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LARGE-SCALE BIOLOGY ARTICLE
Genomic Distribution of Maize Facultative Heterochromatin
Marked by Trimethylation of H3K27
W
Irina Makarevitch,a,b Steven R. Eichten,b Roman Briskine,c Amanda J. Waters,b Olga N. Danilevskaya,d
Robert B. Meeley,d Chad L. Myers,c Matthew W. Vaughn,e and Nathan M. Springerb,1
a Biology
Department, Hamline University, Saint Paul, Minnesota 55104
and Plant Genomics Institute, Department of Plant Biology, University of Minnesota, Saint Paul, Minnesota 55108
c Department of Computer Science and Engineering, University of Minnesota, Minneapolis, Minnesota 55455
d DuPont Pioneer Ag Biotech Research, Johnston, Iowa 50131
e Texas Advanced Computing Center, University of Texas, Austin, Texas 78758
b Microbial
Trimethylation of histone H3 Lys-27 (H3K27me3) plays a critical role in regulating gene expression during plant and animal
development. We characterized the genome-wide distribution of H3K27me3 in five developmentally distinct tissues in maize
(Zea mays) plants of two genetic backgrounds, B73 and Mo17. There were more substantial differences in the genome-wide
profile of H3K27me3 between different tissues than between the two genotypes. The tissue-specific patterns of H3K27me3
were often associated with differences in gene expression among the tissues and most of the imprinted genes that are
expressed solely from the paternal allele in endosperm are targets of H3K27me3. A comparison of the H3K27me3 targets in
rice (Oryza sativa), maize, and Arabidopsis thaliana provided evidence for conservation of the H3K27me3 targets among plant
species. However, there was limited evidence for conserved targeting of H3K27me3 in the two maize subgenomes derived
from whole-genome duplication, suggesting the potential for subfunctionalization of chromatin regulation of paralogs.
Genomic profiling of H3K27me3 in loss-of-function mutant lines for Maize Enhancer of zeste-like2 (Mez2) and Mez3, two of
the three putative H3K27me3 methyltransferases present in the maize genome, suggested partial redundancy of this gene
family for maintaining H3K27me3 patterns. Only a portion of the targets of H3K27me3 required Mez2 and/or Mez3, and there
was limited evidence for functional consequences of H3K27me3 at these targets.
INTRODUCTION
Packaging of DNA into chromatin is essential for the regulation of
genome function in eukaryotes. Nucleosomes, the basic units of
chromatin, are composed of ;147 bp of DNA that is wrapped
around an octomer built by four types of histones. Covalent
modifications of histones, along with other factors, such as DNA
methylation, chromatin-remodeling proteins, and small RNAs,
contribute to the regulation of gene expression (Berger, 2007;
Kouzarides, 2007; Berr et al., 2011). Heterochromatin is often
used to refer to the parts of the genome that contain chromatin
modifications associated with repression of gene expression.
Constitutive heterochromatin describes regions of the genome
that are stably maintained as heterochromatin throughout development and is often associated with 5-methylcytosine and
H3K9me2 in plants (Bernatavichute et al., 2008). By contrast,
facultative heterochromatin describes regions of the genome that
exist as heterochromatin in some cell types and as euchromatin in
1 Address
correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Nathan M. Springer
([email protected]).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.112.106427
other cells. Facultative heterochromatin is often associated with
developmental regulation of gene expression and involves a set of
chromatin modifications, including trimethylation of histone H3
Lys-27 (H3K27me3) (Schuettengruber et al., 2007; Zheng and
Chen, 2011).
In plants, H3K27me3 modifications have been implicated in
regulation of seed development, flowering time, vernalization, and
organ identity (reviewed in Kohler and Villar, 2008; Schatlowski
et al., 2008; Feng et al., 2010; Zheng and Chen, 2011). In both
plants and animals, a remarkable number of transcription factors
are targeted by H3K27me3 (Boyer et al., 2006; Pasini et al., 2007;
Zhang et al., 2007; Kharchenko et al., 2011), further supporting the
role of H3K27me3 in regulation of gene activity and development.
