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RESEARCH ARTICLE 1

The Chromatin Remodelers PKL and PIE1 Act in an Epigenetic Pathway that 2

Determines H3K27me3 Homeostasis in Arabidopsis 3

Benjamin CarterA, Brett Bishop

A, Kwok Ki Ho

A, Ru Huang

B, Wei Jia

B, Heng Zhang

B, Pete E. 4

PascuzziA,C

, Roger B. DealD , Joe Ogas

A 5

6 ADepartment of Biochemistry, Purdue University, IN, United States 7 BShanghai Center for Plant Stress Biology, Songjiang District, Shanghai, China 8 CPurdue University Libraries, Purdue University, IN, United States 9 DDepartment of Biology, Emory University, Atlanta, GA, United States 10 *To whom correspondence should be addressed.1 11 12 Short title: Epigenetic pathway links H2A.Z and H3K27me3 13

One-sentence summary: The chromatin remodelers PKL and PIE1 and the histone methyltransferase 14 CLF act in an epigenetic pathway that promotes and maintains the repressive epigenetic modification 15 H3K27me3 in Arabidopsis. 16 17 The author(s) responsible for distribution of materials integral to the findings presented in this article in 18 accordance with the policy described in the Instructions for Authors (www.plantcell.org) is (are): Joseph 19 P. Ogas (ogas@purdue.edu). 20 21 22

ABSTRACT 23

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Selective, tissue-specific gene expression is facilitated by the epigenetic modification 25

H3K27me3 (trimethylation of lysine 27 on histone H3) in plants and animals. Much remains to 26

be learned about how H3K27me3-enriched chromatin states are constructed and maintained. 27

Here we identify a genetic interaction in Arabidopsis thaliana between the chromodomain 28

helicase DNA-binding chromatin remodeler PICKLE (PKL), which promotes H3K27me3 29

enrichment, and the SWR1-family remodeler PHOTOPERIOD INDEPENDENT EARLY 30

FLOWERING 1 (PIE1), which incorporates the histone variant H2A.Z. Chromatin 31

immunoprecipitation-sequencing and RNA-sequencing reveal that PKL, PIE1, and the H3K27 32

methyltransferase CURLY LEAF act in a common gene expression pathway and are required for 33

H3K27me3 levels genome-wide. Additionally, H3K27me3-enriched genes are largely a subset of 34

H2A.Z-enriched genes, further supporting the functional linkage between these marks. We also 35

found that recombinant PKL acts as a prenucleosome maturation factor, indicating that it 36

promotes retention of H3K27me3. These data support the existence of an epigenetic pathway in 37

which PIE1 promotes H2A.Z, which in turn promotes H3K27me3 deposition. After deposition, 38

PKL promotes retention of H3K27me3 after DNA replication and/or transcription. Our analyses 39

thus reveal roles for H2A.Z and ATP-dependent remodelers in construction and maintenance of 40

H3K27me3-enriched chromatin in plants. 41

Plant Cell Advance Publication. Published on May 25, 2018, doi:10.1105/tpc.17.00867

©2018 American Society of Plant Biologists. All Rights Reserved

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INTRODUCTION 43

The ability to establish and maintain distinct chromatin states in eukaryotes allows 44

genetically identical cells to express different sets of genes and thereby differentiate. Different 45

chromatin states are associated with enrichment of specific epigenetic marks including histone 46

variants, histone tail post-translational modifications, and DNA methylation. In Arabidopsis 47

thaliana, genome-wide analyses have identified nine chromatin states based on enrichment of 48

sixteen epigenetic marks (Sequeira-Mendes et al., 2014). How chromatin states are both 49

constructed and maintained, particularly in dividing cells, is unclear. 50

Chromatin states associated with silencing of tissue-specific genes are commonly 51

enriched for the repressive histone tail modification trimethylation of histone H3 at lysine 27 52

(H3K27me3) (Lafos et al., 2011). In Arabidopsis, loss of H3K27me3 results in derepression of 53

lineage-specific genes, failed development, and progressive degeneration into a disorganized 54

callus (Bouyer et al., 2011). H3K27me3 is thought to silence gene expression in combination 55

with other epigenetic machinery by excluding activating epigenetic marks and by promoting a 56

condensed chromatin environment that is refractory to transcription (Aranda et al., 2015; Del 57

Prete et al., 2015). Deposition of H3K27me3 is catalyzed by the Enhancer of zeste (E(z)) family 58

of histone methyltransferases that are components of the Polycomb Repressive Complex 2 59

(PRC2) (Khan et al., 2015; Kim and Sung, 2014; Margueron and Reinberg, 2011; Mozgova et 60

al., 2015). CURLY LEAF (CLF), MEDEA (MEA), and SWINGER (SWN) are the three E(z) 61

family members in Arabidopsis (Mozgova et al., 2015). These enzymes function in distinct 62

PRC2 complexes and deposit H3K27me3 within differing subsets of genes during different 63

stages of development (de Lucas et al., 2016). MEA contributes to imprinted gene expression in 64

the endosperm and is necessary for seed viability, whereas CLF and SWN cooperatively promote 65

tissue identity in seedlings and adult plants. Seedlings lacking both CLF and SWN degenerate 66

into callus (Chanvivattana et al., 2004; Muller-Xing et al., 2014) reminiscent of plants in which 67

H3K27me3 deposition is abolished (Bouyer et al., 2011). 68

Three of the nine characterized chromatin states in Arabidopsis exhibit strong 69

H3K27me3 enrichment (Sequeira-Mendes et al., 2014), corresponding to approximately 17% of 70

genes in ten-day-old seedlings (Zhang et al., 2007). Notably, these chromatin states are also 71

enriched for the histone variant H2A.Z (Sequeira-Mendes et al., 2014), which plays a role in 72

3

making chromatin dynamic (Subramanian et al., 2015). In particular, genome-wide 73

characterization of H2A.Z enrichment in yeast and animals reveals that the transcription start 74

sites (TSS) of many genes are enriched for H2A.Z, where it is thought to reduce the energy 75

requirement for RNA polymerase II to pass the +1 nucleosome during transcription 76

(Subramanian et al., 2015; Weber et al., 2014). H2A.Z also has been linked to a variety of 77

processes related to gene expression where it is thought to alter nucleosome composition, 78

modification state, and/or stability and thereby alter nucleosome dynamics and chromatin 79

accessibility (Subramanian et al., 2015). For example, H2A.Z facilitates nucleosome depletion 80

and binding of the transcription factor Foxa2 to its targets during mouse embryonic stem cell 81

differentiation (Li et al., 2012). Notably, H3K27me3 and H2A.Z co-localize at bivalent TSS in 82

mouse and human embryonic stem cells (Ku et al., 2012), which are poised to be repressed or 83

activated. In contrast, H2A.Z is absent from H3K27me3-enriched promoters that are stably 84

repressed (Ku et al., 2012). In Arabidopsis, H2A.Z is enriched in the TSS of many genes as was 85

observed in yeast and animals (Coleman-Derr and Zilberman, 2012). In addition, H2A.Z is 86

enriched in the bodies of many genes associated with stimulus-response, where its presence 87

correlates with reduced expression and is necessary for normal transcriptional induction of these 88

loci in response to environmental and developmental cues (Coleman-Derr and Zilberman, 2012). 89

These studies suggest that H2A.Z enrichment in the gene body in plants both enables 90

transcriptional activation in the presence of the appropriate stimulus and contributes to 91

transcriptional repression in the absence of appropriate inductive signals. 92

The molecular machinery associated with incorporation of H2A.Z into chromatin has 93

been characterized in yeast and animals and to a lesser extent in plants. The SWR1 class of ATP-94

dependent remodelers is named after Swr1p in Saccharomyces cerevisiae, which incorporates 95

H2A.Z into nucleosomes as part of a multisubunit complex via histone dimer exchange (Krogan 96

et al., 2003; Lu et al., 2009; Mizuguchi et al., 2004). The animal SWR1 family member SRCAP 97

is a subunit of a similar complex that also promotes incorporation of H2A.Z (Lu et al., 2009; 98

Morrison and Shen, 2009; Ruhl et al., 2006; Wong et al., 2007). In Arabidopsis, incorporation of 99

H2A.Z is promoted by the SWR1-family chromatin remodeler PHOTOPERIOD 100

INDEPENDENT EARLY FLOWERING 1 (PIE1), which likely also functions as a subunit of a 101

complex that is similar to the yeast and animal SWR1/SRCAP-containing complexes 102

(Bieluszewski et al., 2015; Choi et al., 2005; Deal et al., 2005; Deal et al., 2007; March-Diaz et 103

4

al., 2007; Noh and Amasino, 2003). Loss of PIE1 results in phenotypes that are consistent with 104

those of Swr1p-related remodelers and H2A.Z in animals and yeast such as deficiencies in DNA 105

damage responses (Rosa et al., 2013; Shaked et al., 2006). In addition, pie1 plants exhibit defects 106

in transcriptional regulation of genes in stimulus response pathways including effector-triggered 107

immunity (Berriri et al., 2016). Such gene expression defects are consistent with the observation 108

that H2A.Z contributes to proper transcriptional regulation of genes that are responsive to 109

environmental or developmental cues (Coleman-Derr and Zilberman, 2012). pie1 plants do not 110

fully phenocopy seedlings severely depleted in H2A.Z (Berriri et al., 2016; March-Diaz et al., 111

2008), suggesting that other factors can contribute to deposition of H2A.Z or that PIE1 has 112

additional roles beyond promoting H2A.Z deposition. Taken together, these studies raise the 113

prospect that PIE1 and/or H2A.Z contribute to transcriptional regulation of H3K27me3-enriched 114

loci, particularly given that such loci are likely to be enriched for H2A.Z in the gene body and 115

are developmentally regulated. 116

Another ATP-dependent chromatin remodeler that is strongly associated with epigenetic 117

control of gene expression in Arabidopsis is the chromodomain helicase DNA-binding (CHD)-118

family remodeler PICKLE (PKL) (Ho et al., 2013). PKL facilitates H3K27me3-related gene 119

expression during multiple developmental processes (Jing et al., 2013; Zhang et al., 2014; Zhang 120

et al., 2012; Zhang et al., 2008), and loss of PKL results in reduced levels of H3K27me3 at 121

numerous loci (Zhang et al., 2008). However, the mechanism by which PKL promotes 122

H3K27me3 is unknown. We undertook a candidate-based reverse genetic approach that 123

identified a strong genetic interaction between PKL and PIE1. Subsequent RNA-seq analysis 124

revealed that PKL, PIE1, and CLF act in a common gene expression pathway. Further, ChIP-seq 125

analysis revealed that H3K27me3-enriched genes are a subset of H2A.Z-enriched genes and that 126

PIE1 acts with PKL and CLF to promote H3K27me3 at a common set of genes. These findings 127

suggest that H2A.Z facilitates deposition of H3K27me3 at these loci. Biochemical 128

characterization of PKL reveals that it promotes formation of mature nucleosomes from 129

prenucleosomes, suggesting a role for PKL in retention of H3K27me3 rather than deposition. 130

Our combined analyses support the existence of a new epigenetic pathway that contributes to 131

both generation and maintenance of H3K27me3-enriched chromatin in plants. 132

133

RESULTS 134

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PKL and PIE1 exhibit a genetic interaction. 135

To investigate the possibility that other factors work with PKL to promote development, 136

we undertook a candidate-based reverse genetic approach and examined the phenotype of plants 137

that carried different mutant alleles of PKL and PIE1, which encodes another epigenetic factor. 138

One of these lines, with concurrent impairment of PKL and PIE1 gene function, exhibited severe 139

developmental defects (Figure 1). Due to the poor fertility of homozygous pie1 plants (March-140

Diaz et al., 2008), this genetic interaction was characterized using the segregating F3 progeny of 141

pkl-10 pie1-5/PIE1 plants. We observed a pronounced developmental delay in 25% of the F3 142

seedlings, consistent with Mendelian segregation of a recessive trait. Compared to wild-type 143

(WT) and pkl-10 seedlings (Figure 1A and B), these seedlings exhibited sharply reduced or 144

absent organogenesis from both the root and shoot meristems (Figure 1C and D). PCR analysis 145

revealed that ten out of ten seedlings exhibiting the severe phenotype were homozygous for pie1-146

5, whereas none out of ten seedlings exhibiting organogenesis was homozygous for pie1-5. 147

148

PKL, PIE1, and CLF act in a common gene expression pathway. 149

The severe synthetic phenotypes of pkl-10 pie1-5 seedlings suggested that PKL and PIE1 150

affect expression of a common subset of genes. To examine this possibility, we undertook RNA-151

seq analysis of WT, pkl-1, and pie1-5 shoot tissue using three independent biological replicates 152

for each genotype (ENCODE, 2016). As a comparative control for pkl-1 plants, we included 153

plants defective in CLF, which encodes a histone methyltransferase that promotes trimethylation 154

of H3K27 (Schmitges et al., 2011; Schubert et al., 2006). Sample quality was assessed using 155

plots generated with the DESeq2 and limma packages in Bioconductor (Supplemental Figure 1) 156

(Love et al., 2014; Ritchie et al., 2015). In total, approximately 700 to 2,000 DEGs were 157

identified for each sample (Figure 2, Supplemental Data Set 1). 158

We analyzed the intersections between sets of DEGs to determine if the various mutants 159

exhibited significant overlap in gene expression outcomes (Figure 3A and B). Consistent with 160

their common role in promoting H3K27me3, we observed that the intersections between DEGs 161

in clf-28 and in pkl-1 are significantly larger than predicted by chance. Based on the synthetic 162

phenotypes of the pkl-10 pie1-5 seedlings, we hypothesized that the intersection between DEGs 163

in pkl-1 and pie1-5 would also exhibit significant overlap. We observed statistically significant 164

intersections not only between pie1-5 and pkl-1 but also, surprisingly, between pie1-5 and clf-28. 165

6

In contrast, intersections between DEG sets that are differentially expressed in opposite 166

directions were not significant (Supplemental Table 1). 167

To further investigate whether pkl-1, clf-28, and/or pie1-5 have common effects on gene 168

expression, we examined the correlation between differential expression of genes in each mutant 169

using linear regression. This analysis revealed varying degrees of correlation for each pairwise 170

combination (Figure 3C-E). DEGs in pkl-1 and clf-28 plants exhibited a high level of correlation 171

(R2 = 0.78), whereas DEGs in pkl-1 and pie1-5 plants were only weakly correlated (R

2 = 0.29). A 172

moderate correlation was also present between DEGs in clf-28 and pie1-5 plants (R2 = 0.46). 173

Identification of pairwise interactions between DEGs in all three of these mutants raised the 174

prospect that PKL, CLF, and PIE1 act in a common pathway. To explore this possibility, we 175

examined the three-way intersections between DEGs exhibiting increased or decreased 176

expression in pkl-1, pie1-5, and clf-28 plants (Figure 3F-G). These intersections were much 177

larger than predicted by chance both for DEGs with increased expression (70 observed vs. 0.5 178

predicted, p < 10-130

) and for DEGs with decreased expression (42 observed vs. 0.8 predicted, p 179

< 10-57

), revealing that impairment of PKL, PIE1, or CLF gene function altered expression of a 180

common subset of genes. Taken together, these analyses indicate that PKL, PIE1, and CLF act in 181

one or more common gene expression pathways. 182

183

H3K27me3-enriched genes are largely a subset of H2A.Z-enriched genes. 184

PKL, PIE1, and CLF have been linked to distinct epigenetic pathways. CLF and PKL 185

promote the repressive epigenetic modification H3K27me3, whereas PIE1 promotes 186

incorporation of the histone variant H2A.Z in chromatin (Deal et al., 2007; Schubert et al., 2006; 187

Zhang et al., 2008). The observation that PKL, PIE1, and CLF affect expression of a common 188

subset of genes raised the prospect that these factors might additionally contribute to one or more 189

common epigenetic pathways. We performed ChIP-seq analysis on WT, pkl-1, clf-28, and pie1-5 190

shoot tissues using two independent biological replicates for each genotype (ENCODE, 2017) to 191

examine the effects of these mutations on levels of H3K27me3 and H2A.Z in chromatin. The 192

sets of genes identified as enriched for H3K27me3 or H2A.Z in WT plants exhibited a high 193

degree of overlap with enriched gene sets from previous analyses (Figure 4A) (Bouyer et al., 194

