of the PRE-Mcp element.

However, derepression of the white gene was observed in gpp 1A mutant background. Also, some of the heteroallelic combinations of grappa mutants showed arista-to-leg transformation. These phenotypes placed Grappa as a PcG gene 82 . However, some transheterozygote of grappa mutations also displayed TrxG phenotype, such as decoloration of posterior male tergites, an indication of abdominal transformation into anterior identity 82 . H3K79me level was developmentally regulated. It is undetectable in stage 4 Drosophila embryos by immunostaining and gradually accumulates in the later stage of embryogenesis 82 .

Genomic distribution of H3K79me3 correlates well with active transcription 83 . In line with its genomic profile, mammalian DOT1L homolog was isolated in a complex with P-TEFb 84 . A recent ChIP-seq study using FACS-sorted Drosophila mesodermal cells suggested that H3K79me3 cover the gene body of active genes as a broad peak. In addition, enrichment of H3K79me3, along with high level of H3K27Ac and Pol II occupancy appeared to be the signature of active enhancers in Drosophila [START_REF] Bonn | Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development[END_REF] .

In yeast, H2B mono-ubiquitination is required for trimethylation of H3K79 85 . This mechanism seems to be conserved in Drosophila. In homozygous mutant embryo of dBre1, the Drosophila E3 ligase for H2B, H3K79me3 was reduced while mono-and dimethylation at H3K79 were not affected 86 .

Studies from yeast and mammals suggest that H379me is cell cycle regulated 87 . Finally, the identity of H3K79-specific demethylase remains unknown.

5 H3K27me

Catalysis

Four independent research effort identified PRC2 as the H3K27-specific HMT [88][89][90][91] . In

Drosophila

Substrate preference of PRC2 -H3.2 vs. H3.3

A recent study in plants revealed enzymes (ATXR5, ATXR6) dedicated to monomethylation of H3K27

H3K27me is essential for gene repression.

Since mutations of all PRC2-specific components lead to Hox gene derepressions and homeotic transformations [94][95][96][97]103 , it is suspected that H3K27me3 is the key substrate for PRC2 to elicit gene repression. This idea cannot be vigorously tested until the introduction of Drosophila histone replacement system 104 (See Chapter II, p.94) in which transgenes carrying various histone mutants are introduced at ectopic sites to replace the endogenous source of histones. As expected, Drosophila H3K27R mutant phenocopied E(z) mutant and shared the same Hox derepression profiles. This study consolidated the role of H3K27me3 as a key repressive mark 100 . It is believed that H3K27me3 recruit PRC1 to assemble higher order chromatin and inhibit transcription [88][89][90][91]105,106 . Recent advancement of chemical mimics on histone methylations 107 and full reconstitution of chromatintemplated in vitro transcription assay could further specify the mechanistic role of H3K27me3 in gene repression.

H3K27me has been implicated in other functions. The H3K27me1-specific reduction in plant atxr5/6 double mutant is associated with chromatin decondensation, derepression of heterochromatic transposable elements 98 and rereplication of heterochromatin 108 .

H3K27me3 readers

The chromodomain of Pc specifically recognizes H3K27me3 90,91,105,106 and mutation on such chromodomain reduced polycomb association on polytene 109,110 and lead to a lost of polycomb accumulation as nuclear bodies 111 . Consistent with its role of reading repressive histone methylations, these mutants displayed homeotic transformation 111 . Based on Pc recognition of PRC2-deposited H3K27me3, the field has been dominated by the dogma of one direction PRC2-PRC1 chromatin recruitment model, which is recently challenged.

Using reconstituted oligonucleosomes decorated with PRC1 cognate histone mark H2AK119ub as bait, the entire PRC2 complex can be affinity purified. This suggests the opposite recruitment hierarchy 112 .

The beta-propeller domain of ESC can also recognize H3K27me3, and such interaction robustly stimulates PRC2 HMT activity. This constitutes an enzymatic positive-feedback loop that is believed to be important to preserve PcG repressive chromatin during DNA replication 113 (See Chapter I, p.75).

Genomic distribution profile of H3K27me

Array-based and deep-sequencing ChIP experiments revealed H3K27me3 covered the Drosophila genome as broad domains encompassing PRE, transcriptional units and regulatory regions of repressed genes [START_REF] Schuettengruber | Cooperativity, specificity, and evolutionary stability of polycomb targeting in Drosophila[END_REF][START_REF] Schuettengruber | Functional anatomy of polycomb and trithorax chromatin landscapes in Drosophila embryos[END_REF][114][115][116] . In contrast, genomic profiles of PRC1 and PRC2 components appeared as sharp peaks at PREs within H3K27me3 domains. Pc and H3K27me3 occupy over 200 domains across the Drosophila genome, their distribution negatively correlates well with gene activity.

Extensive epigenome studies in Drosophila suggest that repressive H3K27me3 is almost mutually exclusive from active mark such as H3K4me3 [START_REF] Schuettengruber | Cooperativity, specificity, and evolutionary stability of polycomb targeting in Drosophila[END_REF][START_REF] Schuettengruber | Functional anatomy of polycomb and trithorax chromatin landscapes in Drosophila embryos[END_REF][114][115][116]

H3K27me demethylases

Guided with sequence homology with the JmjC domain 75 , the proteins UTX, JMJD3 and UTY are suspected to be the H3K27 demethylases. Based on in-vitro demethylation assay, UTX and JMJD3 were shown to possess H3K27me3 and H3K27me2 demethylation activity [124][125][126][127][128] while UTY was proved to be inactive 126,127 Depending on protein purification method and the type of histone substrates, UTX is show in some case to demethylase H3K27me1 on peptide or calf bulk histones 126,127 .

ChIP profile reveals UTX enrichment in human HOX loci as sharp peaks downstream of TSS of both active and silent HOX genes. Consistent with its role as a demethylase, UTX binding profile matches a sharp drop of H3K27me3 level, even within the broad coverage of H3K27me3 domain 126 .

Overexpression of UTX led to the drop of both H3K27me2 and H3K27me3 levels [125][126][127] . In ES cells, RA-induced differentiation is accompanied with activation of some Hox genes.

The promoter regions of these genes recruit UTX and lose H3K27me3 and E(z)

occupancy, and at the same time H3K4me3 level accumulates. However, upon RNAi against UTX, H3K27me3 and H2AK119Ub levels increase at their promoter region, resulting in a drop of HOX gene expression 125,128 .

Notably, morpholinos knockdown of UTX in zebrafish embryo resulted in developmental defects in posterior somite regions where the responsible HOX genes expression levels are diminished 126 . Also, mutation in one of the worm JMJD3 homologues displayed gonad disorganization 125 . These highlight the important role of H3K27me demethylase in metazoan development.

dUtx

The Drosophila genome encodes a conserved UTX gene with the characteristic TRP and JmjC domain. Purified dUtx from insect cells only demethylates H3K27me3 and H3K27me2 but not H3K27me1 and other repressive or active histone marks. Consistent with studies in mammalian models, such in-vitro demethylase activity requires intact JmjC domain 129 . However, there are discrepancies between publications concerning the characterization of dUtx in Drosophila.

Discrepancy on the effect of dUtx on H3K27me3 level: Loss of function study RNAi against dUtx in S2 cells [START_REF] Gutierrez | The role of the histone H2A ubiquitinase Sce in Polycomb repression[END_REF] or in flies 129 with the use of ubiquitous Act5C-Gal4

6 H3K27Ac

Drosophila CBP is responsible for H3K27Ac. Recombinant CBP acetylates both H3K27 and H3K18 in vitro. In agreement with the in-vitro data, RNAi against CBP in S2 cells leads to reduction of both H3K27Ac and H3K18Ac 134 .

HDAC activity is found to be associated with the immunoprecipitates of EED and Ezh2 135 , and Drosophila HDAC1 Rpd3 was co-purified with PRC2 components 89,92,136 . Subsequent experiment using RNAi against Rpd3 in S2 cells showed an increase in H3K27Ac. These

7 H2AK119Ub

In mammalian cells, H2A is ubiquitinated at K119 while Drosophila H2A is modified at K118. To avoid confusion, I omit the specification of the amino acid residue and use H2Aub for both systems.

In an effort to identify H2A-specific E3 ligase, an in vitro ubiquitination assay was set up, with the use of E1, E2, ATP, FLAG-Ubiquitin, nucleosomal substrates and HeLa cells nuclear extracts. Exhaustive chromatographic purification isolated potential H2A E3 ligases, which were found by mass-spectrometry to be Ring1 (herein Ring1a) and Ring2

(herein Ring1b), the homolog of Drosophila dRing/SCE 142 . Subsequent IP-MS approach using anti-Ring1a antibody identified Ring1a, Ring1b, Bmi1 (homolog of Drosophila PSC) and HPH2 (homolog of Drosophila Ph). Notably PC is not isolated in such complex. The role of dRing as the main H2A E3 ligase is also supported by in vivo studies. RNAi against dRing in Drosophila larva abolished global level of H2AUb 132 . Importantly, dRing is found to associated in an alternative complex known as dRAF (dRING-associated factors), which is composed of three core components: dRing, Psc and dKDM2, a H3K36me2 demethylase 77 . Systematic biochemical reconstitution of PRC1 and dRAF demonstrated that dRing alone showed limited ubiquitination activity, which was moderately enhanced by complexed with Psc, probably via heterodimerization of their RING domains 144,145 , and maximal E3 ligase activity is achieved when dKDM2 is added to the PSC-dRing core. Importantly, adding Pc/Ph to the Psc-dRing complex did not show such stimulatory effect 77 . This strongly argues that dRAF, but not PRC1, as the relevant complex catalyzing H2Aub. Consistent with this idea, RNAi against individual dRAF component, but not Pc or PH, abolished H2A ubiquitination 77 . More recently, it is also demonstrated that ectopic tethering of mammalian PRC2 complex at a defined genomic locus results in establishment of H3K27me3 domain and recruitment of canonical PRC1 complex without catalyzing H2A ubiquitination. This argues against the role of canonical PRC1 in H2Aub deposition 81 .

Lost of dRing derepresses key PcG targets such as Hox genes in Drosophila embryo and larval wing discs [START_REF] Gutierrez | The role of the histone H2A ubiquitinase Sce in Polycomb repression[END_REF]146 , and mutation in dKdm2 enhances transformation phenotypes of Pc mutant while alleviates that of trx mutant 77 . This supports the idea that H2Aub as an important repressive histone mark in the PcG system. However, the exact necessity of

H2Aub is yet to be testified by the corresponding histone mutant.

Genomic distribution of H2Aub

In mammalian models, the Bmi1-Ring1B (analogous to Psc-dRing in Drosophila)

represents the basal catalytic core for H2A ubiquitination 144,145

Effect of H2Aub on chromatin structure

It is postulated that the ubiquitin moiety attached on H2AK119 is positioned in close proximity to the linker histone H1 and hence it might impact chromatin folding 153 .

Reconstitution of homogenous 12-mer nucleosomal array using purified H2Aub displayed normal nucleosome assembly and compaction properties as those reconstituted with unmodified H2A. However, H2Aub nucleosomal array requires a lower salt concentration for magnesium-induced oligomerization than the unmodified counterparts. This property indicates that H2Aub chromatin might be more prone for establishing chromosomal contacts 154 . unaffected in dRing [START_REF] Gutierrez | The role of the histone H2A ubiquitinase Sce in Polycomb repression[END_REF] and PR-DUB mutants 156 . More sensitive assay such as ChIP is needed to better assess the locus-specific impact of H2AUb in regulating H3K27me3 level.

H2Aub-mediated establishment of H3K27me3 domain

8 H3T3ph & H2AT120ph

Histone H2A can be phosphorylated at T119 and T120 respectively in Drosophila and mammals. To avoid confusion, they are all referred to H2Aph unless otherwise specified.

Aurora B is a kinase essential for mitotic progression. Intriguingly, both juxtaposed lysine residues of H3T3 and H2AT120 can be modified by important epigenetic regulators as active mark H3K4me3 and repressive H2AK119ub respectively. The potential implication of these "double mark" would be discussed in Chapter III, p.137.

9 H3S10ph

Mitotic progression The Heterochromatin Protein 1 (HP1) chromodomain specifically recognize H3K9me3 of constitutive heterochromatin including those of pericentromeric regions 41,42 . During mitosis, Aurora B kinase phosphorylates H3S10 and such H3K9me3S10ph dual modification serve as a phosphor-methyl switch that evicts pre-bound HP1 proteins 177,178 .

It is thought that the clearance of HP1 proteins might facilitate mitotic chromosomes condensation and segregation. However, this prevailing idea is not rigorously tested as discussed in detail in Chapter III, p.137.

Anti-silencing

JIL-l (the closest Drosophila homolog of mammalian MSK1/2 kinase) is the interphase kinase for H3S10ph 179 . Genetic evidence identified JIL-1 mutant as an enhancer of PEV 180 .

And lost of JIL-1, and hence depletion of interphase H3S10ph, leads to genomewide enrichment of H3K9me2. Based on these evidences, it is proposed that JIL-1 mediated

H3S10ph is important to counteract H3K9me2-associated repression. This seems to be supported by genomewide study.

To avoid contamination by the huge level of H3S10ph from mitotic cells, ChIP-seq was performed with post-mitotic salivary gland from L3 larvae to profile the distribution pattern of interphase H3S10ph. It was shown that H3S10ph is enriched on active genes as a broad peak slightly downstream of TSS. And lost of H3S10ph in JIL-1 mutant relocated H3K9me2, the genes that gain H3K9me2 showed a significant decrease in expression level 181 .

Since JIL-1 associates with the Male Specific Lethal (MSL) dosage compensation complex and it is two-fold enriched on the active genes of male chromosomes 182 , it has been proposed to be important for dosage compensation 183 . Similarly high level of H3S10ph is particularly enriched along the male X chromosomes, it might be important to counteract H3K9me2 silencing and permit higher transcriptional output from the X-linked genes 181,182 .

As discussed above, H3S10ph is linked to chromosome condensation. However, studies in interphase nuclei suggest the otherwise. Ectopic induction of H3S10ph at a defined locus by artificial tethering JIL-1 kinase leads to local decondensation of Drosophila polytene chromatin 184 .

Gene activation

Studies in mammalian model support a role of H3S10ph in gene activation. It is found that the serine/threonine kinase PIM1 form a complex with the transcription factor MYC and is targeted to a subset of MYC target genes to deposit H3S10ph around TSS 185 . H3S10ph in turn recruits a HAT complex via14-3-3, resulting in H4K16ac accumulation. Together with predeposited H3K9ac, the double mark H3K9acS10ph/H4K16ac creates a binding platform for the double bromodomain containing protein BRD4, through which p-TEFb is recruited to promote transcription elongation 186 .

10 H3S28ph

Cell cycle regulation & implication in mitotic progression.

Unlike H3S10ph, H3S28ph probably does not exist in primitive eukaryotes like yeasts 170 .

Interestingly, despite high degree of conservation of histone H3 sequence, the fission yeast H3 is embedded with an alanine at position 28, instead of a serine as in budding yeast and metazoans. Hence, at least in fission yeast histone H3, it does not support H3S28 phosphorylation.

In metazoan, H3S28ph mirrors the cell cycle pattern of H3S10ph which peaks at mitosis

Role in stimuli-responsive counter-silencing / gene activation

In response to stress, mitogen or differentiation stimuli, the MSK1 and MSK2 kinases are activated to phosphorylate nucleosomal H3 and promote transcription of immediate-early genes such as c-JUN and c-FOS 189,190

11 H3T80ph

Four independent mass-spectrometry studies discovered the phosphorylation of H3T80 located at the globular domain of histone H3 26,[192][193][194]

1 PRC1

Drosophila polycomb repressive complex 1 (PRC1) comprises four core components: Pc, Ph, Psc, and dRing 198,199 . In the first attempt of affinity purification, FLAG-tagged Ph and Recent biochemical purification suggests that Psc-dRing assemble as a core platform on which it can partner with components other than Pc &Ph, as discussed in X (dRAF & Mammalian RYBP-KDM2 PRC1).

1. 1 Pc (Polycomb)

The polycomb mutant was first identified by Pamela Lewis in 1947. As its name implies, such mutant is characterized by the growth of additional comb teeth of the male Drosophila sex comb 203,204 . Besides, Pc mutants displayed classical PcG phenotypes such as cuticular transformation of late embryonic thoracic and abdominal segments into the eighth abdominal segment identity 204,205 . Mutant clones of Pc in wing disc also derepressed Hox genes such as Ubx and Abd-B 205 .

Pc, together with another PcG protein polyhomeotic (Ph), accumulates within distinct subnuclear punctate structures known as the polycomb bodies. It is believed that these Pc foci represent the hub of PcG repressions 105,[206][207][208][209] . Interestingly, repressed PcG target such as the Hox genes has a higher frequency of colocalizing within the same Pc body than when they are expressed 210 . Recent effort implemented with genomewide RNAi screen identifies novel factors that regulate Pc foci morphology; among these factors are components of the SUMOylation machinery and cohesion ring complex 211 . Pc has a characteristic chromodomain at the N-terminus that recognizes specifically H3K27me3 90,91,106 . Mutation in chromodomain, such as the I31F mutation in Pc 106 allele abolishes Pc binding to polytene chromosomes and disrupts formation of punctate nuclear Pc bodies 109,111 . Also swapping the chromodomain of Pc with that of heterochromatin protein 1 (HP1) resulted in relocalization of such Pc-chimera to H3K9me2-enriched, DAPI-dense heterochromatin region of Drosophila S2 cells 105 .

Recognition of H3K27me3 peptide by Pc chromodomain is achieved by the assembly of aromatic cage containing residues, including Y26, W47 and W50, into a complementary fitting grove where the extended methyl-lysine of H3K27 resides 105,106 .

Base on this chromodomain recognition module, it has been postulated for a hierarchical recruitment model in which depositions of H3K27me3 mark on PcG targets help Pc binding and hence PRC1 targeting [START_REF] Schuettengruber | Genome regulation by polycomb and trithorax proteins[END_REF][START_REF] Simon | Occupying chromatin: Polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put[END_REF][212][213][214] . This one-way recruitment model has been recently challenged, as discussed in Chapters I, p.20.

1. 2 Ph (polyhomeotic)

The Drosophila Ph protein is encoded by a tandem-duplication of genes know as ph-d (distal) and ph-p (proximal) 215 . Lost of Ph (maternal and zygotic null) lead to drastic abnormalities that is distinctive from other PcG mutants. Notably these null mutants failed to complete gastrulation and displayed epidermal developmental defects. Weak hypomorphic alleles developed into later stage of embryogenesis and showed segmental transformation phenotypes similar to other PcG mutants [215][216][217] .

Besides, accumulating evidence supports an important role of Ph as a tumor suppressor.

Induction of homozygous Ph mutant clones in imaginal disc led to invasive tissue overgrowth phenotypes associated with ectopic Notch and JAK/STAT signaling pathway activities, in addition to Hox genes derepressions 214,[218][219][220][221] .

Intriguingly, the C-terminus of Ph harbors a sterile alpha motif (SAM) domain that can polymerize in vitro via a head-to-tail configuration as a left-handed helical structure 222 .

Functional relevance of such helical structure is unknown, largely because of the lack of in vivo demonstration of the polymer structure. It is postulated that such fibrous and extended structure might help spreading polycomb domain from a chromatin nucleation site.

However, this idea was not favored by the sharp ChIP-on-ChIP peaks profile of Ph [START_REF] Schuettengruber | Cooperativity, specificity, and evolutionary stability of polycomb targeting in Drosophila[END_REF][START_REF] Schuettengruber | Functional anatomy of polycomb and trithorax chromatin landscapes in Drosophila embryos[END_REF]223 .

It still remains possible that such polymer structure might be important for the higher order structure of PcG repressive domains 224 . The SAM domain polymerization properties is conserve in mammalian homolog Phc2. Introducing a presumably dominant negative SAM domain mutant of Phc2 disrupted nuclear clustering PRC1 bodies without compromising PRC1 complex integrity, it also lead to a reduced H3K27me3 deposition and poor binding of Ring1B to PcG targets 224,225 . Mice engineered with such mutation even showed skeletal transformation phenotypes 225 . In Drosophila, tethering WT-Ph, but not the corresponding SAM domain mutant, is capable to repress transcription of a reporter gene. Also, overexpression of SAM mutant, but not WT Ph, leads to Abd-B derepressions in wing disc 226 . These data suggest the importance of Ph SAM domain in gene repressions.

1. 3 Psc (Posterior Sexcomb)

Genetics studies suggested that Psc is related to an adjacent gene Su(z)2. Despite their sequence homology is limited to the C-terminal stretch of 200 amino acids 227 , Su(z)2 is considered as a functional homolog of Psc 228 . In one example, Hox derepression phenotypes in mutant clones deleted for both Psc and Su(z)2 can be rescued by reintroducing either Psc or Su(z)2, suggesting a functional redundancy between the two proteins 205 . Also they share similar biochemical properties on counteracting SWI/SNFmediated chromatin remodeling and on compaction of nucleosomal array. Besides, Su(z)2 can substitute Psc on PRC1 assembly 228 .

Both Psc and dRing posses RING finger domains that are characteristic of the E3 ubiquitin ligase family 229 . When Psc complexes with dRing, the Ring-Ring-heterodimer configuration represents the catalytic core to catalyze H2A ubiquitination 144,145 . It is important to stress that it is the RING finger of dRing that confer E3 ligase activity in this context. However, the RING finger motif of Psc also confers active E3 ligase activity.

Notably, Psc can form an alternative complex with the anaphase-promoting complex (APC) component APC11/LMG to catalyze Cyclin B ubiquitination during mitotic exit 230 .

Mosaic clones of combined Psc-Su(z)2 mutants display tumor-like phenotype with massive invasion into neighboring tissue, also Ubx is strongly derepressed in the wing disc, even in the hinge and notum regions 205,218 . Also genetic evidence suggests that, similar to Ph, Psc can function independent of dRing or Pc to repress some of the PRC1 target genes such as dac [START_REF] Gutierrez | The role of the histone H2A ubiquitinase Sce in Polycomb repression[END_REF] . This might be attributed to the chromatin compaction activity of Psc, which does not required other PRC1 components 202 .

1. 4 dRing / Sce (Sexcomb extra)

dRing (also known as Sce) is an E3-ligase catalytic subunit of dRAF (see p.37) which confers H2A ubiquitination activity [START_REF] Gutierrez | The role of the histone H2A ubiquitinase Sce in Polycomb repression[END_REF]77,142 . It is also a component of canonical PRC1 complex 200 . Mutation of dRing such as Sce 1 , which encodes a C-terminus truncated protein, displayed homeotic phenotypes. Maternal and zygotic Sce 1 mutant derived from germline clones gives raise to defective embryogenesis typical of PcG mutants, with derepressions of canonical PcG target genes, such as Ubx from the Hox cluster. These mutant embryos were characterized by cuticular transformation of thoracic and abdominal segments into the eighth abdominal segment identity [START_REF] Gutierrez | The role of the histone H2A ubiquitinase Sce in Polycomb repression[END_REF]146 . Mutant clones of Sce 1 induced in wing imaginal disc strongly derepressed Ubx and Abd-B 146 . Despite these typical PcG phenotypes, several observations set dRing apart from other PRC1 members. For the noncanonical PcG target genes such as dachshund (dac), whose promoter is enriched with all PRC1 components including Pho, it is only derepressed in Psc-Su(z)2 or Ph mutants both at embryonic and larval stages. In contrary, lost of dRing does not affect the expression pattern of dac [START_REF] Gutierrez | The role of the histone H2A ubiquitinase Sce in Polycomb repression[END_REF] . Besides, in the cell-lethal mosaic analysis system where mutant clones were highly enriched in an environment lacking cell-cell competition, all mutant of PRC1 components except dRing, displayed tumorigenic over proliferation phenotype in the eye discs [START_REF] Gutierrez | The role of the histone H2A ubiquitinase Sce in Polycomb repression[END_REF]218,219 .