H3K27me3 broadly marks repressed genes (Turck et al., 2007;
Zhang et al., 2007) and is distinct from other epigenetic marks
(Roudier et al., 2011). The expression levels of the H3K27me3modified genes are very low and often exhibit a high degree of
tissue specificity (with the majority of them expressed only in one or
a few tissues), which suggests that repression of these genes by
H3K27me3 is alleviated only in the place(s) where their expression
is needed (Kohler and Villar, 2008; Feng et al., 2010). Recently, the
levels of H3K27me3 were profiled in several plant tissues, including
meristem tissues (Lafos et al., 2011) and roots and shoots (Wang
et al., 2009). These studies revealed large tissue-specific variation
in H3K27me3 levels. Moreover, hundreds of genes were shown to
The Plant Cell Preview, www.aspb.org ã 2013 American Society of Plant Biologists. All rights reserved.
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The Plant Cell
gain or lose H3K27me3 during cell differentiation, demonstrating dynamic regulation of an epigenetic modification in plants. H3K27me3
was shown to correlate with gene repression, and its release
correlated with tissue-specific gene activation, both during differentiation and in mutants for Polycomb group (PcG) proteins
(Lafos et al., 2011).
H3K27me3 is catalyzed by PcG proteins and is thought to
regulate developmental processes in both plants and animals
(reviewed in Schuettengruber et al., 2007; Berr et al., 2011; Zheng
and Chen, 2011). The PcG genes were initially identified through
the characterization of mutants that failed to properly maintain
a repressed state of gene expression for homeotic genes in Drosophila melanogaster (Simon, 1995). A subset of the PcG proteins,
including E(z), esc, Su(z)12, and Nurf55 in Drosophila (Schwartz
and Pirrotta, 2007), form the PRC2 (for Polycomb-repressive
complex) that is involved in catalyzing the methylation of Lys-27 of
histone H3. Many plant species have orthologs for the PRC2
genes (reviewed in Hennig and Derkacheva, 2009; Kohler and
Hennig, 2010; Zheng and Chen, 2011). Homologs of the E(z)-like
gene have been shown to encode H3K27 methyltransferases in
animal species and likely encode the same function in plants
(Schubert et al., 2006). Arabidopsis thaliana contains three genes,
CURLY LEAF (CLF), SWINGER (SWN), and MEDEA (MEA) that are
similar to Drosophila E(Z) (Goodrich et al., 1997; Grossniklaus
et al., 1998; Chanvivattana et al., 2004). MEA appears to function
primarily in gametophyte and seed development, whereas CLF
and SWN are broadly expressed and partially redundant in vegetative
and reproductive development (Chanvivattana et al., 2004; Hennig
and Derkacheva, 2009). The clf swn double mutants in Arabidopsis
show severe developmental abnormalities (Chanvivattana et al.,
2004) and have no H3K27me3 as shown by immunoblot analysis
(Lafos et al., 2011), suggesting that the majority of H3K27me3 in
vegetative tissues of Arabidopsis is catalyzed by PRC2 proteins
and requires at least one copy of clf or swn. The unique contributions of CLF and SWN to H3K27me3 profiles have not been
elucidated at a genome-wide level in Arabidopsis. The maize (Zea
mays) genome encodes three E(z) homologs: Mez1, Mez2, and
Mez3 (Springer et al., 2002). Mez1 is an imprinted gene that is
most closely related to the CLF gene from Arabidopsis (Haun
et al., 2007). Mez2 and Mez3 are highly similar to each other (92%
nucleotide identity), are located in colinear regions of the maize
genome, and are likely paralogs resulting from the ancient allopolyploid event in maize (Springer et al., 2002). These two genes
are more closely related to the SWN/MEA genes in Arabidopsis
(Haun et al., 2007).
Most studies of H3K27me3 in plants have focused on rice (He
et al., 2010) and Arabidopsis (for example, Turck et al., 2007;
Zhang et al., 2007; Ha et al., 2011; Lafos et al., 2011), plants with
relatively small genomes. The relatively large maize genome with
interspersed genes and repetitive sequences (Schnable et al.,
2009) provides an opportunity to further understand the profile of
H3K27me3 in plant genomes. One study assessed the genomewide distribution of H3K27me3 in maize roots and shoots using
chromatin immunoprecipitation (ChIP) sequencing (Wang et al.,
2009). They found a general enrichment for H3K27me3 in genes
relative to transposons and documented differences in the distribution of H3K27me3 relative to DNA methylation. However, detailed characterization of the maize genes targeted by H3K27me3
has not been previously reported. We profiled the H3K27me3
distribution in five developmentally distinct tissues in two genetic
backgrounds, B73 and Mo17, and in mez2/3 mutants. We found
that H3K27me3 targets vary greatly between different tissues and
show limited variation between genetic backgrounds.