2011; Coleman-Derr and Zilberman, 2012). Interestingly, we observed that genes enriched for 195

H3K27me3 were almost exclusively a subset of genes enriched for H2A.Z both at the TSS and in 196

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the gene body (Figure 4B, regions defined in the Figure 4 legend). Only ~10% of H3K27me3-197

enriched genes were not also identified as H2A.Z-enriched. Further, genes that were co-enriched 198

for H2A.Z and H3K27me3 exhibited a lower level of expression on average than genes enriched 199

for only one of these marks (Figure 4C). 200

201

PIE1 promotes H3K27me3. 202

Heat maps were generated to visualize enrichment of H3K27me3 and H2A.Z in WT, pkl-203

1, clf-28, and pie1-5 chromatin (Figure 5). Examination of global levels of H3K27me3 204

enrichment relative to WT reveals a reduction in average levels of this modification not only in 205

pkl-1 and clf-28 plants as predicted (Schmitges et al., 2011; Schubert et al., 2006; Zhang et al., 206

2008), but also in pie1-5 plants (Figure 5A and B). Loss of PKL had the largest effect on the 207

average level of H3K27me3, with enrichment in pie1-5 plants falling somewhere between clf-28 208

and pkl-1 plants. Analysis of genes found to be differentially enriched for H3K27me3 in the 209

various mutants relative to WT confirmed the major role played by PKL in promoting this mark: 210

over 7,200 loci (92% of H3K27me3-enriched genes) exhibited some reduction in H3K27me3 211

enrichment in pkl-1 plants versus ~5,500 in clf-28 plants (Figure 6A). Further, ~2,900 genes 212

exhibited reduced H3K27me3 in pie1-5 plants, confirming that PIE1 promotes H3K27me3 at 213

numerous loci. Many genes exhibiting reduced H3K27me3 did so throughout the coding 214

sequence in each of the lines (Figures 5A-B and 6D). Importantly, transcript levels of known 215

PRC2 subunits were not significantly altered in the pie1-5 mutant relative to WT (Supplemental 216

Table 2), indicating that the effect of ablation of PIE1 on H3K27me3 levels is not due to 217

decreased expression of genes encoding PRC2 machinery. 218

Analysis of relative levels of H2A.Z enrichment revealed distinct roles for PIE1, CLF, 219

and PKL in promoting H2A.Z as compared to H3K27me3. We observed enrichment of H2A.Z in 220

the 5′ region of many genes and throughout the body of a subset of these genes in WT plants 221

(Figure 5C and D). As predicted based on precedent (Deal et al., 2007), loss of PIE1 resulted in a 222

drastic reduction in the average levels of H2A.Z enrichment (Figure 5C and D). In particular, we 223

observed that the 5′ peak of H2A.Z was dramatically reduced in the pie1-5 line, revealing that 224

preferential enrichment of H2A.Z observed at the TSS of genes is largely a PIE1-dependent 225

phenomenon. Surprisingly, clf-28 plants also exhibited reduced H2A.Z, although to a lesser 226

degree than was observed in pie1-5. Further, a peak of H2A.Z enrichment at the TSS of genes 227

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was maintained in clf-28 plants in contrast to its loss in pie1-5 plants, indicating that clf-28 228

affects enrichment of H2A.Z in a different manner than pie1-5. In contrast, loss of PKL had a 229

negligible effect on global levels of H2A.Z. Identification of individual genes with significantly 230

reduced enrichment of H2A.Z either at the TSS or in the gene body confirms widespread 231

reductions in H2A.Z levels in pie1 and clf plants (Figure 6B and C, Supplemental Data Set 1), 232

although loss of PIE1 affects H2A.Z enrichment at many more genes. In total, 73% of identified 233

H2A.Z-enriched TSS (13,656 genes) were significantly reduced for H2A.Z in pie1-5 plants. The 234

extent of the defect in levels of H2A.Z in pie1 plants is consistent with previous characterization 235

suggesting that PIE1 promotes H2A.Z genome-wide (Coleman-Derr and Zilberman, 2012; Deal 236

et al., 2007). The sizable number of loci that do not exhibit PIE1-dependent levels of H2A.Z, 237

however, also provides support for the hypothesis that other factors in addition to PIE1 promote 238

deposition of H2A.Z (Deal et al., 2007). 239

To determine if reductions in H3K27me3 and H2A.Z in pkl-1, pie1-5, and clf-28 were 240

correlated, we examined intersections between gene sets that exhibited reduced levels of the 241

marks in these mutants relative to WT plants (Figure 7A). In pie1-5 and clf-28 plants, we 242

observed that a reduction in one mark was often correlated with a reduction in the other. These 243

correlations raised the possibility that these marks are mutually reinforcing, namely that 244

reduction of H3K27me3 in pie1-5 plants could result from loss of H2A.Z, and reduction of 245

H2A.Z levels in clf-28 plants could result from loss of H3K27me3. Alternatively, either or both 246

of these interactions could be indirect. To investigate these possibilities, we examined if 247

reductions in the non-canonical marks (H3K27me3 for pie1-5 and H2A.Z for clf-28) were 248

correlated with the presence of the canonical marks (H2A.Z for pie1-5 and H3K27me3 for clf-249

28) at a locus. For genes that exhibit PIE1-dependent H3K27me3, we found that 86% of the 250

genes that exhibited decreased H3K27me3 in pie1-5 plants were enriched for H2A.Z, and 65% 251

simultaneously exhibited decreased H2A.Z. Thus, both the presence of H2A.Z and pie1-252

dependent loss of H2A.Z were associated with loss of H3K27me3 in pie1-5 plants, supporting 253

the idea that loss of H3K27me3 was a direct result of loss of H2A.Z in pie1-5 plants. Based on 254

these analyses and on the observation that H3K27me3-enriched genes were largely a subset of 255

H2A.Z-enriched genes (Figure 4), we propose that PIE1 is a component of an epigenetic 256

pathway that directly promotes H3K27me3 via its ability to promote H2A.Z. 257

9

A similar analysis suggests that CLF and/or H3K27me3 are unlikely to be directly 258

required for deposition of H2A.Z. In contrast to the strong correlation between loss of 259

H3K27me3 and loss of H2A.Z in pie1-5 plants, clf-28 plants exhibit a much weaker association 260

between loss of H2A.Z and status of H3K27me3. A metagene profile of H2A.Z levels in clf-28 261

does not reveal preferential loss of H2A.Z at H3K2me3-enriched genes (Supplemental Figure 2). 262

Only 23% of genes exhibiting reduced H2A.Z in clf-28 are enriched for H3K27me3 (p-value > 263

0.94 by Fisher’s exact test), indicating that loss of H2A.Z occurs via a H3K27me3-independent 264

mechanism. In addition, pkl-1 plants exhibit broadly reduced H3K27me3 levels without a 265

concurrent reduction in H2A.Z indicating that wild-type levels of H3K27me3 are generally not 266

necessary for wild-type levels of H2A.Z (Figures 5C-D and 6B-C). Finally, we are unaware of 267

any biochemical data in the literature that directly links E(z) histone methyltransferases to 268

incorporation of H2A.Z. Taken together, these findings strongly suggest that loss of H2A.Z in 269

pie1-5 drives loss of H3K27me3, whereas reductions in H2A.Z levels in clf-28 occur via an 270

uncharacterized mechanism that is not correlated with H3K27me3. 271

272

PIE1, CLF, and PKL act in a common epigenetic pathway to promote H3K27me3 273

To determine if CLF, PIE1, and/or PKL promote H3K27me3 at common subsets of 274

genes, we examined three-way intersections of gene sets that exhibited reduced levels of this 275

modification relative to WT. Surprisingly, this analysis revealed that the vast majority of genes 276

that exhibited PIE1- or CLF-dependent H3K27me3 were also dependent on PKL (> 90% for all 277

intersections examined, Figure 7B). Strikingly, greater than 99% of genes that were both CLF- 278

and PIE1-dependent for H3K27me3 were also PKL-dependent at the TSS and in the gene body, 279

revealing that a large number of genes (1,414) require all three epigenetic factors to maintain 280

H3K27me3 levels. Taken together, these data suggest that genes that exhibited PIE1- and CLF-281

dependent H3K27me3 enrichment were largely (but not entirely) a subset of genes that exhibited 282

PKL-dependent H3K27me3 enrichment and further suggest that these three factors are 283

components of a common epigenetic pathway for a large group of genes. In contrast, there is 284

little evidence that PIE1, CLF, and PKL act in a common pathway to promote H2A.Z. Less than 285

5% of genes that were both CLF- and PIE1-dependent for H2A.Z were also PKL-dependent at 286

the TSS and in the gene body (Figure 7C). 287

288

10

Altered H3K27me3 levels are associated with gene expression phenotypes in pkl, pie1, and 289

clf plants. 290

The significant number of common genes that exhibited reduced H3K27me3 levels in 291

pkl-1, pie1-5, and clf-28 raised the possibility that reduced H3K27me3 levels contributed to the 292

correlation in gene expression outcomes in these mutants (Figure 3). In support of this idea, we 293

observed that genes exhibiting reduced H3K27me3 were highly overrepresented among genes 294

that exhibited differential expression in each of the mutants we examined (Table 1), suggesting 295

that loss of H3K27me3 at these loci contributed to altered expression of these genes. In contrast, 296

altered levels of H2A.Z were generally not strongly associated with changes in gene expression 297

in these lines (Table 1). However, genes exhibiting decreased H2A.Z in pie1-5 and clf-28 plants 298

were more likely than predicted by chance to exhibit increased expression. These results are 299

consistent with possibility that altered levels of H3K27me3 and H2A.Z contributed to reduced 300

expression from these loci (Table 1, Figure 4C). 301

302

PKL is a prenucleosome maturation factor in vitro. 303

The decreased levels of H3K27me3 observed in pkl-1 plants are consistent with a role for 304

PKL in promoting deposition of the mark (e.g. by working with PRC2) or in promoting retention 305

of the mark (e.g. during replication and/or transcription). pkl-1 seedlings exhibited reduced 306

H3K27me3 levels at >45% more loci than clf-28 seedlings (Figure 6A), suggesting that if PKL 307

promotes deposition of H3K27me3, it does so in concert with multiple PRC2 complexes. 308

However, we previously found that PKL primarily exists as a monomer in vivo (Ho et al., 2013), 309

and immunoprecipitation-mass spectrometry experiments do not reveal an association between 310

PKL and subunits of PRC2 (negative data not shown). Based on these observations, we explored 311

the hypothesis that PKL promotes retention of H3K27me3 by playing a role in nucleosome 312

assembly. 313

Re-deposition of nucleosomes after passage of a DNA or RNA polymerase complex has 314

recently been proposed to be a two-step process (Fei et al., 2015). Based on in vitro and in vivo 315

analyses, the histone octamer is first re-deposited by a histone chaperone in the form of a 316

conformational isomer referred to as a prenucleosome. Prenucleosome particles comprise an 317

octamer of core histones but occlude a much smaller length of DNA than do nucleosomes in the 318

canonical conformation (Fei et al., 2015). This is followed by maturation of the prenucleosome 319

11

into a canonical nucleosome by ATP-dependent remodelers. One of the remodelers that was 320

demonstrated to possess prenucleosome maturation activity was CHD1 from D. melanogaster. A 321

high degree of sequence conservation in the ATPase and D domains exists between CHD1 322

remodelers and PKL, and both of these remodelers are primarily found as monomers in vivo (Fei 323

et al., 2015; Ho et al., 2013; Lusser et al., 2005). 324

Based on these observations, we hypothesized that PKL similarly possesses 325

prenucleosome maturation activity and that loss of this activity results in reduced levels of 326

H3K27me3 due to defects in chromatin reassembly. To test if PKL possesses prenucleosome 327

maturation activity, we examined the ability of recombinant PKL to mature prenucleosomes in 328

vitro using the nuclease protection assay established by Fei et al., in which maturation of 329

prenucleosomes into canonical nucleosomes is measured by the appearance of a band that 330

migrates at ~147 bp, the DNA occlusion size of a canonical nucleosome core particle (Fei et al., 331

2015). We found that addition of recombinant PKL and ATP did not alter assembly of the poly-332

prenucleosomal template (Figure 8A). Addition of micrococcal nuclease (MNase) to the 333

polynucleosomal template revealed the presence of prenucleosomes, as indicated by the presence 334

of a band at about 80 bp. Incubation of the template with recombinant PKL and ATP prior to 335

MNase digestion, however, resulted in the appearance of a band slightly shorter than 150 bp, 336

consistent with the size predicted for mature nucleosome particles (Figure 8B lane V). A similar 337

band was not visible if PKL was incubated with the template in the absence of additional ATP 338

(Figure 8B lane VI). These results reveal that recombinant PKL possesses ATP-dependent 339

prenucleosome maturation activity in vitro. Further, they are consistent with the possibility that 340

PKL promotes retention of H3K27me3-enriched nucleosomes in the plant by promoting their re-341

assembly after passage of a DNA and/or RNA polymerase, in a fashion that is consistent with the 342

proposed role of CHD1 in promoting retention of H3K36me3 at transcribed genes (Radman-343

Livaja et al., 2012; Smolle et al., 2012). 344

345

DISCUSSION 346

Chromatin states that are strongly enriched for H3K27me3 are also enriched for H2A.Z 347

in Arabidopsis (Sequeira-Mendes et al., 2014). Our data suggest a functional relationship 348

between these epigenetic features, specifically that H2A.Z underwrites H3K27me3 enrichment at 349

many loci. In agreement with this hypothesis, we found that H3K27me3-enriched genes are 350

12

largely a subset of H2A.Z-enriched genes (Figure 4B). We further observed that PIE1 acts in a 351

common gene expression pathway with CLF and PKL, which are known to contribute to 352

H3K27me3 homeostasis (Figure 3). We also observed that loss of PIE1 resulted not only in a 353

substantial genome-wide decrease in levels of H2A.Z but also in levels of H3K27me3 (Figure 5). 354

Finally, we undertook a biochemical analysis of PKL and found that it possessed prenucleosome 355

maturation activity in vitro, suggesting that potential maturation of H3K27me3-containing 356

prenucleosomes by PKL in vivo helps promote their stability and retention in chromatin (Figure 357

8). 358

Based on these analyses, we propose the existence of an epigenetic pathway by which 359

H3K27me3-enriched chromatin states are constructed and maintained in Arabidopsis (Figure 9). 360

In this pathway, generation of H3K27me3-enriched chromatin is dependent on the prior action of 361

PIE1 and H2A.Z. These H2A.Z- and H3K27me3-enriched chromatin domains are subsequently 362

stabilized during DNA replication and/or transcription by the CHD remodeler PKL, which 363

facilitates retention of epigenetic information after chromatin re-assembly by promoting 364

maturation of prenucleosomes. In support of these factors acting in a common pathway, our 365

RNA-seq analysis reveals that a common set of genes are dependent on PKL, PIE1, and CLF for 366

expression (Figure 3). We found that the changes in gene expression observed in each mutant are 367

strongly associated with reduced levels of H3K27me3 (Table 1). Notably, previous studies have 368

indicated that H2A.Z enrichment in the gene body contributes to transcriptional repression 369

(Coleman-Derr and Zilberman, 2012; Sura et al., 2017). Our results strongly support a role for 370

H2A.Z in transcriptional repression at these loci in part by contributing to enrichment of 371

H3K27me3 (Figure 6). However, since 82% of genes with reduced H2A.Z in the gene body also 372

exhibit reduced H2A.Z at the TSS in pie1-5 plants, it is difficult to determine whether 373

enrichment specifically at one or both of these regions drives the observed changes in expression 374

and/or H3K27me3 levels. 375

This model provides a simple explanation for the reduced H3K27me3 levels observed in 376

pie1-5 seedlings (Figures 5A-B and 6A): PIE1 indirectly promotes H3K27me3 levels via its role 377

in promoting H2A.Z. This model thus also predicts that deposition of H3K27me3 is dependent 378

on the presence of H2A.Z. There is strong precedent for Arabidopsis H3K27 methyltransferases 379

preferentially acting on nucleosomes that contain specific histone variants. The H3K27 380

methyltransferases that promote H3K27me1 in Arabidopsis, ARABIDOPSIS TRITHORAX-381

13

RELATED PROTEIN 5 (ATXR5) and ATXR6 (Jacob et al., 2009), exhibit much greater activity 382

in vitro in the presence of nucleosomes containing the histone variant H3.1 rather than H3.3 383