It remains unclear on the essential role of H2Aub in PcG repression. It is surprised to know that mutation in dRing (I48A), which supposedly hampers its interaction with E2 ubiquitin-conjugating enzyme and hence reduce its E3 ligase activity 144,145 , can actually support Hox repressions. However, such E3 ligase-dead mutant is lethal. Limited by the availability of a reliable antibody that specifically recognizes H2AK118Ub in Drosophila, the E3 ligase dead nature of dRing I48A mutation is yet to be verified 147 .

1. 5 Scm (Sexcomb on midleg)

Scm is considered as a sub-stoichiometric component of PRC1 200 . Similar to other PcG components, late embryos derived from maternal and zygotic null Scm mutant displayed cuticular transformation where the thoracic and abdominal segments acquired features characteristic of the A8 abdominal segment 231 .

Scm protein consists of two MBT repeats that is implicated in methyl-lysine recognition 232,233 . Isothermal titration calorimetry (ITC) assay showed that SCM MBT repeats had a modest binding preference on monomethylated lysine on H3K4, H3K9, H3K27, H3K36 and H4K20, when compared to their unmodified or higher methylations states. Structural study confirmed a more stable MBT-monomethyl-lysine binding configuration when compared to their higher order of methylation derivatives 233 .

Hypomorphs carrying missense mutations at the MBT1 domain, such as Scm KM23 , gave mild derepression phenotype of Hox genes in embryo. Notably these MBT1 mutants showed stronger genetic interaction with other PcG mutants than Scm-null alleles 232 . As discussed in Chapter I, p.38, Scm was suggested to play a role in PcG recruitment.

Reintroduction of WT Scm rescued Hox derepression in

2 dRAF (dRing Associated Factors)

Affinity capture of dRing interacting partners using Drosophila nuclear extracts immunodepleted of Pc identifies PRC1 subcomplex known as dRAF. Core members of dRAF include Psc, dRing and dKdm2, which harbors H3K36 demethylase activity.

Structural studies suggest that the RING fingers from Psc and dRing heterodimerize to confer E3 ligase activity on nucleosomal H2A. Notably, biochemical reconstitution experiment showed that dKdm2, when added to the Psc-dRing complex, strongly stimulated H2A ubiquitination while adding equimolar of Pc-Ph gave limited stimulatory effect on ubiquitination 77 . Consistent with this observation, individual knockdown of dKdm2, Psc and dRing, but not that of Pc and Ph, abolished H2A ubiquitination in S2 cells. These data argue that dRAF, but not canonical PRC1, as the major H2A E3 ligase in Drosophila 77 . dRAF-like complex is conserved in mammals. Mammalian Kdm2B complexes with PCGF1 and Ring1B, along with some other factors, as a non-canonical PRC1 complex 236 . Interestingly, Kdm2B harbors a CXXC domain that specifically recognizes non-methylated CpG islands and artificial tethering of Kdm2B to a defined genomic locus can nucleate de novo H2A ubiquitination and subsequent recruitment of PRC2 and establishment of H3K27me3 domain 81 . This suggests a role of H2Aub in PRC2 recruitment. In support of this idea, both flies and mammalian PRC2 components can be affinity captured on H2Aub-containing oligo-nucleosomal array. And together with the auxiliary factors Jarid2 and AEBP2, PRC2 exhibits a stronger HMT activity on H2Aub nucleosomal substrate than unmodified ones 112 . These observations call for a positive feedback model in which Kdm2B-PRC1 catalyzed H2Aub stimulates PRC2 deposition of H3K27me3, which in turn recruits canonical Pc/CBX containing PRC1 complex.

However, to what extent such positive feedback loop is required for PcG silencing is not well defined. In Drosophila, lost of dRing abolish H2Aub, but it seemed not to alter global H3K27me3 level [START_REF] Gutierrez | The role of the histone H2A ubiquitinase Sce in Polycomb repression[END_REF] . And reintroducing E3-ligase dead mutant of dRing (I48A) can rescue Hox derepression phenotypes of dRing-null mutant 147 . Also, knockdown of dKdm2 in S2 cells 77 and larva homozygous dKdm2 mutants retained normal level of H3K27me3 level 237 . It is thus hypothesized that H2Aub might be specifically required during early embryogenesis when H3K27me3 domains are first established. Embryos derived from germline clones with E3 ligase-dead dRing mutant or establishing histone H2A mutant devoided of ubiquitination site would address this question. Also, genomic profile of H2Aub in Drosophila embryos and larval tissues would help to better assess the relevant role of this histone modification in PcG silencing.

3 Pho-RC (Pho Repressive Complex)

Pho and Phol encode similar proteins and genetic evidence suggests that they function redundantly in PcG repression 238 . Importantly they both have of 4 zinc-finger motifs with overall 80% sequence identity and all the important amino acid residues predicted to contact DNA are fully conserved 238 . This is in good agreement with Gel-shift assay where Phol zinc-finger specifically retards the migration of DNA fragment containing Phobinding site 238 . Homozygous mutant of Pho showed subtle Ubx derepression on the wing pouch whereas Ubx remained repressed in homozygous Phol mutant background.

However, combined Pho and Phol double homozygous mutant synergized Ubx derepression in the wing discs 238 . Pho is the only PcG protein with well-defined DNA binding sequence that is embedded in most PREs [START_REF] Schuettengruber | Functional anatomy of polycomb and trithorax chromatin landscapes in Drosophila embryos[END_REF]239,240 . Since Pho-binding sites in PRE are essential for PcG repressions [241][242][243][244] and given that Pho also physically interacts with PRC1 245 and PRC2 246 components, and lost of Pho abolishes binding of Pc and E(z) binding to bxd 246 , it is thus attributed to function as a recruiter to guide binding of PcG components to their target genes 247 . However, polytene staining of Pho and Phol double mutant revealed substantial retention of PRC1 and PRC2 components on chromatin and only on a few cytological sites where PcG binding is affected 238 . It is possible that other factors such as SCM 247,248 , buffers the lost of Pho/Phol in PcG recruitment. Notably, Ph remained associated to those PcG target sites even when the binding of other PRC1 and PRC2 components are affected in the double mutant background 238 .

Proteomics approach using transgenic Pho-TAP as affinity bait identified Scm-related gene containing four mbt domains (dSfmbt) and dINO80 complex. Owning to its important function in PcG repression, Pho-dSfmbt is referred as Pho-repressive complex (Pho-RC).

Notably, Pho-RC can be reconstituted as a stable complex that can resist ionic strength in 2M KCl containing buffer 249 . dSfmbt is structurally related to Scm protein, since it bears four MBT domains and a C-terminus SAM domain. The 4MBT domain displayed a low affinity binding preference to mono or di-methylated histone at H3K9 or H4K20 249 .

Structural data shows that a specialized Pho spacer region binds stably with the 4MBT domains of dSfmbt, and MBT deletion allele or missense mutants of dSfmbt that disrupt such interaction compromise PcG repression 250 . Consistent with the observation that dSfmbt and Scm could be reconstituted as a complex in vitro 251 and their co-occupancy on a candidate collection of PREs 249,251 , it was found that combined mutations on both Scm and dSfmbt synergized derepressions of PcG targets than individual mutant alone 251 .

Notably, dSfmbt binding to PRE required intact Pho-binding site. Using the PRE bxd as a study model, it was shown that Pho and Scm binding to PRE in S2 cells was independent of each other, but RNAi against either of them abolished binding of PRC1 and PRC2 components 248 . Together with the genetic interaction between Scm and dSfmbt mentioned above, it is thus hypothesized that Pho-RC and Scm act in parallel for PcG recruitment 247 .

These observations call for the quest of genomewide distribution profile of Scm to better access its role in PcG recruitment.

4 PRC2

Ever since the first discovery of histone methylation catalyzed by the SET-domain containing Su(var)3-9 protein 40 , it paved a way to look for novel histone methyltransferase (HMT) activity on known chromatin regulators. Not surprisingly, E(z) as a SET domain containing protein, is suspected to catalyze histone methylation. Indeed affinity purification of FLAG-Esc in Drosophila embryo extracts 88 and FLAG-EED in human embryonic kidney 293 cell nuclear extracts 91 , together with extensive chromatographic purification of HMT activity containing fractions from Drosophila embryo extracts 89 and HeLa cell nuclear pellet 90 unequivocally identified PRC2 and established its core components as ESC, E(z), Su(z)12 and Nurf55. The Zn-finger containing protein AEBP2 90 and HDAC Rpd3 89,136 , was also occasionally copurified with PRC2 components. Studies with immunoprecipitate of PRC2 ( 91), native chromatographic fraction of PRC2 90 and reconstitution of the 4-component PRC2 88 demonstrated its specific HMT activity on H3K27. And guided by the model of HP1 recruitment to Su(var)3-9 catalyzed H3K9me3 41,42 , it was found that the chromodomain of Pc of the PRC1 component specifically recognized H3K27me3 deposited by PRC2 90,91,105,106 , hence the field established a one-way model of PRC2-PRC1 recruitment hierarchy, which is recently subjected to revision 81,252 .

4. 1 Esc (Extra Sexcombs) & Escl (Esc-like)

Embryos derived from parents that were transheterozygous for esc-null alleles (esc 2 /esc 10 ) lacked H3K27me3 94 , they also showed strong transformation phenotype typical of other PcG mutants. Notably, cuticle of these late embryos displayed 12 dendritic belts characteristic of the A8 abdominal segment 103 . Similar to the Psc-Su(z)2 locus, an Esc-like (Escl) isoform with 60% sequence identity to Esc exits and resides ~150 kb away from the esc gene. Similar to Esc 88,89 , Escl can assemble with E(z) Su(z)12 and Nurf55 to form PRC2, with the same HMT activity towards H3K27, despite at a reduced efficiency 253 .

Escl also bind to PRE such as bxd, and combined mutations of Esc and Escl resulted in phenotypic enhancement with more severe transformation phenotypes [253][254][255] . In Drosophila S2 and Kc cells, only simultaneous, but not individual, knockdown of Esc and Escl would deplete H3K27me3 level 254,255 . It is thus believed that Esc and Escl are functionally redundant. In fact, Esc expression level peaks at embryogenesis and then dramatically decreased in later stage of development. Escl show somewhat a reciprocal expression pattern, in which peak expression is achieved at post-embryonic stage and Escl level persist in adulthood 253,254 . Hence Escl may substitute the function of Esc in later stages of development.

Early study showed that Esc-E(z) interacts with each other 256 . And based on this observation, subsequent FLAG-Esc based affinity purification identified PRC2 components and its HMT activity 88,91 . Esc WD40 forms a beta-propeller structure, and study from the human homolog EED suggests that the bottom of the beta-propeller constitutes a peptide-binding groove for interaction with Ezh2. Owning to good conservation of critical residues at the binding interface, it is predicted that Drosophila Esc-E(z) adopts a similar interaction mechanism 257 . More importantly, it is found that EED beta-propeller domain can recognize H3K27me3 and stimulate PRC2 HMT activity. Hence it is proposed that EED-H3K27me3 interaction constitutes a positive feedback loop to enhance the spreading of broad H3K27me3 repression domain, and might serve as an important mechanism to maintain PcG repression during DNA replication 113 . Interestingly, transgenic Esc carrying mutations that abolish H3K27me3 recognition failed to recue the lost of H3K27me3 and lethality associated with esc escl double null mutant, demonstrating the in vivo functional relevance of Esc-H3K27me3 interaction/stimulatory loop 113 .

4. 2 E(z) (Enhancer of zeste)

E(z) was classified as a PcG gene based on the lost of function studies of its temperaturesensitive alleles, which derepressed Ubx during embryogenesis and showed cuticular transformation of late embryonic segment acquiring A8 abdominal identity 258,259 . These E(z) ts alleles also show adult transformation phenotype when they are raised in semipermissive temperature. Notably, ectopic sex combs are found on the second and third legs and male anterior abdominal segments acquired patchy pigmentation reminiscent of the posterior counterparts 90,91 . Similar to other PcG genes, clones of E(z)-null mutants derepressed Hox genes in wing imaginal discs 100,205 .

Since E(z) is conserved with a SET domain which is also found in Su(var)3-9 and Trithorax protein 260 . Following the demonstration that Su(var)3-9 as a SET domaindependent histone methyltransferase 40 , subsequent purification of PRC2 established E(z)/Ezh2 as the catalytic subunit for specific catalysis of H3K27 methylation [88][89][90][91] . E(z)/Ezh2 alone does not possess HMT activity 40,90,91,94,261 . Crystallography study suggested that the post-SET domain of Ezh2 folds backward and block the substratebinding site, hence potentially exhibits an auto-inhibitory effect 262 . Also, cofactor-binding pocket for the methyl-donor SAM is not observed within the Ezh2 SET domain alone, In Drosophila, PRC2 is the sole HMT responsible for H3K27 methylations [94][95][96]205 . Since there are accumulating evidence for the existence of Ezh2 non-histone substrates 266,267 , it might call in to question on whether H3K27 is the key substrate to elicit PRC2-mediated gene repression. Study on Drosophila histone mutant provides a neat answer 100 . Mosaic analysis on Drosophila histone mutant carrying H3K27R mutation depleted H3K27me3 and phenocopied the loss of E(z), hence it strongly suggests that the function of E(z) or PRC2 as a whole is to catalyze H3K27me for gene repressions 100 .

In recent years, a number of reports showing oncogenic mutations of Ezh2 SET domain in a variety of cancers with elevated level of H3K27me3 [268][269][270][271][272][273][274] . WT PRC2 catalyzes efficient monomethylation at H3K27 and exhibit slow conversion of higher methylation states 268 .

The oncogenic mutation hotspot resides at residue Tyrosine 641 of Ezh2 SET domain, and reconstitution of PRC2 using Ezh2 with Y641 mutation showed elevated catalytic activity for converting H3K27me1 into H3K27me2 and H3K27me3. Hence heterozygous cancer patients carrying both WT and Y641 mutant alleles of Ezh2 exhibit marked increase in H3K27me3 level 268,270,271 . Recent structural data shows that Y641 is positioned to form hydrogen bond with the extended side chain of the substrate lysine (H3K27), mutation such as Y641F would alleviate conformational constraints imposed on the dimethylated state of H3K27 and hence allow better accessibility to the methyl-donor S-Adenosyl methionine (SAM) 262 . Besides, such mutation also allows provide additional space to accommodate trimethylated form of lysine. As such, Ezh2 Y641 mutants are more competent for H3K27 methylations. Similar mutation exists in Drosophila. The E(z) Trm A proposed model based on EM-reconstitution of human PRC2-AEBP2 complex docking on a di-nucleosome. In such configuration, the PRC2 complex is docking on the grove between two adjacent nucleosomes such that EED-H3 interaction from one nucleosome positions EZH2 SET domain in close proximity to the H3 tail from another nucleosome. Please refer to texts for details. Drosophila homologs of PRC2 components are highlighted with blue texts. Nurf55 is a common components shared by different chromatin modifying complexes such as CAF1, HAT1, NURF and NuRD 277,278 . In addition, it is also a core PRC2 subunit [89][90][91] .

Notably, Drosophila with reduced expression of Nurf55 displayed aristapedia transformation and mosaic clones of Nurf55 mutants in imaginal discs showed reduced H3K27me3 level, supporting its essential role in PcG repression 97 .

Nurf55 possess WD40 repeats that assemble into a 7-blade beta-propeller structure in which an acidic pocket binds the first 14 amino acid residues of H3 N-terminal tail. Such Nurf55-H3 binding module is compatible with the interaction with Su(z)12, this is consistent with the observation that Nurf55 and Su(z)12 are required for PRC2 to bind nucleosome 261 . Importantly, Nurf55-Su(z)12 binding to H3 tail is abolished when H3K4 is methylated 264 . And H3K4me3 containing nucleosome appeared to be a poor substrate for PRC2, despite PRC2-binding to such modified nucleosome was not affected. Biochemical analysis suggests a reduction of catalytic turnover or processivity when PRC2 acts on H3K4me3 containing nucleosomes 264 . Similar inhibitory effect is observed with H3K36me2 and H3K36me3 nucleosomes. These active mark inhibitions of PRC2 activities are conserved in fly, human and plants. Interestingly, a variant PRC2 in plants with a another Su(z)12 paralog VRN2 is insensitive to H3K4me3 and H3K36me2,3 inhibition. Hence it is suspected that Su(z)12, likely via its conserved VEFS domain, can sense active chromatin mark to modulate PRC2 activity.

Recently, it is shown that PRC2 favored chromatin substrates with higher nucleosomal density 265 . And addition of peptide containing H3 31-42 can mimic high nucleosomal density, and stimulate higher PRC2 activity on loosely packed oligonucleosomes.

Interestingly, deletion mapping and crosslinking chemistry showed that such stimulatory peptide directly interacts with a Su(z)12 fragment containing the conserved acidic residues within the VEFS domain. Strikingly, mutation of some of these acidic residues E548A/E550A/D552A abolished PRC2 responsiveness to nucleosome density without compromising complex integrity and stimulation by the EED-H3K27me3 axis 265 . Also, expression of WT Su(z)12, but not the corresponding allele with the same mutations, rescued H3K27me3 level in a Su(z)12-null ES cell background. It demonstrates the in vivo relevance of the nucleosome-density sensing function of PRC2 265 .

5 PRC2 auxiliary factors

Pcl (Polycomb-like)

Mutation in Pcl caused ectopic growth of sexcombs on both second and third legs, anterior abdominal tergites of these adult flies also gained patchy pigmentation resembling the posterior compartments 279 . Based on early observations of these homeotic phenotypes, Pcl was classified as a PcG gene.

Pcl has an atypical Tudor domain and two PHD fingers. Subsequent yeast-two-hybrid and in vitro pull down assays suggested direct interactions between Pcl PHD fingers and E(z) 280 . Indeed reciprocal tandem affinity purifications using Pcl-TAP and E(z)-TAP isolated Pcl-PRC2 complex from Drosophila embryo extracts 95 .

Interestingly, both Drosophila 95 and mammalian Pcl-PRC2 281,282 displayed a higher catalytic activity particularly for H3K27 trimethylation. In agreement with this enzymatic property, mosaic clone of Pcl mutant in wing discs showed marked reduction in H3K27me3 and Hox genes derepressions without affecting H3K27me1 95 . This is in contrary to clones of E(z) or Su(z)12 mutant, in which both methylation states were severely affected. Hence it is proposed that Pcl acts as a PRC2 stimulatory cofactor to specifically boost H3K27 trimethylation 95 .

ChIP assay on Hox PREs demonstrated specific reduction on H3K27me3 in Pcl mutant embryos, while both H3K27me2 and H3K27me1 levels were reciprocally increased.

Importantly, PRE-binding of PRC2 components such as Su(z)12 was also decreased in the lack of Pcl, while that of Pho and Ph were unaffected 95 Intriguingly, the potential function of Pcl in PRC2 recruitment is elaborated by several studies on mammalian Pcl homologs. All mammalian Pcl homologs including PHF1, PHF19 and MTF2 possess a conserved Tudor domain that specifically interacts with H3K36me3 71-74 . Hence it is proposed that PCL is specifically needed to recruit PRC2 at active genes for de-novo repression during differentiation transitions [71][72][73][74] . Since two critical aromatic residues (C361 and S387) within the Tudor domain of Drosophila Pcl are not conserved, it lacks an intact binding pocket and does not recognize methylated lysine or arginine 284 . However, it seems that when including the PHD1 domain, Drosophila Pcl Tudor domain can act together bind to H3K4me3 and H3K36me3 71 . Further biochemical studies would be needed to clarify this point.

5. 2 Jarid2 (Jumonji, AT Rich Interactive Domain 2)

Jarid2 belongs to the Jumonji histone demethylase family 75 . Since the amino acid critical for catalyzing demethylation is not conserved within Jarid2 Jumonji C domain, hence it is catalytically inactive for histone demethylation [285][286][287][288][289] .

Affinity purification of different epitope-tagged [286][287][288][289] or in vivo biotinylation of mammalian PRC2 components 285 robustly identified Jarid2 as a novel component of PRC2. The presence of PRC2-Jarid2 complex was confirmed by gel filtration where Jarid2 comigrated with PRC2 components [285][286][287] . Using individually purified recombinant proteins, it was found that Jarid2 can directly interacts with Ezh2 287 or Suz12 proteins 286 .

Consistent with the formation of a Jarid2-PRC2 complex, genomewide ChIP assay revealed a striking overlapping (>90%) of Jarid2 occupancy with PcG targets in ES cells [285][286][287][288][289][290] . Since the lost of Jarid2 reduced PRC2 binding to target genes and vice versa, such interdependency suggests a cooperative mode of binding between Jarid2 and PRC2 to their target sites. The PRC2-recruitment function of Jarid2 might be mediated by its DNA 287 and nucleosome 291 binding activity.

With the use of a GAL4-tethering luciferase reporter system, artificial targeting of Jarid2 or Ezh2 results in mutual recruitment and productive catalysis of H3K27me3 associating with silencing of the reporter gene 287,288 . This implies a positive role of Jarid2 in PRC2 activity.

Consistent with the reporter assay, it was shown that Jarid2 could stimulate PRC2 HMT activity 287 . Ironically, there were also studies demonstrating Jarid2 as an inhibitor on PRC2 catalysis 285,286 . The reasons for such discrepancy remained unknown, methods of PRC2 reconstitution, the form of histone H3 substrates and other assay conditions might contribute to different results 123,292 . Similar discrepancy was also reflected on the effect of Jarid2 on H3K27me3 level. Some studies observed reduction of H3K27me3 287-289 whereas others reported unchanged 286 or even increase 285 in H3K27me3 on PcG targets upon the lost of Jarid2 in ES cells.

Nonetheless, a subset of PcG target genes were not properly silenced during differentiation upon knock down of Jarid2 or in a heterozygous genetic background with one Jarid2 allele disrupted by gene trap insertion [286][287][288][289] . PRC2 activity is required for ES cells differentiation [293][294][295] . Similarly, the lost of Jarid2 impaired ES cells differentiation [287][288][289] , partly because of delayed silencing of PcG targets involved in pluripotency such as Oct4 and Nanog 287,288 .

In view of the emerging role of long non-coding RNA (lncRNA) in targeting PcG components [START_REF] Margueron | The Polycomb complex PRC2 and its mark in life[END_REF][296][297][298] , the role of Jarid2 in PRC2 recruitment is elaborated by the observation that Jarid2 and Ezh2 share common pool of direct IncRNA interacting partners 299 . In the case of lncRNA MEG3, which is involved in imprinting 300 and pluripotency 301 , Ezh2-MEG3 interaction depends on Jarid2 299 . Binding studies suggested that Jarid2-Ezh2 interaction is enhanced with the supply of lncRNA.

Despite the role of lncRNA in PcG recruitment, lncRNA and nascent RNA also inhibit PRC2 HMT activity 123,299 . Based on such "recruitment without activation" function of RNA, it entertained an idea where PRC2 activity remained poised and productive H3K27me3 only follows transcriptional repression elicited by other factors upon differentiation 123 . Intriguingly, Jarid2 was shown to alleviate RNA-inhibition of PRC2 activity and might be involved to elevate PRC2 activity upon differentiation 302 .

Study on Drosophila Jarid2 (dJarid2) does not fully recapitulate those functions on mammalian models. Despite dJarid2-PRC2 interaction is conserved in Drosophila, mosaic clones of dJarid2 in eye disc showed a specific increase of H3K27me3 level while overexpression of dJard2 conversely led to a global decrease of H3K27me3 303 . These results seemed to suggest dJarid2 as a negative regulator on H3K27me3 level. Importantly, like the mammalian homolog, the amino acid residue within the JmjC domain critical for histone demethylation is not conserved in dJarid2 303 . And hence dJarid2 should be catalytic inactive for histone demethylation. Biochemical data would need to confirm this idea.

