bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Title: H3.1K27me1 maintains transcriptional silencing and genome stability by preventing GCN5-mediated

Authors: Jie Dong1#, Chantal LeBlanc1#, Axel Poulet1#, Benoit Mermaz1, Gonzalo Villarino1, Kimberly M. Webb2, Valentin Joly1, Josefina Mendez1, Philipp Voigt2 and Yannick Jacob1*

Affiliations: 1 Yale University, Department of Molecular, Cellular and Developmental Biology, Faculty of Arts and Sciences; 260 Whitney Avenue, New Haven, Connecticut 06511, United States.

2 Wellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, United Kingdom.

# These authors contributed equally to this manuscript. * To whom correspondence should be addressed. Email: [email protected]

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1 Abstract 2 In plants, genome stability is maintained during DNA replication by the H3.1K27 3 methyltransferases ATXR5 and ATXR6, which catalyze the deposition of K27me1 on replication- 4 dependent H3.1 variants. Loss of H3.1K27me1 in atxr5 atxr6 double mutants leads to 5 heterochromatin defects, including transcriptional de-repression and genomic instability, but the 6 molecular mechanisms involved remain largely unknown. In this study, we identified the conserved 7 histone acetyltransferase GCN5 as a mediator of transcriptional de-repression and genomic instability 8 in the absence of H3.1K27me1. GCN5 is part of a SAGA-like complex in plants that requires ADA2b 9 and CHR6 to mediate the heterochromatic defects of atxr5 atxr6 mutants. Our results show that 10 Arabidopsis GCN5 acetylates multiple residues on H3.1 variants in vitro, but that H3.1K27 11 and H3.1K36 play key roles in inducing genomic instability in the absence of H3.1K27me1. Overall, 12 this work reveals a key molecular role for H3.1K27me1 in maintaining genome stability by restricting 13 histone acetylation in plants.

14 Keywords (3-10): 15 Transcriptional de-repression, genome instability, histone and acetylation, 16 heterochromatin

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17 Introduction 18 Genome and epigenome instability have been implicated in many human diseases, including cancer 19 and neurodegenerative disorders. In proliferating cells, key mechanisms are required to properly copy 20 DNA and different epigenetic states of the genome in the context of ongoing transcription and DNA 21 repair. replication is therefore a complex molecular operation that can lead to genomic 22 rearrangements and other types of deleterious mutations in the absence of mechanisms preserving 23 genome stability (1, 2).

24 Epigenetic information plays multiple regulatory roles during S phase of the cell cycle that are 25 required to maintain genome stability in eukaryotes. In plants, one of the most well-studied genome 26 maintenance pathways involves the histone post-translational modification (PTM) H3K27me1. The 27 loss of H3K27me1 results in the loss of transcriptional silencing at heterochromatic loci and defects 28 in the structural organization of heterochromatin (3, 4). In addition, decreased levels of H3K27me1 29 induce genome instability characterized by the presence of an excess of repetitive DNA (e.g. 30 transposons) mainly in pericentromeric heterochromatin (hereafter referred to as heterochromatin 31 amplification) (5). H3K27me1 is catalyzed by the plant-specific histone methyltransferases ATXR5 32 and ATXR6 (ATXR5/6), which are recruited to replication forks during DNA replication (3, 6, 7). 33 Biochemical and structural studies have revealed that the SET domains of ATXR5/6 can methylate 34 replication-dependent H3.1 variants, but not replication-independent H3.3 variants (8). These 35 observations indicate that ATXR5/6 maintain H3K27me1 by methylating newly synthesized H3.1 36 variants (H3.1K27me1) during DNA replication, which protects against transcriptional de-repression 37 and heterochromatin amplification. The precise molecular mechanism responsible for 38 heterochromatin amplification in the absence of H3.1K27me1 remains unknown. However, a 39 previous study suggested that transcriptional de-repression in the heterochromatin of atxr5/6 double 40 mutant plants is the cause of the genomic instability phenotype, potentially by inducing collisions 41 between the transcription machinery and replication forks, and/or through R-loop formation (9). 42 Based on this model, it is predicted that ATXR5/6-catalyzed H3.1K27me1 plays a key role in 43 blocking transcriptional activity in the heterochromatin of plants.

44 Many PTMs on function as recruitment signals for chromatin “reader” proteins, which 45 promote specific cellular activities like transcription at genomic regions enriched in these histone

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46 PTMs (10). Multiple studies have shown that methylation at H3K27 regulates transcriptional activity 47 through various mechanisms, which are related to the specific methylation level (i.e., me1, me2 or 48 me3) at K27. For example, H3K27me3 is involved in the recruitment of the repressive PRC1 complex 49 in animals (11), and this role is conserved in plants (12). In contrast to H3K27me3, H3K27me1 and 50 H3K27me2 are not as well characterized in animals, but they have specific effects on the regulation 51 of transcriptional activity that do not appear to involve recruitment of chromatin readers. In mouse 52 embryonic stem cells (ESCs), H3K27me2 is present on the majority of total in chromatin 53 and safeguards against unintended transcription by blocking CBP/p300-mediated H3K27 acetylation 54 (H3K27ac) at non-cell-type-specific enhancers (13). In contrast, H3K27me1 is present at less than 55 5% of total H3s in ESCs, and is associated with transcriptionally active genes and contributes to their 56 expression (13). However, the mechanism by which H3K27me1 performs this function remains 57 unknown. Predicting the role of ATXR5/6-catalyzed H3K27me1 in plants based on comparative 58 analysis with H3K27me1/me2 in animals is challenging, as it shares the same methylation level of 59 transcriptionally-permissive H3K27me1, but its function in heterochromatin silencing in plants 60 suggests properties related to H3K27me2. An additional similarity between plant H3K27me1 and 61 animal H3K27me2 is that these histone PTMs are widely distributed and very abundant in their 62 respective genomes. In Arabidopsis, H3K27me1 was estimated to be present on more than 50% of 63 total H3 in inflorescence tissues (14), and it is enriched in transcriptionally silent regions of the 64 genome (5). These observations suggest that H3.1K27me1 in plants may serve to block H3.1K27ac, 65 providing a potential molecular mechanism for the role of ATXR5/6 in preventing transcriptional de- 66 repression and genomic instability in plants.

67 In this work, we identify the conserved histone acetyltransferase GCN5 as a mediator of 68 transcriptional de-repression and heterochromatin amplification in the absence of H3.1K27me1 in 69 plants. GCN5 cooperates with the transcriptional co-activator ADA2b and the chromatin remodeler 70 CHR6 to induce these heterochromatic phenotypes. Our results also show that H3.1K36 plays a key 71 role in inducing genome instability and transcriptional de-repression in the absence of H3.1K27me1, 72 and that H3.1K27me1 interferes with GCN5-mediated acetylation at both H3.1K27 and H3.1K36. 73 Overall, these results demonstrate the key role played by GCN5-mediated histone acetylation in 74 contributing to the heterochromatin phenotypes observed in the absence of ATXR5 and ATXR6 in 75 plants.

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76 Results 77 Transcriptional de-repression and heterochromatin amplification in the absence of 78 H3.1K27me1 are suppressed in gcn5 mutants 79 One mechanism by which H3.1K27me1 might interfere with transcription in heterochromatin of 80 plants is by preventing the deposition of H3.1K27ac, as methylation and acetylation at H3K27 have 81 been shown to act antagonistically in other biological systems (15, 16). H3K27ac is catalyzed by 82 multiple histone acetyltransferases in eukaryotes, including the widely conserved protein GCN5 (17- 83 21). The Arabidopsis genome contains a single gene encoding a GCN5 ortholog (22). To assess if 84 Arabidopsis GCN5 mediates the heterochromatin phenotypes associated with loss of H3.1K27me1, 85 we created an atxr5/6 gcn5 triple mutant by crossing a T-DNA insertion allele (SALK_030913) of 86 GCN5 into the hypomorphic atxr5/6 mutant background (Supplemental Figure 1A) (3). Flow 87 cytometry analyses showed strong suppression of heterochromatin amplification in the triple mutant 88 as represented by the loss of the characteristic broad peaks corresponding to 8C and 16C 89 endoreduplicated nuclei in atxr5/6 mutants (Figure 1A). We also observed by microscopy that the 90 heterochromatin decondensation phenotype of atxr5/6 plants is suppressed in the atxr5/6 gcn5 triple 91 mutant (Figure 1B, Supplemental Figure 1B). A role for GCN5 in inducing genomic instability in 92 atxr5/6 was confirmed by using a different mutant allele of gcn5 generated by temperature-optimized 93 CRISPR/Cas9 (Supplemental Figure 1A, C and D) (23).

94 To measure the impact of GCN5 on transcriptional de-repression in atxr5/6 mutants, we performed 95 RNA-seq analyses and observed widespread suppression of transposable element (TE) reactivation 96 in the atxr5/6 gcn5 triple mutants compared to atxr5/6, although some TEs remained de-repressed 97 compared to Col (Figure 1C and Supplemental Table 1). Although GCN5 has a genome-wide impact 98 on transcription as shown by the 1781 misregulated genes in gcn5 single mutants (Figure 1D, 99 Supplemental Table 2), none of the known transcriptional suppressors of atxr5/6 mutants (SERRATE 100 [SE], AtTHP1, AtSAC3B, AtSTUbL2, AtMBD9 and DDM1) are downregulated in gcn5 mutants or 101 atxr5/6 gcn5 triple mutants (Supplemental Figure 1E) (9, 24), indicating that suppression of the 102 heterochromatin phenotypes in atxr5/6 gcn5 is not the result of decreased expression levels of these 103 genes.

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104 GCN5 functions with ADA2b and CHR6 to disrupt heterochromatin in the absence of 105 H3.1K27me1 106 GCN5 is a member of the multi-subunit SAGA complex which acts as a transcriptional coactivator 107 in yeast and animals in part by modifying chromatin (25). Key components of this complex are the 108 proteins GCN5, ADA2, ADA3 and SGF29, which form the histone acetylation module of SAGA 109 (Figure 2A). The Arabidopsis genome contains single genes encoding GCN5 and ADA3 and two 110 genes each encoding ADA2 (ADA2a and ADA2b) and SGF29 (SGF29a and SGF29b) (26). gcn5 and 111 ada2b single mutants show pleiotropic phenotypes, which are also shared by the atxr5/6 gcn5 and 112 atxr5/6 ada2b mutants, respectively (Supplemental Figure 2A) (27). To test if ADA2b is also required 113 for inducing the heterochromatin phenotypes of atxr5/6 mutants, we created an atxr5/6 ada2b triple 114 mutant and observed, similarly to atxr5/6 gcn5 mutants, that genomic instability is suppressed in that 115 background (Figure 2B). This result is supported by altered expression of BRCA1, which functions in 116 eukaryotes as a DNA-damage response gene involved in maintaining genome stability (28, 29). As 117 previously reported, BRCA1 levels are upregulated in atxr5/6 (4), and our results show that both 118 ADA2b and GCN5 are required for this induction (Figure 2C and Supplemental Figure 2B). Similarly 119 to gcn5, an ada2b mutation in atxr5/6 suppresses transcriptional de-repression of the heterochromatic 120 TSI DNA repeat (Figure 2D and Supplemental Figure 2C).