RESULTS
Profiling H3K27me3 Levels throughout the Maize Genome
We profiled the distribution of H3K27me3 in several tissues and
genotypes of maize through Chromatin immunoprecipitation
(ChIP) with H3K27me3 antibodies followed by hybridization to
a microarray (ChIP-chip). Three independent biological replicates
of ChIP-chip were performed for five B73 tissues: deetiolated
maize seedlings 12 d after sowing, 3- to 4-cm immature ears, 15to 20-cm tassels, embryos 14 d after pollination, and endosperm
14 d after pollination (see Methods for details on plant growth and
tissue collection). H3K27me3 distribution was also profiled in
Mo17 for the same tissues, except for tassels. The microarray
platform used for this experiment contains ;1.4 million probes
that are restricted to single-copy sequences in the B73 reference
genome (Eichten et al., 2011). Seven sequences that exhibited
significant enrichment for H3K27me3 in at least one tissue and
four sequences that did not exhibit evidence for H3K27me3
were selected for validation by quantitative PCR (qPCR) (see
Supplemental Data Set 1 and Supplemental Figure 1 online). The
presence and tissue-specific patterns of H3K27me3 found in
ChIP-chip experiments were confirmed by the qPCR assays.
There was also significant overlap in the profile of H3K27me3
in our seedling tissue and that reported by Wang et al. (2009)
(see Supplemental Figure 2 online).
H3K27me3 was found more frequently in the gene-dense
chromosome arms and was less prevalent in the pericentromeric
regions (Figure 1A). This is distinct from the abundance of the
marks associated with constitutive heterochromatin, such as 5methylcytosine and H3K9me2 (Eichten et al., 2011, 2012), which
are enriched in the gene-poor pericentromeric regions (Figure 1A).
This genomic distribution of H3K27me3 and the other marks is
quite similar to observations in Arabidopsis (Zhang et al., 2007).
Whereas H3K27me3 was more prevalent in gene-rich portions of
the maize genome, it was not necessarily enriched over transcribed sequences (Figure 1B; see Supplemental Table 1 online).
H3K27me3 was most abundant in sequences that are immediately
59 or 39 of genic sequences and was enriched in exons relative to
introns (see Supplemental Table 1 online).
Whereas the analysis of individual probes can provide information on the distribution of H3K27me3, it is often more informative to identify regions of enrichment for a particular
modification. DNAcopy (Venkatraman and Olshen, 2007) followed
by expectation maximization analysis (Dempster et al., 1977) was
used to identify segments within the genome that include multiple
adjacent probes with enrichment for H3K27me3 (Table 1; see full
details in Supplemental Data Sets 2A to 2E online). This analysis
identified over 1000 regions of H3K27me3 in each of the profiled
tissues. In total, in B73, the H3K27me3-enriched fraction accounted for 0.13% (ear) to 1.91% (seedling) of the genome. In
Genomic Profiling of H3K27me3 in Maize
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Figure 1. The Genomic Distribution of H3K27me3 and DNA Methylation in Different Tissues of B73 and Mo17.
ChIP-chip analysis was used to profile the genomic distribution of chromatin modifications in maize.
(A) Enrichment for H3K27me3 (red), H3K9me2 (blue) (Eichten et al., 2012), and DNA methylation (blue) (Eichten et al., 2011) was visualized along each of
the 10 maize chromosomes. The relative gene density is shown in black.
(B) A more detailed view of the H3K27me3 profile is shown for 400 kb of chromosome 9.
general, the tissues with higher portions of undifferentiated meristematic cells had fewer regions with H3K27me3, while more
differentiated tissues had more prevalent H3K27me3. The median
segment length for H3K27me3-enriched regions was 1 to 2 kb in
each of the tissues, which implies modification of multiple adjacent
nucleosomes. A more detailed analysis of the higher resolution
(due to higher density of probes) data for chromosome 9 did not
reveal a large number of small regions, which would be expected if the modification were limited to a single nucleosome. A
substantial portion of the H3K27me3 segments (24%) did not
contain any genes and contained only single-copy intergenic
sequences, and 84% of the remaining segments were restricted
to a single gene (see Supplemental Figure 3 online). We did not
find any examples in which two adjacent genes marked by
H3K27me3 exhibited identical patterns of H3K27me3 in all tissues profiled.