Although dJarid2 overexpression abolished H3K27me3 level and Su(z)12 binding to polytene chromosomes, lost of function studies using Jarid2 mutant did not affect H3K27me3 level nor Su(z)12 binding pattern 303 . Also dJarid2 polytene pattern is not affected in E(z) mutant background. As opposed to the mammalian model, these data suggest that chromatin-targeting of dJarid2 and PRC2 are independent of each other despite of their interaction and substantial degree of overlap on genomewide ChIP profile 303 . It should be noted that dJarid2 is not enriched in canonical PcG targets such as the Hox clusters 303 , and it remained unknown whether any homoeotic transformation phenotypes were detectable in dJarid2 mutant background.

Nonetheless, affinity bait made of oligonucleosomal array assembled with H2Aub can effectively capture PRC2 with dJarid2 and AEBP2/Jing 112 . And biochemical assay suggested that PRC2 supplemented with both dJarid2 and Jing achieved higher HMT activity than PRC2 alone on H2Aub nucleosomal substrate 112 . Currently, the physiological role of such enhanced PRC2 activity is not known.

5. 3 Jing/AEBP2 (AE-1-Binding Proteins 2)

From the original identification of HMT activity on mammalian PRC2 activity 90 to the discovery of mammalian Jarid2 285-289 , affinity-capture of PRC2 components also copurified AEBP2 304 . (Drosophila AEBP2 homolog is known as Jing) AEBP2 is a zing-finger containing protein that can bind DNA directly 305 .

ChIP-cloning (similar to regular ChIP protocol except an additional restriction enzyme digestion step is performed after immunoprecipitation, this helps to shorten ChIP fragment size for better prediction of binding motif of chromatin-associating factors) of AEBP2bound genomic regions showed that >40% AEBP2 enriched peaks mapped closely to known PcG targets in mouse 306 . Motif analysis revealed that AEBP2 binding sites were enriched with an unusual bipartite structure 306 . Whether such DNA-binding property function in PRC2 recruitments awaits further investigations.

AEBP2 also functions as an allosteric activator. It was shown that AEBP2 enhanced mammalian PRC2 HMT activity 304 . Such stimulatory effect is even more prominent in the presence of Jarid2 on H2Aub-containing oligonucleosomes. Molecular mechanism of such allosteric stimulation is still lacking 112 . Recent EM-reconstitution of human AEBP2-PRC2 complex showed that AEBP2 interacted extensively with other PRC2 subunits and may help to coordinate and stabilize subunit configurations. Such properties might stimulate productive H3K27 methylation by PRC2 263 .

Jing is also copurified with PRC2 and dJarid2 on oligonucleosomal array assembled with H2Aub from Drosophila embryos 112 . Importantly, Jing genetically interacts with Pc, as evident by the enhanced wing-disc Ubx derepression in a transheterozygous jing 22F3 /+; Pc XT109 /+ background 307 . These data highlight the accessory roles of Jing/AEBP2 in facilitating PcG repression.

6 PR-DUB (Polycomb Repressive Deubiquitinase)

Identification of a novel PcG gene with ubiquitin C-terminal hydrolase (UCH) domain paves the way in understanding the regulation of H2Aub homeostasis 217 . This gene is known as Calypso. Through IP-MS approach, Calypso is found to associate with Additional Sex Comb (Asx) and together they form the Polycomb Repressive Deubiquitinase Complex, PR-DUB, which is conserved from Drosophila to mammals 156 . Biochemical reconstitution of both human and fly PR-DUB complexes showed that they have robust deubiquitinase activities on H2Aub, but not H2Bub. Lost of PR-DUB led to a bulk increase in H2Aub level in Drosophila embryo. Also, mosaic clones of both Calypso and Asx mutant derepressed Hox genes in larval wing discs 156 . These data present a seemingly dilemma on why both positive (dRing) and negative (PR-DUB) regulators of H2Aub are required for efficient PcG silencing. It might suggest an optimal level of H2Aub is essential for gene repression. One could imagine that over ubiquitination of H2A in PR-DUB mutants might titrate away limited readers to elicit its function on specific location. On the other hand, this might suggest a high turnover of H2Aub is required for efficient PcG repression 147,308 . Similarly, high turn over of H2Bub, achieved by active ubiquitination-deubiquitination cycle, has been proposed for efficient gene activation 153 .

Mechanism of PcG Repression

Despite With the same assay on chromatin template, subsequent study also showed that reconstituted mammalian PRC1 behave similar inhibitory effect on in vitro transcription 310 .

Besides, it was found with, a similar assay, that mammalian PRC2-Ezh1 also repressed transcription, independent of H3K27 methylation activity 311 . Drosophila PRC2 has yet to be assayed on the same setting. In addition, tethering wild-type Ezh1 and even its SET domain derivative can repress transcription output from a luciferase reporter gene in vivo 311 .

More recently, in vitro transcription assay has been revisited using H3K27-MLA 107 assembled nucleosome as a way to recruit PRC1. With this template immobilized on magnetic beads, associating factors can be compared in transcriptionally active vs.

repressed states 312 . It was found that incubation with reconstituted PRC1 abolished the assembly of Pre-Initiation complex (PIC) in which TFIID (except TBP) and mediator components were occluded from the transcription template 312 . Given that TFIID components can be co-purified with PRC1 in Drosophila 199 , perhaps PRC1-TFIID interaction is not compatible with PIC formation.

Study of bivalent genes in mouse ES cells showed that their promoters were enriched with Ser-5 phosphorylated poised RNA Pol II. And upon conditional deletion of Ring1b in a Ring1A-null background, H2Aub drops drastically and these bivalent genes derepressed 313 .

Notably, there were no significant changes in the RNAP phosphorylation profile of these genes. Hence the authors proposed that Ring1b-mediated H2Aub helped to restrain Pol II in a previously unrecognized form that is not processive for active transcription 313 .

Using the Drosophila inducible heat shock promoter hsp26 as a model, transgenic fly carrying ectopic insertion of PRE-hsp26 promoter modules show PcG-dependent repression of downstream reporter genes. Importantly, such repression does not affect binding of TFIID and Pol II. Rather, it restrained Pol II in state refractory to processive transcriptional elongation 314 .

PcG proteins also affect Pol II recruitment. Genomewide nuclear run on (GRO-seq) and

ChIP-seq of Pol II in Drosophila embryos revealed that, upon perturbation of H3K27me3 in esc -/-mutant (obtained by self-crossing esc [START_REF] Margueron | The Polycomb complex PRC2 and its mark in life[END_REF] , b, pr / CyO, esc 2 ), a large sets of genes (accounts for ~ 25% of all Drosophila genes) acquired significant increase of promoterproximal Pol II occupancy 315 . When compared to WT, these genes can be divided into two classes. In class I genes, Pol II is recruited already in WT condition and the lost of esc further enhance the binding level. While in class II, Pol II occupancy occurs de-novo.

Interestingly, it was found that more class I genes bear signature motifs around their transcription start sites that might favored Pol II binding even in the presence of PcG proteins 315 .

Other than modulation on RNAP, it was shown in macrophages that H2Aub, deposited by another E3 ligase known as hRUL138, is required to repress cytokine genes expressions, potentially by weakening the binding of FACT to H2Aub and hence hamper transcription elongation 316 . Whether polycomb-related H2Aub also counteracts FACT recruitment is yet to be tested.

As discussed above, polycomb repression system could affect the transcription cycle at various stages. These mechanisms might not be generalized and it might vary, depending cell types and structural features of the target genes. Full reconstitution of in vitro transcription reaction on synthetic chromatin template with defined histone marks would be an invaluable tool to dissect the effect of different PcG proteins on transcription.

3 Chromatin cross talks -

Inhibition of active chromatin marks

Accumulating evidence demonstrating that PcG repressive activities are very sensitive to local chromatin environment. Likewise, there is a growing appreciation on the inhibitory effect of PcG repressive chromatin on counteracting active chromatin signatures [START_REF] Di Croce | Transcriptional regulation by Polycomb group proteins[END_REF][START_REF] Simon | Occupying chromatin: Polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put[END_REF]212,317,318 .

H2Aub inhibits H3K4 and H3K36 methylation

Of note, in vitro HMT assays using oligonucleosomes assembled with H2Aub is refractory for H3K36 methylations. Specifically, activities of H3K36 HMTase including Ash1L, HYBP, NSD1 and NSD2 were inhibited, while other HMTase such as PRC2, G9a and Pr-Set7 were not affected 152 . Similarly, H2Aub-comtaining recombinant chromatin inhibits MLL3 for deposition of H3K4me2 and H3K4me3. Treatment of chromatin with H2A-DUB restored MLL3 HMT activity 151 .

H3K27me3 inhibits H3K4 methylation

Vertebrate H3K4me3 is catalyzed by different HMTases such as Set1A, Set1B and a family of Set1-like complexes containing MLL1-4 319 . All these complexes share common subunits WDR5 and RBBP5, whose binding to H3 tail is strongly inhibited when H3K27 is trimethylated. Consequently, H3K4 methylation activity of Set1A/B and MLL1-containing complexes were attenuated by H3K27me3 320 . Hence it suggests that H3K27 demethylation is required to install H3K4me3 by these complexes. Indeed H3K27 demethylase UTX is component of the MLL3/4 Set1-like complex 319 . Whether similar regulation exists in Drosophila awaits further investigations.

Counteracting active histone marks

Some PcG components possess histone-modifying activities against active chromatin marks. For example, a component of dRAF, dKdm2 is active for demethylation on H3K36me. Although dKdm2 JmjC mutant did not compromise its stimulatory role for dRAF-mediated H2Aub, the functional relevance of H3K36 demethylation in PcG silencing awaits in vivo verification 77 . Similarly, JmjC domain of mammalian Kdm2B is dispensable for the Kdm2B-PRC1-PRC2 recruitment axis 81 .

In another case, immunoprecipitates of PRC2 was detected with HDAC activity 135 . Indeed, Rpd3/HDAC1, which is reactive on H3K27 deacetylation, was reported to be co-purified with PRC2 in Drosophila embryos 92,136 . Given the mutually exclusive nature of alternate modified state (methylation/ acetylation) of lysine residues, it is postulated that deacetylation as a prerequisite step for PRC2 methylation on H3K27 134 . Nonetheless, transheterozygous combination of Rpd3 and PcG mutants enhanced Hox derepression and transformation phenotypes 92,137 .

Mammalian Pcl homologs were shown to recognize H3K36me3 via their Tudor domain 71- 74 (see Chapter I, p.46). Such binding is proposed to recruit PRC2 to active genes to elicit de-novo gene repression. Supporting this idea, PHF19 was show to interacts with the

4 Chromatin Compaction

As detected by atomic force microscopy, the 12-mer nucleosomal arrays appear to adopt a "beads on the string" conformation. Ph alone can repress these genes, which indeed is the case as tested by in-vitro transcription assay 201 . This might be supported by the chromatin compaction activity of Psc. How Ph elicits repression on these genes remained unknown. Especially Ph is dispensable for compaction in reconstituted PRC1 202 . However, it is well documented that the SAM domain of Ph can form helical polymer 222,224,225 , and its potential impact on chromatin structure remain untested both in vitro and in vivo.

5 3D genome organization -Formation of repressive topologically associating domains (TAD)

The polycomb-responsive element is characterized as the DNA sequence essential and sufficient to elicit polycomb complex recruitment and gene repression 326,327 . Fab-7

represents one of the best-characterized PRE-containing regulatory elements that control the expression of the Hox gene Abd-B in the bithorax complex (BX-C) 326,327 . Transgenic fly carrying ectopic insertion of Fab-7 recruits PcG proteins and typically gives variegated repressions of a downstream reporter gene 328 . Importantly, as tested on both endogenous and transgenic PREs, silencing of reporter gene is enhanced when the inserted construct is in a homozygous state. This phenomenon is referred as pair-sensitive silencing 326,327 .

Intriguingly, Fab-7 repression on transgene is weakened when the endogenous Fab-7

element is deleted 328 . These genetic evidences infer the possibility that PRE elements cluster to act concertedly for synergized PcG repression.

Indeed, with a series of transgenic assays in Drosophila, it was shown that two Fab

Mechanisms for Derepression of PcG Target

Genes

Guided by comprehensive genomewide studies, it becomes evident that polycomb targets comprise a much broader category beyond the canonical Hox genes. These include signal transduction components, stress-response gene networks, cell cycle regulators and more.

Hence there is an emerging idea that in addition to confer stable repression of the Hox clusters, PcG system also contributes to dynamic and switchable gene expressions in response to developmental cues and environmental stimuli [335][336][337] . Central to this regulation is the mechanism of derepressing PcG target genes, which is the key focus of this chapter

(Fig V).
Of note, mechanism of general active transcription will not be covered.

1 ZRF1 (Zuotin-related factor 1)

ZRF1 was identified as a reader of mono-ubiquitinated H2A by affinity purification.

Genomewide study revealed that a number of ZRF1 binding sites overlapped with PRC1 and H2Aub target genes, comprising key developmental and differentiation regulators.

Importantly, biochemical study suggested that ZRF1 and Ring1b competed to bind H2Aub.

Indeed, ZRF1 is recruited to a subset of H2Aub sites during differentiation to displace Ring1b binding, and hence derepresses of PRC1 targets. In addition to PRC1 eviction, in vitro assay showed that ZRF1 also synergized the deubiquitination efficiency of H2A-DUB Ups21 338,339 .

2 Interphasic H3S28ph

Analogous to the first phosphor-methyl switch where H3K9me3S10ph occludes HP1 binding 177,178,340 , it is found that similar dual modifications such as H3K27me3S28ph could reject binding of PcG components. Notably histone peptide with H3K27me3 can affinitycapture PRC2 and Cbx8 from HeLa cell nuclear extracts 189 . But when imposing a juxtaposed phosphorylation in the form of H3K27me3S28ph, the enriched bindings between H3K27me3 to PcG components were lost. It is believed that the huge charge and atomic size of the phosphate group abolish interaction between methylated lysine to the chromodomain of Pc homologs. Similarly, H3S28ph might also interfere binding of EED to H3K27me3.

A vast array of signaling inputs from differentiation, cellular stress and dopamine neurotransmitter activate the MAP kinase-signaling pathway. These signals are funneled to activate MSK1/2, which are recruited to a subset of PcG target genes related to downstream responses [189][190][191]341 . MSK1/2 phosphorylates H3S28 and the double mark H3K27me3S28ph evicts both PRC1 and PRC2 from chromatin and leads to derepression of PcG targets genes to cope with stress or differentiation 189,190 . During this process, H3K27me3 level and PcG occupancies decrease while a reciprocal increase in H3K4me3

and Pol II binding occurs on these signaling genes [189][190][191] . Of note, prolong stimulation might lead to acetylation at H3K27 and increased H3K27AcS28ph level 190 .

It is important to point out that H3K27me3S28ph is refractory to demethylation by both JMJD3 342 and UTX 343 . Hence in addition to evict PRC1/2, H3S28ph also protects H3K27me3. Owning to the versatility of phosphorylation kinetics, it is proposed that H3S28ph offers a transient and reversible mode of repression. In this model, removing

H3S28ph would allow rapid rebinding of PcG components and robust resumption of repressed chromatin states so that the system retains responsiveness to initial stimuli 343 .

Of note, H3S28ph is cell cycle regulated and is highly enriched in mitotic cells [187][188][189]344 , its role in modulating chromatin-binding behavior of PcG components during mitosis will be discussed in Chapter III, p.134.

3 Aurora B-mediated inhibition of Ring1b-dependent H2Aub.

In resting B cells, Aurora B (AurB) kinase associates with Ring1b and they are required to sustain Pol II occupancy and expressions of a subset of genes. Intriguingly, PRC1 and

AurB are enriched at the TSS of these active genes where only low level of Ezh2 and H3K27me3 were detected 345 . It is proposed that AurB phosphorylates H3S28 and inhibits

Ring1b-dependent H2A ubiquitination to enhance Pol II recruitment and transcription.

Specifically, AurB enhances the activity of DUB USP16 in H2Aub deubiquitination.

Besides, AurB also phosphorylates E2-conjugating enzyme UBE2D3 (UbcH5c) and hampers its activity in Ring1b-dependent H2A ubiquitination 345 . Whether AurB also directly modulates Ring1b is unknown. It is important to note that H3S28ph does not evict nor recruit Ring1b on these genes 345 . It is proposed that some unknown transcription factors are required for selective recruitment of Ring1b and H3S28ph might serve to repel PRC2 to facilitate active transcription 345 .

4 H3K27 demethylation

UTX and JMJD3 belong to the KDM6 demethylase family which specifically remove diand tri-methylation at H3K27 with the conserved Jumonji C (JmjC)-domain [125][126][127][128] (see Chapter I, p.14).

UTX is required to sustain expression of active Hox genes 125,126,128 . RNAi against UTX leads to accumulation of H3K27me2 and H3K27me3 at Hox promoters and enrichment of PRC1 and H2Aub level 128 , resulting in decrease transcriptional repression.

During retinoic acid induction of differentiation, some of the repressed Hox genes such as For example, Utx recruits Brg1, the ATPase subunit of the SWI/SNF remodelers to cardiac enhancer independently of its demethylation activity 342 . It is thus tempting to speculate that the robust demethylation activity of Utx might be specifically required for a subset of genes such as those required during stress responses.

5 DUB (Deubiquitinase)

While PR-DUB complex is essential for PcG repression despite its enzymatic function is to removal H2Aub

Remarks

As listed from the above examples, it becomes clear that several modes of derepressing PcG target genes exist and such a list would probably keep expanding. This calls for a quest to understand the advantage to have a vast array of mechanisms in overriding PcG silencing. Clearly, we lack the detailed kinetics of each derepression mechanism.

Genomewide studies would help to catalog the genes utilizing distinct PcG-counteracting methods, and from the derived gene ontologies we might be able to appreciate more dynamic interplay between PcG repression and derepression.

Modulation of PcG Repression Activity

Thanks to the advancement of assessing histone-modifying activity using synthetic chromatin template with defined histone marks, we now realize that PcG complexes are very sensitive to local chromatin setting. With the help of structural data, we now have a better understanding on how PcG components integrate and channel chromatin environment to modulate its repressive activity. This chapter mainly focuses on how chromatin features affect PcG activity (Fig VI ), and post-translation modification of PcG components will not be discussed.

1 H3K27me3

Stimulation on PRC2 HMT activity

Recruitment mark for canonical PRC1

Analogous to the HP1 chomodomain-H3K9me3 binding module 41,42 , chromodomain of Pc also recognizes H2K27me3 90,91,105,106 . The prevailing idea is that such interaction contributes to PRC1 recruitment to PcG target genes, despite that H3K27me3-independent mode of recruitment also exists. Apart from this, it is not known whether H3K27me3 can modulate PRC1 activity. At least in Drosophila, it is known that dRAF is the dominant form of E3 ligase for H2A ubiquitination and the canonical 4-component PRC1 barely possesses H2A E3 ligase activity 77 . It is known that dRAF and its mammalian paralogous complex prefer to act on nucleosomal H2A 142,144,145,351 , it would be interesting to test whether H3K27me3 could enhance their E3 ligase activities.

2 H2Aub Stimulation on PRC2 HMT activity

Nucleosomal arrays assembled with H2Aub affinity-purify PRC2 components and accessory factors AEBP2 (Drosophila homolog known as Jing) and Jarid2 112 . Intriguingly, the 4-component PRC2 displays higher HMT activity when it is supplied with Jarid2 and AEBP2. Remarkably, even stronger H3K27 methylation efficiency is achieved when the 6component PRC2 is working on H2Aub-containing chromatin template 112 . Hence in addition to H3K27me3, H2Aub also stimulates PRC2 HMT activity. In line with this, ectopic nucleation of H2Aub recruits PRC2 components and establishes H3K27me3 domains de novo. These observations suggest an alternative recruitment hierarchy in which H2Aub deposition by PRC1 helps the subsequent binding of PRC2 to PcG target sites.

In addition to PRC2 components, the H2Aub nucleosomal array also captures PRC1 112 .

And indeed H2Aub was found to be associated with Ring1b 339 . Hence it is proposed that PRC1-H2Aub binding might also constitute a positive feedback loop to reinforce H2A ubiquitination efficiency.

H2Aub and nucleosome density

Some studies suggested that H2Aub might facilitate the association of linker histone H1 to nucleosomes 316,325 . Hence H2Aub chromatin might attain a high nucleosomal density configuration that is stimulatory for PRC2 HMT activity, as discussed below.

3 Polycomb-like

Pcl can be co-purified with PRC2 95,281,282 and it is essential to sustain high activity of PRC2 in catalyzing H3K27me3 95,282 . This is consistent with in vitro HMT assay, where it is demonstrated that both Drosophila Pcl and the corresponding mammalian homolog PHF1 can stimulate high catalytic efficiency of PRC2 95,281,282 . Notably, all mammalian Pcl homologs including PHF1, PHF19 and MTF2 possess a conserved Tudor domain that specifically interacts with H3Kme36 71-74 . Hence it raises an attractive model for mammalian PCL to anchor PRC2 at active genes to elicit de novo repression during differentiation. In line with this idea, the H3K4 and H3K36 dual specific demethylase NO66 321 can complex with PHF19-PRC2. As such, robust removal of active marks alleviates their inhibitions on PRC2 HMT activity and hence permits efficient establishment of H3K27me3 domains 72 . However, since two critical aromatic residues within Tudor domain of Drosophila Pcl are not conserved, it is not expected to bind H3K36me3 71,73,284 .

4 Nucleosome density

In vitro HMT assay with oligonucleosomal arrays of defined linker space demonstrated that PRC2 caters more efficient H3K27 methylation on dense chromatin 265 . As discussed in Chapter I, p.45, the acidic residue within the conserved VEFS motif of Su(z)12 is essential for sensing nucleosome density.

Modeling the EM structure of PRC2-AEBP2 suggests that the complex can adopt a conformation to simultaneous interacts with two adjacent nucleosome without steric hindrance 263 . As such, EED binding on one nucleosome can position Ezh2 SET domain in face of the H3 tail of the adjacent nucleosome 263 . Haveing a higher inter-nucleosome density might stabilize such confirmation for robust PRC2 HMT activity.

5 H3K4me3 & H3K36me3 inhibit PRC2 activity

Chromatin templates pre-coated with active marks such as H3K4me3 or H3K36me3 are refractory to PRC2 methylation in flies, mammals and plants 264,352 . Crystallography data suggest that the side chain of H3K4 is engaged in interaction with the Nurf55 betapropeller domain 264,353 . Hence increasing the methylation states at H3K4 would progressively introduce steric clashes to the binding pocket in Nurf55. Indeed, the binding of Su(z)12-Nurf55 complex to H3 peptide is abolished upon methylation on H3K4 264,353 .

Given that VEFS is the main motif of Su(z)12 in contact with E(z) 354 and EM structural data reveal its close proximity to the catalytic SET domain of Ezh2 263 , it is proposed that the VEFS motif of Su(z)12 can sense and channel chromatin features to modulate PRC2

HMT activity. Consistent with this idea, plant PRC2 with a Su(z)12 variant (VRN2) can remarkably override the inhibitory effect imposed by H3K4me3 and H3K36me3 28 .

6 H3K27Ac

The side chain of a lysine residue cannot support coexistence of both methylation and acetylation. Owning to this chemical constraint, acetylated lysine occludes t 102 he potential of methylation on the same residue. Hence H3K27ac is not a compatible substrate for PRC2 HMT activity. Besides, it remains possible that H3K27ac might have a mild inhibitory effect on PRC2 102 .

7 H3S28ph

Since the first discovery of dually modified H3K9me3S10ph histone mark 177,178 , there are growing examples of phosphor-methyl switches in the eviction of pre-bound chromatin effectors (Chapter I, p.30; Chapter III, p.137). As discussed in Chapter I, p.26, various signaling inputs can activate MSK1/2 to phosphorylate H3S28 on PcG targets [189][190][191]341 . The resulting H3K27me3S28ph double mark evicts PRC1 and PRC2 components and leads to derepression of PcG target genes. Of note the binding affinity of EED beta-propeller domain 113 and Pc Chromodomain 105,106 to H3K27me3 is relatively weak (5-36 uM), and hence these interactions are believed to be disrupted by the huge charge and atomic size imposed by the phosphate group 189,190 .