121 Next, we generated an atxr5/6 ada3 triple mutant, but unlike atxr5/6 ada2b, it did not suppress the 122 genome instability phenotype associated with the atxr5/6 double mutant (Figure 2B). The reported 123 ADA3 protein in Arabidopsis displays low similarity to the ADA3 orthologs from yeast and human 124 (26.3% and 16.3%, respectively, compared to >35% similarity for GCN5 and ADA2b (30)), and 125 might therefore have diverged and not be required for GCN5 and ADA2b to acetylate histones in 126 plants. To further investigate whether other modules of SAGA mediate the heterochromatin 127 phenotypes associated with the loss of H3.1K27me1, we created triple mutant combinations between 128 atxr5/6 and mutated alleles of genes proposed to subunits of the deubiquitination module of 129 SAGA in plants (Figure 2A). Our results show that the Arabidopsis orthologs of ENY2, UBP22 and 130 SGF11 are not required for inducing heterochromatin amplification, indicating that the 131 heterochromatin phenotypes of atxr5/6 are independent of the deubiquitination function of SAGA 132 (Supplemental Figure 2D). Finally, we created triple mutant combinations between atxr5/6 and chr5 133 or chr6. CHR5 and CHR6 are both chromatin remodeling that have been proposed to be

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134 present in the SAGA complex in plants (Figure 2A). CHR5 is the most closely related plant protein 135 to CHD1-type chromatin remodelers that are part of the SAGA complex in yeast and mammals (26, 136 30), while CHR6 (also known as CHD3/PICKLE) has been shown to co-purify with SAGA subunits 137 from Arabidopsis tissue (31). Our results show that heterochromatin amplification is suppressed in 138 the atxr5/6 chr6 triple mutant, but not atxr5/6 chr5 (Figure 2E). Overall, these results indicate 139 essential roles for ADA2b and CHR6 in mediating the heterochromatic phenotypes observed in the 140 absence of H3.1K27me1.

141 GCN5-mediated H3.1K27ac induces the heterochromatin defects associated with loss of 142 H3.1K27me1 143 The GCN5 orthologs in yeast and mammals have been shown to acetylate multiple lysine residues of 144 histone H3 (i.e., K9, K14, K18, K23, K27 and K36) in vitro, however, substrate specificity in the 145 context of different histone H3 variants has never been tested for any GCN5 ortholog (18, 20). In 146 addition, while the Arabidopsis GCN5 has been shown to acetylate H3K9 and H3K14 on H3 peptides 147 in vitro (32), acetylation at H3K27 by the Arabidopsis GCN5 ortholog has not been tested.

148 To investigate the substrate specificity of GCN5, we performed in vitro histone lysine 149 acetyltransferase (HAT) assays using recombinant containing either plant histone H3.1 150 or H3.3 variants. We recombinantly expressed and purified an Arabidopsis protein complex 151 composed of GCN5 and ADA2b (Supplemental Figure 3). Our results show that GCN5 has HAT 152 activity at K9, K14, K18, K23, K27 and K36 of histone H3 (Figure 3A). In contrast to ATXR5/6, the 153 enzymatic activity of GCN5 at H3K27 is not regulated by H3 variants, as H3.1 and H3.3 nucleosomes 154 show equivalent acetylation levels in our HAT assays (Figure 3A). As controls for these results, we 155 used H3.1K27ac and H3.3K27ac peptides to validate that the H3K27ac antibody used did not show 156 preference for H3.1 or H3.3 (Figure 3B), and we validated the specificity of this antibody by using 157 H3K27M nucleosomes (Figure 3C). Similarly to H3K27, we did not observe any major difference in 158 histone acetyltransferase activity between H3.1 and H3.3 nucleosomes at the other lysine substrates 159 of Arabidopsis GCN5 (Figure 3A). We also confirmed that H3.1K27me1 prevents acetylation by 160 GCN5 at K27 by using recombinant nucleosomes mono-methylated at K27 (Figure 3D). To assess if 161 H3.1K27ac mediates the heterochromatin phenotypes present in atxr5/6 mutants in vivo, we

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162 introduced into wild-type plants an H3.1K27Q transgene. Replacement of lysine (K) with glutamine 163 (Q) in histones has been used for in vivo chromatin studies to partially mimic the acetylated state of 164 histone lysine residues (33-35). Our analyses of first-generation transformed (T1) plants show that 165 expression of H3.1K27Q in wild-type plants is sufficient to induce transcriptional de-repression of 166 the heterochromatic TSI repeat (Figure 3E) and activation of the genome instability marker BRCA1 167 (Figure 3F), which are both specifically upregulated in atxr5/6 mutants. Overall, these results support 168 a specific role for GCN5-mediated H3.1K27ac in inducing the heterochromatic phenotypes associated 169 with loss of H3.1K27me1 in atxr5/6 mutants.

170 H3.1K36 is required to induce genome instability in the absence of H3.1K27me1 171 Our in vitro results suggest that, in addition to K27, other lysine residues on H3.1 could contribute to 172 GCN5-mediated genomic instability in the absence of H3.1K27me1. To assess this, we set up a 173 suppressor screen based on in vivo replacement of histone H3.1 with the point mutant H3.1S28A. 174 Replacement of serine with alanine on H3.1 variants at position 28 (H3.1S28A) generates H3.1 175 substrates that cannot be methylated by ATXR5/6 (Figure 4A) (36). In contrast, H3.1S28A can still 176 be methylated at K27 by plant PRC2-type complexes and acetylated by the GCN5-ADA2b complex, 177 albeit at lower efficiencies (Supplemental Figure 4A-B). We transformed the H3.1S28A transgene 178 into a mutant Arabidopsis background expressing a reduced amount of endogenous histone H3.1 (i.e., 179 h3.1 quadruple mutant (8)) and observed in T1 plants the phenotypes associated with loss of 180 H3.1K27me1, including genomic instability as detected by flow cytometry and increased levels of 181 the genome instability marker gene BRCA1 (Figure 4B-C), and transcriptional de-repression of the 182 heterochromatic TSI DNA repeat (Figure 4D). These results indicate that expression of H3.1S28A in 183 plants generates phenotypes similar to atxr5/6 mutants due to loss of H3.1K27me1. We then 184 introduced a series of H3.1S28A expression constructs containing a second mutation (Lys to Arg 185 replacement) at a residue known to be acetylated by GCN5 into the h3.1 quadruple mutant, and T1 186 plants were assessed for the phenotypes associated with loss of H3.1K27me1. This targeted screen 187 identified H3.1K36 as being essential for inducing genome instability, as flow cytometry analyses 188 demonstrated that H3.1S28A K36R suppresses heterochromatin amplification, while the other 189 targeted mutations do not (Figure 4B). The H3.1S28A K36R replacement line also rescued the 190 increased expression of BRCA1 (Figure 4C) and transcriptional de-repression of TSI (Figure 4D).

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191 Furthermore, expression of the H3.1S28A K36R mutant did not generate a serrated leaves phenotype 192 as seen in all the other H3.1S28A lines (Supplemental Figure 5). As mutations at K9, K14, K18 and 193 K23 on the H3.1 variant did not suppress the phenotypes associated with the H3.1S28A mutation, 194 these results indicate a specific role for H3.1K36 in inducing genome instability in the absence of 195 H3.1K27me1.

196 GCN5-mediated acetylation of H3.1K36 could be required to induce the heterochromatin defects of 197 atxr5/6 mutants. One prediction from this model is that increasing histone methylation at H3.1K36 198 (H3.1K36me) would result in the suppression of the atxr5/6 mutant phenotypes, as H3.1K36me 199 would antagonize H3.1K36 acetylation by GCN5. To test this, we constitutively expressed (using the 200 35S promoter) all five Arabidopsis H3K36 methyltransferases (SDG4, SDG7, SDG8, SDG24, and 201 SDG26) in atxr5/6 mutants (37, 38). We performed flow cytometry analyses on T1 plants and found 202 that overexpression of SDG24 (SDG24-OX) strongly suppresses the heterochromatin amplification 203 phenotype (Figure 4E). We did not observe a similar effect in T1 lines overexpressing SDG4, SDG7, 204 SDG8 or SDG26 (Supplemental Figure 6). The ability of SDG24-OX to suppress heterochromatin 205 amplification is dependent on SDG24 having a functional methyltransferase (SET) domain, as 206 overexpression of an SDG24 variant containing a point mutation (Y140N) in a conserved residue 207 essential for SET domain activity does not suppress the phenotype (Figure 4E) (5, 39). We performed 208 ChIP-qPCR experiments with SDG24-OX plants and detected an increase in levels at 209 heterochromatic regions (Ta3, At1G38250, At4G06566) known to be transcriptionally de-repressed 210 in atxr5/6 mutants (Figure 3F). Taken together, these results suggest that H3.1K36ac is required to 211 induce transcriptional de-repression and heterochromatin amplification in the absence of 212 H3.1K27me1.

213 Loss of H3.1K27me1 in plants increases H3K27ac and H3K36ac deposition in heterochromatin 214 Our results support a model in which GCN5 acetylates both H3.1K27 and H3.1K36 in the absence of 215 H3.1K27me1 to induce the heterochromatin phenotypes of atxr5/6 mutants. To assess if H3.1K27me1 216 depletion leads to an increase of H3K27ac and H3K36ac in vivo, we performed ChIP-seq with 217 reference exogenous genome (ChIP-Rx) for H3K27ac and H3K36ac in Col (WT), atxr5/6, gcn5 and 218 atxr5/6 gcn5 (40). We found that both histone marks are enriched at the 5’ end of protein-coding

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219 genes after the transcriptional start site (TSS) in Arabidopsis (Figure 5A), and this spatial distribution 220 correlates with transcriptional activity, albeit not in a linear relationship (Supplemental Figure 7) (41, 221 42). Comparative analysis of H3K27ac and H3K36ac in Col and gcn5 single mutants demonstrates 222 that loss of GCN5 results in a decrease of H3K27ac and H3K36ac at euchromatic genes (Figure 5A).

223 Focusing on heterochromatin, which we defined based on previously identified chromatin states in 224 Arabidopsis (Supplemental Table 3) (43), we identified multiple regions that were enriched in both 225 H3K27ac and H3K36ac in atxr5/6 but not in Col plants (Figure 5B-C and Supplemental Table 4). 226 H3K27ac and H3K36ac enrichment in heterochromatin was greatly decreased in atxr5/6 gcn5 triple 227 mutants (Figure 5B-C), suggesting that higher levels of H3K27ac and H3K36ac in heterochromatic 228 regions of atxr5/6 are almost completely dependent on GCN5. We next tested if the de-repressed 229 TEs in atxr5/6 identified in by RNA-seq overlap or are in close proximity (+/- 3kb) to the genomic 230 regions showing increased levels of H3K27ac and H3K36ac in atxr5/6. We observed a large overlap 231 between transcriptionally de-repressed genomic regions and regions enriched in H3K27ac and 232 H3K36ac in atxr5/6 mutants (Figure 5D). The regions shown in Figure 5D likely represent a low 233 estimate of the total overlap between H3K27ac/H3K36ac regions and transposon reactivation due to 234 the inherent lack of sensitivity of ChIP-seq and RNA-seq experiments in backgrounds showing low- 235 level TE de-repression, like atxr5/6 mutants. For example, we found that a 5-fold increase in 236 sequencing depth (75 versus 15 million reads) in our RNA-seq experiments resulted in a 43% increase 237 in the number of de-repressed TEs identified in atxr5/6 (446 TEs versus 312 TEs) (Supplemental 238 Table 1). To further demonstrate the sensitivity issue associated with low-level de-repression in 239 atxr5/6, we performed RT-qPCR on multiple TEs that showed an increase in H3K27ac in atxr5/6, 240 but were not identified as differently expressed by RNA-seq. For many of these TEs, including 241 At1g36040 and At5g29602 (Supplemental Figure 8), we observed higher expression levels in atxr5/6 242 compared to wild-type plants, thus confirming the limitations of genome-wide sequencing for 243 detecting low-level TE de-repression in atxr5/6 mutants. Taken together, these results demonstrate 244 that the loss of H3.1K27me1 in atxr5/6 mutants leads to GCN5-dependent increase of H3K27ac and 245 H3K36ac in heterochromatin.