(Jacob et al., 2014). Nucleosomes containing H2A.Z may similarly act as the preferred substrate 384

for Arabidopsis PRC2 complexes. In vitro methylation assays of CLF-containing PRC2 385

complexes (Schmitges et al., 2011) using recombinant plant nucleosomes containing canonical 386

H2A or H2A.Z would provide a robust test of this prediction. 387

Although we observed that clf-28 seedlings exhibit reductions in both H2A.Z and 388

H3K27me3 levels (Figures 5 and 6A-C), it is unlikely that H2A.Z and H3K27me3 are mutually 389

reinforcing as was previously observed for CMT-dependent DNA methylation and KYP-390

dependent H3K9 methylation (Du et al., 2014; Du et al., 2012; Jackson et al., 2002; Johnson et 391

al., 2007). Only a fraction of H2A.Z-enriched genes are also enriched for H3K27me3 (Figure 392

4B), and loss of H2A.Z in clf-28 does not occur preferentially at H3K27me3-enriched genes 393

(Supplemental Figure 2). Additionally, pkl-1 seedlings exhibit broadly reduced H3K27me3 394

levels without a concurrent reduction in H2A.Z, further indicating that H3K27me3 is not 395

necessary for normal levels of H2A.Z at loci where both marks are present (Figure 5). Analysis 396

of transcript level of genes that exhibit CLF-dependent expression did not reveal machinery 397

known to be involved in deposition of H2A.Z (Supplemental Data Set). Thus, the pathway(s) by 398

which loss of CLF perturbs H2A.Z homeostasis remains to be determined. 399

Previous characterization of double mutant plants that lack these or related genes is 400

consistent with our observations. clf pkl plants largely exhibit additive shoot phenotypes whereas 401

swn pkl plants exhibit synergistic shoot phenotypes, particularly with regards to traits related to 402

vegetative phase change (Xu et al., 2016). Similarly, characterization of clf pie1 plants reveals 403

additive shoot phenotypes (Noh and Amasino, 2003). The observation of additive shoot 404

phenotypes in clf pie1 and clf pkl plants is consistent with PIE1, CLF, and PKL acting in a 405

common pathway as proposed here. In contrast, the observation of a synergistic phenotype for 406

swn pkl plants raises the prospect that SWN plays a functionally related role outside of the 407

proposed pathway. Analysis of the molecular traits described here (genome-wide levels of 408

H3K27me3 and H2A.Z) in these double mutants is likely to shed additional light into the 409

respective roles of these factors with regards to these epigenetic phenotypes. 410

A precedent exists for linkage between H2A.Z and Polycomb-group associated 411

phenomena in animals. In female mammalian cells, the majority of H2A.Z incorporated into the 412

14

silent X chromosome is ubiquitylated by PRC1, raising the prospect that ubiquitylated H2A.Z 413

specifically contributes to formation of transcriptionally repressive facultative heterochromatin 414

in animal cells (Sarcinella et al., 2007). As noted earlier, H3K27me3 and H2A.Z are both 415

present at bivalent transcription start sites in mouse and human embryonic stem cells (Ku et al., 416

2012). In light of our data, we speculate that H2A.Z might not only play a role in poising the 417

nucleosome to be dynamic (Subramanian et al., 2015) but also in making the nucleosome a more 418

suitable substrate for PRC2. Conversely, the presence of H2A.Z in H3K27me3-repressed 419

chromatin in plants might contribute to the developmental plasticity commonly observed in this 420

kingdom (Xiao et al., 2017) by providing such chromatin with dynamic potential. 421

Although previous analyses indicated that PKL promotes H3K27me3 (Zhang et al., 422

2008), the mechanism by which this occurred was unclear. In particular, biochemical 423

characterization of PKL indicated that it primarily exists as a monomer in vivo (Ho et al., 2013), 424

and no evidence has been reported that PKL associates with PRC2 machinery. Our combined 425

analyses provide strong evidence for a novel role for PKL in retention of H3K27me3 rather than 426

in deposition. We find that PKL is required for normal H3K27me3 levels at approximately 30% 427

more loci than CLF (Figure 6), suggesting that it contributes to H3K27me3 promoted by PRC2 428

complexes containing CLF or SWN. The discovery that PKL promotes maturation of 429

prenucleosomes in vitro (Figure 8) provides a simple explanation for how it can contribute to 430

global homeostasis of H3K27me3: it may act in vivo to promote retention of H3K27me3 after 431

passage of a DNA and/or RNA polymerase. Given that H3K27me3-enriched genes have such 432

low levels of expression (Figure 4C) and thus are rarely disrupted by passage of RNA 433

polymerase, it is more likely that PKL is primarily required to preserve H3K27me3 after passage 434

of a DNA replication fork. Notably, CHD1 and ISWI remodelers also promote maturation of 435

prenucleosomes and have been strongly implicated in nucleosome retention at transcribed loci in 436

S. cerevisiae (Fei et al., 2015; Smolle et al., 2012). Our analyses suggest that PKL, which 437

belongs to a different subfamily of CHD remodelers than CHD1, plays a specialized role in 438

retention of epigenetic states during DNA replication in plants. It remains to be seen if closely 439

related CHD remodelers or some other actors play such a role in animal systems. 440

In addition to contributing to gene repression, PKL also has been implicated in 441

transcriptional activation of H3K27me3-enriched loci (Jing et al., 2013; Zhang et al., 2014; 442

Zhang et al., 2008). In particular, H3K27me3-enriched genes are overrepresented among those 443

15

genes that exhibit reduced expression in pkl plants. Although our combined characterization of 444

pie1-5, clf-28, and pkl-1 plants reveals pronounced reductions in H3K27me3 and concurrent 445

increased expression of H3K27me3-enriched genes, we also observe a common set of 446

H3K27me3-enriched loci that exhibit decreased expression in each of these mutants. Given 447

previous characterization of CLF and H3K27me3 (Mozgova et al., 2015; Schubert et al., 2006; 448

Wang et al., 2016), it is difficult to generate a simple model by which CLF directly contributes to 449

activation of an H3K27me3-enriched locus. In particular, very little evidence exists for a stable 450

chromatin state containing CLF that enables both positive and negative regulation. Instead, given 451

that many H3K27me3-enriched loci are developmentally regulated (de Lucas et al., 2016; Gan et 452

al., 2015; Wang et al., 2016), increased expression of formerly repressed H3K27me3-enriched 453

loci in clf-28 plants might lead to an altered developmental context that precludes expression of 454

other loci subject to regulation by H3K27me3. Based on this reasoning and on the significant 455

common sets of genes that exhibit decreased expression in pkl-1, and pie1-5, and clf-28 plants 456

(Figure 3G), we propose that the entire set represents an indirect effect of reduced H3K27me3 457

levels. 458

The synthetic growth defect of plants carrying null alleles of both PKL and PIE1 strongly 459

suggests that PKL and PIE1 act redundantly to promote at least one chromatin-based event. Loss 460

of PIE1 results in a severe decrease in the level of incorporation of H2A.Z at a number of loci 461

(Deal et al., 2007). Our analyses support a genome-wide role for PIE1 in promoting H2A.Z, 462

particularly at the TSS of genes (Figure 5C-D). However, not all loci exhibit significantly 463

decreased levels of H2A.Z in pie1-5, indicating that other factors in addition to PIE1 can 464

promote incorporation of this histone variant, at least at some fraction of the genome. In line with 465

this proposition, we observed that H2A.Z levels were lower in pie1-5 plants at H3K27me3-466

enriched genes compared to genes that were not enriched for H3K27me3 in WT (Supplemental 467

Figure 2). Since H3K27me3 enrichment is correlated with very low transcript levels (Figure 4C), 468

these data raise the possibility that PIE1-independent mechanisms exist that promote 469

incorporation of H2A.Z specifically at actively transcribed genes. These data thus also provide a 470

possible rationale for the previous observation that pie1 plants are not phenotypically equivalent 471

to plants that are severely depleted for H2A.Z (Berriri et al., 2016). 472

Given that pkl-1 pie1-5 null plants are inviable, it is possible that PKL acts redundantly 473

with PIE1 to promote H2A.Z. In particular, our model predicts that PKL promotes retention of 474

16

both H3K27me3 and H2A.Z after disruption by a polymerase. In the absence of PIE1, this role 475

for PKL in retention of H2A.Z may be essential for maintaining this histone variant at levels that 476

are conducive to normal chromatin-based processes such as transcription. Given that we only 477

observe loss of H3K27me3 and not H2A.Z in pkl-1 plants, we propose that PIE1 acts 478

subsequently on the resulting replacement nucleosomes in pkl-1 plants to restore H2A.Z. 479

Our findings support the existence of an epigenetic pathway in which PIE1 promotes 480

incorporation of H2A.Z which in turn promotes deposition of H3K27me3 by CLF. PKL acts 481

after deposition to promote retention of H3K27me3 by promoting chromatin assembly after 482

DNA replication and/or transcription. These data thus confirm previous observations of H2A.Z 483

and H3K27me3 co-enrichment in plants (Sequeira-Mendes et al., 2014) and reveal that this co-484

enrichment is likely to reflect how H3K27me3-enriched chromatin is generated. Given the 485

proposed role for H2A.Z in enabling switching of transcriptional states (Subramanian et al., 486

2015), it is possible that the presence of H2A.Z in H3K27me3-enriched chromatin also 487

contributes to plasticity of this state and thus also to the developmental plasticity of plants. 488

In total, our analyses suggest that SWR1 and CHD remodelers play a major role in 489

homeostasis of H3K27me3 in plants and raise the prospect that PKL plays a key role in memory 490

of this epigenetic state. Our results leave open the possibility that PKL also contributes to the 491

maintenance of other epigenetic states. Further functional characterization of these and related 492

remodelers and the corresponding mutants is likely to provide additional insight into the 493

molecular processes underlying maintenance of epigenetic states in plants. 494

495

METHODS 496

Plant lines and growth conditions. All plant lines used in these experiments are in the 497

Columbia background. The previously characterized mutant lines used in this study are pkl-1 498

(Ogas et al., 1997), pkl-10 (Zhang et al., 2012), pie1-5 (Deal et al., 2007), and clf-28 (Doyle and 499

Amasino, 2009). Phenotypic characterization was performed as described previously (Zhang et 500

al., 2012). Plants used in RNA-seq and ChIP-seq analyses were sown in soil pots, cold-treated 501

for three days, and grown for three weeks under 18-hour 170 µE light at 22°C in a Percival 502

AR75 incubator prior to sample collection. Oligonucleotide primers used to genotype the 503

segregating pie1-5 mutant plants were as follows: 5′-CTGAGGATGAGACCGTGAGT-3′, 5′-504

17

AAGGTCATGTGAATGGGTCTC-3′, and 5′-ATTTTGCCGATTTCGGAAC-3′ (SALK 505

LBb1.3 border primer) 506

507

RNA extraction and cDNA synthesis. RNA was extracted from the aerial tissues of three-508

week-old seedlings that had not bolted using an RNeasy Plant Mini Kit (Qiagen catalog # 509

74903). Three biological replicates from different pools of seedlings were collected for each 510

genotype. RNA samples were DNAse treated and concentrated using an RNA Clean & 511

Concentrator kit (Zymo catalog #R1013). First-strand cDNA synthesis was performed using an 512

M-MLV Reverse Transcriptase kit (Thermo Fisher catalog # 28025013). 513

514

Chromatin extraction. Chromatin extraction was performed on aerial tissues of three-week-old 515

seedlings that had not bolted using our published protocol (Zhang et al., 2008) with the following 516

modifications: Extraction Buffer 2 was replaced with 1mL of HBM Buffer (25mM Tris pH 7.6, 517

440mM sucrose, 10mM MgCl2, 0.1% Triton X-100, 10mM 2-mercaptoethanol, 2mM spermine, 518

1mM PMSF, 1µg/mL pepstatin, protease inhibitors), IP Buffer was replaced with 1mL of 519

Nuclear Lysis Buffer (50mM Tris pH 8.0, 10mM EDTA, 0.25% SDS, protease inhibitors), 520

centrifugation following sonication was performed at 12,000G for 5min, and the supernatant was 521

transferred to a new tube and diluted to 3mL using ChIP Dilution Buffer (1.1% Triton X-100, 522

1.2mM EDTA, 16.7mM Tris pH 8.0, 167mM NaCl) to reduce the SDS concentration below 523

0.1%. Chromatin samples were stored at -80°C. Two biological replicates from different pools of 524

seedlings were collected for each genotype. 525

526

Chromatin immunoprecipitation. ChIP was performed on 500µL of the diluted chromatin 527

solutions using the published protocol (Zhang et al., 2008) and the following antibodies: anti-H3 528

(Abcam catalog # ab1791), anti-H3K27me3 (Millipore catalog # 07-449), and anti-H2A.Z (Deal 529

et al., 2007). The following modifications were made to the protocol: solutions were rotated at 530

4°C for 15h after addition of the antibodies, 20µL of a 50% slurry of Dynabeads Protein G 531

(Thermo Fisher catalog # 10004D) equilibrated in ChIP Dilution Buffer was substituted for the 532

Protein G sepharose slurry, the Elution Buffer also contained 250mM NaCl, and the elution time 533

was increased to 30min at 65°C. After elution, the following steps were performed in lieu of the 534

ones described previously: the samples were cooled to room temperature and treated with 535

18

0.1µg/µL RNAse A (Thermo Fisher catalog # EN0531) for 15min at 15°C and subsequently with 536

0.1µg/µL proteinase K (Thermo Fisher catalog # AM2546) for 15h at 65°C. The resulting DNA 537

samples were purified using a QIAquick MinElute PCR purification kit (Qiagen catalog # 538

28004). 539

540

Indexing and sequencing. cDNA library construction was performed using a ThruPLEX DNA-541

seq Kit (Rubicon Genomics). ChIP DNA library construction was performed using an NEB Ultra 542

II DNA Library Prep Kit (NEB catalog # E7645L). Strand-specific sequencing was performed 543

using the Illumina HiSeq platform. 544

545

Differential expression analysis. Short reads (~25 million reads per sample) were trimmed of 546

adapter sequences using Trimmomatic (Bolger et al., 2014) and mapped to the TAIR10 reference 547

genome assembly using NCBI’s Magic-BLAST utility (NCBI, 2017). The resulting BAM files 548

were assessed for sample quality with plots generated using the DESeq2 and limma packages in 549

Bioconductor (Love et al., 2014; Ritchie et al., 2015). DEGs were identified using the edgeR 550

package in Bioconductor and Fisher’s exact test with a Benjamini-Hochberg FDR threshold of < 551

0.05 and a fold change threshold of > 1.5-fold difference relative to WT (Fisher, 1922; Robinson 552

et al., 2010). Gene annotations for differential expression analysis were extracted from the 553

Araport11 genome annotation (Cheng et al., 2017). 554

555

Enrichment and differential enrichment analyses. Short reads were trimmed of adapter 556

sequences using Trimmomatic (Bolger et al., 2014) and mapped to the TAIR10 reference 557

genome assembly using the very sensitive end-to-end mode of Bowtie2 (Langmead and 558

Salzberg, 2012). The resulting BAM files (~25 million mapped reads per sample) were 559

converted to BED files using the bamToBed utility of BEDtools2 (Quinlan, 2014). Reads 560

mapping to the mitochondrial or chloroplast genomes were discarded. Regions enriched for 561

H2A.Z or H3K27me3 were identified in WT seedlings relative to H3 using SICER (Xu et al., 562

2014) with a 200bp window size, a 0.85 effective genome fraction, and a false discovery rate of 563

0.05. Genic regions (whole genes, gene bodies, or TSS) from the Araport11 annotation that 564

overlapped with at least one region of enrichment were identified using the closestBed utility of 565

BEDtools2. Enrichment in whole genes and in gene bodies was determined using a SICER gap 566

19

size of 600bp, whereas enrichment at the TSS was determined using a gap size of 200bp. Genic 567

regions were required to exhibit overlap with at least one region of enrichment in both biological 568

replicates to be considered enriched. Heat maps of enrichment were generated using the 569

computeMatrix and plotHeatmap utilities in deepTools2 (Ramirez et al., 2016). 570