8 H3.3

A study from Drosophila cultured cells estimates that H3.

9 CpG methylation

Mammalian Kdm2B-RYBP containing PRC1 variant can nucleate de novo H2A ubiquitination to establish a chromatin platform for PRC2 recruitment and formation of H3K27me3 domain 81,155 . Central to this alternative recruitment hierarchy is the Kdm2B protein, which possess a CXXC domain whose binding to CpG island is sensitive to cytosine methylation 79,80 . Indeed genomewide distribution of Kdm2B is highly enriched at unmethylated CpG islands in ES cells and is inversely correlated to methylated CpG in differentiated cells 80 .

Given the wide distribution of CpG islands over gene promoters, dKM2B-PRC1-PRC2 may represent a versatile recruitment axis subjected to regulation by CpG methylation in mammalian cells 81,356 . In fact, mouse somatic cells severely compromised in DNA methylation displayed drastic redistribution of global H3K27me3. It is proposed that DNA hypomethylation increases accessible CpG island that titrate PRC2 away from its native targets, resulting in the lost of H3K27me3 and their derepression 155,357 .

RNA inhibition of PRC2 activity

In mouse ES cells model, it was found that PRC2 also occupy promoter regions of active genes with low level of H3K27me3 358,359 . In addition, the use of photo-activatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) documented a catalogue of Ezh2-associating nascent RNA 359 . These observations culminate an idea that PRC2 occupancies at active genes are inhibited by their nascent transcripts. Indeed, in vitro assay showed that PRC2-interacting lncRNA such as HOTAIR and RepA inhibits PRC2

HMT activity in a length dependent manner 123,302,358 . Similar observation was made for Drosophila PRC2 360 . Interestingly, RNA lacking secondary structures such as the extended polyA stretches did not possess inhibitory effect. Using Ntn1 as a model of PRC2-bound active gene, introduction of premature polyadenylation site downstream proximal to Nts1 transcription start site (TSS) effectively reduced transcription output and committedly increased H3K27me3 level at promoter region 123 . As such, PRC2 activity can be regulated at a post-recruitment level. A recent report showing genomewide de-novo recruitment of PRC2 upon transcription inhibition 361 , it would be interesting to know whether H3K27me3 also increases in those active genes pre-bound by PRC2.

Remarks: Emerging concepts on regulating PcG repression

The growing knowledge in how chromatin features affect PcG components begins to culminate the idea that, apart from the regulation by binding compatibility, PcG proteins activities can also be modulated at a post-recruitment level.

For example, it was found that during DNA replication, PcG proteins remained associated with nascent chromatin without apparent activity on catalyzing histone methylations 362 .

Besides Another important concept is to interpret chromatin signal in context. Given the vast number of PcG complex variants, one rule might not apply to all. This is so far best demonstrated by the plants PRC2 variants, whereby assembling PRC2 with an alternative Su(z)12 paralog can override the otherwise inhibitory effect of active chromatin 264 . This highlights the functional diversity of PcG complex variants.

Epigenetic Inheritance of PcG-mediated Gene Repression

Polycomb-mediated repression of Hox genes represents a classical epigenetic phenomenon in which the Hox expression pattern established during early embryogenesis were maintained throughout later developmental stages in the absence of initial transcription repressors [START_REF] Simon | Occupying chromatin: Polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put[END_REF]298,[363][364][365] .

1 DNA replication

During DNA replication, transversal of replication fork can evict pre-bound chromatin factors and de-novo chromatinization inevitably dilutes pre-existing histone modifications.

These factors impose immense challenge to maintain PcG repressive chromatin [366][367][368] .

Persistence of PcG proteins during DNA replication

As demonstrated with in vitro SV40 replication system, reconstituted Drosophila PRC1 or Psc alone remains associated with nascent chromatin 369 . The same result is obtained when using a simple reconstituted T7 bacteriophage replication system devoided of endogenous eukaryotic proteins 370 ; hence it highlights the intrinsic persistent-binding properties of

Restoration of H3K27me3 repressive chromatin

Several observations support the idea that parental histones are partitioned randomly over daughter chromatin, together with the pre-existing histone modifications. Notably, H3K27me3 is detected on nascent chromatin even in the presence of translation inhibitors 371 .

Since H3/H4 is inherited as intact tetramer (no splitting) and over 40% of global H3K27me3 exist as asymmetrically modified nucleosomes 119,121 , it does not favor a semiconservative mode of duplicating histone modifications. Hence it is likely that H3K27me3

is not restored in a nucleosome-resolution fashion 365 . Nonetheless, the inheritance of parental histone and their associated modifications can act as seeding sites that guide the propagation of pre-existing chromatin features 363,365 . In the case of PcG targets, the inherited H3K27me3 can be recognized by the EED subunit of PRC2 113 . Such interaction not only helps PRC2 retention but also allosterically stimulates PRC2 HMT activity, hence it constitutes a positive feedback loop to catalyze robust deposition of H3K27me3 113 (See Chapter I, p.41). In line with the "seeding model", it is observed in Drosophila culture cells that there is an increase in H3K27me3 level at PcG target just before DNA replication. Such predeposition of H3K27me3 is proposed to alleviate the effect of passive dilution during DNA replication 372,373 .

SILAC (Stable Isotope

2 Mitosis

To ensure faithful segregation of the replicated genome, systemic chromatin condensation is achieved during mitosis so that sister chromatids are gradually resolved to facilitate pole-ward separation. To facilitate proper condensation, global transcriptions are shut down 377 and even rDNA activity is halted 378,379 . And persistent rDNA transcription would lead to segregation defects such as anaphase bridges, potentially due to entanglement of the active rDNA locus 379 . Notably, H2A.Z-containing +1 nucleosomes of active genes are upshifted to cover the transcription start sites during mitosis, rendering them incompetent to support transcription 380,381 .

Owning to tremendous shrinkage of the chromatin volume, lots of chromatin-bound molecules such as RNA polymerase II, transcription factors and even non-coding RNA are mostly evicted. This presents a challenge to preserve epigenetic information and cellular identity [382][383][384] .

Mitotic bookmarking as a way to preserve transcription status

Early observations revealed the persistent of DNase hypersensitive sites during mitosis 385 and it is now clear that a subset of transcription factors or chromatin effectors remain associated with mitotic chromosomes despite immense chromatin condensation. They are referred as mitotic bookmarking factors and their persistent binding serve to robustly reactivate specific target genes in early G1 phase of the next cell cycle, and help to restore cell type specific transcriptome [382][383][384] . Importantly, repressive chromatin structures are also disrupted during mitosis, and example of bookmarking repressed genes is also reported 386,387 .

Examples of different mitotic bookmarking mechanisms

Among the growing examples of mitotic bookmarking, it was shown that mitotic retention of Brd4 [388][389][390] and MLL 391 on chromatin is required for robust reactivation of their target genes in next G1. Besides, the TATA-box binding protein (TBP) helped to recruit PP2A to keep condensin at a hypo-phosphorylated and inactive state. As such a local decompacted region can be maintained at TBP-bookmarked sites to facilitate subsequent gene reactivation 392,393 .

Recent ChIP-seq analyses of over a hundred transcription factors identified clustered binding sites with over 100 transcription factor occupancies 394 . Interestingly, these clusters are always anchored with cohesin whose binding persisted during mitosis 394,395 . Normally, bulk cohesin rings are removed from chromosome arms starting from prophase under the control of Aurora B kinase 396 . How cohesin at clustered site counteract such removal pathway is not know, but it is proposed that such cohesion retention help to maintain DNA accessibility to facilitate rebinding of transcription factors during mitotic exit 394,395 .

In one elegant study of the pioneering factor GATA1, which is a hematopoietic transcription factor essential for establishment and maintenance of erythroid lineage, livecell imaging and ChIP-seq of FACS-sorted mitotic cells detected GATA1 persistent binding on mitotic chromosomes 387 . Importantly, the bookmarked GATA1 targets included important hematopoietic regulators and they showed a more robust reactivation kinetics compared to unbound targets. The functional relevance of bookmarking was elegantly demonstrated by fusing GATA1 to the degradation box of Cyclin B to achieve mitosisspecific degradation of GATA1. It was found that GATA1 depletion during mitosis resulted in slower reactivation kinetics of GATA1 bookmarked genes in the next G1 phase, highlighting the essential role of mitotic bookmarking 386,387 . Similar bookmarking function is observed for another pioneering factor FOXA1 397 .

Are PcG targets bookmarked during mitosis? If so, how?

Although the majority of known bookmarking examples come from active genes, preserving epigenetic features of repressive chromatin states during mitosis are also important.

As exemplified by the pioneering factor GATA1, which also functions as a repressor, depletion of GATA1 during mitosis also leads to partial derepression of its target genes 387 .

It demonstrates that despite global transcription inhibition during mitosis, silenced genes were also susceptible to ectopic reactivation if their associated repressive chromatin features are not preserved.

As for Drosophila PcG proteins, whether they bind to mitotic chromosomes are somewhat controversial, largely because of the varied methods of detection. Early observation with indirect immunofluorescence on formaldehyde-fixed embryo showed that virtually bulk proportion of PRC1 components such as Pc, Ph and Psc were dissociated from metaphase chromosomes, and Psc is the only component which begins robust rebinding in anaphase 398 . In contrary, pre-extracting culture cells with detergent-containing buffer followed by methanol/acetone fixation robustly detected substantial amount of dRing, Pc and Psc on mitotic chromosomes 399 . Similarly, squashed samples from Drosophila larval brains also readily detect strong retention of Pc, Ph, Psc and E(z) on mitotic chromosomes 400 .

In addition to microscopy approaches on fixed samples, western blot analysis of chromatin-pellet, defined as the insoluble fraction after cell lysis by non-ionic detergent buffer, detect ~ 10-30% of PcG proteins are retained on mitotic chromatin 398 , although the nocodazole-treated cells only show a mitotic index of ~ 68%. Hence some of the chromatin-bound proteins should come from a G2 population. rely on other PRC1 components or H3K27me3, as persistent binding is still detected even in Rinb1a -/-Ring1b -/-, Bmi1 -/-Mel18 -/-and Eed -/-mouse ES cell knockout backgrounds 403 .

In contrary, other PRC1 components binding to mitotic chromosomes are disrupted in Cbx2 -/-knockout line, demonstrating the pioneering role of Cbx2 in mitotic binding of mammalian PRC1 403 . Given that such mitotic PRC1 retention is not dependent on H3K27me3, it would be interesting to know if it reflects a non-specific binding property of PRC1. ChIP-seq of PRC1 with mitotic samples would be needed to address this question.

Finally whether Cbx2-mediated anchoring of PRC1 on mitotic chromosomes is essential for preserving PcG repressive chromatin awaits further lost of function studies.

Other than microscopy approach, ChIP assay is also used to detect and locate mitotic binding of PcG proteins. Taking advantage of specific enrichment of H3S10ph during mitosis, a FACS-sorting procedure was optimized to isolate large quantities of H3S10phpositive mitotic cells from colchicine-treated cell culture 399 . ChIP-seq on Psc using asynchronized (H3-sorted) and purified mitotic samples (H3S10ph-sorted) revealed that majority of Psc bindings were lost during mitosis, while a selection of them persisted, despite at a lower enrichment than in the asynchronized sample. No novel Psc-binding sites were detected in the mitotic sample 399 . Importantly, canonical PcG targets such as the Hox clusters showed dramatic reduction of Psc occupancy during mitosis. For those mitoticretention targets of Psc, they are enriched with genes involved in cell cycle regulation.

Strikingly, 84-94% of mitotic Psc sites overlapped with insulator binding sites, these included CP190, CTCF, BEAF and chromator 399 . Consistent with this finding, it was found that almost half of the Psc mitotic sites overlapped with domain borders, which is defined as the genomic regions lacking long-range chromatin contacts 332 . Although there are no Psc mitotic sites within the Hox clusters, ChIP-qPRC detects similar enrichment of H3K27me3 in Hox PREs in both asynchronized and G2/M cells, suggesting that H3K27me3 might serve as a bookmark to guide rebinding of PcG protein during mitotic exit 399 . Based on these data, it was proposed that the majority of Psc is dissociated from its target sites during mitosis, and those retained on domain borders could serve as mitotic reservoir which facilitates robust relocation of Psc and other PcG proteins to their targets in the following G1 phase 399 .

Outlook and remarks

The golden standards to testify a mitotic bookmarking function reply on at least three important approaches. First, live imaging of potential bookmarking factors is needed to determine chromatin-binding kinetics during the sub-phases of mitosis. FRAP experiments would also be informative on the change of chromatin residence time between interphase and mitosis. Second, ChIP-seq profiling of mitotic chromosome occupancies. With the increasing ease of FACS-sorting H3S10ph-containing mitotic cells 404 , it is important to compare if specific targets are bookmarked, these data would also guide later lost of function studies. Third, selective ablation of bookmarking factors during mitosis ultimately addresses the necessity of their mitotic retention in the resumption of target genes expression status in the subsequent cell cycle.

Unfortunately, previous studies on PcG mitotic bookmarking have yet to incorporate all the above approaches. Particularly, owning to a diversified method of detection, there is a lack of consensus on the mitotic binding behavior of PcG components on Drosophila fixed samples. And although live imaging is more reliable, a comprehensive PcG-GFP fusion lines are still lacking. Despite the observation that a subset of Psc binding sites are retained on mitotic chromosomes, their essential role in maintaining PcG repression awaits further lost of function study. Finally, despite the fact that FACS-sorting mitotic cells are getting more and more efficient, ChIP-seq profiles of any histone modifications during mitosis are still awaiting 384 . This is particularly interesting since mitotic chromosomes are highly enriched with H3S28ph, and H3K27me3S28ph double mark is shown to evict PcG components and refractory to UTX-mediated demethylation 342,343 , it would be of great interest to see how their function interplay is reflected on a genomic scale.

Results

Guided by the studies in mammalian models, interphase H3S28ph deposited as H3K27me3S28ph dual modification was shown to counteract chromatin binding of both PRC1 and PRC2 components 189,190 . This serves as a mechanism to derepress PcG targets in

Principle of the histone replacement system in Drosophila

The

Principle of mosaic analysis of histone mutants in Drosophila larval tissues

Previous report on Drosophila H3K27R mutant showed that the limited number of cell division after cellularization of blastoderm failed to completely replace the chromatinized maternal core histones 100 . Hence a mosaic analysis platform of histone mutant was established in larval imaginal discs where typically larva were dissected 3-4 days after clone induction 36,100 . With the use of this system, it was shown that H3K27me3 and Repressive Deubiquitinase (PR-DUB) 156 . Enhancer of zeste, E(z), the catalytic subunit of PRC2 deposits H3K27me3 on targets genes and dRing is an active E3 ligase subunit in dRAF that monoubiquitinates nucleosomal H2A. In the canonical model, H3K27me3binding by the chromodomain of polycomb (Pc) helps to recruit PRC1 90,91,105,106 . Recent studies suggest an alternative recruitment hierarchy whereby H2Aub initiates PRC2 chromatin targeting and the establishment of H3K27me3 domain 81,112,155 . PcG repressive activity is modulated by local chromatin environment 212,317 . For example, both H3K27me3

113 and H2Aub 112 stimulate PRC2 activity, while active histone marks such as H3K4me3 and H3K36me3 264 show inhibitory effects. Notably, the serine residue juxtaposed to H3K27 can be phosphorylated 187 , and interphase H3S28ph was shown to counteract mammalian PRC1 and PRC2 chromatin binding to derepress PcG target genes in response to stress and developmental signalings 189,190 , as well as to activate stress response genes via displacement of HDAC corepressor complexes 407 . Equally important, biochemical studies showed that H3K27me3S28ph was refractory to UTX 343 and JMJD3 342 demethylations. Hence it seems that H3S28ph on one hand evicts PcG proteins and on the other hand preserves H3K27me3. More intriguingly, H3S28ph is deposited by the mitotic Aurora B kinase and, like H3S10ph, it is highly enriched during the course of mitosis 187 .

As such, H3S28ph might represent a cell-cycle link to PcG repression and epigenetic inheritance. To access the importance of the highly conserved H3S28 residue and the physiological relevance of mitotic H3S28ph in modulating PcG repression, we have established an in vivo Drosophila model where the endogenous source of histone H3 is replaced by a non-phosphorylatable H3S28A mutant with a serine to alanine mutation.

Results & Discussion

Establishment of Drosophila H3S28A and H3K27R histone mutants

The endogenous source of canonical nucleosomal and linker histones are encoded from the histone gene unit (His-GU), which is organized as a multicopy array residing at a single genetic locus in the Drosophila genome known as the histone gene cluster (HisC).

Previous studies showed that embryos carrying homozygous HisC deletion (∆HisC) died in late blastoderm, after exhaustion of maternally deposited histones reserves 104 . A histone replacement genetic platform is developed based on the fact that reintroduction of ∆HisC mutant with 12 copies of wild-type transgenic His-GU is sufficient to rescue lethality.

Likewise, His-GU carrying different point mutations can be introduced to replace endogenous histones to study the biological functions of specific histone modifications 104 .

In the previous study of H3K27R mutant 100 , it was reported that the limited number of cell divisions during late embryogenesis was not sufficient to completely replace wild-type chromatinized maternal histones, resulting in chromatin with a mixture of wild-type and mutant histones. To circumvent this limitation, mosaic analysis systems of histone mutants were developed, such as FLP-FRT mediated recombination which generates homozygous ∆HisC clones that can be supplemented with 12xHis-GU of either WT or mutated histones in larval tissues 36,100 . We adopted the same system to generate clonal histone mutants in larval imaginal discs. In this study, we generate H3S28A mutant with an alanine substitution over the native serine residue, rendering it unable to be phosphorylated. In parallel, histone replacement lines carrying WT and H3K27R histone mutants were established as controls. To simplify the nomenclature, homozygous ∆HisC clones supplemented with different transgenic 12xHis-GU are referred as WT, H3S28A and H3K27R lines, unless otherwise stated.

Consistent with previous reports 36,100 , homozygous ∆HisC clones induced with a heat shock pulse of FLP died and were replaced by neighboring tissues (Supplementary Fig S1).

This is evident by the presence of cells with double GFP staining levels, while the twin clones lacking GFP that would correspond to ∆HisC mutant did not develop.

H3S28A and H3K27R mutants shared similar Hox gene derepression profiles

It has been shown that in various PRC2 mutants 88,205,276 and H3K27R histone mutant 100 clones where H3K27me3 level is compromised, canonical PcG targets such as Hox genes are derepressed. Hence we probed for Hox gene derepression in H3S28A mutant, together with H3K27R mutant as a positive control.

Endogenous expression of Scr is limited to a patch of cells located at the base of antennal discs (arterisks) and is absent within the antennal disc proper 408 . As shown in Fig. M3A, Scr was derepressed across the entire antennal disc in in both H3S28A and H3K27R clones.

Low level of Scr expression were reported in the adepithelial cells of the 2nd and 3rd leg discs (arterisks) 409 . Similar to PcG mutants, Scr was strongly derepressed in H3S28A and H3K27R clones of these tissues (Supplementary Fig S3). Also, in both histone mutant clones, relatively weak notum-restricted Scr derepression was occasionally detected in the wing discs, which was in agreement with a previous report in Pc [START_REF] Strahl | The language of covalent histone modifications[END_REF] seem to support this idea, since the side chain of H3S28 is pointing away from, and hence does not contribute to, the interaction with the Esc beta-propeller domain 113,212 .

However, when the same assay was performed with H3S28A mutant nucleosomes, PRC2

HMT activities were considerably reduced, with or without supplementation of stimulatory H3K27me3-containing peptides (Fig M4B). Similar to WT nucleosomal substrates, the PRC2-stimulatory effects of H3K27me3 and H3K27me3S28A were similar on H3S28A

nucleosomes. As such, it appears that H3S28A mutation introduces structural intolerance that renders it a suboptimal substrate. Although crystal structure of human Ezh2 SET domain is available 411 , the exact contribution from the hydroxyl group of H3S28 for H3K27 methylation remains unknown.

In this report, we have established a H3S28A histone mutant in Drosophila in which mitotic H3S28ph is depleted. H3S28A nucleosomes appear to be a suboptimal substrate for in vitro PRC2 HMT activity. Consistent with this, H3S28A mutant exhibits defects in H3K27 methylation and shows similar but milder Hox derepression profiles and transformation phenotypes to that of H3K27R mutant flies. Currently, we cannot segregate the differential contributions from the structural intolerance versus the lack of H328 phosphorylation associated with the H3S28A mutant to the observed in vivo phenotypes.

The structural role of H3S28 in PRC2 catalysis remains to be determined. A likely possibility is that the hydroxyl group of the serine residue might be needed to interact with the SET domain of E(z) for efficient K27 methylation.

Previous immunostaining detections of Drosophila PcG proteins on mitotic chromosomes showed discrepancies depending on staining protocols and tissue types 344,[398][399][400] .

Nonetheless, live imaging of Pc-GFP, Ph-GFP and E(z)-GFP in early Drosophila embryos 402,412 all suggested that majority of these PcG components were dissociated from mitotic chromosomes. Since stress-induced interphase H3S28ph evicts PcG complexes 190,264 , one might expect rebinding of PcG proteins on mitotic chromosomes depleted of H3S28ph. (A) In vitro HMT assay using reconstituted Drosophila PRC2 complex and WT nucleosomes supplied with different trans-acting histone peptides. Consistent with previous reports, the H3K27me3 peptide stimulates PRC2 activity. Such stimulatory effect is unaffected in H3K27me3S28A, but is lost in H3K27me3S28ph. (B) In vitro PRC2 HMT assay using nucleosomal substrates assembled with either WT-H3 or H3S28A mutant. PRC2 activity is reduced on H3S28A nucleosomes even when it is supplied with stimulatory trans-acting H3K27me3 peptides. Coomassie stainings of histones were used as loading controls.

Extended results

Phenotyping H3S28A mutant in dUtx∆ background

As mentioned previously, H3S28ph blocks both mammalian PcG components 189,190 and demethylases 342,343 As for histone marks, it appeared that for all methylation states of H3K27, there were no notable differences between H3S28A and combined dUtx∆, H3S28A clones (Fig 7B). Surprisingly, adult flies derived from dUtx∆, H3S28A mutant displayed more severe antenna-to-leg transformation than H3S28A mutant alone (Fig 7C ), they showed larger ectopic tissue outgrowth from antennal segment 3. At present, it is not known if dUtx is the only H3K27me3 demethylase in Drosophila. And as discussed in the Chapter I, p.14, its demethylation function in larval stage is yet to be confirmed. Currently there is no explanation for the phenotypic enhancement in H3S28A, dUtx∆ double mutant, it would be informative to know whether similar or even stronger additive effect is observed in H3K27R, dUtx∆ background.

Potential role of H3S28ph in the epigenetic inheritance of PcG repression through mitosis

Studies from mammalian cells showed that interphase H3S28ph on one hand repelled PcG proteins from binding chromatin 189,190 while on the other hand shielded H3K27me3 from demethylases 342,343 . How are these seemingly contrasting properties channeled with epigenetic inheritance? Also, the emerging functions of mitotic bookmarking stress the importance of mitotic

Phospho-methyl switches and mitotic progression

As exemplified by the case of H3K27me3S28ph, addition of a phosphate group renders the methylated lysine irresponsive to both positive 189,190 and negative 342,343 regulators. This

property is yet to be generalized to other mitotic "phospho-methyl" dual modifications, particularly on its role in resisting demethylations.