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246 H3.1K27me1 regulates the deposition of H3.1K36ac by GCN5 247 Methylation and acetylation at H3K27 have an antagonistic relationship in the genome of animals, 248 which is mediated by the interplay between the H3K27 methyltransferase complex PRC2 (H3K27me) 249 and the histone acetyltransferases p300 and CBP responsible for H3K27ac (15, 16). Our work 250 supports a similar relationship in plants at K27 on H3.1 variants that is mediated by different enzymes, 251 with ATXR5/6-catalyzed H3.1K27me1 preventing the acetylation of H3.1K27 by GCN5. Interactions 252 between post-translational modifications on different histone residues also contribute to chromatin 253 regulation in eukaryotes. One example of this is the inhibition of PRC2 activity towards H3K27 when 254 H3K36 is di- or trimethylated on the same histone (44-46). This suggests that the activity of other 255 chromatin-modifying enzymes may be affected by crosstalk between modified forms of H3K27 and 256 H3K36. To assess if acetylation of H3.1K36 by GCN5 is regulated by H3.1K27me1, we performed 257 in vitro HAT assays using recombinant plant nucleosomes containing either unmodified H3.1 or 258 H3.1K27me1. In these assays, we consistently observed a 40% decrease in the levels of acetylation 259 at H3.1K36 on nucleosomes mono-methylated at H3.1K27 compared to unmodified H3.1 (Figure 260 6A-B). This effect of H3.1K27me1 on Arabidopsis GCN5 activity appears to be specific to H3.1K36, 261 as GCN5-mediated acetylation of H3.1K9 was not affected by mono-methylation at K27. We also 262 tested if reciprocally, methylation at H3.1K36 would affect acetylation at K27 by GCN5. We did not 263 observe any difference in acetylation levels at K27 using K36me0 and K36me3 nucleosomes (Figure 264 6C). Overall, these results suggest that ATXR5/6-catalyzed H3.1K27me1 in plants interferes with 265 GCN5-mediated acetylation at both H3.1K27 and H3.1K36.

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266 Discussion 267 Previous work had suggested that transcriptional reactivation of heterochromatic regions is 268 responsible for inducing genomic instability in the absence of H3.1K27me1 in plants (9). However, 269 the mechanism by which H3.1K27me1 prevents transcriptional de-repression in heterochromatin had 270 remained unknown. Our study supports a model where ATXR5/6-mediated H3.1K27me1 serves to 271 prevent a SAGA-like complex that includes GCN5, ADA2b and CHR6 from acetylating the H3.1 272 variant and initiating transcriptional de-repression (Figure 6D). K27me1 is the most abundant post- 273 translational modification on H3.1K27 in plants (14), and our results suggest that it plays a role 274 analogous to the one proposed for PRC2-catalyzed H3K27me2 in animals, which is present on 50- 275 70% of total histone H3 in mouse embryonic stem cells, blocks H3K27ac, and prevents spurious 276 transcription (13, 47, 48). In animals, p300 and CBP are the main histone acetyltransferases 277 contributing to H3K27ac in the absence of PRC2-mediated H3K27 methylation (15, 16). Our results 278 indicate that in plants, GCN5 plays this role. However, transcriptional de-repression is not completely 279 abolished in gcn5 mutants (Figure 1C), thus suggesting that at least one of the five p300/CBP 280 orthologs in Arabidopsis (HAC1/2/4/5/12 (32, 49)) may also contribute to higher histone acetylation 281 levels in the absence of H3.1K27me1.

282 Our work shows that GCN5-catalyzed histone acetylation plays a key role in mediating transcriptional 283 activation in atxr5/6 mutants. The role of GCN5 as a transcriptional co-activator in other biological 284 systems is well defined, thus supporting a conserved function for GCN5 in all eukaryotes. In 285 mammals, H3K27ac is found at active transcriptional enhancers (50, 51), but a recent study in mouse 286 ESCs showed that H3K27ac depletion at enhancers does not affect gene expression (52). This 287 suggests that H3K27ac works redundantly or synergistically with other chromatin features to specify 288 active enhancers. In maize and rice, approximately 30% of H3K27ac sites are found in intergenic 289 regions (42, 53), which support a conserved role for H3K27ac in marking enhancers and contributing 290 to their activity in eukaryotes. However, a recent report has shown that only a small subset of 291 H3K27ac sites (0.5%-3%) are located upstream of the TSS in Arabidopsis, which argues for plant 292 species-specific variation in the types of chromatin features defining active enhancers (54). Aside 293 from marking enhancers, H3K27ac has also been found to be enriched close to the TSS of 294 transcriptionally active protein-coding genes in mammals, maize, rice and Arabidopsis (42, 53-55), a 295 result that we confirmed for Arabidopsis in our ChIP-Rx experiments. H3K36ac has also been shown

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296 in multiple biological systems to co-localize with H3K27ac at the TSS of transcriptionally-active 297 regions of the genome (41, 55). These observations suggest important roles for TSS-localized 298 H3K27ac and H3K36ac in mediating transcriptional activity. Precisely mapping the H3K27ac and 299 H3K36ac regions in the heterochromatin of atxr5/6 mutants in relation to the TSS of de-repressed 300 TEs is challenging, as TSSs are not well defined for TEs. Nevertheless, we did observe H3K27ac and 301 H3K36ac peaks in atxr5/6 at the 5’end of annotated TEs (Figure 5C, Supplemental Figure 8), which 302 would support a similar mode of action for H3K27ac/H3K36ac in regulating transcription of genes 303 and TEs.

304 Yeast and animal GCN5 have been shown to have the ability to acetylate multiple (K9, K14, 305 K18, K23, K27 and K36) in the N-terminal tail of histone H3 (18, 20). Our in vitro results using 306 recombinant nucleosomes suggest that the GCN5 ortholog in Arabidopsis also has broad substrate 307 specificity. However, the specificity of ATXR5/6 for H3K27 and results from this study suggest a 308 critical role for K27 over other target sites of GCN5 on H3.1 variants. One observation supporting a 309 unique role for H3.1K27ac over other acetylated lysines of H3 in Arabidopsis comes from 310 experiments showing that increased levels of cytosolic acetyl-CoA (the essential cofactor for protein 311 acetylation) increase H3 acetylation in plants (17). Results from these experiments show that H3K27 312 is predominantly acetylated over other lysine residues of H3 (i.e. H3K9, H3K14 and H3K18; H3K36 313 was not assessed in that study), in a manner dependent on GCN5. Higher levels of H3K27ac are 314 observed in genic regions, and this correlates with higher transcriptional levels for genes showing 315 gains in H3K27ac (17). Similarly to H3.1K27ac, our in vitro and in vivo results implicate H3.1K36ac 316 as playing a key role in mediating the heterochromatin phenotypes of atxr5/6. However, these results 317 do not rule out the possibility that other acetylated sites (e.g. K9, K14, K18 and K23) on H3.1 also 318 contribute to mediating transcriptional de-repression and genomic instability in plants, for example 319 by acting in a functionally redundant manner. Our in vitro histone acetyltransferase assays indicate 320 that deposition of H3K36ac by GCN5 is negatively regulated by H3.1K27me1, although the 321 molecular mechanism responsible for this crosstalk remains unknown. Previous structural work 322 characterizing a protein complex composed of the histone acetyltransferase (HAT) domain of GCN5 323 from Tetrahymena thermophila and a phosphorylated histone H3 peptide (aa. 5-23) showed that the 324 HAT domain interacts with the sidechain of glutamine 5 (Q5), located 9 amino acids upstream of the 325 target lysine (K14) on the H3 peptide (56). As H3K27 is similarly located 9 amino acids upstream

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326 H3K36, this suggests that the HAT domain of GCN5 in Arabidopsis may interact with the sidechain 327 of H3K27 to regulate the catalytic activity of GCN5 at H3K36. Structural studies of the HAT domain 328 of Arabidopsis GCN5 will be needed to validate this model.

329 The catalytic specificity of ATXR5/6 for replication-dependent H3.1 variants together with the 330 observation that heterochromatin amplification is suppressed when the H3.1 CAF-1 is 331 mutated have led to a model where the H3.1 variant plays a specific role in maintaining genome 332 stability (8). One mechanism that could explain the requirement for H3.1 variants to induce the 333 atxr5/6 mutant phenotypes would be if GCN5, similarly to ATXR5/6, specifically acetylates K27 in 334 H3.1 variants. However, our results show no difference in enzymatic activity for GCN5 on H3.1 and 335 H3.3 variants (Figure 3A). Therefore, GCN5 is unlikely to be directly involved in mediating the H3.1 336 requirement for inducing the atxr5/6 mutant phenotypes. An alternative mechanism to explain the 337 role for H3.1 variants in this process could be that downstream chromatin readers mediating 338 transcriptional de-repression and heterochromatin amplification interact with H3.1K27ac and/or 339 H3.1K36ac, but not H3.3K27ac and/or H3.3K36ac. Another possibility is that transcriptional de- 340 repression mediated through GCN5 is not dependent on H3.1 variants, but heterochromatin 341 amplification is. Previous results have shown that expression of ATXR5/6-resistant H3.1A31T 342 (which partially mimics the N-terminal tail of H3.3 variants) in plants generates very low-level 343 transcriptional de-repression in heterochromatin (which is supported by GCN5 being active on H3.3 344 variants), but genomic instability in the H3.1A31T lines is not detected (8). Therefore, 345 heterochromatin defects in atxr5/6 mutants could be due to H3.1-independent transcriptional de- 346 repression mediated by GCN5-catalyzed H3K27ac and H3K36ac, coupled to another H3.1 variant- 347 specific process that would lead to even higher levels of transcriptional de-repression and 348 heterochromatin amplification. In this two-step model, transcriptional de-repression in 349 heterochromatin via GCN5 in the absence of H3K27me1 would be the initial trigger leading to H3.1- 350 dependent genomic instability. More work will be needed to fully understand the relationship between 351 H3 variants, transcriptional de-repression, and genomic instability.