Developmental Variation for H3K27me3 Marks
We proceeded to identify the genes that were marked with
H3K27me3 in at least one tissue. In total 12,266 (11%) genes in
the working gene set (WGS; n = 110,028; maize genome annotation 5b.60) were marked by H3K27me3 in at least one of the
tissues. These included 6337 genes (16%) of the filtered gene
set (FGS; n = 39,585; Figure 2; see Supplemental Data Set 3
online), which represents a significant (P < 0.01) enrichment for
H3K27me3 in the FGS relative to the WGS. The WGS included
all putative genes in maize, while the FGS was filtered to remove
gene fragments, pseudogenes, and transposon-related sequences (Schnable et al., 2009). The genes that were modified
with H3K27me3 exhibited substantial variation among the five
profiled tissues (Figure 2). A large portion of the H3K27me3marked genes were marked by H3K27me3 only in one (40.6%)
or two (21%) of the five tissues. Only 8.2% of the H3K27me3
target genes contained the chromatin modification in all five
tissues. Clustering of H3K27me3 enrichment levels for all segments in these five tissues revealed that the triploid endosperm
was most different from the other tissues, exhibiting many
uniquely marked genes (Figure 2A). When we looked at the
variation among the diploid vegetative tissues, we found that
30.4% of the genes with H3K27me3 were marked by H3K27me3
only in one of the four tissues (Figure 2B) and 17.9% were
marked in all four tissues. The level of tissue-specific gene expression for the genes marked by H3K27me3 was assessed
using data from a maize gene expression atlas (Sekhon et al.,
2011). High levels of Shannon entropy (Schug et al., 2005;
Zhang et al., 2007) indicate genes that have constitutive expression whereas low levels of entropy reflect tissue-specific
expression. The genes that were marked with H3K27me3 in one
or two of the tissues were enriched for those with lower levels of
entropy compared with all maize genes, reflecting some enrichment for genes with tissue-specific expression. The genes
that were marked by H3K27me3 in four or five tissues were even
more biased toward higher levels of tissue-specific expression
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The Plant Cell
Table 1. Characteristics of H3K27me3 Marked Segments in Maize Epigenome
Tissue
No. of Segmentsa
Total Length (Mb)
Average Length
Median Length
Proportion of Genome
Seedlings, all
Seedlings, Mez2/3 dependent
Seedlings, Mez2/3 independent
Immature Ear
Tassel
Endosperm
Embryo
4374
742
3530
1183
3960
3392
4307
37.15
1.97
34.96
2.49
18.39
19.37
20.95
6890
2661
9903
2107
4645
5710
4863
1800
600
3600
1200
2000
1860
1400
1.91%
0.10%
1.79%
0.13%
0.92%
0.96%
1.07%
a
The number of segments with a segment mean [log2(H3K27me3 IP/input)] > 1.
(Figure 2C). There were some examples of differences in H3K27me3
levels between B73 and Mo17 (Figure 2D). However, a comparison
of H3K27me3 levels in multiple tissues of B73 and Mo17 suggests
that there are more differences between tissues than between
genotypes (Figure 2D). Our analyses use probes designed from
the reference B73 genome sequence and have omitted probes
that show high levels of CGH variation, which may result in an
underestimation of the level of H3K27me3 variation among
genotypes.
exhibiting tissue-specific expression that was associated with
H3K27me3 were observed to have similar patterns of chromatin
modification and transcript abundance in both B73 and Mo17
(Figure 3B). There were genes with tissue-specific expression
that did not contain H3K27me3 in any of the tissues that were
studied, and there were examples of tissue-specific H3K27me3
patterns that were not well correlated with tissue-specific expression levels. It is likely that H3K27me3 provides a component
of tissue-specific gene regulation but is certainly not the only
factor regulating tissue-specific expression.