It was shown that H3T3ph repelled binding of TFIID (TAF3) 421 , MLL5 166,422 and CHD1 27 to H3K4me3. H3S10ph blocks HP binding to H3K9me3 177,178 . H3S28ph shields H3K27me3 away from PcG components 189,190 and demethylases 342,343 . H2AK119ub and H2AT120 exists individually, but their presences as dually modified form on the same tail remained to be verified. Similarly, H3K79me3S80ph coexistence remained to be confirmed. Except for H3T3ph and H2AT120ph [162][163][164] , which has proved to activate Aurora B kinase for mitotic progression, the necessity of other mitotic phosphorylations has yet to be verified. Lack of H3S10ph in budding yeast H3S10A mutant is viable with no mitotic defects 170 ; it only displays condensation phenotype for an artificially lengthened chromosome. This is different for eukaryotes that support H3K9me3. In the case of fission yeast 173,174 and Tetrahymena 172 , H3S10A mutation led to various mitotic defects. Notably, pericentromeric occupancy of H3K9mes dropped significantly in fission yeast H3S10A mutant 174 , it would be interesting to know if it is caused by excessive demethylation or inefficient catalysis by Clr4, homolog of Su(var)3-9, on H3S10A nucleosome.

Interestingly, primitive eukaryotes that lack H3K27me3 seem not to support H3S28ph.

There are no reports on the existence of H3S28ph in budding yeast so far, and the combined H3S10A, H3S28A mutant is viable with no defects in mitosis and meiosis 169 . It is more extreme in the case fission yeast, whose native genome encodes histone H3 with an alanine at position 28. Hence the endogenous wild type H3 is not phosphorylatable for H3S28ph. Sophisticated histone mutagenesis and mitotic chromatin-tethering tools would be needed to systematically address the roles of these dual modifications in mitosis.

Alternative method to address the function of mitotic H3S28ph

Since H3S28A mutant introduces structural intolerance that hampers PRC2 HMT activity, it is difficult to explain the observed phenotype as a result of the lack of H3S28ph or reduced H3K27me3-deposition efficiency on H3S28A mutant. If we know exactly how H3S28 contributes in PRC2 catalysis, it might be possible to design complementing suppressor mutation on E(z) to override H3S28A inhibitory effect. Assuming the hydroxyl group of H3S28 is essential for hydrogen bonding, the best amino-acid substitute that ablates phosphorylation potential by serine/threonine kinase while retaining side-chain polarity would be 2, 3-diaminopropanoic acid (Dap). Of note, DAP differ from a serine residue by replacing the side chain hydroxyl group with an amino group. In fact Dap exists as non-proteinogenic amino acid in cellular metabolites. It can be incorporated during artificial peptide synthesis and hence compatible with in vitro studies. However, tools for in vivo orthogonal labeling of Dap is not yet available. Apart from optimizing the mutation on histones, lost of function approaches can still be applied to Aurora B kinase. Since Integration plasmid:

• pfC31attB3xHisGU

Since WT-H3 and H3K27R mutant derivatives of His-GU is requested from Alf Herzig lab, only the molecular cloning of H3S28A is described as follow:

Gene Synthesis

The following histone H3 fragment is synthesized by Eurofins Scientific to exchange the codon AGT->GCC for H3S28A serine to alanine substitution. This fragment is 815 bp and is flanked by two restrictions site for subsequent subcloning steps. 5' restriction site: NcoI, 3' restriction site: SacI. Coding region of histone H3, start codon and the introduced mutation codon is highlighted in grey, blue and red respectively. The fragment is synthesized and cloned into pEX-A vector for amplification.

Construction of pENTR221-HisGU-H3S28A

NcoI-SacI digestion is applied to pENTR221-HisGU to release the WT 815 bp fragment.

Vector backbone is gel-purified and ligated to the corresponding H3S28A 815 bp fragment released from the gene synthesis pEX-A vector. This gives rise to pENTR221-HisGU-H3S28A.

Construction of pENTRL4R1-HisGU-H3S28A and pENTRR2L3-

HisGU-H3S28A

AgeI-Acc65I sequential digestion is applied to pENTRL4R1-HisGU to release the WT-HisGU fragment (~5 kb), the resulting vector backbone (~2.7 kb) is gel-purified and ready for ligation. Similarly, the vector backbone of pENTRR2L3-HisGU with AgeI-Acc65I ends is prepared. To prepare the HisGU-H3S28A insert (~5kb), the same restriction digestion is applied to pENTR221-HisGU-H3S28A, and in this case, the vector backbone is discarded, while the HisGU-H3S28A fragment is gel-purified and ligated to pENTRL4R1 and pENTRR2L3 vector backbones.

Recombineering

Recombineering follows the instructions of the commercial kit (Invitrogen 12537-023, MultiSite Gateway Three-Fragment Vector Construction Kit). The following integration plasmids are generated:

• pfC31attB3xHisGU (~23 kb) • pfC31attB3xHisGU-H3S28A (~23 kb) • pfC31attB3xHisGU-H3K27R (~23 kb)

Generation of the histone locus deficiency line

The histone deficiency alleles used in this study is Df(2L)BSC104 (BL#8670

Scanning electron microscopy

Adult fly heads were fixed as described 423 using the critical-point-dried method. Briefly, dissected heads were fixed at 1% glutaraldehyde, 1% formaldehyde and 1 M sodium cacodylate (pH 7.2) for 2 hr at room temperature. Samples were washed once in water and dehydrated through incubations at increasing ethanol concentrations, starting from 25%, 50%, 75% and twice in 100%. Each incubation step lasted for at least 4 hr. After criticalpoint-dried, the samples were then sputter-coated with a 10 nm thick gold film and examined under a scanning electron microscope (Hitachi S4000) using a lens detector with an acceleration voltage of 10 kV at calibrated magnifications.

In vitro PRC2 histone methyltransferase assay

In vitro PRC2 HMT assay was performed by our collaborator -

Nucleosome and Chromatin Reconstitution

Nucleosomes and chromatin arrays were reconstituted by salt gradient dialysis as described 424 , using recombinant Xenopus laevis core histones. Nucleosomes contained a 187bp long DNA fragment with the 601 DNA sequence at its center 425 . Histones mutant H3S28A/C110A and was generated by site-directed mutagenesis. All reconstitutions were analyzed by native gel electrophoresis.

H3 Peptides Synthesis

H3 peptides corresponding to H3 amino acids 21-33 were synthesized according to standard procedures. A non-natural tyrosine was added at the C-terminus for quantification. Peptides carrying no modification (H3 unmod), H3S28ph, H3K27me3, H3K27me3S28ph and H3K27me3S28A were generated.

HMT Assays

Histone methyltransferase (HMT) reactions were performed using 1 µg nucleosomes in a total volume of 15 µl for 1 h at 30°C. HMT reactions were carried out in presence of 0.8 µCi [methyl-

Fly Stocks

The following fly lines were used in this study Miscellaneous starter lines for the mosaic histone replacement system y, w, hsp70-flp; FRT40A; TM2/TM6B (Erika Bach) y, w; P{Ubi-GFP. Margueron and Reinberg, 2011; Schuettengruber et al., 2007; Simon and Kingston, 2013).

Drosophila PcG proteins assemble into at least five different multiprotein complexes, namely Polycomb Repressive Complex 1 (PRC1) (Shao et al., 1999), PRC2 (Cao et al., 2002; Czermin et al., 2002; Kuzmichev et al., 2002; Muller et al., 2002), Pho-Repressive Complex (Pho-RC) (Klymenko et al., 2006), dRing Associated Factor (dRAF) (Lagarou et al., 2008) and Polycomb Repressive Deubiquitinase (PR-DUB) (Scheuermann et al., 2010). Enhancer of zeste, E(z), the catalytic subunit of PRC2 methylates the lysine 27 residue of histone H3 (H3K27me) on target chromatin regions. dRing is an active E3 ligase subunit in dRAF that monoubiquitinates nucleosomal H2A. In the canonical model, H3K27me3-binding by the chromodomain of polycomb (Pc) helps to recruit PRC1 to target genes (Cao et al., 2002; Fischle et al., 2003; Kuzmichev et al., 2002; Min et al., 2003). Recent studies suggest an alternative recruitment hierarchy whereby H2Aub initiates PRC2 chromatin targeting and the establishment of H3K27me3 domains (Blackledge et al., 2014; Cooper et al., 2014; Kalb et al., 2014).

The local chromatin environment modulates the repressive functionality of PcG complexes (Muller and Verrijzer, 2009; O'Meara and Simon, 2012). For example, both H3K27me3 (Margueron et al., 2009) and H2Aub (Kalb et al., 2014) stimulate PRC2 activity, whereas active histone marks such as H3K4me3 and H3K36me3 (Schmitges et al., 2011) inhibit its function. Notably, the serine residue juxtaposed to H3K27, H3S28, is widely conserved and present in every species that has a canonical PRC2-dependent silencing system (Figure S1A). In contrast, it is absent in species like fission yeast that do not possess PRC2. H3S28 is mainly phosphorylated during mitosis (Giet and Glover, 2001), but whether this mitotic phosphorylation is required for mitosis in metazoans is unknown. Interphase H3S28ph has also been detected in mammals, where it was shown to counteract mammalian PRC1 and PRC2 chromatin binding. This mark derepresses PcG target genes in response to stress and developmental signaling (Gehani et al., 2010; Lau and Cheung, 2011) and activates stress response genes via displacement of HDAC corepressor complexes (Sawicka et al., 2014).

However, biochemical studies showed that H3K27me3S28ph is refractory to demethylation by UTX (Sengoku and Yokoyama, 2011) and JMJD3 (Kruidenier et al., 2012). Hence, on one hand H3S28ph might evict PcG proteins and on the other hand it might preserve H3K27me3. H3S28ph is established by the mitotic Aurora B kinase and, like H3S10ph, it is highly enriched during the course of mitosis (Giet and Glover, 2001).

As such, H3S28ph might play a role in PcG repression and epigenetic inheritance.

However, whether this is the only function of H3S28 remains unknown. In particular, the putative in vivo function of its non-phosphorylated state has not been investigated.

To assess the importance of the highly conserved H3S28 residue and to investigate the physiological relevance of mitotic H3S28ph in modulating PcG repression, we have established an in vivo Drosophila model where the endogenous source of histone H3 is replaced by a non-phosphorylatable serine 28 to alanine mutation. Surprisingly, this mutation did not significantly affect mitosis. However, methylation of H3K27 was severely impaired and Polycomb-mediated silencing of Hox genes was partially lost, resulting in homeotic transformations in the adults. The deregulation of PcG silencing associated with H3S28A mutation is independent of active demethylation and cannot be recapitulated upon the loss of Aurora B kinase. These observations are consistent with in vitro experiments showing that PRC2 activity is impaired upon H3 Serine to Alanine mutation at position 28 (H3S28A). Collectively, our data suggest that one main function of H3S28 is to support Polycomb-mediated silencing via PRC2-dependent methylation of H3K27.

Results

All H3K27 methylation states are reduced in the H3S28A mutant

In the Drosophila genome, canonical core and linker histones are encoded by the histone gene unit (HisGU), which is organized as a multicopy gene array residing at a single locus known as the histone gene cluster (HisC). Previous studies showed that embryos carrying homozygous HisC deletion (ΔHisC) die in late blastoderm, after exhaustion of maternally deposited histone reserves (Gunesdogan et al., 2010; McKay et al., 2015). A histone replacement genetic platform has been developed based on the fact that reintroduction of a minimum of 12 copies of wild-type transgenic HisGU is sufficient to rescue lethality of the ΔHisC mutant. Likewise, HisGU carrying different point mutations can be introduced in this system to study the biological functions of specific histone residues and modifications thereof (Gunesdogan et al., 2010). In a previous study a H3K27R mutant was analyzed (McKay et al., 2015; Pengelly et al., 2013). It was reported that the limited number of cell divisions during late embryogenesis was not sufficient to completely replace wild-type, chromatinized maternal histones, resulting in chromatin with a mixture of wild-type and mutant histones. To circumvent this limitation, mosaic analysis of histone mutants was developed with the use of FLP-FRT mediated recombination (Figure S1B). This way, homozygous ΔHisC clones are detected when they are supplemented with 12xHisGU of either WT or mutated histones in larval tissues (Hodl and Basler, 2012; Pengelly et al., 2013). We adopted the same system to generate a H3S28A mutant. In parallel, histone replacement lines carrying WT and H3K27R histone mutants were established as controls.

This system replaces all canonical histones, leaving the endogenous genes coding for the histone variant H3.3 (His3.3A and His3.3B) intact. To simplify the nomenclature, we refer to homozygous ΔHisC clones supplemented with different transgenic 12xHisGU as WT, H3S28A and H3K27R lines, unless otherwise stated.

Consistent with previous reports (Hodl and Basler, 2012; Pengelly et al., 2013), homozygous ΔHisC clones (lacking GFP) induced with a heat shock pulse of FLP died (data not shown). Reintroducing 12xHisGU in the form of WT-H3, H3S28A or H3K27R mutants displayed DAPI and H3 staining patterns similar to the neighboring GFP-positive tissues (Figure 1 and Figure S1C). Importantly, H3S28A clones specifically lost the mitotic H3S28ph signal, while they retained normal levels of H3S10ph (Figure 1B). Likewise and as previously reported (McKay et al., 2015; Pengelly et al., 2013), H3K27R clones almost completely lost the H3K27me3 mark, without affecting neighboring mitotic phosphorylation at H3S28 (Figure S1D). These results imply that the native pool of histone variant H3.3 neither supports mitotic H3S28ph nor H3K27me3. This may be linked to the transcription-coupled deposition pathway of H3.3, which restricts its distribution to active promoters, resulting in genomic distributions that might not be compatible with PRC2 catalysis. Importantly, mitotic H3S10ph, H3S28ph and H3K27me3 signals were not affected in WT clones (Figure 1A, 1C and S1D).

Since H3K27 and H3S28 are adjacent to each other, and as H3S28ph was shown to evict PcG (Gehani et al., 2010; Lau and Cheung, 2011) and to prevent H3K27me demethylation (Kruidenier et al., 2012; Sengoku and Yokoyama, 2011), we asked whether the loss of H3S28ph in H3S28A clones affects H3K27 methylation. Strikingly, H3K27me3 and H3K27me1 were strongly reduced in H3S28A clones (Figure 1D). Considerable reduction of H3K27me2 was also detected. This was in stark contrast to active chromatin marks, where H3K27ac was only mildly decreased and H3K4me3 was unaffected in H3S28A

clones. As a control, WT clones showed normal staining levels indistinguishable from neighboring tissues for all histone marks examined (Figure 1C). These findings show that H3S28 is specifically required for PRC2 function in vivo.

H3S28A and H3K27R mutants both cause derepression of Hox genes

In various PRC2 mutants (Beuchle et al., 2001; Birve et al., 2001; Muller et al., 2002), as well as in the H3K27R histone mutant clones where H3K27me3 levels are compromised (McKay et al., 2015; Pengelly et al., 2013), canonical PcG targets such as Hox genes are derepressed. Hence, we analyzed Hox gene expression in the H3S28A mutant in comparison to the H3K27R mutant.

Endogenous expression of Scr is limited to a patch of cells located at the base of antennal discs (asterisks) and is absent within the antennal disc proper (Abzhanov et al., 2001). Scr was derepressed across the entire antennal disc in in both H3S28A and H3K27R mutant clones (Figure 2A). In line with a previous report (Pattatucci and Kaufman, 1991), low levels of endogenous Scr expression were observed in the adepithelial cells of the 2nd and 3rd leg discs (Figure S2A, asterisks). We observed strong derepression of Scr in H3S28A

and H3K27R mutant clones of these tissues (Figure S2A). Moreover, Scr derepression was occasionally detected in the notum region of wing discs in both histone mutant clones (Figure S2A), similar to what was previously reported in Pc 3 /+ heterozygous mutant background (Pattatucci and Kaufman, 1991).

We also detected strong derepression of Ubx in antennal and wing discs of both H3S28A

and H3K27R mutant clones (Figure 2A and S2A). Ubx derepression was mainly restricted to the wing pouch region at a level comparable to its native expression in the haltere disc.

For Abd-B, strong derepression was detected in H3K27R clones across the entire eye-antennal and wing discs (Pengelly et al., 2013), whereas H3S28A clones showed no obvious derepression in the same tissues (Figure 2A and S2A), suggesting that the partial loss of H3K27me3 was not sufficient to induce Abd-B derepression in H3S28A clones. No changes in expression of Hox genes were detected in the imaginal discs of WT-H3 clones.

In addition to the aforementioned Hox genes, we observed derepression of Antp in the antennal discs of H3S28A and H3K27R mutant clones (Figure 2B and S2A). Similar to the Antp Ns mutant with ectopic expression of Antp in the antennal disc (Figure S2B), GAL4driven overexpression of various Hox genes in the antennal disc are known to silence the antennal selector gene Hth and cause antenna-to-leg transformation (Yao et al., 1999). In agreement with this observation, the H3S28A and H3K27R clones also showed Hth silencing, which was most prominent in the H3K27R mutant (Figure 2B). recombining dUtxΔ mutation (Copur and Muller, 2013) with ΔHisC, we generated double homozygous mutants by recombination over a single FRT element. As shown in Figure S3A, the dUtxΔ, H3S28A mutant clone is depleted of dUtx. However, all H3K27 methylation states are not rescued under such condition (Figure 3A). We also tested the possibility that the H3S28A phenotype is a consequence of the absence of H3S28 phosphorylation. We therefore induced RNAi against the Aurora B kinase in the wing imaginal disc. This resulted in robust depletion of Aurora B (Figure S3B) and mitotic

H3S10ph and H3S28ph (Figure S3C). Consistent with previous reports, loss of Aurora B caused aneuploidy and enlarged nuclei (Giet and Glover, 2001). If the decrease in H3K27me and the loss of Hox gene silencing observed in the H3S28A mutant was depending on the absence of H3S28 phosphorylation, one would expect to observe these phenotypes upon loss of Aurora B. In contrast, we did not observe reduction of H3K27me3 levels, nor Ubx derepression in the knockdown region of wing discs (Figure 3B). Taken together, these data suggest that demethylation by dUtx and the loss of H3S28 phosphorylation per se do not compromise Polycomb silencing in the H3S28A mutant.

H3S28A impairs H3K27-methylation of nucleosomes by PRC2

Together, the results described above suggested the possibility that the serine-to-alanine substitution per se might affects PRC2 activity. To directly test this hypothesis, we performed in vitro histone methyltransferase (HMT) assays using a reconstituted, 4component core Drosophila PRC2 complex that includes Esc, Su(z)12, E(z) and Nurf55 (Muller et al., 2002; Schmitges et al., 2011). As shown by previous reports (Margueron et al., 2009; Schmitges et al., 2011), when assayed on WT nucleosomes supplied with a trans-acting histone peptide carrying H3K27me3, PRC2 activity was robustly enhanced in a dose-dependent manner (Figure 4). Only basal HMT activities were detected when H3S28ph, unmodified or no histone peptides were added to the reaction mixtures.

Interestingly, the stimulatory effect of H3K27me3 was retained when combined with the H3S28A mutation (H3K27me3S28A, Figure 4A). By contrast, the PRC2-stimulatory activity was abolished in context of the H3K27me3S28ph "double mark" (Figure 4A).

Available structural data shows that the stimulatory effect relies on the binding of H3K27me3 to the beta-propeller structure of Esc with the side chain of H3S28 pointing away from the binding side (Margueron et al., 2009; O'Meara and Simon, 2012).

Apparently, this interaction is not affected by the loss of the hydroxyl group in the serineto-alanine substitution in H3K27me3S28A peptides. However, the introduction of the more bulky and charged phosphate group on H3S28 prevents any stimulatory effect. We assume that this is due to hampered binding of the H3K27me3S28ph peptide to Esc.

We then performed PRC2 HMT assays with H3S28A mutant nucleosomes. In this context, enzymatic activity was considerably reduced, with or without supplementation of stimulatory H3K27me3-containing peptides in trans (Figure 4B). Similar to WT nucleosomal substrates, the stimulatory effect of H3K27me3 and H3K27me3S28A peptides was comparable on H3S28A nucleosomes. Therefore, the H3S28A effect can be ascribed to inhibition of PRC2-dependent methylation of H3K27.

Discussion

In this report, we have established a H3S28A histone mutant in Drosophila. In theory, this mutation could have two different effects on the polycomb system. i) it could be that PcG proteins are not evicted from H3K27me3 binding sites in the absence of H3S28ph and, thus, PcG target genes might become ectopically repressed or ii) the mutation at H3S28 or the absence of H3S28ph could compromise PcG functions, resulting in derepression of PcG target genes. We found no evidence for the first possibility, although it is formally possible that H3S28 is phosphorylated under certain developmental conditions or in response to particular stimuli to counteract Polycomb silencing. Instead, our data points to an inhibition of PRC2 activity by the H3S28A mutation. This inhibition is independent of active H3K27 demethylation by dUtx. Besides, RNAi against Aurora B kinase and hence depletion of H3S28ph, did not hamper polycomb silencing. On the other hand, H3S28A nucleosomes proved to be a suboptimal substrate for in vitro PRC2 HMT activity.

Although a 3D structure of the human Ezh2 SET domain is available (Antonysamy et al., 2013), the exact contribution of the hydroxyl group of H3S28 for H3K27 methylation is difficult to deduce from the available data. vSET, the only other protein capable of H3K27 methylation in the absence of PRC2 subunits, does not require H3S28 for catalysis, while it does use H3A29 to define substrate specificity (Wei and Zhou, 2010). Clearly, more work will be required to determine the exact structural and biochemical role of H3S28 in PRC2 catalysis. Consistent with the in vitro HMT assays, in vivo the H3S28A mutant exhibits defects in H3K27 methylation and shows similar, though milder, Hox derepression profiles and transformation phenotypes to those observed in H3K27R mutant flies.

Interestingly, the "KS" module is frequently found in Ezh2 substrates other than K27S28 of histone H3. These include K26S27 of human histone H1 variant H1b (H1.4), K38S39 of the nuclear orphan receptor RORα and K180S181 of STAT3 (Kim et al., 2013; Kuzmichev et al., 2004; Lee et al., 2012; Pasini et al., 2004). Whether these serine residues act similarly to H3S28 to support methylation of the adjacent lysine residue remains unknown.

Of note, some other Ezh2 substrates can be methylated despite the lack of a "KS" module.

These include K26 of mouse histone H1 variant H1e, K49 of STAT3 and K116 of Jarid2 (Dasgupta et al., 2015; Kuzmichev et al., 2004; Sanulli et al., 2015), where the lysine residue is followed by an alanine, glutamate and phenylalanine respectively. Moreover, the link between peptide sequence and enzymology of Ezh2 was shown to differ in nonhistone substrates (Lee et al., 2012). Hence the role of serine following the Ezh2 methylation target amino acid might not be extrapolated to all other Ezh2 substrates, and should be tested individually.

Previous reports revealed discrepancies in Drosophila PcG protein localization on mitotic chromosomes depending on staining protocols and tissue types (Buchenau et al., 1998; Fanti et al., 2008; Follmer et al., 2012; Fonseca et al., 2012). Nonetheless, live imaging of Pc-GFP, Ph-GFP and E(z)-GFP in early Drosophila embryos (Cheutin and Cavalli, 2012; Steffen et al., 2013) all suggested that the majority of these PcG components are dissociated from mitotic chromosomes. Since stress-induced H3S28ph evicts PcG complexes during interphase (Lau and Cheung, 2011; Schmitges et al., 2011), one might expect rebinding of PcG proteins on mitotic chromosomes depleted of H3S28ph. While we did observe loss of Ph from mitotic chromosomes in WT background, we did not observe significant Ph association in H3S28A mutant condition (Figure S4). The reduced levels of H3K27me3 in the H3S28A mutant could contribute to this observation. Alternatively, other mechanisms might operate to dissociate the majority of PcG proteins during mitosis.

The establishment of the histone replacement system in Drosophila has proven to be an important tool to complement functional characterization of chromatin modifiers (Hodl and Basler, 2012; McKay et al., 2015; Pengelly et al., 2013). While depletion of H3K27 methylation, either by mutation of the histone mark writer E(z) or by mutation of the histone itself in the H3K27R mutant, leads to similar loss of Polycomb-dependent silencing (McKay et al., 2015; Pengelly et al., 2013), other histone mutations revealed different phenotypes than the loss of their corresponding histone mark writers. For example, H3K4R mutations in both H3.2 and H3.3, hence a complete loss of H3K4 methylation, did not hamper active transcription (Hodl and Basler, 2012). Also, the loss of H4K20 methylation upon H4K20R mutation unexpectedly supports development and does not phenocopy cell cycle and gene silencing defects reported upon the loss of the H4K20 methylase PR-Set7 (McKay et al., 2015). Here, by comparing the phenotype of Aurora B knockdown and H3S28A mutation in vivo, together with in vitro HMT assay, we specifically attribute the requirement of the unmodified H3S28 residue in supporting PRC2-deposition of H3K27 methylation.