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352 Materials and Methods 353 Plant materials 354 Arabidopsis plants were grown under cool‐white fluorescent lights (approximately 100 μmol m−2 s−1) 355 in long‐day conditions (16 h light/8 h dark). The atxr5/6 double mutant was described previously (3). 356 gcn5 (At3g54610, SALK_030913), ada2b (At4g16420, SALK_019407), ada3 (At4g29790, 357 SALK_042026C), sgf11 (At5g58575, SAIL_856_F11), eny2 (At3g27100, SALK_045015C), ubp22 358 (At5g10790, GK-263H06), chr5 (At2g13370, SAIL_504_D01) and chr6 (At2g25170, GK-273E06) 359 are in the Col-0 genetic background and were obtained from the Arabidopsis Biological Resource 360 Center (Columbus, Ohio). Temperature-optimized CRISPR/Cas9 was used to generate additional 361 mutant alleles of GCN5 (in Col-0 and atxr5/6) used in this study (23). The h3.1 quadruple mutant was 362 described previously (8). Transgenic plants expressing WT H3.1 (At5g65360), H3.1S28A, H3.1K9R, 363 H3.1S28A K9R, H3.1K14R, H3.1S28A K14R, H3.1K18R, H3.1S28A K18R, H3.1K23R, H3.1S28A 364 K23R, H3.1K36R, H3.1S28A K36R were made by transforming the h3.1 quadruple mutant 365 background.

366 Constructs 367 Cloning of the catalytic fragment of ATXR6 (a.a. 25-349) and the plant PRC2 complexes for protein 368 expression and in vitro methyltransferase assays was described previously (3, 8). The histone H3.1 369 gene (At5g65360) and its promoter (1167 bp upstream of the start codon) were cloned into pENTR/D- 370 TOPO (ThermoFisher Scientific, Waltham, MA) and then sub-cloned using Gateway Technology 371 into the plant binary vectors pB7WG (57). Site-directed mutagenesis to generate the different H3.1 372 point mutant constructs was performed using QuikChange II XL Site-Directed Mutagenesis Kit 373 (Agilent Technologies, Santa Clara, CA). The ADA2b coding sequence was cloned into pETDuet-1 374 (Millipore, Burlington, MA) vector using the SalI and NotI restriction sites, yielding pETDuet-1- 375 ADA2b. The GCN5 coding sequence was cloned into pETDuet-1-ADA2b plasmid using the EcoRV 376 and PacI restriction sites, yielding pETDuet-1-ADA2b-GCN5. The cloning procedure used to make 377 the CRISPR construct targeting GCN5 in Arabidopsis was performed as described previously (58).

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378 Protein expression and purification 379 Expression and purification of the ATXR6 protein and the plant PRC2 complexes CURLY LEAF 380 and MEDEA has been described previously (3, 8). For the GCN5-ADA2b protein complex, pETDuet- 381 1-ADA2b-GCN5 was transformed into BL21 (DE3) E. coli (Millipore), cultured in LB and induced 382 to express proteins by adding 1 mM IPTG. Cells were then pelleted by centrifugation, resuspended

383 in NPI-10 buffer (50mM NaH2PO4, 300mM NaCl, 10mM Imidazole, pH 8), and lysed by sonification. 384 After centrifugation to remove cell debris, Ni-NTA agarose (Qiagen, Hilden, Germany) was added to 385 the supernatant and rotated at 4 ̊C for 2 hours. The Ni-NTA agarose was washed 3 times using NPI-

386 20 buffer (50mM NaH2PO4, 300mM NaCl, 20mM imidazole, pH 8), and the protein complex was

387 eluted in NPI-250 buffer (50mM NaH2PO4, 300mM NaCl, 250mM imidazole, pH 8). The buffer was 388 changed to 1×PBS (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4) containing 10% glycerol 389 using an Amicon Ultra-0.5 Centrifugal Filter Unit (30kDa cutoff). The proteins were aliquoted, flash- 390 frozen in liquid nitrogen, and then stored at -80 ̊C.

391 The protocols to generate the H3K27me1 and H3K36me2 methyl-lysine analog-containing histones 392 and to make the recombinant chromatin used in the in vitro histone modification assays (methylation 393 and acetylation) was described previously (45).

394 Histone lysine methyltransferase (HMT) and acetyltransferase (HAT) assays 395 The general procedure used to perform the in vitro histone modification assays presented in this study 396 were described in detail in a previous publication (59).

397 For the radioactive HMT assays, 0.5 μg of ATXR6, 1.5 μg of MEA or 1.5 μg of CLF (PRC2) 398 complexes were incubated with 1 μg of Histone H3 peptides (GenScript, Piscataway, NJ) and 1.5 μCi 399 of 3H-SAM (Perkin Elmer, Waltham, MA) in a 25 μl reaction. The histone methyltransferase buffer

400 contained 50 mM Tris pH 8.0, 2.5 mM MgCl2 and 4 mM DTT. The methylation reactions were 401 incubated at 22°C for 2 hours. The samples were pipetted onto Whatman P-81 filter paper and dried

3 402 for 15 minutes. The free H-SAM was removed by washing 3 x 30 minutes in 50 mM NaHCO3 pH 403 9.0. The filter paper was dried and added to a vial containing Opti-FluorÒ O (Perkin Elmer). 404 Radioactivity on the filter papers was determined using a liquid scintillation counter (Perkin Elmer).

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405 For the HAT assays with antibody detection, 1 µg of recombinant nucleosomes and 2 µg of the GCN5- 406 ADA2b complex were incubated in 50 µl histone acetyltransferase (HAT) buffer (1 mM HEPES pH 407 7.3, 0.02% BSA) containing 50 mM acetyl co- A (Acetyl-CoA; Sigma) at 23 ̊C for 3 hours 408 (wild type H3.1, H3.1K27M and H3.3 nucleosomes) or 5 hours (H3K27me0, H3K27me1, H3K36me0 409 and H3K36me3 nucleosomes). The reactions were stopped by adding 4X Laemmli Sample Buffer 410 (Bio-Rad) and boiling at 95 ̊ C for 5 min. The samples were resolved by 15% SDS-PAGE gel, 411 transferred to PVDF membrane, and western blot was performed using anti-H3K9ac (Cell Signaling 412 Technology, Danvers, MA: 9649), anti-H3K14ac (Active Motif, Carlsbad, CA: 39698), anti-

413 H3K18ac (Active Motif : 39588), anti-H3K23ac (Active Motif : 39132), anti-H3K27ac (Active Motif: 414 39135), anti-H3K36ac (Active Motif: 39379) or anti-H3 antibodies (Abcam: ab1791) and a secondary 415 anti-Rabbit HRP-labeled antibody (Sigma).

416 For the radioactive HAT assays, 1 µg of peptides and 1 µg of GCN5-ADA2 complex were incubated 417 in 25 µl HAT buffer containing 0.625 µCi 3H-Acetyl-CoA (PerkinElmer) at 23 ̊ C for 2 hours. 418 Reactions were stopped by pipetting onto Whatman P-81 filter paper and activity (cpm) was measured 419 using a liquid scintillation counter (Perkin Elmer).

420 Chromatin 421 ChIP was performed as described previously (60), with some modifications. Leaves from three-week- 422 old plants were fixed in 1% formaldehyde. Immunoprecipitation was performed using protein A 423 magnetic beads (New England BioLabs, Ipswich, MA). Following the Proteinase K treatment of each 424 sample, immunoprecipitated DNA was purified using ChIP DNA Clean & Concentrator kit (Zymo 425 Research, Irvine, CA). 2 μl of Histone H3 antibody (Abcam: ab1791), 2.5 μl of H3K27ac antibody 426 (Active Motif: 39135), 5 μl of H3K36ac antibody (Active Motif: 39379) or 2.5 μl of H3K36me3 427 (Abcam: ab9050), was used per immunoprecipitation (750 μl of chromatin solution). For the 428 H3K27ac and H3K36ac ChIP experiments, ChIP with exogenous genome (ChIP-Rx) was performed 429 in order to properly normalize the data (40). For each sample, an equal amount of drosophila 430 chromatin (Active Motif: 53083) was added prior to chromatin shearing.

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431 Nuclei DAPI staining 432 Leaves from four-week-old plants were fixed in 3.7% formaldehyde in cold Tris buffer (10 mM Tris- 433 HCl pH 7.5, 10 mM NaEDTA, 100 mM NaCl) for 20 minutes. Formaldehyde solution was removed, 434 and leaves were washed twice for 10 minutes in Tris buffer. The leaves were then finely chopped 435 with razor blade in 500 μl LB01 buffer (15 mM Tris-HCl pH7.5, 2 mM NaEDTA, 0.5 mM spermine- 436 4HCl, 80 mM KCl, 20 mM NaCl and 0.1% Triton X-100). The lysate was filtered through a 30 µm 437 mesh (Sysmex Partec, Gorlitz, Germany). 5 μl of lysate was added to 10 μl of sorting buffer (100 mM

438 Tris-HCl pH 7.5, 50 mM KCl, 2mM MgCl2, 0.05% Tween-20 and 5% sucrose) and spread onto a 439 coverslip until dried. Cold methanol was added onto each coverslip for 3 min, then rehydrated with 440 TBS-Tx (20 mM Tris pH 7.5, 100 mM NaCl, 0.1% Triton X-100) for 5 min. The coverslips were 441 mounted onto slides with Vectashield mounting medium DAPI (Vector Laboratories, Burlingame, 442 CA). Nuclei were imaged on a Nikon Eclipse Ni-E microscope with a 100X CFI PlanApo Lamda 443 objective (Nikon, Minato City, Tokyo, Japan). Digital images were obtained using an Andor Clara 444 camera. Z-series optical sections of each nucleus were obtained at 0.3 μm steps. Images were 445 deconvolved by imageJ using the deconvolution plugin.

446 RT-qPCR 447 Total RNA was extracted from three-week-old leaf tissue using TRIzol (Invitrogen, Carlsbad, CA). 448 Samples were treated with RQ1 RNase-free DNase (Promega, Madison, WI) at 37°C for 30 min. 449 SuperScript II Reverse Transcriptase (Invitrogen) was used to produce cDNA from 1 µg of total RNA. 450 Reverse transcription was initiated using oligo dT primers. Quantification of cDNA was done by real‐ 451 time PCR using a CFX96 Real‐Time PCR Detection System (Bio‐Rad, Hercules, CA) with KAPA 452 SYBR FAST qPCR Master Mix (2×) Kit (Kapa Biosystems, Wilmington, MA). Each primer pair was

453 assessed for efficiency of amplification. Relative quantities were determined by the Ct method (61). 454 Actin was used as the normalizer. At least three biological samples were used for each experiment.

455 Flow cytometry 456 Rosette leaves from three-week-old plants were finely chopped in 0.5 ml Galbraith buffer (45 mM

457 MgCl2, 20 mM MOPS, 30 mM sodium citrate, 0.1% Triton X-100, 40 μg/μl RNase A) using a razor

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458 blade. The lysate was filtered through a 30 µm mesh (Sysmex Partec). Propidium iodide (Sigma, St. 459 Louis, MO) was added to each sample to a concentration of 20 µg/ml and vortexed for 3 seconds. 460 Each sample was analyzed using a BD FACS LSR Fortessa X20 (Becton Dickinson, Franklin Lakes, 461 NJ). Quantification (nuclei counts and robust CV values) was performed using Flowjo 10.0.6 (Tree 462 Star, Ashland, Oregon).

463 Next-generation sequencing library preparation 464 RNA samples were prepared from three-week old leaf tissue using the RNeasy Plant Mini Kit 465 (Qiagen). RNA and ChIP sequencing libraries were prepared at the Yale Center for Genome Analysis 466 (YCGA). RNA samples were quantified and checked for quality using the Agilent 2100 Bioanalyzer 467 Nano RNA Assay. Library preparation was performed using Illumina’s TruSeq Stranded Total RNA 468 with Ribo-Zero Plant in which samples were normalized with a total RNA input of 1 µg and library 469 amplification with 8 PCR cycles. ChIP library preparation was performed using TruSeq Library Prep 470 Kit (Illumina, San Diego, CA). Libraries were validated using Agilent Bioanalyzer 2100 High 471 sensitivity DNA assay and quantified using the KAPA Library Quantification Kit for Illumina® 472 Platforms kit. Sequencing was done on an Illumina NovaSeq 6000 using the S4 XP workflow.