Relationship between Gene Expression and H3K27me3
H3K27me3 can play a role in contributing to tissue-specific regulation of gene expression. To understand the effects of H3K27me3
on maize gene expression, we performed RNA sequencing for
tissues that were similar (seedling, embryo, and endosperm were
from different biological samples but were similarly harvested)
or identical (immature ear and tassel) to the tissues used for
H3K27me3 profiling. The genes that were marked with H3K27me3
were significantly (P < 0.01) enriched for genes with low or no expression (<1 gene-specific reads per kilobase gene length per
million reads [RPKM]) in all tissues (Table 2; see Supplemental
Table 2 online). Analysis of the genes with at least 1 RPKM revealed
that the genes marked by H3K27me3 were enriched for those with
lower expression levels (Figure 3A; see Supplemental Figure 4
online). However, there were also some genes marked with
H3K27me3 that were expressed at moderate to high levels. Each of
these tissues represents a complex mixture of cell types that might
include some cell types with the H3K27me3 modification and
others that lack the modification. This complexity of cell types may
interfere with the ability to correlate H3K27me3 and gene expression in these tissues. These analyses suggest that in many cases
the presence of H3K27me3 is associated with reduced levels of
gene expression but that there are some cases in which the
presence of H3K27me3 is not associated with low expression.
The effects of H3K27me3 on developmental gene regulation
were investigated by comparing the presence of the modification
and expression in each of the five tissues. One-quarter (25.1%) of
the 6337 genes exhibited a negative correlation (r # 0.5) between
expression level and H3K27me3 presence among these five tissues. In many of these examples, the loss of H3K27me3 in one or
several tissues was associated with activation of expression (Figure
3B). This suggests that H3K27me3 is associated with transcription
in maize and that the developmental variation in H3K27me3 levels
can be associated with tissue-specific expression. These genes
Characterization of H3K27me3 Targets
The genes that are targeted by H3K27me3 were further characterized to understand potential functions and conservation among
species. Putative Gene Ontology (GO) annotations were assigned
for maize genes based on orthology to Arabidopsis. Enrichments
for functional GO annotations were assessed in a variety of gene
lists using BinGO (Maere et al., 2005). The biological process and
molecular function GO annotations were assessed for all 6337
H3K27me3 targets as well as several subsets that exhibited different tissue-specific patterns (see Supplemental Figures 5 and 6
online). There was significant (P = 6*10292) enrichment for genes
annotated as having transcription factor activity in most of the
lists of H3K27me3 targets (see Supplemental Figure 5 online). The
H3K27me3 targets were also significantly enriched for biological
process annotations of “developmental process” and “transcription, DNA-dependent” (see Supplemental Figures 5 and 6 online).
The level of enrichment for specific GO terms varies for genes that
were marked by H3K27me3 in one to two tissues relative to those
marked in four to five tissues. Genes that were constitutively
marked by H3K27me3 (targets in four or five tissues) showed very
strong overrepresentation of transcription factors and genes involved in developmental processes, while the genes that were
targets for H3K27me3 in only one or two tissues did not show
such overrepresentation (see Supplemental Figure 5 online).
There is evidence that some families of maize transcription factors
are enriched for H3K27me3 targets relative to other families (see
Supplemental Figure 6B online). Certain families of transcription
factors, including YABBY, MADS, G2-like, Homeobox, NAC, and
C2C2-DOF, were significantly enriched (P < 0.01) for genes
marked by H3K27me3. The genes marked by H3K27me3 were
also significantly enriched for classical maize genes (Schnable
and Freeling, 2011) that were defined by forward genetics (see
Supplemental Figure 7 online).
Genomic Profiling of H3K27me3 in Maize
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Figure 2. Developmental Variation in H3K27me3 Marks in Five Maize Tissues in B73 and Mo17.
(A) Hierarchical clustering (Ward’s method) of 6337 filtered genes with H3K27me3 marks in at least one of five tissues of B73.
(B) Distribution of 5690 filtered genes marked with H3K27me3 in at least one of four diploid tissues of B73.
(C) Tissue specificity of gene expression in B73 for H3K27me3 target genes marked in one to two tissues (black dotted line), four to five tissues (orange
dashed line), and non-H3K27me3 target genes (blue line), measured by Shannon entropy values (low entropy values = high tissue specificity).
(D) Hierarchical clustering of segments marked by H3K27me3 in at least one of the tissues of B73 or Mo17. The labels indicate the tissue and genotype
(blue, B73; red, Mo17). The color intensity reflects the level of H3K27me3 enrichment.