Differential enrichment of H2A.Z or H3K27me3 in the various mutants was determined 571

relative to WT using SICER-df with H3 as the reference treatment. Parameters used were 572

identical to those listed above with the additions of a fold change threshold of > 1.2-fold 573

difference relative to WT and a false discovery rate of 0.05 for WT vs. mutant. Genic regions 574

were required to overlap with at least one region of differential enrichment in both biological 575

replicates to be considered differentially enriched. 576

577

Prenucleosome maturation assay. Generation of recombinant PKL was performed as described 578

previously (Ho et al., 2013). Reconstitution of mono-prenucleosomes by salt dialysis and ligation 579

of mono-prenucleosomes to free DNA in the presence of an ATP regeneration system were 580

conducted as described by Fei et al. using an 80 bp DNA fragment containing a 601 nucleosome 581

positioning sequence (Fei et al., 2015). The poly-prenucleosomal templates were diluted 1:1 and 582

incubated with recombinant PKL as described by Fei et al. (Fei et al., 2015), with the exception 583

that additional ATP was sometimes omitted from the maturation reaction as indicated in the text. 584

MNase digestion of the assembled nucleosomes was conducted according to Torigoe et al. 585

(Torigoe et al., 2011). Following digestion, the samples (per 50 µl) were de-proteinated by 586

mixing with 5µl of 3M sodium acetate pH 5.5 and 55mg of guanidine HCl and centrifuged 587

through a QIAquick column (Qiagen catalog # 28104). The column was washed with 750µl of 588

PE buffer (Qiagen) and the DNA was eluted from the column using TE buffer. DNA fragments 589

were analyzed by electrophoresis on a 3% agarose gel, and bands were visualized using ethidium 590

bromide as done in Torigoe et al (Torigoe et al., 2011). 591

592

Accession numbers. RNA-seq and ChIP-seq data from this article can be found in the Gene 593

Expression Omnibus data library under accession number GSE103361. These data include 594

regions of H2A.Z and H3K27me3 differential enrichment and the corresponding statistics, which 595

are provided as processed data files. 596

597

20

Supplemental Data: 598

Supplemental Figure 1: Quality assessment of RNA-seq data. 599

Supplemental Figure 2: Association between H2A.Z levels and H3K27me3 enrichment status. 600

Supplemental Table 1: Statistical analysis of inverse intersections between clf, pie1, and pkl 601

DEGs 602

Supplemental Data Set 1: Lists of genes determined to be differentially expressed (RNA-seq) 603

or differentially enriched (ChIP-seq). 604

605

ACKNOWLEDGEMENTS 606

We acknowledge the Arabidopsis Biological Resource Center and the Salk Institute Genomic 607

Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants 608

used in this study. We thank Nadia Atallah for her valuable input with regard to ChIP-seq data 609

analysis. We also thank Craig Peterson for many thoughtful and productive discussions and Nick 610

Carpita for critical insight and feedback. 611

612

AUTHOR CONTRIBUTIONS 613

BC was principally responsible for study design, data acquisition and analysis, and manuscript 614

preparation and editing (with JO). BB and KKH participated in study design and data 615

acquisition. RH and WJ participated in data acquisition and analysis. HZ, PEP, and RBD 616

participated in data analysis and manuscript preparation and editing. JO participated in study 617

design, data analysis, and was principally responsible for manuscript preparation and editing 618

(with BC). 619

620

REFERENCES 621

622

Aranda, S., Mas, G., and Di Croce, L. (2015). Regulation of gene transcription by Polycomb proteins. Sci 623 Adv 1, e1500737. 624 Berriri, S., Gangappa, S.N., and Kumar, S.V. (2016). SWR1 Chromatin-Remodeling Complex Subunits and 625 H2A.Z Have Non-overlapping Functions in Immunity and Gene Regulation in Arabidopsis. Molecular 626 plant 9, 1051-1065. 627 Bieluszewski, T., Galganski, L., Sura, W., Bieluszewska, A., Abram, M., Ludwikow, A., Ziolkowski, P.A., and 628 Sadowski, J. (2015). AtEAF1 is a potential platform protein for Arabidopsis NuA4 acetyltransferase 629 complex. BMC Plant Biol 15, 75. 630

21

Bolger, A.M., Lohse, M., and Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence 631 data. Bioinformatics 30, 2114-2120. 632 Bouyer, D., Roudier, F., Heese, M., Andersen, E.D., Gey, D., Nowack, M.K., Goodrich, J., Renou, J.P., Grini, 633 P.E., Colot, V., et al. (2011). Polycomb repressive complex 2 controls the embryo-to-seedling phase 634 transition. PLoS genetics 7, e1002014. 635 Chanvivattana, Y., Bishopp, A., Schubert, D., Stock, C., Moon, Y.H., Sung, Z.R., and Goodrich, J. (2004). 636 Interaction of Polycomb-group proteins controlling flowering in Arabidopsis. Development 131, 5263-637 5276. 638 Cheng, C.Y., Krishnakumar, V., Chan, A.P., Thibaud-Nissen, F., Schobel, S., and Town, C.D. (2017). 639 Araport11: a complete reannotation of the Arabidopsis thaliana reference genome. The Plant journal : 640 for cell and molecular biology 89, 789-804. 641 Choi, K., Kim, S., Kim, S.Y., Kim, M., Hyun, Y., Lee, H., Choe, S., Kim, S.G., Michaels, S., and Lee, I. (2005). 642 SUPPRESSOR OF FRIGIDA3 encodes a nuclear ACTIN-RELATED PROTEIN6 required for floral repression in 643 Arabidopsis. The Plant cell 17, 2647-2660. 644 Coleman-Derr, D., and Zilberman, D. (2012). Deposition of histone variant H2A.Z within gene bodies 645 regulates responsive genes. PLoS genetics 8, e1002988. 646 de Lucas, M., Pu, L., Turco, G., Gaudinier, A., Morao, A.K., Harashima, H., Kim, D., Ron, M., Sugimoto, K., 647 Roudier, F., et al. (2016). Transcriptional Regulation of Arabidopsis Polycomb Repressive Complex 2 648 Coordinates Cell-Type Proliferation and Differentiation. The Plant cell 28, 2616-2631. 649 Deal, R.B., Kandasamy, M.K., McKinney, E.C., and Meagher, R.B. (2005). The nuclear actin-related 650 protein ARP6 is a pleiotropic developmental regulator required for the maintenance of FLOWERING 651 LOCUS C expression and repression of flowering in Arabidopsis. The Plant cell 17, 2633-2646. 652 Deal, R.B., Topp, C.N., McKinney, E.C., and Meagher, R.B. (2007). Repression of flowering in Arabidopsis 653 requires activation of FLOWERING LOCUS C expression by the histone variant H2A.Z. The Plant cell 19, 654 74-83. 655 Del Prete, S., Mikulski, P., Schubert, D., and Gaudin, V. (2015). One, Two, Three: Polycomb Proteins Hit 656 All Dimensions of Gene Regulation. Genes 6, 520-542. 657 Doyle, M.R., and Amasino, R.M. (2009). A single amino acid change in the enhancer of zeste ortholog 658 CURLY LEAF results in vernalization-independent, rapid flowering in Arabidopsis. Plant physiology 151, 659 1688-1697. 660 Du, J., Johnson, L.M., Groth, M., Feng, S., Hale, C.J., Li, S., Vashisht, A.A., Gallego-Bartolome, J., 661 Wohlschlegel, J.A., Patel, D.J., et al. (2014). Mechanism of DNA methylation-directed histone 662 methylation by KRYPTONITE. Molecular cell 55, 495-504. 663 Du, J., Zhong, X., Bernatavichute, Y.V., Stroud, H., Feng, S., Caro, E., Vashisht, A.A., Terragni, J., Chin, 664 H.G., Tu, A., et al. (2012). Dual binding of chromomethylase domains to H3K9me2-containing 665 nucleosomes directs DNA methylation in plants. Cell 151, 167-180. 666 ENCODE (2016). ENCODE Guidelines and Best Practices for RNA-seq: Revised December 2016 667 (encodeproject.org). 668 ENCODE (2017). ENCODE Guidelines for Experiments Generating ChIP-seq Data: January 2017 669 (encodeproject.org). 670 Fei, J., Torigoe, S.E., Brown, C.R., Khuong, M.T., Kassavetis, G.A., Boeger, H., and Kadonaga, J.T. (2015). 671 The prenucleosome, a stable conformational isomer of the nucleosome. Genes & development 29, 672 2563-2575. 673 Fisher, R.A. (1922). On the interpretation of x(2) from contingency tables, and the calculation of P. J R 674 Stat Soc 85, 87-94. 675 Gan, E.S., Xu, Y., and Ito, T. (2015). Dynamics of H3K27me3 methylation and demethylation in plant 676 development. Plant signaling & behavior 10, e1027851. 677

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Ho, K.K., Zhang, H., Golden, B.L., and Ogas, J. (2013). PICKLE is a CHD subfamily II ATP-dependent 678 chromatin remodeling factor. Biochimica et biophysica acta 1829, 199-210. 679 Jackson, J.P., Lindroth, A.M., Cao, X., and Jacobsen, S.E. (2002). Control of CpNpG DNA methylation by 680 the KRYPTONITE histone H3 methyltransferase. Nature 416, 556-560. 681 Jacob, Y., Bergamin, E., Donoghue, M.T., Mongeon, V., LeBlanc, C., Voigt, P., Underwood, C.J., Brunzelle, 682 J.S., Michaels, S.D., Reinberg, D., et al. (2014). Selective methylation of histone H3 variant H3.1 regulates 683 heterochromatin replication. Science 343, 1249-1253. 684 Jacob, Y., Feng, S., LeBlanc, C.A., Bernatavichute, Y.V., Stroud, H., Cokus, S., Johnson, L.M., Pellegrini, M., 685 Jacobsen, S.E., and Michaels, S.D. (2009). ATXR5 and ATXR6 are H3K27 monomethyltransferases 686 required for chromatin structure and gene silencing. Nat Struct Mol Biol 16, 763-768. 687 Jing, Y., Zhang, D., Wang, X., Tang, W., Wang, W., Huai, J., Xu, G., Chen, D., Li, Y., and Lin, R. (2013). 688 Arabidopsis chromatin remodeling factor PICKLE interacts with transcription factor HY5 to regulate 689 hypocotyl cell elongation. Plant Cell 25, 242-256. 690 Johnson, L.M., Bostick, M., Zhang, X., Kraft, E., Henderson, I., Callis, J., and Jacobsen, S.E. (2007). The SRA 691 methyl-cytosine-binding domain links DNA and histone methylation. Current biology : CB 17, 379-384. 692 Khan, A.A., Lee, A.J., and Roh, T.Y. (2015). Polycomb group protein-mediated histone modifications 693 during cell differentiation. Epigenomics 7, 75-84. 694 Kim, D.H., and Sung, S. (2014). Polycomb-mediated gene silencing in Arabidopsis thaliana. Mol Cells 37, 695 841-850. 696 Krogan, N.J., Keogh, M.C., Datta, N., Sawa, C., Ryan, O.W., Ding, H., Haw, R.A., Pootoolal, J., Tong, A., 697 Canadien, V., et al. (2003). A Snf2 family ATPase complex required for recruitment of the histone H2A 698 variant Htz1. Molecular cell 12, 1565-1576. 699 Ku, M., Jaffe, J.D., Koche, R.P., Rheinbay, E., Endoh, M., Koseki, H., Carr, S.A., and Bernstein, B.E. (2012). 700 H2A.Z landscapes and dual modifications in pluripotent and multipotent stem cells underlie complex 701 genome regulatory functions. Genome biology 13, R85. 702 Lafos, M., Kroll, P., Hohenstatt, M.L., Thorpe, F.L., Clarenz, O., and Schubert, D. (2011). Dynamic 703 Regulation of H3K27 Trimethylation during Arabidopsis Differentiation. PLoS Genet 7, e1002040. 704 Langmead, B., and Salzberg, S.L. (2012). Fast gapped-read alignment with Bowtie 2. Nat Methods 9, 357-705 359. 706 Li, Z., Gadue, P., Chen, K., Jiao, Y., Tuteja, G., Schug, J., Li, W., and Kaestner, K.H. (2012). Foxa2 and H2A.Z 707 mediate nucleosome depletion during embryonic stem cell differentiation. Cell 151, 1608-1616. 708 Love, M.I., Huber, W., and Anders, S. (2014). Moderated estimation of fold change and dispersion for 709 RNA-seq data with DESeq2. Genome biology 15, 550. 710 Lu, P.Y., Levesque, N., and Kobor, M.S. (2009). NuA4 and SWR1-C: two chromatin-modifying complexes 711 with overlapping functions and components. Biochem Cell Biol 87, 799-815. 712 Lusser, A., Urwin, D.L., and Kadonaga, J.T. (2005). Distinct activities of CHD1 and ACF in ATP-dependent 713 chromatin assembly. Nat Struct Mol Biol 12, 160-166. 714 March-Diaz, R., Garcia-Dominguez, M., Florencio, F.J., and Reyes, J.C. (2007). SEF, a new protein required 715 for flowering repression in Arabidopsis, interacts with PIE1 and ARP6. Plant physiology 143, 893-901. 716 March-Diaz, R., Garcia-Dominguez, M., Lozano-Juste, J., Leon, J., Florencio, F.J., and Reyes, J.C. (2008). 717 Histone H2A.Z and homologues of components of the SWR1 complex are required to control immunity 718 in Arabidopsis. The Plant journal : for cell and molecular biology 53, 475-487. 719 Margueron, R., and Reinberg, D. (2011). The Polycomb complex PRC2 and its mark in life. Nature 469, 720 343-349. 721 Mizuguchi, G., Shen, X., Landry, J., Wu, W.H., Sen, S., and Wu, C. (2004). ATP-driven exchange of histone 722 H2AZ variant catalyzed by SWR1 chromatin remodeling complex. Science 303, 343-348. 723 Morrison, A.J., and Shen, X. (2009). Chromatin remodelling beyond transcription: the INO80 and SWR1 724 complexes. Nat Rev Mol Cell Biol 10, 373-384. 725