473 RNA-seq processing and analysis 474 Two independent biological replicates for Col, atxr5/6, gcn5 and atxr5/6 gcn5 were sequenced. 475 Paired-end reads were filtered and trimmed using BBTools (version 38.79) (62). Reads with quality 476 inferior to 20 were removed. The resulting data sets were aligned against the Arabidopis genome 477 (TAIR10) using STAR (version 2.7.2a) allowing 2 mismatches (--outFilterMismatchNmax 2) (63). 478 Consistency between biological replicates was confirmed by Pearson correlation using deepTools2 479 (Supplementary Figure 9) (64). Protein-coding genes and transposable elements (TE) were defined 480 as described in the TAIR10 annotation gff3 file. The program featureCounts (version 1.6.4) (65) was 481 used to count the paired-end fragments overlapping with the annotated protein-coding genes and TEs. 482 Differential expression analysis of protein-coding genes was performed using DESeq2 version 1.26 483 (66) on raw read counts to obtain normalized fold changes (FC) and Padj-values for each gene. Genes 484 were considered to be differentially expressed only if they showed a log2FC >1 and a Padj-values <

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485 0.05. TPM (transcripts per million) values were calculated for TEs. To define TEs as up-regulated in 486 the atxr5/6 mutant, they must show 2-fold up-regulation as compared to Col in both biological 487 replicates and have a value of TPM > 5. The heatmap was drawn with R program (version 3.6.2) (67).

488 ChIP-seq processing and analysis 489 Two independent biological replicates for Col, atxr5/6, gcn5 and atxr5/6 gcn5 were sequenced. In 490 order to properly compare H3K27ac and H3K36ac levels between each genotype, we performed ChIP 491 with reference exogenous genome (ChIP-Rx) (40) using equal amounts of drosophila chromatin in 492 each sample as reference. Paired-end reads were filtered and trimmed using BBTools (62). Reads 493 with quality inferior to 20 were removed. Data sets were aligned against combined genomes of 494 Arabidopsis thaliana (TAIR10) and Drosophila melanogaster (dm6) using bowtie2 (68) with default 495 parameters. Duplicate reads were removed using Picard toolkit (69) (MarkesDuplicates with 496 REMOVE_DUPLICATES=true). Consistency between biological replicates was confirmed by 497 Pearson correlation using deepTools2 (Supplementary Figure 10) (64). To calculate the Rx scaling 498 factor of each biological replicate, Drosophila-derived IP read counts were normalized according to 499 the number of input reads. Spike-in normalization was performed as previously described (70). The 500 Rx factors are presented in Supplementary Table 4. We generated bedgraph files with a bin size of 501 10 bp using deepTools. The bedgraph files were then scaled by adjusting the number of reads in each 502 bin with the Rx factors and therefore generating reference-adjusted reads per million (RRPM). 503 H3K27ac and H3K36ac enriched regions were identified by computing the differential between each 504 bin (± 1kb) to define local maxima.

505 The number of reads corresponding to euchromatic regions was much higher than the ones from 506 heterochromatic regions. To best determine the heterochromatic enrichment of H3K27ac in each 507 genotype of interest, we avoided the “noise” from the euchromatic reads by first defining 508 heterochromatic regions and extracting the corresponding reads from each genotype. We defined the 509 heterochromatic regions based on the chromatin states proposed by Sequeira-Mendes et al., 2014 510 (43). We attributed the value of the state number (1 to 9) for each bin of the Sequeira-Mendes et al. 511 annotation, and averaged them on 100 kb windows. Only the 100 kb windows with a score superior 512 to 7 were considered as heterochromatic regions (Supplementary Table 2). We then generated a bam

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513 file with the reads corresponding to the defined heterochromatic regions. We identified 514 heterochromatic H3K27ac an H3K36ac enriched regions by calculating the log2 ratio between 515 H3K27ac or H3K36ac IP and H3 input using the heterochromatin bam file. The enriched regions 516 were defined with the following criteria: log2 (IP/H3) > 0.3. To compare the H3K27ac and H3K36ac 517 enriched regions between Col and our mutant genotypes, we computed log2 (mutant/Col), using the 518 Rx factor normalized bedgraph file. We considered the levels of H3K27ac and H3K36ac to be 519 differential between genotypes when log2 (mutant/Col) > 0.8. These regions needed to be detected in 520 both replicate in order to be considered.

521 Primers 522 All primers used in this study are listed in Supplemental Table 7.

523 Data availability 524 Sequencing data are available at the Gene Expression Omnibus (GEO) under accession code 525 GSE146126.

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526 Figure Legends

527 Main Figures 528 Figure 1. A mutation in GCN5 suppresses transcriptional de-repression and heterochromatin 529 amplification associated with H3.1K27me1 depletion. (A) Flow cytometry profiles of Col, atxr5/6, 530 gcn5 and atxr5/6 gcn5 nuclei stained with propidium iodide (PI) with 2000 gated events. The numbers 531 below the peaks indicate ploidy levels of the nuclei. The numbers above the 16C peaks indicate the 532 robust coefficient of variation (CV). (B) Leaf interphase nuclei of Col, atxr5/6, gcn5 and atxr5/6 gcn5 533 stained with DAPI. (C) Heat map showing the relative expression levels of atxr5/6-induced TEs 534 (Supplemental Table 1) as measured by TPM (transcripts per million) in Col, atxr5/6, gcn5 and 535 atxr5/6 gcn5. (D) Euler diagrams showing the upregulated and downregulated genes (2-fold change) 536 in atxr5/6, gcn5 and atxr5/6 gcn5 in comparison to Col plants (Padj < 0.05).

537 Figure 2. GCN5 requires ADA2b to induce heterochromatic defects in atxr5/6 mutants. (A) 538 Proposed subunits of the Arabidopsis SAGA complex. HAT: histone acetylation module; DUB: 539 deubiquitination module; SPT: recruiting module; TAF; coactivator architecture module. (B) Flow 540 cytometry profiles of Col, atxr5/6, ada2b, atxr5/6 ada2b, ada3, and atxr5/6 ada3. (C and D) RT- 541 qPCR analyses of BRCA1 (C) and the repetitive element TSI (D) in Col, atxr5/6, ada2b and atxr5/6 542 ada2b. Data represent the mean of three biological replicates and error bars indicate the standard error 543 of the mean (SEM). Unpaired t-test: * p < 0.01, ** p < 0.001. (E) Flow cytometry profiles of Col, 544 atxr5/6, chr5, atxr5/6 chr5, chr6, and atxr5/6 chr6.

545 Figure 3. Arabidopsis GCN5 acetylates H3.1K27 and induces the heterochromatic defects 546 associated with atxr5/6. (A) In vitro HAT assays with the GCN5-ADA2b complex and H3.1 and 547 H3.3 nucleosomes using anti-H3K9ac, anti-H3K14ac, anti-H3K18ac, anti-H3K23ac, anti-H3K27ac, 548 anti-H3K36ac and anti-H3 antibodies. (B) Western blot of H3.1K27ac and H3.3K27ac peptides with 549 H3K27ac antibody. (C) In vitro HAT assay with the GCN5-ADA2b complex and H3K27M 550 nucleosomes. (D) In vitro HAT assays with the GCN5-ADA2b complex and H3K27me0 and 551 H3K27me1 nucleosomes using anti-H3K27ac, anti-H3 antibodies (E and F) RT-qPCR for the 552 heterochromatic transcriptional reactivation marker TSI (E) and the genome stability marker BRCA1

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553 (F) in Col, atxr5/6 and first-generation transformed (T1) plants expressing WT H3.1 or H3.1K27Q. 554 Ten independent T1 plants were used in the experiments. Unpaired t-test: * p < 0.001.

555 Figure 4. Heterochromatin amplification in the absence of H3.1K27me1 requires H3.1K36 (A) 556 In vitro histone lysine methylation assays using H3.1 and H3.1S28A peptide substrates and ATXR6. 557 The average of three experiments and SEM are shown. CPM; counts per minute (B) Robust CV values 558 for 16N nuclei obtained by flow cytometry analysis. For Col and atxr5/6, each dot represents an 559 independent biological replicate. For the H3.1 replacement lines, each dot represents one T1 plant. 560 Horizontal bars indicate the mean. Unpaired t-test: * p < 0.0001 and n.s. = not significantly different. 561 (C and D) RT-qPCR analyses of BRCA1 (C) and the repetitive element TSI (D) in Col, atxr5/6 and 562 H3.1 replacement lines. For Col and atxr5/6, each dot represents an independent biological replicate. 563 For the H3.1 lines, each dot represents one T1 plant. Horizontal bars indicate the mean. Unpaired t- 564 test: * p < 0.01, ** p < 0.0005 (E) Flow cytometry analyses showing robust CV values for 16N nuclei. 565 For the SDG24-OX lines, each dot represents one T1 plant. Horizontal bars indicate the mean. 566 Unpaired t-test: * p < 0.001. (F) H3K36me3 ChIP-qPCR at Ta3, At1g38250, At4g06566 and Actin7. 567 For Col and atxr5/6, each dot represents an independent biological replicate. For the SDG24-OX 568 lines, each dot represents one T1 plant. Bars indicate the mean. Error bars indicate SEM.

569 Figure 5. Mutations in atxr5/6 lead to an increase in H3K27ac and H3K36ac in 570 heterochromatin. (A) Normalized average distribution of H3K27ac and H3K36ac over protein 571 coding genes for Col, atxr5/6, gcn5 and atxr5/6 gcn5 in reference-adjusted reads per million (RRPM). 572 TSS, transcription start site; TES, transcription end site. (B) Normalized average distribution and 573 heatmap of H3K27ac and H3K36ac normalized reads surrounding the H3K27ac/H3K36ac enriched 574 heterochromatic regions in atxr5/6 compared to Col. (C) Genome browser snapshot showing 575 normalized H3K27ac and H3K36ac ChIP-seq data over a region of chromosome 5 that includes TE 576 genes At5g29591 and At5g29602. The y-axis unit is RRPM. (D) Heatmap showing the RNA-seq reads 577 mapping to the region 3 kb around the center of H3K27ac/H3K36ac peaks as measured by RPKM 578 (reads per kilobase million) in Col, atxr5/6, gcn5 and atxr5/6 gcn5.

579 Figure 6. H3K36 acetylation by the GCN5-ADA2b complex is regulated by H3K27me1. (A) In 580 vitro HAT assays with the GCN5-ADA2b complex and H3K27me0 and H3K27me1 nucleosomes.

23 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

581 (B) Data from three technical replicates of HAT assays with the GCN5-ADA2b complex and 582 H3K27me0 and H3K27me1 nucleosomes. Unpaired t-test: * p < 0.05, and n.s. = not significantly 583 different. (C) In vitro HAT assays with the GCN5-ADA2b complex and H3K36me0 and H3K36me3 584 nucleosomes. D) Model depicting the role of H3.1K27me1 in blocking GCN5-mediated acetylation 585 of H3.1K27ac and H3.1K36ac.