While the published data suggest that H3S28 phosphorylation might be important for eviction of PcG components for derepression of PcG target genes upon stimulatory cues (Gehani et al., 2010; Lau and Cheung, 2011; Sawicka et al., 2014), our data reveals a so far unacknowledged function of the unphosphorylated state of H3S28. We show that serine 28 is required to enable proper methylation of H3K27 by PRC2 and thus to establish Polycomb-dependent gene silencing. Serine 28 of histone H3 is universally conserved in species that display canonical PRC2-dependent silencing mechanisms. Given the fact that we find no major mitotic defects upon its mutation, we propose that the major role of this residue is to ensure optimal PRC2 function while facilitating the removal of Polycomb proteins in response to signals that induce phosphorylation.

Supplemental Information for this article is available online.

Experimental Procedures

Plasmids

Construction of integration plasmids pϕC31attB3xHisGU carrying either wild type H3, H3K27R or H3S28A mutation followed published procedures (Gunesdogan et al., 2010).

Entry vectors pENTR221-HisGU, pENTRL4R1-HisGU, pENTRR2L3-HisGU and their H3K27R mutant derivatives, together with the destination vector pDESTR3R4-ϕC31attB

were obtained from Alf Herzig (Max-Planck-Institut für Infektionsbiologie, Berlin, Germany). Gene synthesis (Eurofins Scientific S.E.) of a DNA fragment carrying the codon exchange AGTAEGCC introduced the H3S28A mutation. Subcloning of the H3S28A DNA fragment into entry vectors and subsequent recombineering followed the procedures published for generating the H3K27R mutant (Gunesdogan et al., 2010).

Drosophila Stocks

Site-specific transgenesis and fly genetics were performed as previously described (Gunesdogan et al., 2010). The histone deficiency mutant (ΔHisC) used in this study (Df(2L)BSC104) was obtained from Konrad Basler (Institute of Molecular Life Sciences, Zurich, Switzerland) in the form of Df(2L) BSC104, FRT40A recombinant (Hodl and

In vitro PRC2 histone methyltransferase assay

Nucleosomes and chromatin arrays were reconstituted by the salt gradient dialysis method as described (Luger et al., 1999), using recombinant Xenopus laevis core histones.

Nucleosomes contained a 187bp long DNA fragment with the 601 DNA sequence at its center (Munari et al., 2012). The histone mutant H3S28A was generated by site-directed mutagenesis. All reconstitutions were analyzed by native gel electrophoresis.

H3 peptides corresponding to H3 amino acids 21-33 were synthesized according to standard procedures. A non-natural tyrosine was added at the C-terminus for quantification.

Histone methyltransferase (HMT) reactions were performed using 1 µg nucleosomes in a total volume of 15 µl for 1 h at 30°C. HMT reactions were carried out in presence of More details on the experimental procedures can be found in the Supplemental Information.

Fig 1 .Fig 7 |

 17 Fig 1. Cell cycle dynamics of H3S10ph, H3S28ph and H3K27me3.................................. Fig 2. Cell cycle dynamics of H3S10ph, H3S28ph and Pc................................................. Fig 3. In vivo RNAi against Drosophila Aurora B.............................................................. 91 Fig 4. H3K27me3, H3K27Ac, Pc and Ubx immunostaining patterns were unaffected upon RNAi against Aurora B ..................................................... Fig 5. Antenna duplication defects in flies upon Aurora B knockdown ............................ Fig 6 | Principle of the Drosophila histone replacement systems ...................................... Manuscript Figures Fig M1 | Validation of the mosaic histone replacement system ....................................... Fig M2 | Strong reduction of H3K27 methylations in H3S28A mutant............................ Fig M3 | Deregulation of Hox and antenna selector genes in H3K27R and H3S28A mutants and their associating transformation phenotypes ........................... 109 Fig M4 | H3S28A nucleosome is a suboptimal substrate for PRC2 activity..................... 111 Fig S1. Homozygous ∆HisC clones are not viable in larval tissues ................................. Fig S2. Validation of the mosaic histone replacement system ......................................... Fig S3. Hox genes derepression profiles in H3S28A and H3K27R clones........................ 114 Fig S4. En and Antp were not derepressed in WT, H3S28A and H3K27R clones ........... 116 Fig S5. Ectopic silencing of endogenous expression of PcG target genes in H3K27R clones ................................................................................................................. Fig S6. Heterogeneous Ubx derepression in H3S28A clones............................................ Fig S7. Ectopic expression of Antp repressed of Hth in Antp Ns antenna disc .................. Fig S8. Nuclear staining pattern of Pc and Ph remained unchanged in H3S28A and H3K27R mutant .........................................................................................................Fig 7 | Combining dUtx∆ with H3S28A mutant did not rescue reduction of H3K27 methylations ......................................................................................................... 124 Fig 8 | Mitotic abnormality in H3S28A mutant clones ..................................................... Fig 9 | FACS-sorting of mitotic populations from asynchronous Drosophila S2 cell culture ................................................................................................ Fig 10 | Using secondary antibodies to prevent H3S10ph contamination of ChIP-seq samples ................................................................................. Fig 11 | Potential role of H3S28ph in the epigenetic inheritance of PcG repression through mitosis ................................................................................... 136

  maintaining the repressive states of key developmental genes. Canonical PcG targets incudes Homeotic (Hox) gene complexes, which are clusters of genes encoding transcription factors specifying cell fates along the anterior-posterior axis of segmented animals. Mutations in PcG genes result in derepression of Hox genes, leading to homeosis characterized by the transformation of one body segment into another. Central to the PcG function is the deposition of the repressive histone mark H3K27me3, which can be regulated by a juxtaposed phosphorylation on H3S28. H3S28ph on one hand counteracts chromatin binding of PcG proteins, while on the other hand it shields H3K27me3 from demethylation. Besides, H3S28ph is deposited by the mitotic Aurora B kinase and, like H3S10ph, it is highly enriched during mitosis. As such, H3S28ph might represent a cellcycle link between PcG repression and epigenetic inheritance. To assess the importance of H3S28ph, we have generated a Drosophila mutant model in which the endogenous source of histone H3 is replaced with a non-phosphorylatable form of H3 (H3S28A) where the serine residue is substituted with an alanine. Mosaic analysis of H3S28A clones in larval imaginal discs revealed a near-complete depletion of H3S28ph on mitotic chromosomes while bulk H3 and H3S10ph levels remained unchanged, as compared to the surrounding tissues or clones supplied with wild type H3 transgenes. Strikingly, all methylation states at H3K27 dropped drastically in H3S28A clones, while active chromatin marks such as H3K4me3 are unaffected. Although nuclear staining patterns of polycomb (Pc) and polyhomeotic (Ph) proteins were not affected, H3S28A clones derepressed Hox genes and adult tissues developed homeotic transformations.

  Yuk Kwong YUNG / CAVALLI Lab Les protéines du groupe Polycomb (PcG) sont des modificateurs chromatiniens qui préservent l'identité cellulaire en maintenant la répression de gènes développementaux clés. Les cibles canoniques des protéines PcG incluent les complexes Homéotiques (Hox), des clusters de gènes codant pour des facteurs de transcription établissant l'identité des cellules le long de l'axe antéro-postérieur des animaux segmentés. Des mutations des gènes PcG conduisent à la dérépression des gènes Hox, entrainant un changement d'identité de segments corporels. Le dépôt de la marque répressive H3K27me3 est le mécanisme fondamental de la répression PcG-dépendante. Cette modification d'histone peut être régulée par la juxtaposition d'une phosphorylation sur le résidu voisin, H3S28. Cette dernière modification, H3S28ph, empêche d'une part la fixation des protéines PcG et, d'autre part, protège la marque H3K27me3 de la déméthylation. H3S28ph est déposée par la kinase Aurora B et, de même que H3S10ph, elle est fortement enrichie au cours de la mitose. H3S28ph pourrait donc établir le lien entre répression dépendante de PcG et mémoire épigénétique à travers la division cellulaire. Afin d'étudier l'importance de H3S28ph, nous avons mis au point un modèle mutant de drosophile dans lequel nous avons remplacé la source endogène d'histone H3 par une forme non-phosphorylable (H3S28A), en substituant le résidu sérine 28 par une alanine. L'analyse des clones mutants H3S28A dans des disques imaginaux d'yeux mosaïques révèle une disparition presque complète de H3S28ph tandis que le niveau global de H3S10ph reste inchangé. De manière frappante, tous les niveaux de méthylation de H3K27 diminuent drastiquement dans les clones mutants H3S28A, tandis que les niveaux des marques de chromatine active telle que H3K4me3 ne sont pas affectés. En conséquence, les gènes Hox sont déréprimés dans les clones mutants H3S28A et les tissus adultes développent des transformations homéotiques, sans que les profils d'immunomarquage nucléaire de polycomb (Pc) et polyhomeotic (Ph) soient affectés.

  in which specific histone modifications acting either individually or in a combinatorial manner to recruit specific trans-acting factors to trigger proper downstream responses. For less than two decades, a number of histone code writers (enzymes catalyze specific histone modifications), readers (proteins with specialized motif recognizing particular histone modification) and erasers (enzymes that remove specific histone modifications) are identified and are implicated in virtually any DNA-templated processes ranging from DNA replication, transcription, DNA repair and more. In Chapter I (p.2-30), a selection of histone modifications implicated in transcriptional control and mitosis are discussed. Apart from histone modifications, nucleosome occupancy, DNA methylation, histone variants, non-coding RNAs and nuclear organizations all together constitute extra layers of chromatin information to regulate various DNA metabolism 1 . Such information plays pivotal roles to instruct cellular differentiation. In metazoans, all cells share the same genomic blueprint and yet specialized cell types are epigenetically defined by the underlying unique transcription programs during differentiation. The polycomb group (PcG) and trithorax group (trxG) proteins, first discovered in Drosophila, represent one of the best-documented examples of epigenetic inheritance 4-8 . Specifically, the Hox genes expression patterns are established during early embryogenesis by specific interactions of transcription regulators. These expression guidelines disappeared at the later stages of development. Since then, PcG proteins maintain repressive chromatin states and trxG sustain active chromatin features of Hox genes through adulthood. Mutations in either PcG or trxG components distort epigenetic memory and lead to homeosis, as characterized by transformation of one body segment identity into another. Introduction of individual PcG components and how their repressive activities are elicited and regulated will be covered in Chapter I (p.31-74). How PcG proteins preserve repressive chromatin states along the cell cycle presents a challenging question that, only until recently, we begin to have molecular description of epigenetic inheritance, please refer to Chapter I (p.75-85) for details. Given

  , immunoprecipitate of ESC was detected with H3-specific HMT activity89 .Chromatography and affinity purification from embryos of either wild-type 89 or FLAG-ESC harboring transgenic flies 88 unequivocally identified Su(z)12, E(z), Nurf55 and Esc as the core components of PRC2. Importantly, E(z) alone is inactive 40 while complete reconstitution of the 4-component dPRC2 from insect cells exhibits robust HMT activity on nucleosomal substrate 88 . The enzyme was not active on H3K27A mutant histone and Edman degradation on PRC2-treated polynucleosomes revealed H3K27 as the radiolabeled residue. These strongly argue H3K27 as the methylation target of dPRC2. Parallel studies in mammalian models using extensive chromatographic fractionation 90 and affinity purification 91 also identified PRC2 as the H3K27 HMT. All studies established that Su(z)12, E(z), Nurf55 and Esc as the conserved core PRC2 components in Drosophila and mammals. Importantly, some accessory factors such as the histone deacetylase Rpd389,92 and AEBP2 90 were also co-purified with PRC2.The highly conserved SET domain of E(z) is essential for H3K27 methylation, mutation of which abolished HMT activity in vitro and failed to restore HOX gene silencing in E(z)null mutant fly 88 . Similarly, heat-inactivation of a temperature-sensitive E(z) 61 mutant results in lost of H3K27me2 ( 90 ) and derepression of HOX genes 93 .Consistent with the in vitro data, lost of function of individual PRC2 component such as E(z), Esc and Su(z)12 lead to reduction of H3K27me and derepression of HOX genes as tabulated below:

suggest

  Rpd3 involvement in H3K27Ac deacetylation 134 . Genetics evidences also suggest a role of Rpd3 in PcG silencing. Ubx PRE-mediated repression of mini-white reporter gene was alleviated in Rpd3 mutant background, the resulting flies showed variegated eye color, as opposed to the strong PcG silencing and white-eye phenotype in wild-type background 136 . Besides transheterozygotes of several Rpd3 alleles and particularly Pc 4 /Hdac1 326 double heterozygotes showed extensive Ubx derepression in larval wing discs 137 . Besides, both Rpd3 and dMi-2 associate as components of the NuRD complex. Importantly, dMi-2 also shows extensive genetic interactions with PcG proteins. Combined dMi-2 and PcG mutants synergize Hox derepression in both embryos and imaginal discs 138 . It is suspected that NuRD/Rpd3-mediated deacetylation of H3K27Ac facilitates PRC2medited H3K27 methylation and gene repressions. This idea is elaborated by a study in mammalian model. In ES cells, a subset of PcG targets with bivalent domains overlap with NuRD, and lost of a NuRD structural component in ES cells reduced PRC2 occupancy at these bivalent promoter 139 , accompanied with a reduction of H3K27me3 level. Genomic distribution of H3K27Ac appeared as broad domains well separated from H3K27me3. In Drosophila cultured Sg4 cells clonally derived from late embryos, Abd-B is active while all others Hox genes within the BX-C cluster are silenced. H3K27Ac covers only Abd-B and neatly separated from the broad repressive domain of H3K27me3 over the more anterior Hox genes 140 . The counteracting nature between H3K27Ac and H3K27me3 is also exemplified in a reverse situation. In parasegment PS7 where only the anterior Hox genes Ubx and abd-A are expressed, they gain H3K27Ac, while the posterior Abd-B is covered with H3K27me3 and remained silenced 114 . In general, binding of TrxG proteins ASH1/TRX-NT are highly enriched with H3K27ac that spreads across both upstream and downstream of target genes. H3K27Ac domains without ASH1/TRX-NT tend to be more confined as narrow peak slightly downstream of TSS. Importantly, active enhancers are particular enriched with H3K27ac, H3K79me3 and Pol II 11 . Lost of individual PRC2 component (Ezh2, SUZ12 or EED) in ES cells 141 or RNAi against E(z) in Drosophila S2 cells 134 depletes H3K27me3 with a concomitant global increase of H3K27Ac. This might suggest H3K27Ac involvement in transcriptional activation. Despite strong association of H3K27Ac with gene activity, its essential role in transcription activation has been challenged with the study of H3K27R histone mutant in Drosophila, where H3K27 can neither be methylated nor acetylation. Give the H3K27R mutant phenocopies the lost of E(z) with the same depression profiles of Hox genes, it suggests that ectopic Hox activation can occur without H3K27Ac. Hence H3K27Ac might function primarily in counteracting PcG silencing, potentially by blocking the lysine residue for methylation. In addition, there is evidence suggesting that H3K27Ac can directly inhibit PRC2 HMT activity 102 .

Fig I . The

 . Fig I. Overview of a selection of histone modifications. A selection of histone modifications and their functions are shown. Of note, many histone marks are juxtaposed to each other as "Phosphor-Methyl" pairs. Of all mitosis-enriched/ specific phosphorylations, those essential for mitotic progression are highlighted. * Mammalian Pcl homologs, not the Drosophila counterparts, recognize H3K36me3. The kinase responsible for mitotic H3T80ph remains unknown.

  Psc transgenic flies were generated and isolated PRC1 complex from Drosophila embryo nuclear extracts that accounted for ~ 2 MDa size. Identifications of the subunit components were hampered by antibodies availability. Nonetheless, western blot analysis detected the presence of Ph, Psc, Pc and sub-stoichiometric amounts of Scm but not E(z) as part of the PRC1 components 200 . Aided by the availability of the first Drosophila genome sequence and better proteincoding gene annotations, together with the advancement of mass-spectrometry, other polypeptides associated with PRC1 were identified, these includes dRing, the DNAbinding factor Zeste and general transcription factor dTAFII. Subsequent biochemical reconstitution defined the core PRC1 particle as Pc, Ph, Psc and dRing 198 . PRC1 is characterized with its ability to stabilize chromatin. Reconstituted PRC1 or recombinant Psc alone counteracts chromatin remodeling mediated by SWI/SNF factors 198 and inhibits transcription in vitro 201 . And examination with electron microscope demonstrated that PRC1 (with or without Ph) could compact 12-mer nucleosomal arrays in vitro, Psc alone also displayed such compaction activities 202 .

  Scm-null clones in wing discs. In contrary, reintroducing MBT deletion or missense mutant of Scm only partially restored Ubx repression; residual Ubx derepression is readily detectable in the wing pouch 233 . Similar to Ph, Scm also possesses a SAM domain at the C-terminus end and Scm-SAM can form helical polymers in vitro. Strikingly, Scm-SAM and Ph-SAM can co-polymerize in vitro 234 . In line with this result, recombinant Scm can co-assemble with the core PRC1 complex 235 . However, only sub-stoichiometric amount of SCM was co-purified with PRC1 complex from Drosophila embryo 200 , indicating that Scm might only interact with a small population of PRC1. Nonetheless, genetics evidence supports the important role of Scm-SAM domain in PcG repressions. Scm-SAM missense mutant that cannot bind full-length Scm failed to rescue Scm-null mutant. Intriguingly, overexpression of a 10-kDa wild type Scm-SAM domain alone can lead to typical PcG transformation phenotypes in adult flies including ectopic sex combs on the second and third legs, antennapedia and wing-tohaltere transformations. It is postulated that the highly abundance SAM domain might compete and titrate interacting partners essential for PcG repressions 235 .

  indicating the essential role for other PRC2 subunit interactions to proper assemble correct catalytic site configuration 262 . Recently, EM reconstruction of the entire human PRC2-AEBP2 complex at 21Å resolution displayed subunit organization among different components (Fig II) 263 . Notably, the SET domain of Ezh2 is in direct contact with the beta-propeller domain of EED and the VEFS (VRN2-EMF2-FIS2-SUZ12) domain of Su(z)12, this might provide mechanistic clue on how these domains integrate the sensing of repressive 113 and active histone marks 264 to allosterically alter the HMT activity of PRC2. Besides, the position of individual subunit can be configured in a way that allows PRC2 to interact simultaneously with two adjacent nucleosomes. As such, EED binding to H3K27me3 of one nucleosome can better orientate H3 tail of the subsequent nucleosome in face of the SET domain of PRC2 263 . This might explain why PRC2 works more efficiently on dense oligonucleosomes array 265 .

(

  Fig II. A proposed possible model for the binding of the PRC2-AEBP2 complex to a dinucleosome.

  Figure modified from Ciferri et al 263 . 3. 4. 3 Su(z)12 (Suppressor of zeste 12) & Nurf55 (Nucleosome Remodeling Factors 55) Su(z)12 is the last core PRC2 components recovered by Drosophila genetics. Homozygous Su(z)12 mutants usually died at L1 or L2 larval stage. Maternal and zygotic null mutant embryos strongly derepress Hox genes with late embryos cuticle showing segmental transformation into A8 identity. Combination of transheterozygous Su(z)12 alleles allow development of pharate adults with severe transformation phenotypes as exemplified by the presence of ectopic sexcombs on both second and third legs, growth of haltere-like wings and development of antennapedia 276 . Biochemical purification identifies Su(z)12 asa core component of PRC2 and it is indispensable for PRC2 HMT activity[88][89][90][91]94 .

  . Similarly, ChIP assay in wing imaginal discs on a PRE element of the Hox gene Ubx (bxd) showed that the lost of Pcl only affects PRE-recruitment of E(z) but not Pho 283 . While simultaneous lost of both Pho and Phol abolished binding of both E(z) and Pcl. This suggests a role for Pcl to mediated PRC2 recruitment downstream of Pho and Phol. The proposed recruitment function was also supported by polytene staining experiment, in which chromatin binding of E(z) is largely abolished upon the lost of Pcl 283 .

  Fig III. Drosophila polycomb group proteins and related components. The five main Drosophila PcG complexes are shown. They include Pho-RC which directly binds DNA and recruits other PcG complexes, dRAF which robustly deposits H2Aubiquitination; PRC2 which catalyzes H3K27 methylations; PRC1 which recognizes H3K27me3 and elicits chromatin compaction and PR-DUB which removes H2Aubiquitination. dUtx is a trithorax group component which demethylates H3K27me3. Auxiliary factors of PRC1 and PRC2 are highlighted in dotted line. Please refer to text for details.

  decades of research in identifying novel PcG components and their associated chromatin-modifying activities, how PcG proteins concertedly elicit gene repression remains a challenging question in the field. In this chapter, potential mechanisms of PcG repressions are discussed, as illustrated in Fig. IV. 4. 1 Counteracting chromatin remodeling PRC1 either purified as immunoprecipitant of FLAG-Ph, FLAG-Psc from Drosophila embryo extracts 200 or reconstituted from recombinant proteins Pc Ph, Psc and dRing 198 , inhibits chromatin remodeling initiated by the SWI/SNF complex on both linear and plasmid nucleosomal arrays. Interestingly, Psc or its paralog Su(z)2 alone also possess such inhibitory function 228 . It is postulated that by resisting nucleosome sliding and hence burying the underlining DNA sequence, PRC1 renders promoter sequence inaccessible to transcription factors for gene activation. Similar study in using mammalian PRC1 components showed that the Pc homolog, M33 (Cbx2) alone or in form of core PRC1 with only Ring1A and Bmi1 was capable of nucleosome remodeling 309 . Notably, Ring1A and Bmi1 protein alone was inactive in resisting remodeling. Hence it is hypothesized that the chromatin stabilization function of Psc in Drosophila is acquired by the Cbx2 subunit in mammals 309 . Deletion mapping of M33 identified a highly basic region required for counteracting remodeling. Intriguingly, components of PRC1 variants carrying similar basic region such as Pc1 can also inhibit remodeling, indicating that it might be a general function of PRC1 309 .4. 2 Interference with the transcription machineryReconstituted chromatin template assembled with Gal4 binding sites positioned and occupied by nucleosome supports transcription upon sequential addition of GAL4-VP16, ATP, SWI/SNF, HeLa nuclear extracts and NTPs. In the reaction, SWI/SNF remodeling exposes GAL4-binding sites and allows access of GAL4-VP16, which recruits transcriptional machinery from the nuclear extracts 201 . With this in vitro transcription assay, reconstituted PRC1 was shown to efficiently repress transcription even at a low molar ratio to the template, and such repression occurred without affecting GAL4-VP16 binding to the template 201 . Importantly, of all individually purified PRC1 components, only Psc and Ph alone can repress transcription on their own, albeit Ph is less efficient. This fits well with the observation that Psc and Ph belong to the PRC1 mutants that give tumorous phenotype and the most severe Hox derepression phenotype 205 . Also Psc and Ph are the only necessary factor of PRC1 to repress a class of non-canonical PcG target genes 205 .

  H3K36me3 demethylase NO66 321 , hence potentially coupling active H3K36 demethylation with productive installment of H3K27me3 during differentiation transitions 71 . Similarly in ES cells, PRC2 was shown to recruit RBP2 (JARID1A) for active H3K4me3 demethylation in exchange for robust deposition of H3K27me3 upon commitment in differentiation 322 .