23

Mozgova, I., Kohler, C., and Hennig, L. (2015). Keeping the gate closed: functions of the polycomb 726 repressive complex PRC2 in development. The Plant journal : for cell and molecular biology 83, 121-132. 727 Muller-Xing, R., Clarenz, O., Pokorny, L., Goodrich, J., and Schubert, D. (2014). Polycomb-Group Proteins 728 and FLOWERING LOCUS T Maintain Commitment to Flowering in Arabidopsis thaliana. The Plant cell 26, 729 2457-2471. 730 NCBI (2017). Magic-BLAST Short Read Mapper 731 (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/magicblast/LATEST/). 732 Noh, Y.S., and Amasino, R.M. (2003). PIE1, an ISWI family gene, is required for FLC activation and floral 733 repression in Arabidopsis. The Plant cell 15, 1671-1682. 734 Ogas, J., Cheng, J.C., Sung, Z.R., and Somerville, C. (1997). Cellular differentiation regulated by gibberellin 735 in the Arabidopsis thaliana pickle mutant. Science 277, 91-94. 736 Quinlan, A.R. (2014). BEDTools: The Swiss-Army Tool for Genome Feature Analysis. Curr Protoc 737 Bioinformatics 47, 11.12.11-34. 738 Radman-Livaja, M., Quan, T.K., Valenzuela, L., Armstrong, J.A., van Welsem, T., Kim, T., Lee, L.J., 739 Buratowski, S., van Leeuwen, F., Rando, O.J., et al. (2012). A key role for Chd1 in histone H3 dynamics at 740 the 3' ends of long genes in yeast. PLoS genetics 8, e1002811. 741 Ramirez, F., Ryan, D.P., Gruning, B., Bhardwaj, V., Kilpert, F., Richter, A.S., Heyne, S., Dundar, F., and 742 Manke, T. (2016). deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic 743 acids research 44, W160-165. 744 Ritchie, M.E., Phipson, B., Wu, D., Hu, Y., Law, C.W., Shi, W., and Smyth, G.K. (2015). limma powers 745 differential expression analyses for RNA-sequencing and microarray studies. Nucleic acids research 43, 746 e47. 747 Robinson, J.T., Thorvaldsdottir, H., Winckler, W., Guttman, M., Lander, E.S., Getz, G., and Mesirov, J.P. 748 (2011). Integrative genomics viewer. Nat Biotechnol 29, 24-26. 749 Robinson, M.D., McCarthy, D.J., and Smyth, G.K. (2010). edgeR: a Bioconductor package for differential 750 expression analysis of digital gene expression data. Bioinformatics 26, 139-140. 751 Rosa, M., Von Harder, M., Cigliano, R.A., Schlogelhofer, P., and Mittelsten Scheid, O. (2013). The 752 Arabidopsis SWR1 chromatin-remodeling complex is important for DNA repair, somatic recombination, 753 and meiosis. The Plant cell 25, 1990-2001. 754 Ruhl, D.D., Jin, J., Cai, Y., Swanson, S., Florens, L., Washburn, M.P., Conaway, R.C., Conaway, J.W., and 755 Chrivia, J.C. (2006). Purification of a human SRCAP complex that remodels chromatin by incorporating 756 the histone variant H2A.Z into nucleosomes. Biochemistry 45, 5671-5677. 757 Sarcinella, E., Zuzarte, P.C., Lau, P.N., Draker, R., and Cheung, P. (2007). Monoubiquitylation of H2A.Z 758 distinguishes its association with euchromatin or facultative heterochromatin. Molecular and cellular 759 biology 27, 6457-6468. 760 Schmitges, F.W., Prusty, A.B., Faty, M., Stutzer, A., Lingaraju, G.M., Aiwazian, J., Sack, R., Hess, D., Li, L., 761 Zhou, S., et al. (2011). Histone methylation by PRC2 is inhibited by active chromatin marks. Molecular 762 cell 42, 330-341. 763 Schubert, D., Primavesi, L., Bishopp, A., Roberts, G., Doonan, J., Jenuwein, T., and Goodrich, J. (2006). 764 Silencing by plant Polycomb-group genes requires dispersed trimethylation of histone H3 at lysine 27. 765 The EMBO journal 25, 4638-4649. 766 Sequeira-Mendes, J., Araguez, I., Peiro, R., Mendez-Giraldez, R., Zhang, X., Jacobsen, S.E., Bastolla, U., 767 and Gutierrez, C. (2014). The Functional Topography of the Arabidopsis Genome Is Organized in a 768 Reduced Number of Linear Motifs of Chromatin States. The Plant cell 26, 2351-2366. 769 Shaked, H., Avivi-Ragolsky, N., and Levy, A.A. (2006). Involvement of the Arabidopsis SWI2/SNF2 770 chromatin remodeling gene family in DNA damage response and recombination. Genetics 173, 985-994. 771

24

Smolle, M., Venkatesh, S., Gogol, M.M., Li, H., Zhang, Y., Florens, L., Washburn, M.P., and Workman, J.L. 772 (2012). Chromatin remodelers Isw1 and Chd1 maintain chromatin structure during transcription by 773 preventing histone exchange. Nat Struct Mol Biol 19, 884-892. 774 Subramanian, V., Fields, P.A., and Boyer, L.A. (2015). H2A.Z: a molecular rheostat for transcriptional 775 control. F1000prime reports 7, 01. 776 Sura, W., Kabza, M., Karlowski, W.M., Bieluszewski, T., Kus-Slowinska, M., Paweloszek, L., Sadowski, J., 777 and Ziolkowski, P.A. (2017). Dual Role of the Histone Variant H2A.Z in Transcriptional Regulation of 778 Stress-Response Genes. The Plant cell 29, 791-807. 779 Torigoe, S.E., Urwin, D.L., Ishii, H., Smith, D.E., and Kadonaga, J.T. (2011). Identification of a rapidly 780 formed nonnucleosomal histone-DNA intermediate that is converted into chromatin by ACF. Molecular 781 cell 43, 638-648. 782 Wang, H., Liu, C., Cheng, J., Liu, J., Zhang, L., He, C., Shen, W.H., Jin, H., Xu, L., and Zhang, Y. (2016). 783 Arabidopsis Flower and Embryo Developmental Genes are Repressed in Seedlings by Different 784 Combinations of Polycomb Group Proteins in Association with Distinct Sets of Cis-regulatory Elements. 785 PLoS genetics 12, e1005771. 786 Wang, M., Zhao, Y., and Zhang, B. (2015). Efficient Test and Visualization of Multi-Set Intersections. Sci 787 Rep 5, 16923. 788 Weber, C.M., Ramachandran, S., and Henikoff, S. (2014). Nucleosomes are context-specific, H2A.Z-789 modulated barriers to RNA polymerase. Molecular cell 53, 819-830. 790 Wong, M.M., Cox, L.K., and Chrivia, J.C. (2007). The chromatin remodeling protein, SRCAP, is critical for 791 deposition of the histone variant H2A.Z at promoters. The Journal of biological chemistry 282, 26132-792 26139. 793 Xiao, J., Jin, R., and Wagner, D. (2017). Developmental transitions: integrating environmental cues with 794 hormonal signaling in the chromatin landscape in plants. Genome biology 18, 88. 795 Xu, M., Hu, T., Smith, M.R., and Poethig, R.S. (2016). Epigenetic Regulation of Vegetative Phase Change 796 in Arabidopsis. The Plant cell 28, 28-41. 797 Xu, S., Grullon, S., Ge, K., and Peng, W. (2014). Spatial clustering for identification of ChIP-enriched 798 regions (SICER) to map regions of histone methylation patterns in embryonic stem cells. Methods in 799 molecular biology 1150, 97-111. 800 Zhang, D., Jing, Y., Jiang, Z., and Lin, R. (2014). The Chromatin-Remodeling Factor PICKLE Integrates 801 Brassinosteroid and Gibberellin Signaling during Skotomorphogenic Growth in Arabidopsis. Plant Cell 26, 802 2472-2485. 803 Zhang, H., Bishop, B., Ringenberg, W., Muir, W.M., and Ogas, J. (2012). The CHD3 remodeler PICKLE 804 associates with genes enriched for trimethylation of histone H3 lysine 27. Plant physiology 159, 418-432. 805 Zhang, H., Rider, S.D., Jr., Henderson, J.T., Fountain, M., Chuang, K., Kandachar, V., Simons, A., Edenberg, 806 H.J., Romero-Severson, J., Muir, W.M., et al. (2008). The CHD3 remodeler PICKLE promotes 807 trimethylation of histone H3 lysine 27. The Journal of biological chemistry 283, 22637-22648. 808 Zhang, X., Clarenz, O., Cokus, S., Bernatavichute, Y.V., Pellegrini, M., Goodrich, J., and Jacobsen, S.E. 809 (2007). Whole-genome analysis of histone H3 lysine 27 trimethylation in Arabidopsis. PLoS biology 5, 810 e129. 811

812

FIGURE LEGENDS 813

Figure 1. pkl-10 pie1-5 seedlings exhibit profound defects in organogenesis. Seedlings were 814

grown on MS medium under 24-hour light and images were collected at two weeks of age. Scale 815

25

bar represents 2mm. Background colors are desaturated for visual clarity. (A) Representative WT 816

seedling. (B) Representative pkl-10 seedling exhibiting the characteristic reduced petiole length 817

and rosette diameter. (C-D) Representative pkl-10 pie1-5 seedlings. 818

819

Figure 2. Summary of differentially expressed genes. Total numbers of genes identified as 820

differentially expressed relative to WT in the indicated samples. Gene sets corresponding to 821

statistically significant increases in expression are shaded yellow, and those corresponding to 822

significant decreases in expression are shaded blue. Differential expression was determined 823

based on mean counts per million using the edgeR package with a Benjamini-Hochberg FDR 824

threshold of < 0.05 and a fold change threshold of ≥ 1.5-fold change relative to WT. 825

826

Figure 3. pkl, pie1, and clf affect expression of common sets of genes. (A-B) Statistical 827

analysis of intersections between sets of DEGs exhibiting increased (A) or decreased (B) 828

expression in the indicated mutants relative to WT. Gene sets are ordered by size. y-axes indicate 829

the size of the indicated gene set (first three columns) or intersection in number of genes. Bars 830

are shaded to reflect the p-value of the intersection obtained using Fisher’s exact test with a null 831

hypothesis of an intersection no greater than predicted by chance. Column end labels indicate the 832

log(p-value) for the indicated intersection. Data visualization was performed using the 833

SuperExactTest package in Bioconductor (Wang et al., 2015). (C-E) Correlation between 834

expression of DEGs in the indicated genotypes. Axes indicate fold change in expression relative 835

to WT plants on a log2 scale. Green points represent genes that are differentially expressed in 836

both genotypes, and grey points represent genes that are differentially expressed in one of the 837

two genotypes. Dotted lines depict linear-fit trendlines of the stated R2 value calculated using838

common DEGs (green points). (F-G) Diagrams of the three-way intersections from panels A and 839

B. Numbers in parentheses indicate the size of the intersection predicted by chance (the product840

of the probabilities of a gene being in each group * population size). 841

842

Figure 4. H3K27me3 and H2A.Z exhibit co-enrichment and are associated with low levels 843

of gene expression. Genes enriched for H3K27me3 and H2A.Z were identified relative to H3 844

using SICER (Xu et al., 2014). (A) Diagrams of intersections between gene sets determined to be 845

enriched for H3K27me3 or H2A.Z in the gene body relative to H3 in our analysis vs. previously 846

26

published analyses (Bouyer et al., 2011; Coleman-Derr and Zilberman, 2012). Gene body is 847

defined as the central region of genes that remain after omitting the terminal 1,000bp ends from 848

the TSS and the transcription termination site (TTS) as annotated in the Araport11 reference 849

genome. Genes shorter than 2.1kb are thus excluded. (Coleman-Derr and Zilberman, 2012) (B) 850

Diagrams of intersections between genes enriched for H2A.Z and/or in H3K27me3 relative to 851

H3 in the transcription start site (TSS, upper left) or in the gene body (lower right). TSS is 852

defined as the first 500bp (from base 0 to base +500) from the start of the mRNA sequence. 853

Genes shorter than 500bp are excluded. (C) Box-and-whisker plot depicting the distributions of 854

gene expression for genes enriched in the gene body in varying combinations of H2A.Z and/or 855

H3K27me3 as indicated. The y-axis represents mRNA mean counts per million values in WT 856

plants. Dashes indicate that all genes were included in the set regardless of enrichment status of 857

the indicated mark. 858

859

Figure 5. H3K27me3 and H2A.Z enrichment patterns in WT, clf-28, pie1-5, and pkl-1. (A-860

D) Enrichment visualizations generated using the deepTools2 package (Ramirez et al., 2016). 861

Gene regions are scaled to 1kb on the x-axes. Samples were normalized using the RPKM 862

method. Displayed genes are restricted to those that contain at least one region of enrichment of 863

the relevant mark relative to H3 as determined by SICER. Data are representative of two 864

independent biological replicates. (A) Heat maps of H3K27me3 enrichment in the indicated 865

genetic backgrounds. (B) Metagene profile of average enrichment data from panel A. (C) Heat 866

maps of H2A.Z enrichment in the indicated genetic backgrounds. (D) Metagene profile of 867

average enrichment data from panel C. 868

869

Figure 6: Summary of differentially enriched genes. (A-D) Summary of differentially 870

enriched genes identified for each mutant line. Differential enrichment was determined relative 871

to H3 using SICER-df (Xu et al., 2014). Differentially enriched gene sets were filtered to contain 872

only genes identified as such in both of two biological replicates. (A) Summary of genes 873

identified as differentially enriched relative to WT for H3K27me3. (B) Summary of genes 874

identified as differentially enriched relative to WT for H2A.Z in the gene body. (C) Summary of 875

genes identified as differentially enriched relative to WT for H2A.Z at the TSS. The gene body 876

and TSS data sets are described in the Figure 4 legend. (D) Two representative H3K27me3-877

27

enriched genes that exhibit reduced levels of H3K27me3 in the indicated genetic backgrounds. 878

Displayed bigwig tracks were normalized using the RPKM method of deepTools2 and visualized 879

using IGV (Robinson et al., 2011). Data are representative of two independent biological 880

replicates. 881

882

Figure 7. Intersection analysis of differentially enriched gene sets. (A) Diagrams of 883

intersections between gene sets exhibiting reduced levels of H3K27me3 and those exhibiting 884

reduced levels of H2A.Z in the indicated mutant lines. Genes were limited to those enriched for 885

both epigenetic marks for this analysis. (B) Venn diagrams depicting the intersections among 886

gene sets exhibiting reduced H3K27me3 in the indicated mutant lines at the TSS (left) or in the 887

gene body (right). (C) Venn diagrams depicting the intersections among gene sets exhibiting 888

reduced H2A.Z in the indicated mutant lines at the TSS (left) or in the gene body (right). 889

890

Figure 8. PKL converts prenucleosomes into canonical nucleosomes. Prenucleosome 891

maturation assay performed as described previously (Fei et al., 2015). Reaction products were 892

de-proteinated and fragments were analyzed using agarose gel electrophoresis. Bands were 893

visualized using ethidium bromide. (A) Assembly of poly-prenucleosomal templates (Prenuc.) in 894

the absence (lane I) or presence (lane II) of recombinant PKL. Mono-prenucleosomes were 895

assembled as previously described (Fei et al., 2015), and poly-prenucleosomes were synthesized 896

by ligating the mono-prenucleosomes using T4 DNA ligase and ATP. (B) Digestion of poly-897

prenucleosomal templates from panel A (lane III) with micrococcal nuclease (MNase), which 898

spares DNA fragments rendered inaccessible due to occlusion by a nucleosome. Prior to 899

digestion, samples were incubated in the absence or presence of recombinant PKL and/or ATP 900

(lanes IV through VI). Generation of mature nucleosomes was assessed by the appearance of 901

~147 bp DNA fragments (indicated with a star) corresponding to the DNA occlusion length of 902

the mature nucleosome core particle. 903

904

Figure 9. Model for H3K27me3 deposition and maintenance. Generation of H3K27me3-905

enriched chromatin is dependent on the prior action of PIE1 and H2A.Z. PKL acts subsequently 906

to maintains this epigenetic state during DNA replication and/or transcription by facilitating 907

nucleosome retention. 908

28

909

TABLES 910

911

Table 1. Statistical analysis of intersections between DEGs and ChIP-seq data. 912

ChIP-seq Set DEG Set Observed Predicted Log(p-value) Significance

H3K27me3 Up in pkl Up in pkl 0 3 0

H3K27me3 Down in pkl Up in pkl 109 50 -15 **

H3K27me3 Up in pkl Down in pkl 2 4 -1

H3K27me3 Down in pkl Down in pkl 162 75 -22 ***

H3K27me3 Up in pie1 Up in pie1 11 8 -1

H3K27me3 Down in pie1 Up in pie1 107 45 -17 **

H3K27me3 Up in pie1 Down in pie1 8 7 -1

H3K27me3 Down in pie1 Down in pie1 43 39 -1

H3K27me3 Up in clf Up in clf 3 4 -1

H3K27me3 Down in clf Up in clf 79 22 -24 ***

H3K27me3 Up in clf Down in clf 20 6 -6 *

H3K27me3 Down in clf Down in clf 75 30 -13 **

H2A.Z Up in pkl Up in pkl 3 2 -1

H2A.Z Down in pkl Up in pkl 5 1 -3

H2A.Z Up in pkl Down in pkl 12 3 -5

H2A.Z Down in pkl Down in pkl 2 2 -1

H2A.Z Up in pie1 Up in pie1 1 0.4 -1

H2A.Z Down in pie1 Up in pie1 226 94 -42 ***

H2A.Z Up in pie1 Down in pie1 1 0.4 -1

H2A.Z Down in pie1 Down in pie1 66 88 0

H2A.Z Up in clf Up in clf 0 0.2 0

H2A.Z Down in clf Up in clf 42 12 -13 **

H2A.Z Up in clf Down in clf 1 0.2 -1

H2A.Z Down in clf Down in clf 19 10 -3

Observed intersections between DEGs and differentially enriched genes of the indicated samples. Gene sets were 913 determined relative to WT. H2A.Z gene sets are the “gene body” sets defined above. Observed: number of genes in 914 common between the indicated gene sets. Predicted: number of genes predicted to be in common by chance. p-915 value: obtained using Fisher’s exact test with a null hypothesis of an intersection that is no greater than expected by 916 chance. Significance: * = α ≤ 10-5, ** = α ≤ 10-10, *** = α ≤ 10-20. 917

918

A B

D

Figure 1. pkl-10 pie1-5 seedlings exhibit profound defects in

organogenesis. Seedlings were grown on MS medium under 24-hour light

and images were collected at two weeks of age. Scale bar represents 2mm.