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The Plant Cell
Table 2. Syntenic Analysis of Genes Targeted by H3K27me3 in B73 Tissues
Features
Genome
Subgenome 1
Subgenome 2
Proportion
Subgenome 1
Proportion
Subgenome 2
All genes
All methylated genes
Proportion of methylated genes
Pairs of local duplicates
Pairs of methylated local duplicates
Only one gene of a local duplicate
pair methylated
Proportion of local duplicates with
methylation marks
Proportion of the pairs with preserved
methylation
Pairs of syntenic genes
Pairs of methylated syntenic genes
Only one gene of a syntenic pair methylated
Proportion of syntenic duplicates with
methylation marks
Proportion of the syntenic pairs with preserved
methylation
110,029
12,266
11.1%
9,669
808
2,071
61,541
7,649
12.4%
5,431
508
1,398
38,923
4,078
10.5%
3,358
268
593
55.9%
62.4%
NA
56.2%
62.9%
67.5%
35.4%
33.2%
NA
34.7%
33.2%
28.6%
29.8%
35.1%
25.6%
NA
NA
28.1%
26.7%
31.1%
NA
NA
3,795
297
773
28.2%
3,795
297
405
18.5%
3,795
297
368
17.5%
NA
NA
52.4%
NA
NA
NA
47.6%
NA
27.8%
42.3%
44.7%
NA
NA
NA, not applicable.
The ancient tetraploid nature of maize results in two subgenomes
that are biased in retention and expression level (Schnable et al.,
2011). The proportion of genes with H3K27me3 was slightly higher
than expected for subgenome 1 (the more retained and expressed
subgenome), but this was not statistically significant. There are
many examples of paralog retention following whole-genome duplication, and we assessed whether these paralogs that are located
in syntenic positions have conserved patterns of H3K27me3.
Among 3795 genes with retained syntenic gene duplications
(Schnable et al., 2012), we identified 1070 (28.2%) pairs of genes
with at least one gene marked by H3K27me3, which is very similar
to the expected proportion. Out of these 1070 pairs, 297 pairs
(27.8%) had both copies marked with H3K27me3, while in the remaining 773 pairs, only one of the genes was marked with
H3K27me3 (Table 2). The tissue-specific pattern of H3K27me3 was
often different in instances in which both paralogs were marked
with H3K27me3. Only ;10% of the 297 pairs of paralogs in which
both were marked by H3K27me3 exhibited the same tissue-specific
pattern of modification. This suggests that paralogs often have
diverged in their regulation by H3K27me3.
The targets of H3K27me3 in maize were compared with the
targets of H3K27me3 in Arabidopsis (Lafos et al., 2011) and rice
(Oryza sativa) (He et al., 2010). Many (34%) of the maize targets
of H3K27me3 that had orthologs in Arabidopsis were marked by
H3K27me3 in both species. The conservation of H3K27me3
targets between maize and rice was even higher (64% for maize
genes marked with H3K27me3 in seedlings). This conservation
was substantially higher for genes marked by H3K27me3 in
multiple maize tissues. For example, rice orthologs for 74% of
maize genes marked by H3K27me3 in all five profiled tissues
were also marked by H3K27me3 in rice seedlings, while only 28
to 38% of the maize genes marked by H3K27me3 in only one of
the tissues had rice orthologs marked by H3K27me3. Given the
fact that different tissues were sampled in the different species,
we would expect some variation in the targets. However, there
was evidence for conservation of the targets of H3K27me3
among plant species.
The Majority of Paternally Expressed Imprinted Genes Are
Marked by H3K27me3
There is evidence that H3K27me3 plays a role in the regulation
of imprinted gene expression in endosperm tissue of Arabidopsis (Wolff et al., 2011) and maize (Haun and Springer, 2008).
A recent RNA sequencing analysis of maize endosperm tissue
identified 100 imprinted maize genes, including 46 paternally
expressed genes (PEGs) and 54 maternally expressed genes
(Waters et al., 2011). Nearly all of the PEGs (41/46; 89%) were
marked by H3K27me3 in endosperm tissue, whereas only four
of 54 maternally expressed genes were marked with H3K27me3.
The allele specificity of H3K27me3 was tested for several of the
PEGs. ChIP of B73xMo17 hybrid endosperm revealed that the
H3K27me3 found in endosperm is restricted to the silenced
maternal allele (see Supplemental Figure 8 online). The comparison of expression patterns and H3K27me3 patterns among
tissues revealed two subgroups of the H3K27me3-marked
PEGs (see Supplemental Figure 8 online). One group (21/41) of
genes was expressed in many plant tissues and contained
H3K27me3 only in endosperm tissue. The genes from the other
group (13/41) were expressed only in endosperm tissue and
marked by H3K27me3 in all tissues studied.