  Upon addition of the reconstituted 4-component PRC1, the linear arrays constrict into aggregates in which individual nucleosome cannot be resolved. These compact structures also occupy much less space, as measured by the minimal encircling diameter. Notably, Psc alone or PRC1 lacking Ph can induce such compaction 202 . Recently characterized self-associating and nucleosome-bridging activities of Psc might be responsible for its compaction function 323 . In mammalian cells, there are two E(z) homologs known as Ezh2 and Ezh1. Both proteins are interchangeable for PRC2 assembly and possess HMT activity towards H3K27 311 . One marked difference between these two mammalian PRC2 variants is that only PRC2-Ezh1 can induce nucleosomal array compaction. And unlike Drosophila PRC1 and Psc 202 , PRC1-Ezh1 compaction required intact histone tails 311 . Similar compaction activity for Drosophila PRC2 is yet to be determined. In Ring1b -/-mouse embryonic stem cells, global H2Aub level drops substantially, Hox genes derepress and the Hox locus becomes decompacted, as measured by FISH detection of interprobe distance of the affected locus 324 . Intriguingly, reintroduction of WT Ring1b but also the E3-ligase dead allele restores the compact structure of the Hox locus. Hence it supports an H2Aub-independent mode of chromatin compaction effected by the PRC1 complex 324 . Nonetheless, H2Aub was shown to facilitate the binding of linker histone H1 to nucleosomal template in vitro 325 . Recent study suggests that chromatin compaction function is conserved among different components of PRC1 variants 309 . Interestingly, genome-wide profiling of Drosophila PRC1 components identify noncanonical PcG targets whose repressions neither require dRing nor Pc. It suggests Psc and

- 7 elements

 7 Fig IV. Mechanisms of PcG-mediated gene repression. Gene repression by PcG proteins are manifested at different levels, from inhibition of nucleosome remodeling and transcription (A), counteracting active histone marks (B), direct compaction of synthetic chromatin (C) to the organization of repressive topologically associating domains (TAD) (D). Please refer to text for details.

  HOXA1 and HOXB1 become transcriptionally active during which UTX accumulates at their promoter, leading to a decrease in H3K27me3 level. At the same time, PRC2 components are gradually replaced with TrxG proteins and H3K4me3 accumulates at the promoter of these genes125,128 . Similar epigenetic changes occur during cardiac differentiation when UTX is recruited to cardiac enhancers to derepress PcG target genes 342 . Besides differentiation, Utx complexes with transcription factors Oct4, Sox2 and Klf4 to derepress potent pluripotency promoting gene during induced cellular reprogramming346 .Of note, it is demonstrated that the programmed lost of H3K27me3 during early embryonic development can occur independently of Utx and JmjD3, indicating that other demethylation mechanisms might exist, or passive dilution through cell division might be sufficient for lowering H3K27me3 347 . Also there is an emerging idea that the catalytic activity of Kdm6 proteins might only play accessory roles. Given the ability to recognize H3K27me3, they might function as a reader to recruit factor essential for gene activation.

  Fig V. Mechanisms for derepression of PcG target genes. Upon stimulation by environmental factors and developmental cues, PcG targets are derepressed by various mechanisms to trigger downstream responses. H3S28ph evicts prebound PRC1 and PRC2 complexes (A). Similarly, ZRF1 displaces Ring1b and hence PRC1 from their targets genes (B). In resting B cells, Ring1b is essential to sustain transcription of a subset of genes in collaboration with AuroraB kinase, which phosphorylates key factors to counteracts H2A ubiquitination (C). Also, derepressions of PcG targets are accompanied with the lost of PcG cognate histone marks (D). Please refer to text for details.

PRC2

  deposition of H3K27me3 interacts with the seven-bladed beta-propeller structure of Esc/EED WD40 repeats and such interaction stimulates PRC2 HMT activity 113 . Hence H3K27me3-Esc/EED stimulatory interaction constitute a positive feedback loop which might be particular important to sustain H3K27me3 repressive domain during DNA replication. Importantly, some phenylalanine residues from the Esc WD40 repeats are critical to contact methylated lysine. Introduction of the corresponding alanine-substitution mutant failed to rescue esc escl null mutant and those Drosophila larva display diminished amount of global H3K27me2 and H3K27me3 level 113 .

  Such epigenetic memory entails mechanisms to preserve repressive chromatin states from one cell cycle to the next. In this Chapter, potential epigenetic inheritance mechanisms in cope with DNA replication (Fig VII) and mitotic chromosomes condensation (Fig IX) will be discussed.

  PRC1 and Psc during the course of replication. Given that Psc can self associates and bridge nucleosomal templates 323 , a model is proposed in which the potentially displaced Psc proteins during replication fork traversal are retained by self-association to those Psc molecules on the parental chromatin. These retained pools of Psc would reassociate with nascent daughter chromatin once they are emerged from the replication fork. This elegant model emphasizes retention of Psc/PRC1 on chromatin template while allowing the passage of replication fork323,363 . The SAM domain containing PcG protein Ph is capable of oligomerization 225 , such property may act similarly to PSC to accommodate replication fork passage. Supporting this persistent binding model, both PRC1 and PRC2 components are detected on nascent chromatin by both mass-spectrometry 371 and microscopy approaches 362 .

  Fig VIII. Buffer model of H3K27me3 levels for effective gene repression despite cell cycle fluctuation. The model suggests that cell cycle dilution of H3K27me3 is not sufficient to trigger derepression so that it permits post-replication full restoration of parental H3K27me3 level. * Figure modified from Huang et al 365 .

  Owning to potential bias introduced to the immunostaining protocol, live-cell imaging is regarded as the golden standard for detecting chromatin-retention of PcG factors during mitosis. Until now several PcG-GFP fusion lines were established, and all of these fusions rescue their corresponding null mutants, indicating that GFP-fusion does not interfere with the protein function. Pc-GFP fusion rescues the chromodomain mutant of Pc but not the corresponding null mutant, nonetheless, Pc-GFP shares same polytene staining pattern as endogenous Pc and hence it is considered that the GFP-fusion can reflect cellular distribution of Pc 401 . With these GFP-fusion lines, Pho, Pc, Ph, E(z) and Esc (unpublished) were found dissociated from chromatin during the course of entire mitosis in syncytial blastoderm 402 . Of note, the Hox gene expression patterns are not yet specified at such early embryonic stage. FRAP study of Pc-GFP showed that the residence time of Pc on mitotic chromosomes increase in later stage of development 344 . Recently a series of inducible PcG-GFP fusion stably cell lines were analyzed in mammalian model 403 , and unlike the tested Drosophila homologs, canonical PRC1 components Cbx2-GFP, Ring1b-GFP, Phc1-GFP (Drosophila Ph homolog) and Mel18-GFP (Drosophila Psc homolog) showed striking association on mitotic chromosomes with a long residence time 403 . Of note, among other Pc homologs, only Cbx2 show exclusive mitotic retention with long residence time. Other Cbx components show much faster exchange rate on mitotic chromosomes. Importantly, mitotic association of Cbx2 does not

1 .Fig 1 | 2 .Fig 3 |Fig 4 |Fig 5 |

 112345 Fig 1 | Cell cycle dynamics of H3S10ph, H3S28ph and H3K27me3. Immunostaining of H3S10ph, H3S28ph and H3K27me3 of Drosophila S2 cells during interphase and mitosis. Since H3S10ph and H3S28ph are undetectable in interphase, the nuclei boundaries of both channels are highlighted with GRWWHG OLQH 6FDOH EDU ƫP

Fig 6 |

 6 Fig 6 | Principle of the Drosophila histone replacement system.(A) The Drosophila genome encodes canonical nucleosomal and linker histones from a single locus (HisC) at chromosome 2L. Homozygous histone deficiency mutant ( HisC) is not viable, but it can be rescued by introducing 12 copies of transgenic histone gene units (His-GU) in chromosome 3. (B) Mosaic analysis of histone mutant begins with parental cells that are heterozygous for the histone locus. Mitotic crossover occurs at FRT40A with the supply of FLP such that homozygous HisC alleles, either in WT or deficiency forms, can be generated. Homozygous HisC clones are marked by the lack of GFP, while that of WT HisC have double GFP expression. Since chromosome 3 is integrated with 12xHisGU, the endogenous source of H3 can be replacement by different H3 alleles in the HisC clones.

  Reintroducing 12xHisGU in the form of WT-H3, H3S28A or H3K27R mutants rescued ∆HisC clone development. As shown in Fig M1A, these histone replacement clones displayed similar DAPI and H3 staining patterns to the neighboring GFP +ve tissues. Importantly, H3S28A clones specifically lost mitotic H3S28ph signal, while retained normal level of H3S10ph (Fig M1B). Likewise and as previously reported 100 , H3K27R clones almost depleted H3K27me3 level, without affecting juxtaposed mitotic phosphorylation at H3S28 (Fig M1C). This indicates that the native pool of histone variant H3.3 neither supports mitotic H3S28ph nor H3K27me3. This may be linked to the transcription-coupled deposition pathway of H3.3, which restricts its distribution on active promoters, resulting in genomic distributions that might not be compatible with PRC2 catalysis. With a similar logic, it might be inferred that mitotic H3S28ph is not randomly distributed on a global genomic scale and the responsible Aurora B kinase might not overlap with H3.3 over active chromatin. As a control, in WT clones, mitotic H3S10ph, H3S28ph and H3K27me3 were not affected (Fig M1B & C).Strong reduction of all H3K27 methylation states in the H3S28A mutantOwning to the close proximity between H3K27me3 and H3S28, and because H3S28ph is shown to both evict PcG components190,264 and prevent H3K27me demethylations342,343 , we asked if the lost of H3S28ph in H3S28A clones would affect H3K27 methylation. Strikingly, H3K27me3 and H3K27me1 dropped drastically in H3S28A clones (Fig M2A). Considerable reduction in H3K27me2 was also detected. This is in contrast to active chromatin marks, where H3K27ac is only mildly decreased and H3K4me3 is unaffected in H3S28A clones. For all histone marks examined, WT clones showed normal staining levels indistinguishable from neighboring tissues (Fig M2B).

*

  /+ heterozygous mutant background409 . Strong Ubx derepression was also detected in both H3S28A and H3K27R mutant clones in antennal and wing discs(Supplementary Fig S3). In both mutants, Ubx derepression tended to be zonally confined within the wing pouch region at a level comparable to its native expression in the haltere disc. Intriguingly, strong Abd-B derepression was only detected in H3K27R clones across the entire wing 100 and eyeantennal discs while H3S28A clones showed no obvious derepression in the same tissues (Fig M3A & Supplementary Fig S3). Hox derepressions were undetectable in WT clones in all the imaginal discs examined. Similar to the Antp Ns mutant with ectopic expression of Antp in the antennal disc (Supplementary Fig S7), GAL4-driven overexpression of various Hox genes in the antennal disc were known to silence the antennal selector gene Hth and caused antenna-toleg transformation 410 . In the antennal discs carrying H3S28A and H3K27R clones, we observed Antp derepression in addition to the aforementioned Hox genes. These clones also showed Hth silencing, most prominent in the H3K27R mutant (Fig M3B). Consistent with the Hox derepression and Hth silencing phenotypes in the larval tissues, adult flies developing from clonal H3S28A and H3K27R mutant backgrounds displayed antenna-toleg transformation. Wild-type Drosophila adults show distinct antenna segmentation into a1-a3 and arista, as exemplified by w1118 in Fig M3C. Notably, H3K27R mutant displayed enlarged a3 antennal segment, from which massive outgrowth developed with leg-like features resembling the antennapedia phenotype of the Antp Ns mutant. H3S28A mutant showed milder transformations, with smaller a3 segment protrusions and thickening of arista resembling the aristapedia phenotype. In contrast, WT histone replacement flies showed normal antenna structures (Fig M3C). In vitro PRC2 HMT assay was performed by our collaborator -Alexandra Stuetzer* & Wolfgang Fischle* * Laboratory of Chromatin Biochemistry, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany. Because H3S28A mutant phenotypes closely resemble H3K27R homeotic transformations, we wondered if the serine-to-alanine substitution per se would affect PRC2 activity. To address this question, in vitro histone methyltransferase (HMT) assay was performed with reconstituted 4-component Drosophila PRC2 complex that includes Esc, Su(z)12, E(z) and Nurf55. As shown by previous reports 113,264 , when assayed on WT nucleosomes supplied with trans-acting histone peptide carrying H3K27me3, PRC2 activity was robustly enhanced in a dose-dependent manner (Fig M4). On the other hand, basal HMT activities were detected when H3S28ph, unmodified or no histone peptides were added in the reaction mixture. Interestingly, the stimulatory effect of H3K27me3 was retained when combined with H3S28A mutation but was abolished in the form of H3K27me3S28ph "double mark" (Fig M4A). Since the stimulatory effect of H3K27me3 relies on its binding to the beta-propeller structure of Esc 113 , this indicates that the lost of hydroxyl group in the serine-to-alanine substitution in H3K27me3S28A does not affect Esc-H3K27me3 recognition while juxtaposed phosphorylation as H3K27me3S28ph does. Structural data

  Fig M2 | Strong reduction of H3K27 methylations in H3S28A mutant.Wing imaginal disc carrying (A) WT or (B) H3S28A clones were immunostained with DAPI and the indicated histone marks antibodies. Clones were marked by the lack of GFP. H3S28A clones showed strong reduction of all H3K27 methylation states (arrowheads) while active histone mark H3K27ac was mildly decreased and H3K4me3 was unaffected. WT clones showed normal levels of all indicated histone marks, which were indistinguishable from the VXUURXQGLQJ *)3 YH WLVVXHV 6FDOH EDU ƫP

  Fig M3 | Deregulation of Hox and antenna selector genes in H3K27R and H3S28A mutants and their associating transformation phenotypes. (C) Electron micrographs of adult Drosophila heads derived from w1118, Antp Ns and the indicated mosaic histone replacement lines. H3K27R mutant displayed antennapedia transformation while H3S28A mutant showed milder transformation resembling aristapedia. WT histone replacement line and w1118 showed normal segmented antenna structures. Antp Ns was VKRZQ DV D SRVLWLYH FRQWURO IRU DQWHQQDWROHJ WUDQVIRUPDWLRQ 6FDOH EDUV ȝP

Fig S1 .

 S1 Fig S1. Homozygous ǻ+LV& clones are not viable in larval tissues.Mosaic clones carrying homozygous ǻ+LV& (GFP -ve) were induced in eye-antennal (upper row) and wing imaginal discs (lower row). Samples were immunostained with DAPI and the indicated antibodies. Twin clones that co-emerged with ǻ+LV& clones were marked with double expression levels of GFP. 6FDOH EDU ȝP

  from accessing H3K27me3. Since H3S28A shows PcG phenotypes that are similar but milder to that of H3K27R mutant, I wonder if the lack of H3S28ph might render H3K27me3 more susceptible to excessive demethylation. This is in line with the in vitro result showing H3S28A nucleosome is a suboptimal substrate of PRC2. Since both factors could, in principle, lead the drop of H3K27 methylation levels observed in larval H3S28A clones. To test this idea, I recombine a molecular null allele of dUtx (dUtx∆, 2L-31C) 132 into ∆HisC background. Since both deficiency alleles reside on chromosome 2L, a single FRT40A recombination element can be used to generate double homozygous mutant via canonical mosaic analysis technique. As such H3S28A mutant can be assayed in dUtx∆ background. As verified in Fig 7A, H3S28A clones (marked by the lack of GFP) combined with homozygous dUtx∆ (molecular null mutant) or dUtx PB (PiggyBac transposon inserted mutant) both displayed drastic reduction in dUtx immunostaining signal. Owning to similar results obtained with dUtx∆ and dUtx PB , only dUtx∆ allele was used in further experiments.

Fig 7 |Fig 7 |Fig 7 |Fig 8 |Fig 9 |

 77789 Fig 7 | Combining G8W[Ƌ with H3S28A mutant did not rescue reduction of H3K27 methylations. (A) Validation of the lost of dUtx in G8W[Ƌ, H3S28A and dUtx PB , H3S28A clones. Wing imaginal discs with clones homozygous of the combined dUtx, H3S28A mutants were stained with DAPI and the indicated antibodies. Clones were marked by the absence of GFP, which correspond to a drastic reduction of dUtx signal in both G8W[Ƌ +6$ and dUtx PB , H3S28A JHQHWLF EDFNJURXQGV 6FDOH EDU RI IXOOGLVF PLFURJUDSK UHSUHVHQWV ƫP DQG WKRVH RI PDJQLILHG UHJLRQV UHSUHVHQWV ƫP

Fig 11 |

 11 Fig 11 | Potential role of H3S28ph in the epigenetic inheritance of PcG repression through mitosis. Extensive chromosomal contacts including polycomb-related topologically associated domains (TAD) are systemically dismantled during the mitosis, as shown in the comparison between interphase and mitotic Hi-C chromosomal contact heat maps 418 . The repulsive property of H3S28ph against PcG-H3K27me3 interaction might facilitate the lost of PcG proteins from mitotic chromosomes and resolution of their associated chromatin looping structures. At the same time, the lost of PcG proteins at their target sites, such as the Hox clusters, during mitosis might render them susceptible to negative regulators. Hence the demethylase-resistant nature of H3K27me3S28ph might help to preserve H3K27me3 level during mitosis. Besides, the displaced population of Psc might make it available to form alternative complex with Apc11, which act together with APC/Cdc20 to ubiquitinate cyclin B and trigger anaphase onset. As such, chromosome segregation is initiated in a condition when sufficient Psc-Apc11 complex is formed. Although a subpopulation of Psc is retained on mitotic chromosomes, their enrichment at domain borders might indicate a less engagement in mediating chromatin contacts. Global histone dephosphorylations along mitotic exit resume H3K27me3-PcG bindings and gradual restoration of their repressive TADs structures in the next G1 phase. * Hi-C maps are modified from Naumova et al.

  Aurora B kinase was shown to tethering to subcellular compartments by different factors 160 , it remains possible that specific components are required for chromatin-targeting of Aurora B. If this is the case, lost of function approach against such factor might uncouple histone phosphorylations from other essential Aurora B-dependent mitotic functions without the need of histone mutation. histone replacement system largely follows a previous report 104 . Generation of Integration plasmid carrying 3xHisGU requires recombineering of three different entry vectors into a destination vector carrying an attB site. Recombineering follows the instructions of the commercial kit (Invitrogen 12537-023, MultiSite Gateway Three-Fragment Vector Construction Kit)

Since

  Hox gene derepression can induce homeotic transformations, we investigated the phenotypic consequence of the induction of H3S28A or H3K27R clones in adults. Consistent with the Hox derepression and Hth silencing phenotypes in larval tissues, adult flies developing from clonal H3S28A and H3K27R mutant backgrounds displayed antennato-leg transformation. Wild-type Drosophila adults showed distinct antenna segmentation into a1-a3 and arista, as annotated on w 1118 in Figure 2C. Notably, the H3K27R mutant displayed an enlarged a3 antennal segment, from which massive outgrowth developed with leg-like features. This resembled the antennapedia phenotype of the Antp Ns mutant. The H3S28A mutant showed milder transformations, with smaller a3 segment protrusions and thickening of arista resembling the aristapedia phenotype. In contrast, WT histone replacement flies showed normal antenna structures (Figure 2C). In summary, the partial loss of H3K27me3 in the H3S28A mutant induces loss of Polycomb silencing, although the effects were milder than those of the H3K27R mutant, as indicated by Abd-B staining which only shows derepression in H3K27R clones (Figure 2A and S2A). Deregulation of PcG silencing in H3S28A mutant is independent of active demethylation and cannot be recapitulated by depletion of Aurora B kinase We investigated the mechanistic basis for the loss of silencing induced by the H3S28A mutation. Since H3S28ph was shown to evict PcG protein binding to H3K27me3 but also to prevent H3K27me3 demethylation, one explanation for the observed deregulation in PcG silencing in H3S28A mutant could be active H3K27me demethylation upon the loss of H3S28ph. If this were correct, combining dUtx null background with the H3S28A mutation should alleviate the reduction of H3K27me levels and PcG silencing defects. By

0. 8

 8 µCi [methyl-3 H]-S-Adenosyl-L-methionine ( 3 H-SAM) and 325 ng PRC2 (75 nM) in HMT buffer (50 mM Tris-HCl (pH = 8.8), 5 mM MgCl 2 , 4 mM DTT). Recombinant Drosophila melanogaster PRC2 complex, comprised of Su(z)12, E(z), Nurf55 and Esc, was a kind gift of Nicolas Thomä (Friedrich Miescher Institute, Basel, Switzerland) and was purified as described(Schmitges et al., 2011). PRC2 HMT assays in presence of H3 21- 33 peptides in trans contained 5 or 40 µM of peptide. Reactions were stopped by addition of SDS sample buffer followed by separation by SDS-PAGE. Reactions were analyzed by fluorography.

Figure 1 .

 1 Figure 1. H3S28A mutant specifically depletes mitotic H3S28ph and compromises all

Figure 2 .

 2 Figure 2. Deregulation of Hox and antenna selector genes in H3K27R and H3S28A

  Fig.1

Figure S1 ,

 S1 Figure S1, Related to Figure 1. Schematic overview of the system for assaying the function of H3S28 and validation on histone replacement clones in larval imaginal discs. (A) The serine residue at position 28 of the histone H3 protein is conserved. Alignment of the N-terminal 1-50 amino acids of H3 from various eukaryotes. H3K27, the methylation target site of PRC2, is highlighted in red; the conserved H3S28 residue is highlighted in yellow and the variant alanine is highlighted in green. (B) Principle of mosaic analysis of Drosophila histone mutants. Inductions of mosaic histone mutant clones begin with parental cells that are heterozygous for the histone locus. Mitotic crossover occurs at FRT40A with the supply of FLP. And upon appropriate segregation of sister chromatids (bottom box in the right part of the figure), homozygous HisC alleles either in WT or deleted forms can be generated. Because of their co-emergence, they are also referred to as twin clones. Homozygous ΔHisC clones are marked by the lack of GFP, while twin WT HisC clones show twice the GFP expression compared to the surrounding heterozygous cells. Since chromosome 3 is integrated with 12xHisGU, the endogenous source of H3 can be replaced by different H3 alleles in the ΔHisC clones. (C) and (D) Wing imaginal discs of homozygous ΔHisC clones supplemented with 12xHisGU transgenes carrying either H3 WT, H3S28A or H3K27R alleles. Immunostaining with the indicated antibodies is shown; DNA was stained with DAPI. Clones of interest are marked by the lack of GFP signal and are indicated by dashed lines. (C) Comparison of H3 staining in WT (GFP positive) tissue with clones containing WT, H3S28A or H3K27R histone replacement. Note that all histone replacement clones displayed normal H3 staining (red) level similar to the surrounding GFP positive (green) cells. (D) Comparison of H3K27me3 (red) levels in GFP-negative (histone replacement) clones with GFP-positive normal tissue. Note that H3K27R mutant cells (bottom row) were depleted of H3K27me3 despite having normal mitotic H3S28ph signal (green). WT clones supported robust H3K27me3 levels indistinguishable from the surrounding GFP positive cells. For clarity of composite micrographs, GFP is pseudocolored to blue in (D). Scale bars correspond to 10 μm.

Figure S2, related to Figure 2 .

 2 Figure S2, related to Figure 2. Hox gene derepression profiles in histone mutants (H3S28A and H3K27R) clones and Antp Ns . (A) Micrographs showing full-scale imaginal discs described in Figure 2 and main text. For each imaginal disc, a selected region (dashed line) is magnified and displayed below the corresponding micrograph. Asterisks mark zones of endogenous expression of Scr and Ubx. Yellow arrowheads highlight weak Scr derepression occasionally detected in the notum region of wing discs in both H3S28A and H3K27R mutant clones. The white arrowhead indicates ectopic silencing of endogenous Ubx expression in the haltere disc carrying H3K27R clones. Scale bars of wing and eye-antenna discs represent 100 μm and those of leg discs represent 50 μm. (B) Antennal discs of w 1118 (top panel) and Antp Ns (bottom panel) were immunostained with the indicated antibodies. DNA was stained with DAPI. Arrowheads indicate ectopic expression of Antp and silencing of the antennal selector gene Hth. Scale bar corresponds to 100 μm.