Background colors are desaturated for visual clarity. (A) Representative

WT seedling. (B) Representative pkl-10 seedling exhibiting the

characteristic reduced petiole length and rosette diameter. (C)

Representative pie1-5 seedling. (D) Representative pkl-10 pie1-5 seedling.

C

0 500 1000 1500 2000 2500

clf

pie1

pkl

Number of DEGs

UpDown488 734

304 418

1,063 914

Figure 2. Summary of differentially

expressed genes. Total numbers of genes

identified as differentially expressed relative to

WT in the indicated samples. Gene sets

corresponding to statistically significant

increases in expression are shaded yellow, and

those corresponding to significant decreases in

expression are shaded blue. Differential

expression was determined based on mean

counts per million using the edgeR package and

Fisher’s exact test using a Benjamini-Hochberg

FDR threshold of < 0.05 and a fold change

threshold of ≥ 1.5-fold change relative to WT.

R² = 0.46

-10

-5

0

5

10

-10 0 10

pie1

log 2

(FC

)

clf log2(FC)

R² = 0.78

-10

-5

0

5

10

-10 0 10

clf

log 2

(FC

)

pkl log2(FC)

R² = 0.29

-10

-5

0

5

10

-10 0 10

pie1

log 2

(FC

)

pkl log2(FC)

pkl

200

400

600

800

1,000 Log(p-value)

0 -65 -131

0

ALog(p-value)

0 -90 -181

B

Size

of

Ge

ne

Se

t

Size

of

Ge

ne

Se

t

Increased Expression Decreased Expression

280

56

7037

836

141

(0.5)

clf

pie1pkl

GF Increased Expression Decreased Expression

C D E

-35-34-181

-57-76

-90

-131-92200

400

600

800

1,000

0

pkl

101 446

50

42164

740

162

(0.8)

clf

pie1pkl

82

Figure 3. pkl, pie1, and clf affect expression of common sets of genes. (A-B) Statistical analysis of

intersections between sets of DEGs exhibiting increased (A) or decreased (B) expression in the

indicated mutants relative to WT. Gene sets are ordered by size. y-axes indicate the size of the indicated

observed gene set (first three columns) or intersection in number of genes. Bars are shaded to reflect the

p-value of the intersection based on Fisher’s exact test. Column end labels indicate the log(p-value) for

the indicated intersection. Data visualization was performed using the SuperExactTest package in

Bioconductor [73]. (C-E) Correlation between expression of DEGs in the indicated genotypes. Axes

indicate fold change in expression relative to WT plants on a log2 scale. Green points represent genes

that are differentially expressed in both genotypes, and grey points represent genes that are

differentially expressed in one of the two genotypes. Dotted lines depict linear-fit trendlines of the

stated R2 value calculated using common DEGs (green points). (F-G) Diagrams of the three-way

intersections from panels A and B. Numbers in parentheses indicate the size of the intersection

predicted by chance (the product of the probabilities of a gene being in each group * population size).

C

A

2,276

4,595

1,039

H3K27me3- enriched

Genes

Bouyer et al.

H2A.Z- enriched

Gene Bodies

Coleman-Derr et al.

1,660

2,883

1,198

B

13,926 4,739 481

H2A.Z- enriched

TSS

H3K27me3- enriched

TSS

3,032 1,511 171

H2A.Z- enriched

Gene Body

H3K27me3- enriched

Gene Body

H2A.Z Enr. Yes Yes Yes No No H3K27me3 Enr. Yes Yes No Yes No # Genes 13,671 4,543 1,682 1,511 3,032 171 8,957

Figure 4. H3K27me3 and H2A.Z exhibit co-enrichment and are associated with low levels of

gene expression. (A) Diagrams of intersections between gene sets determined to be enriched for

H3K27me3 or H2A.Z in the gene body relative to H3 in our analysis vs. previously published

analyses [3, 18]. Gene body is defined as the central region of genes that remain after omitting the

terminal 1,000bp ends from the TSS and the transcription termination site (TTS) as annotated in the

Araport11 reference genome. Genes shorter than 2.1kb are thus excluded. [18] (B) Diagrams of

intersections between genes enriched in H2A.Z and/or in H3K27me3 relative to H3 in the

transcription start site (TSS, upper left) or in the gene body (lower right). TSS is defined as the first

500bp (from base 0 to base +500) from the start of the mRNA sequence. Genes shorter than 500bp

are excluded. (C) Box-and-whisker plot depicting the distributions of gene expression for genes

enriched in the gene body in varying combinations of H2A.Z and/or H3K27me3 as indicated. The

y-axis represents mRNA mean counts per million values in WT plants. Dashes indicate that all

genes were included in the set regardless of enrichment status of the indicated mark.

H3

K2

7m

e3

-En

rich

ed

Ge

ne

s WT

clf

pie1

pkl

A B

C

H2

A.Z

-en

rich

ed

Ge

ne

s

D

WT

clf

pie1

pkl

Avg

. H3

K2

7m

e3

En

rich

me

nt

Avg

. H2

A.Z

En

rich

me

nt

WT clf pkl pie1

Figure 5. H3K27me3 and H2A.Z enrichment patterns in WT, clf-28, pie1-5, and pkl-1. (A-D)

Enrichment visualizations generated using the deepTools2 package [49]. Gene regions are scaled to 1kb

on the x-axes. Displayed genes are restricted to those that contain at least one region of enrichment of the

relevant mark relative to H3 as determined by SICER. Samples were normalized using the RPKM

method. Data are representative of two independent biological replicates. (A) Heat maps of H3K27me3

enrichment in the indicated genetic backgrounds. (B) Metagene profile of average enrichment data from

panel A. (C) Heat maps of H2A.Z enrichment in the indicated genetic backgrounds. (D) Metagene

profile of average enrichment data from panel C.

TSS TTS TSS TTS TSS TTS TSS TTS

20

40

60

80

100

120

WT clf pkl pie1

50

100

150

200

250

TSS TTS TSS TTS TSS TTS TSS TTS

TSS TTS

TSS TTS

0 1000 2000 3000 4000

clf

pie1

pkl

H2A.Z Differentially Enriched Gene Bodies

0 2000 4000 6000 8000

clf

pie1

pkl

H3K27me3 Differentially Enriched Genes

174 7,248

294

407

2,931

5,490

97

10

62

3,466

19 1,134

A

B

Reduced Levels

Increased Levels

D

MEA

WT

clf-28

pkl-1

pie1-5

PKR2

0 5000 10000 15000

clf

pie1

pkl

H2A.Z Differentially Enriched TSS

564

471

40 13,656

65 4,952

C

Figure 6: Summary of differentially enriched genes. (A-D) Summary of differentially enriched genes

identified for each mutant line. Differential enrichment was determined relative to H3 using SICER-df [48].

Differentially enriched gene sets were filtered to contain only genes identified as such in both of two

biological replicates. (A) Summary of genes identified as differentially enriched relative to WT for

H3K27me3. (B) Summary of genes identified as differentially enriched relative to WT for H2A.Z in the

gene body. (C) Summary of genes identified as differentially enriched relative to WT for H2A.Z at the TSS.

The gene body and TSS data sets are described in the Figure 4 legend. (D) Two representative H3K27me3-

enriched genes that exhibit reduced levels of H3K27me3 in the indicated genetic backgrounds. Displayed

bigwig tracks were normalized using the RPKM method of deepTools2 and visualized using IGV [74]. Data

are representative of two independent biological replicates.

pkl

765

57

305

clf pie1

1,411

1 20

117

933

348

Gene Body

pkl

2,180

332

839

clf pie1

4,327

9 121

472

2,770

1,104

TSS

A Reduced H2A.Z Reduced H3K27me3

B

clf pie1

pkl

533 4,226

9,029

14 179

222

56

TSS H2A.Z Down

clf pie1

pkl

338 3

25

2,406 1,336

552

1,223

TSS H3K27me3 Down

clf pie1

pkl

125 2

4

816 432

138

328

Gene Body H3K27me3 Down

C clf pie1

pkl

106 1,004

2,411

0 24

27

11

Gene Body H2A.Z Down

Figure 7. Intersection analysis of differentially enriched gene sets. (A)

Diagrams of intersections between gene sets exhibiting reduced levels of

H3K27me3 and those exhibiting reduced levels of H2A.Z in the indicated mutant

lines. Genes were limited to those enriched in both epigenetic marks for this

analysis. (B) Venn diagrams depicting the intersections among gene sets

exhibiting reduced H3K27me3 in the indicated mutant lines at the TSS (left) or

in the gene body (right). (C) Venn diagrams depicting the intersections among

gene sets exhibiting reduced H2A.Z in the indicated mutant lines at the TSS (left)

or in the gene body (right).

PKL + + + Prenuc.

+ +

III IV

Figure 8. PKL converts prenucleosomes into canonical nucleosomes.

Prenucleosome maturation assay performed as described previously [46].

Reaction products were de-proteinated and fragments were analyzed using

agarose gel electrophoresis. Bands were visualized using ethidium bromide. (A)

Assembly of poly-prenucleosomal templates (Prenuc.) in the absence (lane I) or

presence (lane II) of recombinant PKL. Mono-prenucleosomes were assembled

as previously described [46], and poly-prenucleosomes were synthesized by

ligating the mono-prenucleosomes using T4 DNA ligase and ATP. (B) Digestion

of poly-prenucleosomal templates from panel A (lane III) with micrococcal

nuclease (MNase), which spares DNA fragments rendered inaccessible due to

occlusion by a nucleosome. Prior to digestion, samples were incubated in the

absence or presence of recombinant PKL and/or ATP (lanes IV through VI).

Generation of mature nucleosomes was assessed by the appearance of ~147 bp

DNA fragments (indicated with a star) corresponding to the DNA occlusion

length of the mature nucleosome core particle.

+ + I II

+

200 bp 150 bp 100 bp

PKL MNase

ATP

A B

75 bp 50 bp

250 bp 300 bp

200 bp 150 bp 100 bp 75 bp 50 bp

250 bp 300 bp

V VI

+ +

Polymerase Complex

Pre- nucleosomes

H2A.Z Deposition

PIE1

CLF

H3K27me3 Deposition

Nucleosome Maturation

Chaperone

PICKLE

Figure 9. Model for H3K27me3 deposition and maintenance. Generation of H3K27me3-enriched chromatin is

dependent on the prior action of PIE1 and H2A.Z. PKL acts subsequently to maintains this epigenetic state during

DNA replication and/or transcription by facilitating nucleosome retention.

Parsed CitationsWe acknowledge the Arabidopsis Biological Resource Center and the Salk Institute Genomic Analysis Laboratory for providing thesequence-indexed Arabidopsis T-DNA insertion mutants used in this study. We thank Nadia Atallah for her valuable input with regardto ChIP-seq data analysis. We also thank Craig Peterson for many thoughtful and productive discussions and Nick Carpita for criticalinsight and feedback.

AUTHOR CONTRIBUTIONS

BC was principally responsible for study design, data acquisition and analysis, and manuscript preparation and editing (with JO). BBand KKH participated in study design and data acquisition. RH and WJ participated in data acquisition and analysis. HZ, PEP, and RBDparticipated in data analysis and manuscript preparation and editing. JO participated in study design, data analysis, and was principallyresponsible for manuscript preparation and editing (with BC).

REFERENCES

Aranda, S., Mas, G., and Di Croce, L. (2015). Regulation of gene transcription by Polycomb proteins. Sci Adv 1, e1500737.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Berriri, S., Gangappa, S.N., and Kumar, S.V. (2016). SWR1 Chromatin-Remodeling Complex Subunits and H2A.Z Have Non-overlappingFunctions in Immunity and Gene Regulation in Arabidopsis. Molecular plant 9, 1051-1065.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Bieluszewski, T., Galganski, L., Sura, W., Bieluszewska, A., Abram, M., Ludwikow, A., Ziolkowski, P.A., and Sadowski, J. (2015). AtEAF1 isa potential platform protein for Arabidopsis NuA4 acetyltransferase complex. BMC Plant Biol 15, 75.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Bolger, A.M., Lohse, M., and Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114-2120.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Bouyer, D., Roudier, F., Heese, M., Andersen, E.D., Gey, D., Nowack, M.K., Goodrich, J., Renou, J.P., Grini, P.E., Colot, V., et al. (2011).Polycomb repressive complex 2 controls the embryo-to-seedling phase transition. PLoS genetics 7, e1002014.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Chanvivattana, Y., Bishopp, A., Schubert, D., Stock, C., Moon, Y.H., Sung, Z.R., and Goodrich, J. (2004). Interaction of Polycomb-groupproteins controlling flowering in Arabidopsis. Development 131, 5263-5276.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Cheng, C.Y., Krishnakumar, V., Chan, A.P., Thibaud-Nissen, F., Schobel, S., and Town, C.D. (2017). Araport11: a complete reannotationof the Arabidopsis thaliana reference genome. The Plant journal : for cell and molecular biology 89, 789-804.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Choi, K., Kim, S., Kim, S.Y., Kim, M., Hyun, Y., Lee, H., Choe, S., Kim, S.G., Michaels, S., and Lee, I. (2005). SUPPRESSOR OF FRIGIDA3encodes a nuclear ACTIN-RELATED PROTEIN6 required for floral repression in Arabidopsis. The Plant cell 17, 2647-2660.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Coleman-Derr, D., and Zilberman, D. (2012). Deposition of histone variant H2A.Z within gene bodies regulates responsive genes. PLoSgenetics 8, e1002988.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

de Lucas, M., Pu, L., Turco, G., Gaudinier, A., Morao, A.K., Harashima, H., Kim, D., Ron, M., Sugimoto, K., Roudier, F., et al. (2016).Transcriptional Regulation of Arabidopsis Polycomb Repressive Complex 2 Coordinates Cell-Type Proliferation and Differentiation.The Plant cell 28, 2616-2631.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Deal, R.B., Kandasamy, M.K., McKinney, E.C., and Meagher, R.B. (2005). The nuclear actin-related protein ARP6 is a pleiotropicdevelopmental regulator required for the maintenance of FLOWERING LOCUS C expression and repression of flowering inArabidopsis. The Plant cell 17, 2633-2646.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Deal, R.B., Topp, C.N., McKinney, E.C., and Meagher, R.B. (2007). Repression of flowering in Arabidopsis requires activation ofFLOWERING LOCUS C expression by the histone variant H2A.Z. The Plant cell 19, 74-83.

Pubmed: Author and Title

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Del Prete, S., Mikulski, P., Schubert, D., and Gaudin, V. (2015). One, Two, Three: Polycomb Proteins Hit All Dimensions of GeneRegulation. Genes 6, 520-542.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Doyle, M.R., and Amasino, R.M. (2009). A single amino acid change in the enhancer of zeste ortholog CURLY LEAF results invernalization-independent, rapid flowering in Arabidopsis. Plant physiology 151, 1688-1697.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Du, J., Johnson, L.M., Groth, M., Feng, S., Hale, C.J., Li, S., Vashisht, A.A., Gallego-Bartolome, J., Wohlschlegel, J.A., Patel, D.J., et al.(2014). Mechanism of DNA methylation-directed histone methylation by KRYPTONITE. Molecular cell 55, 495-504.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Du, J., Zhong, X., Bernatavichute, Y.V., Stroud, H., Feng, S., Caro, E., Vashisht, A.A., Terragni, J., Chin, H.G., Tu, A., et al. (2012). Dualbinding of chromomethylase domains to H3K9me2-containing nucleosomes directs DNA methylation in plants. Cell 151, 167-180.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

ENCODE (2016). ENCODE Guidelines and Best Practices for RNA-seq: Revised December 2016 (encodeproject.org).

ENCODE (2017). ENCODE Guidelines for Experiments Generating ChIP-seq Data: January 2017 (encodeproject.org).