586 Supplemental Figures

587 Supplemental Figure 1. Effect of GCN5 on genome stability and transcriptional de-repression. 588 (A) Gene structure for GCN5. Exons are highlighted as black boxes. The location of the mutations in 589 the gcn5 alleles used in this study are shown. (B) Quantification of chromocenter appearance. Shown 590 is the percentage of nuclei that are fully condensed (green), hollow spheres characteristic of the 591 atxr5/6 mutant plants (blue) and irregularly/partially decondensed (grey). At least 25 nuclei for 3 592 biological replicates of each genotype were assessed. Error bars indicate SEM (C) CRISPR-induced 593 mutations of GCN5 in Col and atxr5/6 backgrounds. Mutations (red) and PAM motif (blue) are 594 shown. (D) Flow cytometry profiles of Col, atxr5/6 and the CRISPR-induced knockout allele of gcn5 595 in Col and the atxr5/6 mutant background. The numbers below the peaks indicate ploidy levels of the 596 nuclei. The numbers above the 16C peaks indicate the robust CV. (E) Gene expression levels in our 597 RNA-seq experiments for the known suppressors of transcriptional de-repression in atxr5/6.

598 Supplemental Figure 2. Role of SAGA-related proteins in transcriptional de-repression and 599 genome stability. (A) Growth phenotype of gcn5 and ada2b single mutants and in combination with 600 atxr5/6. (B and C) RT-qPCR for the genome stability marker BRCA1 (B) and the heterochromatic 601 transcriptional reactivation marker TSI (C). The average of three biological replicates and SEM are 602 shown. (D) Flow cytometry profiles of the mutant alleles of genes predicted to code for subunits of 603 SAGA in plants. The Robust CV value calculated for the 16C peak on each plot is used as a measure 604 of heterochromatin amplification.

24 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

605 Supplemental Figure 3. Purification of the GCN5-ADA2b complex. Coomassie-stained gel 606 showing GCN5-ADA2b protein expression either pre- or post-induction in E. coli, or after affinity 607 purification (eluate).

608 Supplemental Figure 4. In vitro histone modification assays. (A) In vitro histone lysine 609 methylation assays at H3.1K27 using peptide substrates, ATXR6 and plant PRC2 complexes. The 610 average of three experiments and SEM are shown. (B) In vitro HAT assays using H3.1 peptides. 611 Lysine (K) to arginine (R) mutations were introduced (blue) on the peptides at other potential targets 612 (H3.1K18, H3.1K23, H3.1K36 and H3.1K37) of GCN5-ADA2b, so that the acetylation signal could 613 be specifically measured at H3.1K27 (red). H3.1S28A is shown in green. The average of three 614 experiments and SEM are shown.

615 Supplemental Figure 5. Growth and developmental phenotypes of T1 plants expressing 616 different H3.1 transgenes.

617 Supplemental Figure 6. Robust CV values for 16N nuclei obtained by flow cytometry analyses. 618 For Col and atxr5/6, each dot represents an independent biological replicate. For overexpression lines, 619 each dot represents one first-generation transformed (T1) plant.

620 Supplemental Figure 7. Average distribution of H3K27ac and H3K36ac over protein- coding 621 genes grouped by their expression levels. TSS, transcription start site; TES, transcription end site.

622 Supplemental Figure 8. Validation of ChIP-seq and RNA-seq analyses. Genome browser 623 snapshots at different TEs showing H3K27ac enrichment in atxr5/6, ChIP-qPCR confirmation of 624 H3K27ac enrichment and expression levels for these TEs. At4g06566 and At1g38250 were detected 625 as de-repressed in atxr5/6 by RNA-seq, but not At1g36040 or At5g29602. Data represents the mean 626 of three biological replicates and error bars indicate SEM. Unpaired t-test: * p < 0.05, ** p < 0.01 and 627 *** p < 0.001.

628 Supplemental Figure 9. Scatterplots and Pearson correlation coefficients for RNA-seq 629 replicates of Col, atxr5/6, gcn5 and atxr5/6 gcn5.

25 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

630 Supplemental Figure 10. Scatterplots and Pearson correlation coefficients for H3K27ac and 631 H3K36ac ChIP-seq replicates of Col, atxr5/6, gcn5 and atxr5/6 gcn5.

632 Supplementary Tables

633 Supplemental Table 1. TEs de-repressed in atxr5/6. TEs highlighted in blue are detected only at 634 the highest sequencing depth. TEs in green are detected only at the lowest sequencing depth. TEs 635 highlighted in gray are detected at the highest and lowest sequencing depths.

636 Supplemental Table 2. Misregulated genes in atxr5/6, gcn5 and atxr5/6 gcn5.

637 Supplemental Table 3. Regions of Arabidopsis genome defined as heterochromatin.

638 Supplemental Table 4. Heterochromatic regions enriched in H3K27ac and H3K36ac in atxr5/6.

639 Supplemental Table 5. TEs that are de-repressed and overlap with heterochromatic regions 640 enriched in H3K27ac and H3K36ac in atxr5/6.

641 Supplemental Table 6. Rx factors for Col, atxr5/6, gcn5 and atxr5/6 gcn5 replicates.

642 Supplemental Table 7. Cloning and PCR primers.

26 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

643 Author Contributions 644 Y.J., J.D., C.L. and A.P. designed the experiments. Y.J. wrote the paper with contributions from J.D., 645 C.L. and A.P. All in vitro assays were performed by J.D. C.L. performed the ChIP experiments. A.P. 646 did the bioinformatics analyses of all ChIP-seq experiments. A.P. and V.J. did RNA-seq analyses. 647 Microscopy was done by C.L. Flow cytometry analyses were performed by C.L., B.M, G.V and J.M. 648 RNA extractions and RT-qPCR were done by C.L. and J.D and G.V. Crosses were done by G.V. and 649 B.M. Genotyping and plant transformations were performed by G.V., J.M., C.L., and B.M. G.V. made 650 the CRISPR/Cas9 mutants. K.M.W. and P.V. made the modified and unmodified nucleosomes used

651 in the in vitro assays.

652 Acknowledgments 653 We thank members of our lab for discussions and advice during the course of this work. We want to 654 acknowledge Christopher Bolick and his staff at Yale for help with plant growth and maintenance. 655 We also thank Jean-François Couture (University of Ottawa) for sending the K27M nucleosomes 656 used in this study, and Kenneth Nelson (Yale University) for technical help with flow cytometry. This 657 project was supported by grant #R35GM128661 from the National Institutes of Health to Y.J. B.M. 658 was supported by a Yale University Brown Fellowship. V.J. is supported by the Fonds de Recherche 659 du Québec-Nature et Technologies (FRQNT) [272565]. Work in the Voigt lab is supported by the 660 Wellcome Trust ([104175/Z/14/Z], Sir Henry Dale Fellowship to P.V.) and the European Research 661 Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (ERC- 662 STG grant agreement No. 639253). The Wellcome Centre for Cell Biology is supported by core 663 funding from the Wellcome Trust [203149]. We are grateful to the Edinburgh Protein Production 664 Facility (EPPF) for their support. The EPPF was supported by the Wellcome Trust through a Multi- 665 User Equipment grant [101527/Z/13/Z]. The authors declare that they have no competing interests.

666 Competing Interests 667 The authors declare that they have no competing interests.

27 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

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33 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A B Col 150 atxr5/6 Col atxr5/6 300 100 200 2.8 15.0 Count 50 100

0 0 2C 4C 8C 16C 2C 4C 8C 16C 400 gcn5 atxr5/6 gcn5 gcn5 250 atxr5/6 gcn5 300 200

200 150 2.9

Count 100 100 2.3 50 0 100 200 300 400 5 um 0 0 2C 2C 4C 4C 8C 8C 16C 16C 2C2C 4C 4C 8C 8C 16C 16C PI PI

C TPM D Upregulated Downregulated 0 100 200 300 400 atxr5/6 gcn5 atxr5/6 gcn5 288 145

TPM 62 46 356 780 48 188 atxr5/6 atxr5/6 163 224 7 12 gcn5 120 gcn5 270

Col atxr5/6 gcn5 atxr5/6 gcn5

Col atxr5/6 gcn5 atxr5/6 gcn5

Figure 1. A mutation in GCN5 suppresses transcriptional de-repression and heterochromatin amplification associated with H3.1K27me1 depletion. (A) Flow cytometry profiles of Col, atxr5/6, gcn5 and atxr5/6 gcn5 nuclei stained with propidium iodide (PI) with 2000 gated events. The numbers below the peaks indicate ploidy levels of the nuclei. The numbers above the 16C peaks indicate the robust coefficient of variation (CV). (B) Leaf interphase nuclei of Col, atxr5/6, gcn5 and atxr5/6 gcn5 stained with DAPI. (C) Heat map showing the relative expression levels of atxr5/6-induced TEs (Supplemental Table 1) as measured by TPM (transcripts per million) in Col, atxr5/6, gcn5 and atxr5/6 gcn5. (D) Euler diagrams showing the upregulated and downregulated genes (2-fold change) in atxr5/6, gcn5 and atxr5/6 gcn5 in comparison to Col plants (Padj < 0.05). bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. A B

100 SAGA 400 Col atxr5/6 14.5 80 complex 300 SPT Module 60 200 TRA1a/b 40 Count HAT module TAF13 100 2.1 20 ADA1a/b ADA2a/b HAF1 0 0 2C 4C 8C 16C 2C 4C 8C 16C GCN5 TAF9 SPT20 SGF29a/b HAF2 400 ADA3 TAF10 TAF5 ada2b atxr5/6 ada2b 300 TAF12a/b 300 TAF6a/b 200 200 ENY2 2.5 UBP22 TAF Module 2.3 SFG11 Count 100 100 CHR5 DUB Module CHR6 0 0 Chromatin Module 2C 4C 8C 16C 2C 4C 8C 16C

300 ada3 100 atxr5/6 ada3 14.6 80 200 60 2.6 Count 100 40 20 0 0 2C 4C 8C 16C 2C 4C 8C 16C PI PI

12 C * D 3000 ** ** 10 *

8 2000

6

4 1000 Expression Expression

2 Relative TSI Relative BRCA1 0 0 Col atxr5/6 ada2b atxr5/6 Col atxr5/6 ada2b atxr5/6 ada2b ada2b

400 E Col 400 chr5 500 chr6 400 300 300 2.0 300 200 200 1.8 200 Count 100 100 2.2 100 0 0 0

150 atxr5/6 200 atxr5/6 chr5 atxr5/6 chr6 300 100 150 200 100 Count 10.8 50 10.4 50 100 2.6

0 0 0 2C 4C 8C 16C 2C 4C 8C 16C 2C 4C 8C 16C PI PI PI

Figure 2. GCN5 requires ADA2b to induce heterochromatic defects in atxr5/6 mutants. (A) Proposed subunits of the Arabidopsis SAGA complex. HAT: histone acetylation module; DUB: deubiquitination module; SPT: recruiting module; TAF; coactivator architecture module. (B) Flow cytometry profiles of Col, atxr5/6, ada2b, atxr5/6 ada2b, ada3, and atxr5/6 ada3. (C and D) RT-qPCR analyses of BRCA1 (C) and the repetitive element TSI (D) in Col, atxr5/6, ada2b and atxr5/6 ada2b. Data represent the mean of three biological replicates and error bars indicate the standard error of the mean (SEM). Unpaired t-test: * p < 0.01, ** p < 0.001. (E) Flow cytometry profiles of Col, atxr5/6, chr5, atxr5/6 chr5, chr6, and atxr5/6 chr6. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. A B