Figure S3 ,

 S3 Figure S3, related to Figure 3. Validation of clone induction of dUtxΔ, H3S28A double mutant and of in vivo RNAi against Aurora B kinase. (A) Wing imaginal discs with clones homozygous of the combined dUtxΔ, H3S28A mutants were immunostained with the indicated antibodies. DNA was stained by DAPI. Clones were marked by the absence of GFP, which correspond to a drastic reduction of dUtx signal. (B) and (C) Drosophila wing imaginal discs of L3 larva with nub-GAL4 directed RNAi against Aurora B at the wing pouch compartment marked by GFP and dotted lines. Wing discs were immunostained with the indicated antibodies and DNA was stained with DAPI. Scale bar of full-disc micrograph represents 100 μm and those of magnified regions in (A) represents 40 μm.

Figure S4, related to Figure 2 .

 2 Figure S4, related to Figure 2. Nuclear staining patterns of Ph remain unchanged in H3S28A mutant. (A) Immunostaining of wing imaginal discs for Ph. WT (top row) and H3S28A (bottom row) clones are identified by the lack of GFP signal and are marked by dashed lines. Samples were stained with DAPI and the indicated antibodies. Regions at the clone borders were further cropped and magnified. (B) Selections of mitotic cells from WT and H3S28A clones in wing imaginal discs were compiled to show the distribution of Ph during mitosis in the corresponding backgrounds. Only GFP-positive internal controls of WT clones are shown. Scale bars in (A) and (B) represent 10 μm and 2.5 μm, respectively.

H3 Serine 28 is essential for efficient PcG- mediated gene repression in Drosophila. Thesis Summary Philip Yuk Kwong YUNG / CAVALLI Lab Polycomb

  group (PcG) proteins are chromatin modifiers that preserve cellular identities by

	PHF19	PHD Finger Protein 19
	Pho	Pleiohomeotic
	Pho-RC	Pho Repressive Complex
	PMSF	phenylmethanesulfonylfluoride
	PP1	Protein Phosphatase 1
	PP2A	Protein Phosphatase 2A
	PR-DUB	Polycomb Repressive Deubiquitinase
	PRC	Polycomb group Repressive Complex
	PRE	Polycomb Responsive Element
	Psc	Posterior Sexcomb
	PWWP	Pro-Trp-Trp-Pro motif
	RA	Retinoic Acid
	RNAi	RNA interference
	RNAPII	RNA Polymerase II
	RNF2	Ring Finger Protein 2
	Rpd3	Reduced Potassium Dependency
	RYBP	RING1 and YY1 binding protein
	SAM domain	domain Sterile Alpha Motif domain
	SAM	S-adenosyl Methionine
	Sce	Sexcomb Extra
	Scm	Sexcomb on Midleg
	Scr	Sexcomb Reduced
	SDS	Sodium Dodecyl Sulfate
	SET	Su(var)3-9 E(z) Trithorax
	SILAC	Stable isotope labeling by amino acids in cell culture
	Su(var)3-9	Suppressor of variegation 3-9
	Su(z)12	Suppressor of zeste 12
	SUMO	Small Ubiquitin-like Modifier
	SWI/SNF	SWItch/Sucrose Non-Fermentable
	TAD	Topologically Associating Domain
	TAP	Tandem Affinity Purification
	TBP	TATA-box binding protein
	TFIID	Transcription Factor II D
	TRR	Trithorax-related
	TRX	Trithorax
	TrxG	Trithorax Group
	TSS	Transcription Start Site
	Ubx	Ultrabithorax
	USP16	Ubiquitin Specific Peptidase 16
	UTX	Ubiquitously transcribed tetratricopeptide repeat, X chromosome
	VEFS	VRN2-EMF2-FIS2-SUZ12 domain
	VRN2	VERNALIZATION2
	ZRF1	Zuotin-related Factor 1

XI

Histone

  98

  Importantly, comparative ChIP assay using wing vs. haltere disc samples showed that all PRC1 components and Su(z)12 binds to PREs at the Ubx locus in both transcriptionally repressed (wing discs) and active states (haltere discs) of Ubx 122 . Similarly, recent ChIPseq experiment using FACS-sorted parasegment PS7-specific nuclei showed that Pc, Ph and Su(z)12 are recruited to actively transcribed Hox genes devoided of H3K27me3 114 .

	This indicates that genomic bindings of Pc and Ph are not the absolute determinant of gene
	repression and they can be recruited to PRE independent of H3K27me3. Since Su(z)12 is
	recruited to PRE without H3K27me3, it might suggest PRC2 associates with chromatin as
	a poised state 123 , and its HMT activity depends on local chromatin environment 114 (See
	Chapter I, p.73).
	. One strong exception is
	exemplified in mammalian embryonic stem cells where both functionally antagonistic
	modifications coexist as "bivalent" domains covering promoter regions of key
	developmental genes 117 . It is proposed that such bivalent signature on one hand represses
	lineage commitment genes to sustain pluripotency, and at the same time potentiates them at
	a poised state competent for robust activation 118 . Recent biochemical effort clarifies the
	molecular nature of bivalency where repressive H3K27me3 and active marks reside on
	separate H3 tails as asymmetrically modified nucleosomes 119-121 .

  132 ! Importantly, only homozygous dUtx1 exhibits very low adult survival rate (<10%), this is in total contrast to other transheterozygous allelic combination, including homozygous dUtx∆, all of which have a adult survival rate over 60%! In light of these discrepancies, it is worth suspecting a 2 nd site mutation in the dUtx 1 background; likely reside on the same chromosome arm as dUtx132 . It is also possible that dUtx 1 is a recessive gain-of-function allele where the premature STOP codon in the JmjC domain encodes a truncated protein that interferes with normal cellular functions.The latest study suggests that the dUtx demethylation activity on H3K27me3 is specifically required during early embryogenesis 132 . Over 60% of homozygous zygotic mutants of dUtx∆ develop into adulthood without recognizable structural or cuticle defects, despite having a short life span. Intriguingly, maternal and zygotic dUtx∆ homozygotes mostly

	Discrepancy on the role of dUtx as a tumor suppressor
	Using the MARCM system (Mosaic analysis with a repressible cell marker) 133 , clones of
	homozygous dUtx 1 mutant showed over-proliferation in the eye-antenna disc and the
	corresponding adult eyes show rough and disorganized ommatidia development. Based on

drivers, both showed a mild increment of H3K27me3 level in cells and adult protein extracts, respectively. Importantly, lethality rates and adult phenotypes of such RNAi experiment were not mentioned! Since once would expect ectopic repression of PcG targets or Trx-like transformation phenotypes as a result of a global increase of H3K27me3 level. Subsequent mosaic analysis dUtx 1 mutant (with a premature STOP codon within the JmjC domain) clones also find increased H3K27me3 level in eye disc 130 and testis 131 . However, re-evaluation of the mosaic analysis of dUtx 1 mutant and a newly generated molecular null mutant dUtx∆, suggested that global levels of H3K27me3, H3K27Ac and H3K4me1 were indistinguishable from neighboring WT cells in wing discs 132 . Besides, western blot analysis using serially diluted samples from larval imaginal discs and brain extracts of dUtx 1 /Df(2L)BSC143 and dUtx∆/Df(2L)BSC143 transheterozygotes, failed to detect any noticeable differences in global levels of H3K27me3, H3K27Ac and H3K4me1, when compared to control wild-type extracts 132 . Discrepancy on the effect of dUtx on H3K27me3 level: Gain of function study Western blot analysis detected a drop of H3K27me3 level in adult extracts upon ubiquitous overexpression of dUtx with strong tub-Gal4 driver 16 . However, en-Gal4 driven overexpression of dUtx in the posterior compartment of wing disc fails to cause any noticeable changes in H3K27me3 level 130 . these data, the authors claimed that dUtx as a potential tumor suppressor 130 . However, MARCM study of the same dUtx 1 allele or another molecular null allele dUtx∆ failed to show over-proliferation died at the end of embryogenesis with ectopic repression of Abd-B, those survived to L3 larval stage displayed patchy repression of Ubx in haltere discs. Unfortunately for both conditions of ectopic repression of Hox genes in embryos and imaginal discs, the level of H3K27me3 is not determined 132 . It is then difficult to reconcile these phenotypes with the suspected demethylase activity of dUtx. Notably, dUtx∆ oocyte fertilized by WT dUtx sperm completely rescued lethality with a significant fraction (~30%) of the adult males displayed lost of A5 abdominal tergites pigmentation, indicating an A5 to A4 transformation reminiscent of trx-like phenotype 132 . Based on these data, the authors propose that dUtx is specifically required during early embryogenesis to oppose PcG silencing and it's role in H3K27me demethylation diminishes in later stage of development 132 . This early time window requirement of dUtx might explain the fact that no mutation on dUtx has even been identified as modifier of PcG silencing in classical genetic screens. Alternatively, the lack of trx-phenotypes in zygotic dUtx∆ mutant might suggest the existence of another H3K27me demethylase operates in later stage of development.

Effect on H2Aub in transcription

	In mouse ES cells of Ring1A and Ring1B double knockout background, reintroducing
	catalytically dead Ring1b transgene restored PRC1 occupancy on its target genes and
	restored chromatin compaction of the Hoxb cluster, despite the lack of H2Aub. However
	these genes nevertheless gained H3K4me3 and Pol II binding, and derepressed. This
	suggests that the H2Aub modification is crucial for gene repression 150 . Supporting the
	reciprocal relationship between H2Aub and active chromatin marks, isolated
	mononucleosomes from Drosophila did not show coexistence of H2Aub and H3K36me2 77 .
	Besides, it was shown that chromatin template reconstituted with H2Aub was inactive
	substrates for MLL3-mediated H3K4 di-and trimethylation and was refractory to
	transcription initiation in vitro 151 . More recently, H2Aub was show to inhibit a broad panel
	of H3K36 methyltransferases 152 (See Chapter I, p.55).
	. Using Bmi1-null and wild-
	type MEF cells, ChIP-seq analysis was performed to define the genomic distribution of
	Bmi1-dependent H2Aub. H2Aub level peaks over TSS of target genes and is enriched in
	those promoters covered with H3K27me3. Notably, there are still substantial fraction of
	H2Aub regions did not overlap with H3K27me3. H2Aub target genes exhibit low very
	level of transcription and are derepressed upon the lost of Bmi1 149 . In mouse ES cells,
	ChIP analysis defines a set of H2Aub target genes that are significantly occupied by
	Ring1B and H3K27me3. These include the Hox genes and many key developmental
	regulators 150 . At present, there is no data available regarding to the genomic distribution
	profile of H2Aub in Drosophila.

  147,[156][157][158][159] , some DUB enzymes are involved in the derepression of PcG

	target genes.
	In mouse ES cells, the H2A-deubiquitinase USP16 displays an anti-correlated genome-
	distribution profile with Ring1b. USP16 is enriched with active genes while Ring1b and
	H2Aub accumulate on repressed genes such as the Hox clusters. ES cells lacking USP16
	displayed genomewide increase of H2Aub occupancy and they failed to differentiate. It
	was found that USP16 is relocated to a number of novel targets during which a global H2A
	deubiquitination occurs, which is important to activate developmental regulators to commit
	differentiation 348 .
	Biochemical data showed that H2Aub nucleosomal template did not support H3K4
	methylation by MLL3 and it is incompetent for in vitro transcription initiation 151 . During
	hepatocyte regeneration, global H2Aub level drops. And gene activations were associated
	with the lost of H2Aub and a reciprocal gain of H3K4me2 and H3K4me3. Based on
	differential gene expression data during hepatocyte regeneration, USP21 was identified as
	the H2A deubiquitinase that specifically acted on nucleosomal H2Aub to promote gene
	activation 151 .
	2A-DUB/KIAA1915/MYSM1 is yet another H2Aub-specific deubiquitinase. It was
	identified as a positive regulator for Androgen-signaling genes, which are also subjected to
	PcG repression 349 . Interestingly, 2A-DUB forms a complex with a histone acetyltransferase
	known as p300/CBP-associating factor (p/CAF), and its deubiquitination efficiency is
	enhanced on acetylated nucleosomal template 350 . Besides, H2AK119R mutant associates
	with less linker histone H1 when compared to WT H2A. Hence it is suspected that the lack
	of H2Aub might favors H1-phoshorylation and eviction from chromatin. Upon addition of
	androgen-agonist, the downstream signaling target genes PSA is derepressed. During this
	process, androgen receptor together with 2A-DUB and p/CAF are recruited to PSA
	promoter, on which the level of H3 acetylation increases with a concomitant drop of
	H2Aub and H1 occupancy 350 .

  , by isolating tissues with differential expression states of the Hox gene Ubx, it was shown that PRC1 and PRC2 components bind to PRE of Ubx disregarded of its expression status 122 . Similarly, FACS-sorting parasegment-specific nuclei from Drosophila embryos with differential Hox expression states demonstrated the presence of Su(z)12 at active Hox genes without apparent level of H3K27me3 114 . More recently, it was shown in mouse ES cells that nascent RNA transcripts emanating from PcG binding site can inhibits PRC2 HMT activity without affecting their binding 123 . As such, it becomes evident that the PcG

occupancy at target sites might not necessarily coincide with the presence of their cognate histone modifications. It is thus proposed that, like RNA polymerase, PcG complexes can be held in a poised state competent for robust establishment of repressive chromatin states upon the presence of transcriptional repressors 123 .

  Drosophila histone genes encoding canonical nucleosomal and linker histones are organized as a histone gene unit (His-GU) as shown in Fig 6A.There are 23 annotated His-GU in the haploid Drosophila genome, all of them cluster at a single locus at chromosome 2L, known as the Histone complex (HisC). With the availability of two FRT-containing pelement transposable insertions flanking HisC, the entire histone cluster is deleted via FLP-FRT recombination, resulting the histone deficiency (∆HisC) allele known as Df(2L)BSC10436 . Homozygous ∆HisC mutant dies during embryogenesis after exhaustion of maternally deposited histone pools 104 . However it has been shown that a total of 12 copies of wild-type His-GU transgenes are sufficient to rescue lethality associated with histone deficiency 104 . Using site-specific plasmid integration with the ΦC31 integrase system 406 , the transgenesis was achieved by inserting plasmid with 3xHisGU into two genomic loci on chromosome III. One located on chromosome 3L at 68E, and the other resides at 86Fb. Hence recombinant carrying 3xHisGU at both 68E and 86Fb would attain 12xHisGU at homozygous state. With the same transgenesis scheme, transgene carrying wild type or mutant HisGU can generate the corresponding histone replacement lines 104 .

  Because of their large sizes, it is difficult to verify plasmid purification by midi-or maxipreps. Hence it is useful to verify plasmid identity by restriction digestion with NcoI-BastXI, which yield restriction fragments of 8370 bp, 2x 5082 bp, 3603 bp abd 641 bp.

	Drosophila Transgenesis

ϕC31-mediated site-specific transgenesis is performed by the BestGene Inc. company, using the attP landing sites reside at cytological location 68E and 86Fb. Positive and stable transformants are selected based on the mini-white marker, which is subsequently removed using Cre-Lox recombination. Hence the resulting 3xHisGU transgenic lines at both 68E and 86Fb lack phenotypic markers, detection of transgenes insertions require PCR. 68E and 86Fb transgenic lines carrying the same HisGU alleles are recombined (confirmed by PRC) and balanced. The resulting line would have 12xHisGU on chromosome III at homozygous state.

sorting of mitotic population from S2 cell culture

	Drosophila S2 cell culture conditions follow standard guidelines from the Drosophila
	RNAi Screening Center (DRSC, Harvard Medical School). S2 cells are cultured in
	Schneider's medium (Sigma S0146) supplemented with heat-inactivated 10% Fetal Bovine
	Serum (Life technologies, 10500064) and Penicillin-Streptomycin (Invitrogen, 15070-
	063).
	S2 culture is kept at a cell density within 1-5 x 10 6 /ml in order to attain relatively higher
	mitotic index (1-2%) from asynchronous population. Prepare sufficient culture accounts
	for 120-200 x 10 6 cells. Harvest cells by scrapping and resuspend them in aliquots. Fix
	cells in medium at final 1% formaldehyde (Thermo Scientific, 28908) for 10 min at room
	temperature. Quench fixation by adding glycine to a final concentration of 130mM,
	incubate for 5 min at room temperature. Wash cells with ice-cold PBS and permeabilize at
	0.5% saponin/PBS for 15 min at 4°C. Block cells with 1% BSA/0.5% saponin/PBS for 30
	min at 4°C. Pellet cells and adjust cell density at 20 x 10 6 cells/ml with 0.5% saponin/PBS
	and add anti-H3S10ph (Millipore, Mouse Monoclonal, 05-806) at 0.5ug/ml, incubation for
	1 hr at 4°C. Similarly, add secondary antibody donkey anti-mouse Alexa-647 at 2ug/ml
	incubation for 1 hr at 4°C. Allow thorough washes with 0.5% saponin/PBS after incubation
	of primary and secondary antibodies. Adjust sample to 20 x 10 6 cells/ml, protect them from
	light and set on ice before use. When the cell sorter is ready, pass sample 10 times though
	18-20G needle and pass them through 40 µm cell strainer (BD Falcon, 352340) to disrupt
	cell clumps.
	3 H]-S-Adenosyl-L-methionine ( 3 H-SAM) and 325 ng PRC2 The following inhibitors are applied after fixation: Complete Protease inhibitor, EDTA
	(75 nM) in HMT buffer (50 mM Tris-HCl pH 8.8, 5 mM MgCl 2 , 4 mM DTT). Free (Roche, 04693132001), 1 mM PMSF, 2 mM EDTA, PhosSTOP phosphatase
	Recombinant Drosophila melanogaster PRC2 complex, comprised of Su(z)12, E(z), inhibitors (Roche, 04906837001).
	Nurf55 and ESC, was a kind gift of Nico Thomä (FMI Basel) and was purified as
	described 264 . PRC2 HMT assays in presence of H3 21-33 peptides in trans contained 5 or 40
	µM of peptide. Reactions were stopped by addition of SDS sample buffer followed by
	separation by SDS-PAGE. Reactions were analyzed by fluorography.

FACS-

Mosaic analysis of histone mutants compatible with live-imaging

  PcG) proteins are epigenetic regulators essential for repression of key developmental genes. Canonical targets of PcG proteins include the Hox genes, which specify the identities of body segments along the anterior to posterior axis. PcG mutants fail to maintain repressive chromatin states, leading to derepression of Hox genes and to homeotic transformations(Di Croce and Helin, 2013; Grossniklaus and Paro, 2014;

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Drosophila histone mutant model

Acknowledgements

We are grateful to Natalia Azpiazu, Konrad Basler, Walter Gehring, David Glover, Peter J. Harte, Alf Herzig, Martina Hödl, Ginés Morata, Jürg Müller, Nicolas Thomä and Feng Tie, for providing research reagents. Boyan Bonev, Thierry Cheutin, Satish Sati and Bernd Schuettengruber provided helpful comments on the manuscript. We thank Chantal Cazevieille for assistance with scanning electron microscopy and in the use of the Montpellier RIO imaging facility. Research in the laboratory of G.C. was supported by grants from the European Research Council (ERC-2008-AdG No 232947), the CNRS, the European Network of Excellence EpiGeneSys, the Agence Nationale de la Recherche, and the Fondation ARC pour la Recherche sur le Cancer. Funding of P.Y.K.Y. was supported by the Croucher Foundation and La Ligue contre le Cancer. Research in the laboratory of W.F. was supported by the Max Planck Society.

Drosophila Genetics

Mosaic analysis and Immunostaining

Egg depositions were allowed over a 24 hr time window at 25°C. Larva developed for two days before they were heat shocked at 37°C for 1 hr. L3 larva emerged 3 days after heat shock, and they were selected for the lack of green balancer before dissection. Inverted larva heads were fixed in 4% formaldehyde/PBS for 20 min at room temperature. After washing with PBS, the samples were permeabilized twice with 0.3% TritonX-100/PBS for 30 min at room temperature. After blocking with 3% BSA 0.03% TritonX-100/PBS for 1 hr, the samples were incubated with primary antibodies in 0.03% Triton X-100/PBS overnight at 4°C. Samples were then incubated with Alexa (Invitrogen) fluorophores conjugated secondary antibodies before DAPI staining. Thorough washes were applied in between incubations. Imaginal discs were carefully removed and mounted with ProLong

Mosaic analysis and Immunostaining

Egg depositions were allowed over a 24 hr time window at 25°C. Larva developed for two days before they were heat shocked at 37°C for 1 hr. L3 larva emerged 3 days after heat shock, and they were selected for the lack of green balancer before dissection. Inverted larvae heads were fixed in 4% formaldehyde in PBS for 20 min at room temperature. After washing with PBS, the samples were permeabilized twice with 0.3% TritonX-100 in PBS

for 30 min at room temperature. After blocking with 3% BSA 0.03% TritonX-100 in PBS for 1 hr, the samples were incubated with primary antibodies in 0.03% Triton X-100 in PBS overnight at 4°C. Samples were then incubated secondary antibodies conjugated to Alexa (Invitrogen) fluorophores before DAPI staining. Thorough washes were applied in between incubations. Imaginal discs were carefully removed and mounted with ProLong Gold (Invitrogen) antifade reagent. Micrographs were acquired on a Zeiss LSM 780 inverted confocal microscope. Raw micrographs were processed with ImageJ and assembled using Adobe Illustrator.

Scanning electron microscopy

Adult fly heads were fixed as described (Wolff, 2011) using the critical-point-dried method. They were then sputter-coated with a 10 nm thick gold film and examined under a scanning electron microscope (Hitachi S4000) using a lens detector with an acceleration voltage of 10kV at calibrated magnifications.

Conflict of interest

The authors declare that they have no conflict of interest. See also Figure S3. )LJXUH

Supplemental Information

Supplemental Figures S1-S4

Supplemental Experimental Procedures

Drosophila Stocks

The following fly lines were used in this study y, w; P{Ubi-GFP.D}33, P{Ubi-GFP.D}38, FRT40A (Bloomington #BL5189) y, w, hsp70-flp; Df(2L) BSC104, FRT40A/CyO, twist-Gal4, UAS-GFP; MKRS/TM6B (Hodl and Basler, 2012) y, w; dUtxΔ, FRT40A/CyO, twist-Gal4, UAS-GFP (Copur and Muller, 2013) y, w, hsp70-flp122; P{Ubi-GFP.D}33, P{Ubi-GFP.D}38, FRT40A; 6xHisGU y, w, hsp70-

Mosaic analysis and Immunostaining

Primary antibodies used for immunostaining in this study: Hox antibodies were purchased from the Developmental Studies Hybridoma Bank (DSHB). Mouse anti-Scr (DSHB 6H4.1, 1:20), mouse anti-Antp (DSHB 8C11, 1:20), mouse anti-Ubx (DSHB FP3.38, 1:20), mouse anti-Abd-B (DSHB 1A2E9, 1:10), mouse anti-En (DSHB 4D9, 1:20), mouse anti-H3 (ActiveMotif 39763, 1:500), mouse anti-H3S10ph (Millipore 05-806, 1:1000), rat anti-H3S28ph (Millipore MABE76, 1:1000), rabbit anti-H3K27me3 (Millipore 07-449, 1:500), rabbit anti-H3K27me2 (Upstate 07-452, 1:500), rabbit anti-H3K27me1 (Millipore 07-448, 1: 250), rabbit anti-H3K27ac (Abcam Ab4729, 1:500), rabbit anti-H34me3 (Millipore 04-745, 1:500), rabbit anti-Pc ( (Grimaud et al., 2006), 1:500), rabbit anti-Ph ( (Grimaud et al., 2006), 1:500), guinea pig anti-Hth (a gift from Ginés Morata, 1:200), rabbit anti-Aurora B (a gift from David Glover, 1: 200), rabbit anti-dUtx (a gift from Feng Tie and Peter J. Harte, 1: 200) chicken anti-GFP (Invitrogen A10262, 1:500).

Supplemental References

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Supplemental Information Inventory

Supplemental Figures S1-S4 x List of primary antibodies used in this study.

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