Fei, J., Torigoe, S.E., Brown, C.R., Khuong, M.T., Kassavetis, G.A., Boeger, H., and Kadonaga, J.T. (2015). The prenucleosome, a stableconformational isomer of the nucleosome. Genes & development 29, 2563-2575.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Fisher, R.A. (1922). On the interpretation of x(2) from contingency tables, and the calculation of P. J R Stat Soc 85, 87-94.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Gan, E.S., Xu, Y., and Ito, T. (2015). Dynamics of H3K27me3 methylation and demethylation in plant development. Plant signaling &behavior 10, e1027851.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ho, K.K., Zhang, H., Golden, B.L., and Ogas, J. (2013). PICKLE is a CHD subfamily II ATP-dependent chromatin remodeling factor.Biochimica et biophysica acta 1829, 199-210.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Jackson, J.P., Lindroth, A.M., Cao, X., and Jacobsen, S.E. (2002). Control of CpNpG DNA methylation by the KRYPTONITE histone H3methyltransferase. Nature 416, 556-560.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Jacob, Y., Bergamin, E., Donoghue, M.T., Mongeon, V., LeBlanc, C., Voigt, P., Underwood, C.J., Brunzelle, J.S., Michaels, S.D.,Reinberg, D., et al. (2014). Selective methylation of histone H3 variant H3.1 regulates heterochromatin replication. Science 343, 1249-1253.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Jacob, Y., Feng, S., LeBlanc, C.A., Bernatavichute, Y.V., Stroud, H., Cokus, S., Johnson, L.M., Pellegrini, M., Jacobsen, S.E., andMichaels, S.D. (2009). ATXR5 and ATXR6 are H3K27 monomethyltransferases required for chromatin structure and gene silencing. NatStruct Mol Biol 16, 763-768.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Jing, Y., Zhang, D., Wang, X., Tang, W., Wang, W., Huai, J., Xu, G., Chen, D., Li, Y., and Lin, R. (2013). Arabidopsis chromatin remodelingfactor PICKLE interacts with transcription factor HY5 to regulate hypocotyl cell elongation. Plant Cell 25, 242-256.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Johnson, L.M., Bostick, M., Zhang, X., Kraft, E., Henderson, I., Callis, J., and Jacobsen, S.E. (2007). The SRA methyl-cytosine-bindingdomain links DNA and histone methylation. Current biology : CB 17, 379-384.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Khan, A.A., Lee, A.J., and Roh, T.Y. (2015). Polycomb group protein-mediated histone modifications during cell differentiation.Epigenomics 7, 75-84.

Pubmed: Author and Title

Google Scholar: Author Only Title Only Author and Title

Kim, D.H., and Sung, S. (2014). Polycomb-mediated gene silencing in Arabidopsis thaliana. Mol Cells 37, 841-850.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Krogan, N.J., Keogh, M.C., Datta, N., Sawa, C., Ryan, O.W., Ding, H., Haw, R.A., Pootoolal, J., Tong, A., Canadien, V., et al. (2003). A Snf2family ATPase complex required for recruitment of the histone H2A variant Htz1. Molecular cell 12, 1565-1576.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ku, M., Jaffe, J.D., Koche, R.P., Rheinbay, E., Endoh, M., Koseki, H., Carr, S.A., and Bernstein, B.E. (2012). H2A.Z landscapes and dualmodifications in pluripotent and multipotent stem cells underlie complex genome regulatory functions. Genome biology 13, R85.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Lafos, M., Kroll, P., Hohenstatt, M.L., Thorpe, F.L., Clarenz, O., and Schubert, D. (2011). Dynamic Regulation of H3K27 Trimethylationduring Arabidopsis Differentiation. PLoS Genet 7, e1002040.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Langmead, B., and Salzberg, S.L. (2012). Fast gapped-read alignment with Bowtie 2. Nat Methods 9, 357-359.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Li, Z., Gadue, P., Chen, K., Jiao, Y., Tuteja, G., Schug, J., Li, W., and Kaestner, K.H. (2012). Foxa2 and H2A.Z mediate nucleosomedepletion during embryonic stem cell differentiation. Cell 151, 1608-1616.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Love, M.I., Huber, W., and Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome biology 15, 550.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Lu, P.Y., Levesque, N., and Kobor, M.S. (2009). NuA4 and SWR1-C: two chromatin-modifying complexes with overlapping functions andcomponents. Biochem Cell Biol 87, 799-815.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Lusser, A., Urwin, D.L., and Kadonaga, J.T. (2005). Distinct activities of CHD1 and ACF in ATP-dependent chromatin assembly. NatStruct Mol Biol 12, 160-166.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

March-Diaz, R., Garcia-Dominguez, M., Florencio, F.J., and Reyes, J.C. (2007). SEF, a new protein required for flowering repression inArabidopsis, interacts with PIE1 and ARP6. Plant physiology 143, 893-901.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

March-Diaz, R., Garcia-Dominguez, M., Lozano-Juste, J., Leon, J., Florencio, F.J., and Reyes, J.C. (2008). Histone H2A.Z andhomologues of components of the SWR1 complex are required to control immunity in Arabidopsis. The Plant journal : for cell andmolecular biology 53, 475-487.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Margueron, R., and Reinberg, D. (2011). The Polycomb complex PRC2 and its mark in life. Nature 469, 343-349.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Mizuguchi, G., Shen, X., Landry, J., Wu, W.H., Sen, S., and Wu, C. (2004). ATP-driven exchange of histone H2AZ variant catalyzed bySWR1 chromatin remodeling complex. Science 303, 343-348.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Morrison, A.J., and Shen, X. (2009). Chromatin remodelling beyond transcription: the INO80 and SWR1 complexes. Nat Rev Mol CellBiol 10, 373-384.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Mozgova, I., Kohler, C., and Hennig, L. (2015). Keeping the gate closed: functions of the polycomb repressive complex PRC2 indevelopment. The Plant journal : for cell and molecular biology 83, 121-132.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Muller-Xing, R., Clarenz, O., Pokorny, L., Goodrich, J., and Schubert, D. (2014). Polycomb-Group Proteins and FLOWERING LOCUS TMaintain Commitment to Flowering in Arabidopsis thaliana. The Plant cell 26, 2457-2471.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

NCBI (2017). Magic-BLAST Short Read Mapper (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/magicblast/LATEST/).

Noh, Y.S., and Amasino, R.M. (2003). PIE1, an ISWI family gene, is required for FLC activation and floral repression in Arabidopsis. ThePlant cell 15, 1671-1682.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ogas, J., Cheng, J.C., Sung, Z.R., and Somerville, C. (1997). Cellular differentiation regulated by gibberellin in the Arabidopsis thalianapickle mutant. Science 277, 91-94.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Quinlan, A.R. (2014). BEDTools: The Swiss-Army Tool for Genome Feature Analysis. Curr Protoc Bioinformatics 47, 11.12.11-34.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Radman-Livaja, M., Quan, T.K., Valenzuela, L., Armstrong, J.A., van Welsem, T., Kim, T., Lee, L.J., Buratowski, S., van Leeuwen, F.,Rando, O.J., et al. (2012). A key role for Chd1 in histone H3 dynamics at the 3' ends of long genes in yeast. PLoS genetics 8, e1002811.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ramirez, F., Ryan, D.P., Gruning, B., Bhardwaj, V., Kilpert, F., Richter, A.S., Heyne, S., Dundar, F., and Manke, T. (2016). deepTools2: anext generation web server for deep-sequencing data analysis. Nucleic acids research 44, W160-165.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ritchie, M.E., Phipson, B., Wu, D., Hu, Y., Law, C.W., Shi, W., and Smyth, G.K. (2015). limma powers differential expression analyses forRNA-sequencing and microarray studies. Nucleic acids research 43, e47.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Robinson, J.T., Thorvaldsdottir, H., Winckler, W., Guttman, M., Lander, E.S., Getz, G., and Mesirov, J.P. (2011). Integrative genomicsviewer. Nat Biotechnol 29, 24-26.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Robinson, M.D., McCarthy, D.J., and Smyth, G.K. (2010). edgeR: a Bioconductor package for differential expression analysis of digitalgene expression data. Bioinformatics 26, 139-140.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Rosa, M., Von Harder, M., Cigliano, R.A., Schlogelhofer, P., and Mittelsten Scheid, O. (2013). The Arabidopsis SWR1 chromatin-remodeling complex is important for DNA repair, somatic recombination, and meiosis. The Plant cell 25, 1990-2001.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ruhl, D.D., Jin, J., Cai, Y., Swanson, S., Florens, L., Washburn, M.P., Conaway, R.C., Conaway, J.W., and Chrivia, J.C. (2006). Purificationof a human SRCAP complex that remodels chromatin by incorporating the histone variant H2A.Z into nucleosomes. Biochemistry 45,5671-5677.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Sarcinella, E., Zuzarte, P.C., Lau, P.N., Draker, R., and Cheung, P. (2007). Monoubiquitylation of H2A.Z distinguishes its association witheuchromatin or facultative heterochromatin. Molecular and cellular biology 27, 6457-6468.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Schmitges, F.W., Prusty, A.B., Faty, M., Stutzer, A., Lingaraju, G.M., Aiwazian, J., Sack, R., Hess, D., Li, L., Zhou, S., et al. (2011). Histonemethylation by PRC2 is inhibited by active chromatin marks. Molecular cell 42, 330-341.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Schubert, D., Primavesi, L., Bishopp, A., Roberts, G., Doonan, J., Jenuwein, T., and Goodrich, J. (2006). Silencing by plant Polycomb-group genes requires dispersed trimethylation of histone H3 at lysine 27. The EMBO journal 25, 4638-4649.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Sequeira-Mendes, J., Araguez, I., Peiro, R., Mendez-Giraldez, R., Zhang, X., Jacobsen, S.E., Bastolla, U., and Gutierrez, C. (2014). TheFunctional Topography of the Arabidopsis Genome Is Organized in a Reduced Number of Linear Motifs of Chromatin States. The Plantcell 26, 2351-2366.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Shaked, H., Avivi-Ragolsky, N., and Levy, A.A. (2006). Involvement of the Arabidopsis SWI2/SNF2 chromatin remodeling gene family inDNA damage response and recombination. Genetics 173, 985-994.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Smolle, M., Venkatesh, S., Gogol, M.M., Li, H., Zhang, Y., Florens, L., Washburn, M.P., and Workman, J.L. (2012). Chromatin remodelersIsw1 and Chd1 maintain chromatin structure during transcription by preventing histone exchange. Nat Struct Mol Biol 19, 884-892.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Subramanian, V., Fields, P.A., and Boyer, L.A. (2015). H2A.Z: a molecular rheostat for transcriptional control. F1000prime reports 7, 01.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Sura, W., Kabza, M., Karlowski, W.M., Bieluszewski, T., Kus-Slowinska, M., Paweloszek, L., Sadowski, J., and Ziolkowski, P.A. (2017).Dual Role of the Histone Variant H2A.Z in Transcriptional Regulation of Stress-Response Genes. The Plant cell 29, 791-807.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Torigoe, S.E., Urwin, D.L., Ishii, H., Smith, D.E., and Kadonaga, J.T. (2011). Identification of a rapidly formed nonnucleosomal histone-DNA intermediate that is converted into chromatin by ACF. Molecular cell 43, 638-648.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Wang, H., Liu, C., Cheng, J., Liu, J., Zhang, L., He, C., Shen, W.H., Jin, H., Xu, L., and Zhang, Y. (2016). Arabidopsis Flower and EmbryoDevelopmental Genes are Repressed in Seedlings by Different Combinations of Polycomb Group Proteins in Association with DistinctSets of Cis-regulatory Elements. PLoS genetics 12, e1005771.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Wang, M., Zhao, Y., and Zhang, B. (2015). Efficient Test and Visualization of Multi-Set Intersections. Sci Rep 5, 16923.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Weber, C.M., Ramachandran, S., and Henikoff, S. (2014). Nucleosomes are context-specific, H2A.Z-modulated barriers to RNApolymerase. Molecular cell 53, 819-830.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Wong, M.M., Cox, L.K., and Chrivia, J.C. (2007). The chromatin remodeling protein, SRCAP, is critical for deposition of the histonevariant H2A.Z at promoters. The Journal of biological chemistry 282, 26132-26139.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Xiao, J., Jin, R., and Wagner, D. (2017). Developmental transitions: integrating environmental cues with hormonal signaling in thechromatin landscape in plants. Genome biology 18, 88.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Xu, M., Hu, T., Smith, M.R., and Poethig, R.S. (2016). Epigenetic Regulation of Vegetative Phase Change in Arabidopsis. The Plant cell28, 28-41.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Xu, S., Grullon, S., Ge, K., and Peng, W. (2014). Spatial clustering for identification of ChIP-enriched regions (SICER) to map regions ofhistone methylation patterns in embryonic stem cells. Methods in molecular biology 1150, 97-111.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Zhang, D., Jing, Y., Jiang, Z., and Lin, R. (2014). The Chromatin-Remodeling Factor PICKLE Integrates Brassinosteroid and GibberellinSignaling during Skotomorphogenic Growth in Arabidopsis. Plant Cell 26, 2472-2485.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Zhang, H., Bishop, B., Ringenberg, W., Muir, W.M., and Ogas, J. (2012). The CHD3 remodeler PICKLE associates with genes enrichedfor trimethylation of histone H3 lysine 27. Plant physiology 159, 418-432.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Zhang, H., Rider, S.D., Jr., Henderson, J.T., Fountain, M., Chuang, K., Kandachar, V., Simons, A., Edenberg, H.J., Romero-Severson, J.,Muir, W.M., et al. (2008). The CHD3 remodeler PICKLE promotes trimethylation of histone H3 lysine 27. The Journal of biologicalchemistry 283, 22637-22648.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Zhang, X., Clarenz, O., Cokus, S., Bernatavichute, Y.V., Pellegrini, M., Goodrich, J., and Jacobsen, S.E. (2007). Whole-genome analysisof histone H3 lysine 27 trimethylation in Arabidopsis. PLoS biology 5, e129.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Aranda, S., Mas, G., and Di Croce, L. (2015). Regulation of gene transcription by Polycomb proteins. Sci Adv 1, e1500737.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Berriri, S., Gangappa, S.N., and Kumar, S.V. (2016). SWR1 Chromatin-Remodeling Complex Subunits and H2A.Z Have Non-overlappingFunctions in Immunity and Gene Regulation in Arabidopsis. Molecular plant 9, 1051-1065.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Bieluszewski, T., Galganski, L., Sura, W., Bieluszewska, A., Abram, M., Ludwikow, A., Ziolkowski, P.A., and Sadowski, J. (2015). AtEAF1 isa potential platform protein for Arabidopsis NuA4 acetyltransferase complex. BMC Plant Biol 15, 75.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Bolger, A.M., Lohse, M., and Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114-2120.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Bouyer, D., Roudier, F., Heese, M., Andersen, E.D., Gey, D., Nowack, M.K., Goodrich, J., Renou, J.P., Grini, P.E., Colot, V., et al. (2011).Polycomb repressive complex 2 controls the embryo-to-seedling phase transition. PLoS genetics 7, e1002014.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Chanvivattana, Y., Bishopp, A., Schubert, D., Stock, C., Moon, Y.H., Sung, Z.R., and Goodrich, J. (2004). Interaction of Polycomb-groupproteins controlling flowering in Arabidopsis. Development 131, 5263-5276.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Cheng, C.Y., Krishnakumar, V., Chan, A.P., Thibaud-Nissen, F., Schobel, S., and Town, C.D. (2017). Araport11: a complete reannotationof the Arabidopsis thaliana reference genome. The Plant journal : for cell and molecular biology 89, 789-804.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Choi, K., Kim, S., Kim, S.Y., Kim, M., Hyun, Y., Lee, H., Choe, S., Kim, S.G., Michaels, S., and Lee, I. (2005). SUPPRESSOR OF FRIGIDA3encodes a nuclear ACTIN-RELATED PROTEIN6 required for floral repression in Arabidopsis. The Plant cell 17, 2647-2660.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Coleman-Derr, D., and Zilberman, D. (2012). Deposition of histone variant H2A.Z within gene bodies regulates responsive genes. PLoSgenetics 8, e1002988.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

de Lucas, M., Pu, L., Turco, G., Gaudinier, A., Morao, A.K., Harashima, H., Kim, D., Ron, M., Sugimoto, K., Roudier, F., et al. (2016).Transcriptional Regulation of Arabidopsis Polycomb Repressive Complex 2 Coordinates Cell-Type Proliferation and Differentiation.The Plant cell 28, 2616-2631.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Deal, R.B., Kandasamy, M.K., McKinney, E.C., and Meagher, R.B. (2005). The nuclear actin-related protein ARP6 is a pleiotropicdevelopmental regulator required for the maintenance of FLOWERING LOCUS C expression and repression of flowering inArabidopsis. The Plant cell 17, 2633-2646.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Deal, R.B., Topp, C.N., McKinney, E.C., and Meagher, R.B. (2007). Repression of flowering in Arabidopsis requires activation ofFLOWERING LOCUS C expression by the histone variant H2A.Z. The Plant cell 19, 74-83.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Del Prete, S., Mikulski, P., Schubert, D., and Gaudin, V. (2015). One, Two, Three: Polycomb Proteins Hit All Dimensions of GeneRegulation. Genes 6, 520-542.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Doyle, M.R., and Amasino, R.M. (2009). A single amino acid change in the enhancer of zeste ortholog CURLY LEAF results invernalization-independent, rapid flowering in Arabidopsis. Plant physiology 151, 1688-1697.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Du, J., Johnson, L.M., Groth, M., Feng, S., Hale, C.J., Li, S., Vashisht, A.A., Gallego-Bartolome, J., Wohlschlegel, J.A., Patel, D.J., et al.(2014). Mechanism of DNA methylation-directed histone methylation by KRYPTONITE. Molecular cell 55, 495-504.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Du, J., Zhong, X., Bernatavichute, Y.V., Stroud, H., Feng, S., Caro, E., Vashisht, A.A., Terragni, J., Chin, H.G., Tu, A., et al. (2012). Dualbinding of chromomethylase domains to H3K9me2-containing nucleosomes directs DNA methylation in plants. Cell 151, 167-180.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

ENCODE (2016). ENCODE Guidelines and Best Practices for RNA-seq: Revised December 2016 (encodeproject.org).