No enzyme GCN5-ADA2b H3.1K27ac H3.3K27ac Peptide Peptide H3.1 H3.3 H3.1 H3.3 2 0.5 2 0.5 (µg)

H3K9ac H3K27ac

H3K14ac Coomassie

H3K18ac C No enzyme GCN5-ADA2b WT K27M K27MWT H3K23ac H3K27ac

H3K27ac

H3 H3K36ac

H3 D No Enzyme GCN5-ADA2b K27me0 K27me1 K27me0 K27me1

H3K27ac

H3

E F 3000 20

15

ss io n 2000

10 * TSI ex pre

1000

5 tive Rela tive

* Relative BRCA1 expressio n

0 0 Col atxr5/6 H3.1 H3.1K27Q H3.1S28A Col atxr5/6 H3.1 H3.1K27Q H3.1S28A

Figure 3. Arabidopsis GCN5 acetylates H3.1K27 and induces the heterochromatic defects associated with atxr5/6. (A) In vitro HAT assays with the GCN5-ADA2B complex and H3.1 and H3.3 nucleosomes using anti-H3K9ac, anti-H3K14ac, anti-H3K18ac, anti-H3K23ac, anti-H3K27ac, anti-H3K36ac and anti-H3 antibodies. (B) Western blot of H3.1K27ac and H3.3K27ac peptides with H3K27ac antibody. (C) In vitro HAT assay with the GCN5-ADA2B complex and H3K27M nucleo- somes. (D) In vitro HAT assays with the GCN5-ADA2B complex and H3K27me0 and H3K27me1 nucleosomes using anti-H3K27ac, anti-H3 antibodies (E and F) RT-qPCR for the heterochromatic transcriptional reactivation marker TSI (E) and the genome stability marker BRCA1 (F) in Col, atxr5/6 and first-generation transformed (T1) plants expressing WT H3.1 or H3.1K27Q. Ten independent T1 plants were used in the experiments. Unpaired t-test: * p < 0.001. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made A available under aBCC-BY 4.0 International license.

20 n.s. * 6000 n.s. H3.1 n.s. 5000 H3.1S28A 15 n.s. n.s. 4000 * 10 3000 Robust CV CPM 2000 5 1000

0 0 No Enzyme ATXR6

Col WT K9R S28A K14R K18R K23R K36R atxr5/6 S28A K9R S28A K14R S28A K18R S28A K23R S28A K36R C D

60 * * 400 * n.s. n.s. 300 40 * * Expression 200 * 20 100

0 Relative TSI Expression 0 Relative BRCA1 Col K9R Col K9R S28A K14R K18R K23R K36R S28A K14R K18R K23R K36R atxr5/6 atxr5/6 H3.1 WT H3.1 WT S28A K9R S28A K9R S28A K14R S28A K18R S28A K23R S28AK36R S28A K14R S28A K18R S28A K23R S28AK36R E F

20 Col 15 atxr5/6 SDG24-OX in atxr5/6 * 15

10 10

5 Robust CV Percent Input (%) Percent Input 5

0 0 Col Ta3 At1g38250 At4g06566 Actin 7 atxr5/6 Heterochromatin euchromatin SDG24-OXatxr5/6 atxr5/6 in SDG24-OX (Y140N) in Figure 4. Heterochromatin amplification in the absence of H3.1K27me1 requires H3.1K36 (A) In vitro histone lysine methyla- tion assays using H3.1 and H3.1S28A peptide substrates and ATXR6. The average of three experiments and SEM are shown. CPM; counts per minute (B) Robust CV values for 16N nuclei obtained by flow cytometry analysis. For Col and atxr5/6, each dot represents an independent biological replicate. For the H3.1 replacement lines, each dot represents one T1 plant. Horizontal bars indicate the mean. Unpaired t-test: * p < 0.0001 and n.s. = not significantly different. (C and D) RT-qPCR analyses of BRCA1 (C) and the repetitive element TSI (D) in Col, atxr5/6 and H3.1 replacement lines. For Col and atxr5/6, each dot represents an indepen- dent biological replicate. For the H3.1 lines, each dot represents one T1 plant. Horizontal bars indicate the mean. Unpaired t-test: * p < 0.01, ** p < 0.0005 (E) Flow cytometry analyses showing robust CV values for 16N nuclei. For the SDG24-OX lines, each dot represents one T1 plant. Horizontal bars indicate the mean. Unpaired t-test: * p < 0.001. (F) H3K36me3 ChIP-qPCR at Ta3, At1g38250, At4g06566 and Actin7. For Col and atxr5/6, each dot represents an independent biological replicate. For the SDG24-OX lines, each dot represents one T1 plant. Bars indicate the mean. Error bars indicate SEM. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which A was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Genes 400 H3K27ac 600 H3K36ac Col 500 atxr5/6 300 gcn5 400 atxr5/6 gcn5 200 300 RRPM 200 100 100

-1.0kb TSS TES 1.0kb -1.0 TSS TES 1.0Kb

B Col atxr5/6 gcn5 atxr5/6 gcn5 H3K27ac H3K36ac H3K27ac H3K36ac H3K27ac H3K36ac H3K27ac H3K36ac 100 80 60 40 RRPM 20 -3.0Kb 0 3.0Kb 80

60 RRPM

40

20 -3.0Kb 0 3.0Kb C D 11,272,500 bp 11,273,500 bp 11,274,500 bp Col atxr5/6 gcn5 atxr5/6 gcn5 50 Col [0-150]

atxr5/6 [0-150] 40

gcn5 [0-150]

H3K27ac 30 atxr5/6 gcn5 [0-150]

20 RPKM Col [0-150]

atxr5/6 [0-150] 10

gcn5 [0-150]

H3K36ac 0 atxr5/6 gcn5 [0-150] Figure 5. Mutations in atxr5/6 lead to an increase in H3K27ac and H3K36ac in heterochromatin. (A) Normalized average distribution of H3K27ac and H3K36ac over protein coding genes for Col, atxr5/6, gcn5 and atxr5/6 gcn5 in reference-adjusted reads per million (RRPM). TSS, transcrip- At5G29591 At5G29602 tion start site; TES, transcription end site. (B) Normalized average distribution and heatmap of H3K27ac and H3K36ac normalized reads surrounding the H3K27ac/H3K36ac enriched heterochromatic regions in atxr5/6 compared to Col. (C) Genome browser snapshot showing normalized H3K27ac and H3K36ac ChIP-seq data over a region of chromosome 5 that includes TE genes At5g29591 and At5g29602. The y-axis unit is RRPM. (D) Heatmap showing the RNA-seq reads mapping to the region 3 kb around the center of H3K27ac/H3K36ac peaks as measured by RPKM (reads per kilobase million) in Col, atxr5/6, gcn5 and atxr5/6 gcn5. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A B C No Enzyme GCN5-ADA2b No Enzyme GCN5-ADA2b

K27me0 K27me1 K27me0 K27me1 K36me0 K36me3 K36me0 K36me3

1.5 H3K27me0 nucleosomes H3K36ac H3K27ac H3K27me1 nucleosomes

n.s. * H3 H3K9ac 1.0

H3 H3K36ac 0.5

H3 Relative acetylation level 0.0 H3K36Ac H3K9Ac

D ATXR5 Col ATXR5 atxr5 atxr6

ATXR6 K27 me ATXR6 K27 ac K36 K36 ac GCN5 H3.1 H3.1 Ada2B GCN5 Ada2B

Transcriptional silencing Transcriptional de-repression Genome stability Heterochromatin amplification

Figure 6. H3K36 acetylation by the GCN5-ADA2B complex is regulated by H3K27me1. (A) In vitro HAT assays with the GCN5-ADA2b complex and H3K27me0 and H3K27me1 nucleosomes. (B) Data from three technical replicates of HAT assays with the GCN5-ADA2b complex and H3K27me0 and H3K27me1 nucleosomes. Unpaired t-test: * p < 0.05, and n.s. = not significantly different. (C) In vitro HAT assays with the GCN5-ADA2b complex and H3K36me0 and H3K36me3 nucleosomes. D) Model depicting the role of H3.1K27me1 in blocking GCN5-mediated acetylation of H3.1K27ac and H3.1K36ac. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A 1 bp insertion (atxr5 atxr6 gcn5) SALK_030913 4 bp deletion (gcn5) ATG Stop

GCN5 (At3g54610)

B C Fully condensed 120 Hollow spheres 1 bp insertion (atxr5 atxr6 gcn5) Irregularly/partially decondensed 100 24-AACGCCGCCAAATCGTTCCCGCAGTTCTCAGACTC gcn5

80 CC : AATCGTTCCCGCAGTTCT gRNA

60

40 4 bp deletion (gcn5) Percentage (%) 20 238-CGATGAACTCGAGAGCATCTC :::: CGCGGCGCC gcn5 0 Col atxr5/6 gcn5' atxr5/6 gcn5 CTCGAGAGCATCTCCGCTCG gRNA

D E Col 4.4 atxr5 atxr6 12.2 9 8 7 6

Count 5

TPM 4 3 2 2C 4C 8C 16C 2C 4C 8C 16C 1 gcn5 atxr5 atxr6 gcn5 0 (CRISPR allele) (CRISPR allele) SE AtTHP1 AtSAC3B AtStubl2 AtMBD9 DDM1 3.7 3.0 Col atxr5/6 gcn5 atxr5/6 gcn5 Count

2C 4C 8C 16C 2C 4C 8C 16C PI PI

Supplemental Figure 1. Effect of GCN5 on genome stability and transcriptional de-repression. (A) Gene structure for GCN5. Exons are highlighted as black boxes. The location of the mutations in the gcn5 alleles used in this study are shown. (B) Quantification of chromocenter appearance. Shown is the percentage of nuclei that are fully condensed (green), hollow spheres characteristic of the atxr5/6 mutant plants (blue) and irregularly/partially decondensed (grey). At least 25 nuclei for 3 biological replicates of each genotype were assessed. Error bars indicate SEM (C) CRISPR-in- duced mutations of GCN5 in Col and atxr5/6 backgrounds. Mutations (red) and PAM motif (blue) are shown. (D) Flow cytometry profiles of Col, atxr5/6 and the CRISPR-induced knockout allele of gcn5 in Col and the atxr5/6 mutant background. The numbers below the peaks indicate ploidy levels of the nuclei. The numbers above the 8C peaks indicate the robust CV. (E) Gene expression levels in our RNA-seq experiments for the known suppressors of transcriptional de-repression in atxr5/6. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A D

Col atxr5 atxr6 2.3 atxr5 Col 11.5

atxr6 Count

atxr5 2C 4C 8C 16C 2C 4C 8C 16C gcn5 atxr6 gcn5 eny2 atxr5 atxr6 eny2 atxr5 ada2b atxr6 ada2b 2.0 12.7 Count

2C 4C 8C 16C 2C 4C 8C 16C

B BRCA1 ubp22 atxr5 atxr6 ubp22 12

10 10.0 8 Count 2.5 6 2C 4C 8C 16C 2C 4C 8C 16C

BRCA1 Expression 4 2 sgf11 atxr5 atxr6 sgf11

Relative 0 4.4 Col atxr5/6 gcn5 atxr5/6 gcn5 13.8 Count C TSI 2C 4C 8C 16C 2C 4C 8C 16C 50 PI PI