Mutations in Mez2 and Mez3 Reduce H3K27me3 in Some
Regions but Not Others
Evidence from Arabidopsis suggests that the majority of
H3K27me3 in plants is provided by E(z) homologs (Lafos et al.,
2011). The three maize genes with homology to E(z), namely,
Genomic Profiling of H3K27me3 in Maize
Figure 3. H3K27me3 Negatively Correlates with Gene Expression Levels.
(A) Expression level of H3K27me3 target genes (blue and purple) compared with non-H3K27me3 target genes (red and black) in tassel and
immature ear tissue. Only FGS genes with expression of at least 1 RPKM
were used in this comparison. The numbers in the legend indicate the
number of expressed genes with (target) or without (nontarget) H3K27me3
in the indicated tissue.
(B) H3K27me3 (red indicates positive values, while blue indicates negative values) and RNA sequencing profiles (height of black bars reflects
number of reads) are shown for two examples of genes that exhibit
a negative correlation between H3K27me3 and expression levels. Black
bars at the bottom indicate 1-kb scale.
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Mez1, Mez2, and Mez3, were expressed in the five tissues that
were analyzed (Figure 4A). Several mu-insertion alleles were
obtained via reverse-genetic screening, including mez3-m1,
mez3-m4, and mez2-m4 (Figure 4B). Although Mu insertions
were identified in sequences upstream of Mez1 (Haun et al.,
2009), we did not find any Mez1 insertions that resulted in lossof-function alleles. Mez2 and Mez3 mutant alleles were backcrossed into B73 for at least seven generations. The effect of
these insertions upon the transcript was assessed through
quantitative RT-PCR (qRT-PCR) and RNA sequencing experiments. qRT-PCR experiments amplifying the regions located 39
of the insertion detected severely reduced transcript levels in
homozygous mutant individuals (Figure 4C) and RNA sequencing data from the mutant lines revealed dramatically reduced
expression through the whole transcript (for mez2-m4 and
mez3-m1 alleles) and frequent mis-splicing of the affected exons
(for mez2-m4 and mez3-m4 alleles). There were no striking
phenotypic differences in homozygous mutant plants for each of
the three alleles or a mez2-m4 mez3-m4 double mutant relative
to wild-type siblings. These mutant lines tended to be somewhat
smaller and may have minor quantitative phenotypes but were
fully fertile and had a fairly normal morphology.
Despite the lack of a striking morphological phenotype, we were
interested in monitoring the molecular phenotypes in these mutant
lines. H3K27me3 profiling was performed using ChIP-chip on
a pool of homozygous mutant plants for each of the three alleles
and for homozygous mez2-m4 mez3-m4 double mutants along
with a B73 control to assess the role of these genes in the genome-wide distribution of H3K27me3 in maize (see Supplemental
Data Sets 2A and 3 online). RNA sequencing was performed on
these same tissues to assess transcript abundance in these mutant genotypes. A simple profile of H3K27me3 abundance over
genes revealed that the levels of H3K27me3 in the promoter regions were reduced in each of the single mutants and were most
strongly affected in the double mutant (see Supplemental Figure
9A online). A set of 4374 segments enriched for H3K27me3 was
detected in the B73 control seedlings that were grown and sampled at the same time as the mutant stocks (see Supplemental
Table 1 online). The level of H3K27me3 for these segments in each
of the genotypes was used to perform hierarchical clustering
(Figure 4D). There was clear evidence that a subset of regions with
H3K27me3 was affected in the mez2 and/or mez3 mutants but
that many regions with H3K27me3 were not affected by these
mutations. The regions that were affected in the mez2/3 mutants
were referred to as Mez2/3 dependent, and the segments not affected in mez2/3 mutants were termed Mez2/3 independent. The
742 Mez2/3-dependent segments (17% of the total number of
H3K27me3-marked segments) include 21 that were only mez2
dependent, 315 that were only Mez3 dependent, 90 that were
affected only in the double mutant, and 316 that were dependent
on either of the mutations (Figure 4E, Table 1; see Supplemental
Data Set 2A online). The effects of the two mez3 mutant alleles
were quite similar. At least one genomic region showing each of
these patterns of mez2/3 dependence or independence was selected and used for qPCR validation (see Supplemental Figures 9B
and 9C and Supplemental Data Set 1 online). The patterns observed in ChIP-chip were confirmed by qPCR. There was no evidence for genomic clustering of the Mez2/3-dependent regions.