ENCODE (2017). ENCODE Guidelines for Experiments Generating ChIP-seq Data: January 2017 (encodeproject.org).

Fei, J., Torigoe, S.E., Brown, C.R., Khuong, M.T., Kassavetis, G.A., Boeger, H., and Kadonaga, J.T. (2015). The prenucleosome, a stableconformational isomer of the nucleosome. Genes & development 29, 2563-2575.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Fisher, R.A. (1922). On the interpretation of x(2) from contingency tables, and the calculation of P. J R Stat Soc 85, 87-94.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Gan, E.S., Xu, Y., and Ito, T. (2015). Dynamics of H3K27me3 methylation and demethylation in plant development. Plant signaling &behavior 10, e1027851.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ho, K.K., Zhang, H., Golden, B.L., and Ogas, J. (2013). PICKLE is a CHD subfamily II ATP-dependent chromatin remodeling factor.Biochimica et biophysica acta 1829, 199-210.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Jackson, J.P., Lindroth, A.M., Cao, X., and Jacobsen, S.E. (2002). Control of CpNpG DNA methylation by the KRYPTONITE histone H3methyltransferase. Nature 416, 556-560.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Jacob, Y., Bergamin, E., Donoghue, M.T., Mongeon, V., LeBlanc, C., Voigt, P., Underwood, C.J., Brunzelle, J.S., Michaels, S.D.,Reinberg, D., et al. (2014). Selective methylation of histone H3 variant H3.1 regulates heterochromatin replication. Science 343, 1249-1253.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Jacob, Y., Feng, S., LeBlanc, C.A., Bernatavichute, Y.V., Stroud, H., Cokus, S., Johnson, L.M., Pellegrini, M., Jacobsen, S.E., andMichaels, S.D. (2009). ATXR5 and ATXR6 are H3K27 monomethyltransferases required for chromatin structure and gene silencing. NatStruct Mol Biol 16, 763-768.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Jing, Y., Zhang, D., Wang, X., Tang, W., Wang, W., Huai, J., Xu, G., Chen, D., Li, Y., and Lin, R. (2013). Arabidopsis chromatin remodelingfactor PICKLE interacts with transcription factor HY5 to regulate hypocotyl cell elongation. Plant Cell 25, 242-256.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Johnson, L.M., Bostick, M., Zhang, X., Kraft, E., Henderson, I., Callis, J., and Jacobsen, S.E. (2007). The SRA methyl-cytosine-bindingdomain links DNA and histone methylation. Current biology : CB 17, 379-384.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Khan, A.A., Lee, A.J., and Roh, T.Y. (2015). Polycomb group protein-mediated histone modifications during cell differentiation.Epigenomics 7, 75-84.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Kim, D.H., and Sung, S. (2014). Polycomb-mediated gene silencing in Arabidopsis thaliana. Mol Cells 37, 841-850.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Krogan, N.J., Keogh, M.C., Datta, N., Sawa, C., Ryan, O.W., Ding, H., Haw, R.A., Pootoolal, J., Tong, A., Canadien, V., et al. (2003). A Snf2family ATPase complex required for recruitment of the histone H2A variant Htz1. Molecular cell 12, 1565-1576.

Pubmed: Author and Title

Google Scholar: Author Only Title Only Author and Title

Ku, M., Jaffe, J.D., Koche, R.P., Rheinbay, E., Endoh, M., Koseki, H., Carr, S.A., and Bernstein, B.E. (2012). H2A.Z landscapes and dualmodifications in pluripotent and multipotent stem cells underlie complex genome regulatory functions. Genome biology 13, R85.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Lafos, M., Kroll, P., Hohenstatt, M.L., Thorpe, F.L., Clarenz, O., and Schubert, D. (2011). Dynamic Regulation of H3K27 Trimethylationduring Arabidopsis Differentiation. PLoS Genet 7, e1002040.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Langmead, B., and Salzberg, S.L. (2012). Fast gapped-read alignment with Bowtie 2. Nat Methods 9, 357-359.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Li, Z., Gadue, P., Chen, K., Jiao, Y., Tuteja, G., Schug, J., Li, W., and Kaestner, K.H. (2012). Foxa2 and H2A.Z mediate nucleosomedepletion during embryonic stem cell differentiation. Cell 151, 1608-1616.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Love, M.I., Huber, W., and Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome biology 15, 550.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Lu, P.Y., Levesque, N., and Kobor, M.S. (2009). NuA4 and SWR1-C: two chromatin-modifying complexes with overlapping functions andcomponents. Biochem Cell Biol 87, 799-815.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Lusser, A., Urwin, D.L., and Kadonaga, J.T. (2005). Distinct activities of CHD1 and ACF in ATP-dependent chromatin assembly. NatStruct Mol Biol 12, 160-166.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

March-Diaz, R., Garcia-Dominguez, M., Florencio, F.J., and Reyes, J.C. (2007). SEF, a new protein required for flowering repression inArabidopsis, interacts with PIE1 and ARP6. Plant physiology 143, 893-901.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

March-Diaz, R., Garcia-Dominguez, M., Lozano-Juste, J., Leon, J., Florencio, F.J., and Reyes, J.C. (2008). Histone H2A.Z andhomologues of components of the SWR1 complex are required to control immunity in Arabidopsis. The Plant journal : for cell andmolecular biology 53, 475-487.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Margueron, R., and Reinberg, D. (2011). The Polycomb complex PRC2 and its mark in life. Nature 469, 343-349.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Mizuguchi, G., Shen, X., Landry, J., Wu, W.H., Sen, S., and Wu, C. (2004). ATP-driven exchange of histone H2AZ variant catalyzed bySWR1 chromatin remodeling complex. Science 303, 343-348.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Morrison, A.J., and Shen, X. (2009). Chromatin remodelling beyond transcription: the INO80 and SWR1 complexes. Nat Rev Mol CellBiol 10, 373-384.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Mozgova, I., Kohler, C., and Hennig, L. (2015). Keeping the gate closed: functions of the polycomb repressive complex PRC2 indevelopment. The Plant journal : for cell and molecular biology 83, 121-132.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Muller-Xing, R., Clarenz, O., Pokorny, L., Goodrich, J., and Schubert, D. (2014). Polycomb-Group Proteins and FLOWERING LOCUS TMaintain Commitment to Flowering in Arabidopsis thaliana. The Plant cell 26, 2457-2471.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

NCBI (2017). Magic-BLAST Short Read Mapper (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/magicblast/LATEST/).

Noh, Y.S., and Amasino, R.M. (2003). PIE1, an ISWI family gene, is required for FLC activation and floral repression in Arabidopsis. ThePlant cell 15, 1671-1682.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ogas, J., Cheng, J.C., Sung, Z.R., and Somerville, C. (1997). Cellular differentiation regulated by gibberellin in the Arabidopsis thalianapickle mutant. Science 277, 91-94.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Quinlan, A.R. (2014). BEDTools: The Swiss-Army Tool for Genome Feature Analysis. Curr Protoc Bioinformatics 47, 11.12.11-34.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Radman-Livaja, M., Quan, T.K., Valenzuela, L., Armstrong, J.A., van Welsem, T., Kim, T., Lee, L.J., Buratowski, S., van Leeuwen, F.,Rando, O.J., et al. (2012). A key role for Chd1 in histone H3 dynamics at the 3' ends of long genes in yeast. PLoS genetics 8, e1002811.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ramirez, F., Ryan, D.P., Gruning, B., Bhardwaj, V., Kilpert, F., Richter, A.S., Heyne, S., Dundar, F., and Manke, T. (2016). deepTools2: anext generation web server for deep-sequencing data analysis. Nucleic acids research 44, W160-165.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ritchie, M.E., Phipson, B., Wu, D., Hu, Y., Law, C.W., Shi, W., and Smyth, G.K. (2015). limma powers differential expression analyses forRNA-sequencing and microarray studies. Nucleic acids research 43, e47.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Robinson, J.T., Thorvaldsdottir, H., Winckler, W., Guttman, M., Lander, E.S., Getz, G., and Mesirov, J.P. (2011). Integrative genomicsviewer. Nat Biotechnol 29, 24-26.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Robinson, M.D., McCarthy, D.J., and Smyth, G.K. (2010). edgeR: a Bioconductor package for differential expression analysis of digitalgene expression data. Bioinformatics 26, 139-140.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Rosa, M., Von Harder, M., Cigliano, R.A., Schlogelhofer, P., and Mittelsten Scheid, O. (2013). The Arabidopsis SWR1 chromatin-remodeling complex is important for DNA repair, somatic recombination, and meiosis. The Plant cell 25, 1990-2001.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ruhl, D.D., Jin, J., Cai, Y., Swanson, S., Florens, L., Washburn, M.P., Conaway, R.C., Conaway, J.W., and Chrivia, J.C. (2006). Purificationof a human SRCAP complex that remodels chromatin by incorporating the histone variant H2A.Z into nucleosomes. Biochemistry 45,5671-5677.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Sarcinella, E., Zuzarte, P.C., Lau, P.N., Draker, R., and Cheung, P. (2007). Monoubiquitylation of H2A.Z distinguishes its association witheuchromatin or facultative heterochromatin. Molecular and cellular biology 27, 6457-6468.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Schmitges, F.W., Prusty, A.B., Faty, M., Stutzer, A., Lingaraju, G.M., Aiwazian, J., Sack, R., Hess, D., Li, L., Zhou, S., et al. (2011). Histonemethylation by PRC2 is inhibited by active chromatin marks. Molecular cell 42, 330-341.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Schubert, D., Primavesi, L., Bishopp, A., Roberts, G., Doonan, J., Jenuwein, T., and Goodrich, J. (2006). Silencing by plant Polycomb-group genes requires dispersed trimethylation of histone H3 at lysine 27. The EMBO journal 25, 4638-4649.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Sequeira-Mendes, J., Araguez, I., Peiro, R., Mendez-Giraldez, R., Zhang, X., Jacobsen, S.E., Bastolla, U., and Gutierrez, C. (2014). TheFunctional Topography of the Arabidopsis Genome Is Organized in a Reduced Number of Linear Motifs of Chromatin States. The Plantcell 26, 2351-2366.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Shaked, H., Avivi-Ragolsky, N., and Levy, A.A. (2006). Involvement of the Arabidopsis SWI2/SNF2 chromatin remodeling gene family inDNA damage response and recombination. Genetics 173, 985-994.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Smolle, M., Venkatesh, S., Gogol, M.M., Li, H., Zhang, Y., Florens, L., Washburn, M.P., and Workman, J.L. (2012). Chromatin remodelers

Isw1 and Chd1 maintain chromatin structure during transcription by preventing histone exchange. Nat Struct Mol Biol 19, 884-892.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Subramanian, V., Fields, P.A., and Boyer, L.A. (2015). H2A.Z: a molecular rheostat for transcriptional control. F1000prime reports 7, 01.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Sura, W., Kabza, M., Karlowski, W.M., Bieluszewski, T., Kus-Slowinska, M., Paweloszek, L., Sadowski, J., and Ziolkowski, P.A. (2017).Dual Role of the Histone Variant H2A.Z in Transcriptional Regulation of Stress-Response Genes. The Plant cell 29, 791-807.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Torigoe, S.E., Urwin, D.L., Ishii, H., Smith, D.E., and Kadonaga, J.T. (2011). Identification of a rapidly formed nonnucleosomal histone-DNA intermediate that is converted into chromatin by ACF. Molecular cell 43, 638-648.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Wang, H., Liu, C., Cheng, J., Liu, J., Zhang, L., He, C., Shen, W.H., Jin, H., Xu, L., and Zhang, Y. (2016). Arabidopsis Flower and EmbryoDevelopmental Genes are Repressed in Seedlings by Different Combinations of Polycomb Group Proteins in Association with DistinctSets of Cis-regulatory Elements. PLoS genetics 12, e1005771.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Wang, M., Zhao, Y., and Zhang, B. (2015). Efficient Test and Visualization of Multi-Set Intersections. Sci Rep 5, 16923.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Weber, C.M., Ramachandran, S., and Henikoff, S. (2014). Nucleosomes are context-specific, H2A.Z-modulated barriers to RNApolymerase. Molecular cell 53, 819-830.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Wong, M.M., Cox, L.K., and Chrivia, J.C. (2007). The chromatin remodeling protein, SRCAP, is critical for deposition of the histonevariant H2A.Z at promoters. The Journal of biological chemistry 282, 26132-26139.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Xiao, J., Jin, R., and Wagner, D. (2017). Developmental transitions: integrating environmental cues with hormonal signaling in thechromatin landscape in plants. Genome biology 18, 88.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Xu, M., Hu, T., Smith, M.R., and Poethig, R.S. (2016). Epigenetic Regulation of Vegetative Phase Change in Arabidopsis. The Plant cell28, 28-41.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Xu, S., Grullon, S., Ge, K., and Peng, W. (2014). Spatial clustering for identification of ChIP-enriched regions (SICER) to map regions ofhistone methylation patterns in embryonic stem cells. Methods in molecular biology 1150, 97-111.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Zhang, D., Jing, Y., Jiang, Z., and Lin, R. (2014). The Chromatin-Remodeling Factor PICKLE Integrates Brassinosteroid and GibberellinSignaling during Skotomorphogenic Growth in Arabidopsis. Plant Cell 26, 2472-2485.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Zhang, H., Bishop, B., Ringenberg, W., Muir, W.M., and Ogas, J. (2012). The CHD3 remodeler PICKLE associates with genes enrichedfor trimethylation of histone H3 lysine 27. Plant physiology 159, 418-432.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Zhang, H., Rider, S.D., Jr., Henderson, J.T., Fountain, M., Chuang, K., Kandachar, V., Simons, A., Edenberg, H.J., Romero-Severson, J.,Muir, W.M., et al. (2008). The CHD3 remodeler PICKLE promotes trimethylation of histone H3 lysine 27. The Journal of biologicalchemistry 283, 22637-22648.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Zhang, X., Clarenz, O., Cokus, S., Bernatavichute, Y.V., Pellegrini, M., Goodrich, J., and Jacobsen, S.E. (2007). Whole-genome analysisof histone H3 lysine 27 trimethylation in Arabidopsis. PLoS biology 5, e129.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

DOI 10.1105/tpc.17.00867; originally published online May 25, 2018;Plant Cell

Deal and Joe OgasBenjamin Carter, Brett Bishop, Kwok Ki Ho, Ru Huang, Wei Jia, Heng Zhang, Pete E Pascuzzi, Roger

H3K27me3 Homeostasis in ArabidopsisThe Chromatin Remodelers PKL and PIE1 Act in an Epigenetic Pathway that Determines

 This information is current as of March 26, 2021

 

Supplemental Data /content/suppl/2018/06/14/tpc.17.00867.DC2.html /content/suppl/2018/05/25/tpc.17.00867.DC1.html

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