40

30 Expression

TSI 20

10

Relative 0 Col atxr5/6 gcn5 atxr5/6 gcn5

Supplemental Figure 2. Role of SAGA-related proteins in transcriptional de-repression and genome stability. (A) Growth phenotype of gcn5 and ada2b single mutants and in combination with atxr5/6. (B and C) RT-qPCR for the genome stability marker BRCA1 (B) and the heterochromatic transcriptional reactivation marker TSI (C). The average of three biological replicates and SEM are shown. (D) Flow cytometry profiles of the mutant alleles of genes predicted to code for subunits of SAGA in plants. The Robust CV value calculated for the 16C peak on each plot is used as a measure of heterochromatin amplification. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

(kDa) Marker Pre Post Eluate

80 GCN5 58 His-ADA2b

46

37

25 22

Supplemental Figure 3. Purification of the GCN5-ADA2b complex. Coomassie-stained gel showing GCN5-ADA2b protein expression either pre- or post-induction in E. coli, or after affinity purification (eluate). bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A B

25 000 H3.1 peptide H3.1S28A peptide H3.1 15-APRRQLATRAARKSAPATGGVRRPHR-35 No peptide 20 000 H3.1S28A 15-APRRQLATRAARKAAPATGGVRRPHR-35

15 000 CPM

10 000 CPM (x 1000) 5000

0 _ _ No enzyme ATXR6 FIE-MSI1 FIE-MSI1 GCN5-ADA2b + + MEA-EMF2 CLF-EMF2 H3.1 peptide H3.1S28A peptide

Supplemental Figure 4. In vitro histone modification assays. (A) In vitro histone lysine methylation assays at H3.1K27 using peptide substrates, ATXR6 and plant PRC2 complexes. The average of three experiments and SEM are shown. (B) In vitro HAT assays using H3.1 peptides. Lysine (K) to arginine (R) mutations were introduced (blue) on the peptides at other potential targets (H3.1K18, H3.1K23, H3.1K36 and H3.1K37) of GCN5-ADA2b, so that the acetylation signal could be specifically measured at H3.1K27 (red). H3.1S28A is shown in green. The average of three experiments and SEM are shown. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Col

H3.1

Col atxr5/6

H3.1 H3.1S28A

H3.1K9R H3.1S28A K9R

H3.1K14R H3.1S28A K14R

H3.1K18R H3.1S28A K18R

H3.1K23R H3.1S28A K23R

H3.1K36R H3.1S28A K36R

Supplemental Figure 5. Growth and developmental phenotypes of T1 plants expressing different H3.1 trans- genes. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

15

10

Robust C V 5

0

Col atxr5/6 SDG4-OXSDG7-OXSDG8-OX SDG26-OX

Supplemental Figure 6. Robust CV values for 16N nuclei obtained by flow cytometry analyses. For Col and atxr5/6, each dot represents an independent biological replicate. For overexpression lines, each dot represents one first-generation transformed (T1) plant. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

H3K27ac over genes based on expression levels H3K36ac over genes based on expression levels

Col (0 to 1 TPM) 1000 Col (1 to 5 TPM) 500 Col (5 to 10 TPM) Col (10 to 20 TPM) 800 Col (up 20 TPM) 400 600 300 RRPM 200 400

100 200

-1.0kb TSS TES 1.0kb -1.0kb TSS TES 1.0kb

Supplemental Figure 7. Average distribution of H3K27ac and H3K36ac over protein- coding genes grouped by their expression levels. TSS, transcription start site; TES, transcrip- tion end site. bioRxiv preprint was notcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade 0.05, **p<0.01and***0.001. At5g29602. Datarepresentsthemeanofthreebiologicalreplicatesand errorbarsindicateSEM.Unpairedt-test:* these TEs. showing H3K27acenrichmentin Supplemental Figure 8. Validation ofChIP-seqandRNA-seqanalyses. atxr5/6 gcn5 gcn5 atxr5/6 Col atxr5/6 gcn5 gcn5 atxr5/6 Col atxr5/6 gcn5 gcn5 atxr5/6 Col atxr5/6 gcn5 gcn5 atxr5/6 Col doi: At4g06566 andAt1g38250weredetectedasde-repressedin https://doi.org/10.1101/2020.07.17.209098 At1g36040 At1g38250 At4g06566 atxr5/6, ChIP-qPCRconfirmationofH3K27acenrichmentandexpression levelsfor At5g29602 available undera ; CC-BY 4.0Internationallicense this versionpostedJuly17,2020. [0-150] [0-150] [0-150] [0-150] [0-300] [0-300] [0-300] [0-300] [0-150] [0-150] [0-150] [0-150] [0-75] [0-75] [0-75] [0-75]

% Input % Input % Input % Input 0.5 1.5 0.5 1.5 2.5 0 1 3 4 0 2 5 0 1 1 2 3 1 2 2 0 atxr5/6 byRNA-seq,butnotAt1g36040or Col Col Col Col ** * tr/ c5atxr5/6 gcn5 atxr5/6 * H3K27ac ChIP * * Genome browsersnapshotsatdifferent TEs H3K27ac ChIP H3K27ac ChIP H3K27ac ChIP tr/ c5atxr5/6 gcn5 atxr5/6 * tr/ c5atxr5/6 gcn5 atxr5/6 tr/ c5atxr5/6 gcn5 atxr5/6 . * * * The copyrightholderforthispreprint(which ** ** * * gcn5 gcn5 gcn5 gcn5

Relative Expression Relative Expression Relative Expression

Relative Expression 10 20 30 40 50 0 4 0 0 1 1 2 3 2 3 4 10 20 30 40 50 0 Col Col Col Col ** ** tr/ c5atxr5/6 gcn5 atxr5/6 tr/ c5atxr5/6 gcn5 atxr5/6 * * * tr/ c5atxr5/6 gcn5 atxr5/6 atxr5/6 gcn5 atxr5/6 RT-qPCR RT-qPCR RT-qPCR RT-qPCR * * ** ** * * * gcn5 gcn5 gcn5 gcn5 p < bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Col Rep 1 atxr5/6 Rep 1

Col Rep 2 atxr5/6 Rep 2 gcn5 Rep 1 atxr5/6 gcn5 Rep 1

gcn5 Rep 2 atxr5/6 gcn5 Rep 2

Supplemental Figure 9. Scatterplots and Pearson correlation coefficients for RNA-seq replicates of Col, atxr5/6, gcn5 and atxr5/6 gcn5. bioRxiv preprint was notcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade Col rep1 Col rep1 H3K36ac ChIP-seq H3K27ac ChIP-seq pearson=0.90 pearson=0.99

Col rep2 5

H3K36ac ChIP-seqreplicates ofCol, Supplemental Figure 10.ScatterplotsandPearsoncorrelation coefficientsfor H3K27ac and 5 doi: Col rep2 https://doi.org/10.1101/2020.07.17.209098

10 10 4 6 8 10 12 2 4 6 8 10 12

atxr5/6 rep1 atxr5/6 rep1 available undera atxr5/6 5.0 pearson=0.96

5.0 pearson=0.98 atxr5/6

7.5 7.5 rep2 rep2

10.0 10.0 atxr5/6, gcn5andatxr5/6. ; CC-BY 4.0Internationallicense

12.5 12.5 this versionpostedJuly17,2020. 4 6 8 10 12 12 4 6 8 10

gcn5 rep1 gcn5 rep1

5.0 5.0 pearson=1.00 pearson=0.99 gcn5 gcn5

7.5 7.5 rep2 rep2

10.0 10.0 . 12.5

12.5 The copyrightholderforthispreprint(which 12 6 8 10 4 6 8 10 12

atxr5/6 gcn5 rep1 atxr5/6 gcn5 rep1 atxr5/6 gcn5 atxr5/6 5.0 gcn5 atxr5/6 5.0 pearson=0.99 pearson=0.99

7.5 7.5

10.0 10.0 rep2 rep2

12.5 12.5 4 6 8 10 12 4 6 8 10 12 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Col Col atxr5/6 atxr5/6 gcn5 gcn5 atxr5/6 atxr5/6 Rep1 Rep2 Rep1 Rep2 Rep1 Rep2 gcn5 gcn5 Rep1 Rep2 D. melanogaster 51265 134411 69402 104229 114932 100768 69215 115431 unique H3 reads A. thaliana unique H3 30827170 57924375 39621511 50359868 54725117 44476014 49878156 44258118 reads % of D. melanogaster 0.16629811 0.23204566 0.17516243 0.20696837 0.21001691 0.22656707 0.13876816 0.26081317 H3 reads D. melanogaster 73203 154549 69048 88588 211097 172676 161463 171638 H3K27ac reads A. thaliana H3K27ac 35425890 53958274 39611214 38922755 34654299 36321638 50898781 35808072 reads

H3K27ac Rx 2.2717389 1.50143749 2.53682114 2.33630259 0.99488344 1.31209355 0.85944248 1.51955376 factor D. melanogaster 100430 134550 120870 126640 355181 302930 35828 223401 H3K36ac reads A. thaliana H3K36ac 40539185 47383778 38801208 41274576 20973432 19928195 25937684 18896708 reads

H3K36ac Rx 1.65586086 1.72460545 1.44918033 1.63430491 0.59129545 0.74791888 0.413212005 1.167466434 factor

Supplemental Table 6. Rx factors bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209098; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Gene Forward Primer Reverse Primer

Cloning ADA2B into caggtcgacATGGGTCGCTCTCGAGGG cttgcggccgcTTAAAGTTGAGCAATACCCTTCTT pETDuet-1 AACTTC CAC

GCN5 into ttggatatcg agcttaattaaCTATTGAGATTTAGCACCAGATTG pETDuet-1 ATGGACTCTCACTCTTCCCAC ADA2B

RT-qPCR

BRCA1 CATGTGCCTTTTGTCAGTGTTC-3’ TGGAGCCCATTCAGCACAGTTT

TSI ATCCAGTCCGAAGAACGCGAACTA TCACTTGTGAGTGTTCGTGAGGTC

Actin TCGTGGTGGTGAGTTTGTTAC CAGCATCATCACAAGCATCC

At1G38250 CAACAAGCCGCCAAGTTCCTA AGGGTTTAGTGGCTCTTGGAG

At4G06566 GTGAAACATATCCCACGCACT GGATAGTTATGAGCAAGTGGT

At1G36040 CTTTGACTCAGCGCAAGAGA GAGGCAGATCGGTTGGATTT

At5G29602 ACAGAGGACTCGTCAGTCG ATGGTGAGCTCTCAACAATCTC

ChIP-qPCR

Ta3 GAGAGCAAACAGAGTGAGGCTCGT TCGTCGCCATCGATCAGAATCAAG

Actin 7 GCTGCTCGTTAGTCGTTATTGTTG CAAGCACGGATCGAATCACATAAC

At1G38250 GCTTCACCATGAGTTCAAACAG CAAGCCTAGTAGACCAAGATTCA

At4G06566 GTGAAACATATCCCACGCACT GGATAGTTATGAGCAAGTGGT

At1G36040 CTTTGACTCAGCGCAAGAGA GAGGCAGATCGGTTGGATTT

At5G29602 ACAGAGGACTCGTCAGTCG ATGGTGAGCTCTCAACAATCTC

Supplemental Table 7. Cloning and PCR primers