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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 ([email protected]). 20 21 22
ABSTRACT 23
24
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
5
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
7
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
8
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
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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).
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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
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