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Figure 4. Distribution of H3K27me3 in Mez2 and Mez3 Mutants.
(A) Expression levels of three mez genes in five tissues profiled.
(B) Mutant alleles of mez3 and mez2 genes used in the study. Mu insertions in mez genes are shown by black triangles. Exons are shown in blue
rectangles, while introns are shown as blue lines. One of the introns in each of the two genes is very long and is not shown completely.
(C) Expression of mez2 and mez3 in mutants used in the study (the level of expression was analyzed by qRT-PCR and normalized to expression levels
of mez2 and mez3 genes in B73; GAPC was used as a control gene for normalizations). Error bars show SD between the technical replicates.
(D) Hierarchical clustering of mez2/3-dependent and mez2/3-independent segments. Each row represents a segment marked by H3K27me3 in B73
seedlings and each column represents a different genotype. Red or black indicate tissues in which a particular segment exhibited high or low levels of
H3K27me3, respectively.
(E) Examples of mez2/3-dependent and mez2/3-independent genes targeted by H3K27me3 in B73 seedlings. Black bars indicate 1-kb scale.
While there was a set of genomic regions that required Mez2/3
for modification by H3K27me3, we did not find evidence that
this modification was functionally important at these regions.
There were 659 FGS genes located in Mez2/3-dependent regions, and these were enriched for nonsyntenic genes or for
instances of H3K27me3 that was not conserved among species.
These genes did not show GO enrichments or tissue-specific
expression patterns similar to those observed for the mez2/3-
independent regions (see Supplemental Figures 10 and 11A
online). RNA sequencing was performed to assess expression
changes in the Mez2/3 mutants relative to wild-type seedlings.
Although there were a number of genes (738) with twofold differences in expression levels in mez2-m4 mez3-m4 double
mutants relative to the wild type, these genes were not enriched
for targets of Mez2/3-dependent H3K27me3. Instead, the majority of the genes with differential expression was not targets of
Genomic Profiling of H3K27me3 in Maize
H3K27me3 or had Mez2/3-independent H3K27me3 (see
Supplemental Figure 11B online). Analysis of the expression
levels of all genes with mez-dependent H3K27me3 did not
reveal evidence for altered expression levels in the mutants
relative to the wild type (see Supplemental Figure 11C online),
suggesting that the Mez2/3-dependent H3K27me3 does not
play a significant role in transcriptional regulation in seedling
tissue.
DISCUSSION
In Arabidopsis, H3K27me3 has been shown to play an important
role in the regulation of gene expression by providing facultative
heterochromatin that can reinforce silencing of gene expression in
some tissues (Kohler and Villar, 2008; Schatlowski et al., 2008;
Feng et al., 2010; Zheng and Chen, 2011). Relatively little is known
about the role of H3K27me3 in other plant species. An analysis of
several chromatin modifications in rice revealed that H3K27me3 is
present in many genes and is often associated with lower gene
expression (He et al., 2010). Analysis of H3K27me3 in maize roots
and shoots demonstrated that low expressed genes are enriched
for H3K27me3 and that very few genomic regions are enriched for
both DNA methylation and H3K27me3 (Wang et al., 2009). Here,
we profiled the genomic distribution of H3K27me3 in several tissues of two maize genotypes and compared the variation in
H3K27me3 distribution among tissues and genotypes to understand the role that H3K27me3 might play in regulation of tissue-specific gene expression and in gene expression variation due
to different genetic backgrounds. This study also developed
a catalog of H3K27me3 targets that can be exploited to identify
the role of H3K27me3 in developmental regulation of gene expression. Finally, the profiling of H3K27me3 and gene expression
in mutants for several maize H3K27 methyltransferases revealed
relatively minor contributions of these genes to developmental
regulation of gene expression.
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found, while variation in DNA methylation levels was much more
common between the two subspecies (He et al., 2010). Our results
suggest that H3K27me3 likely plays an important role in regulating
gene expression during development and that these H3K27me3
patterns are mostly conserved among maize inbreds (see
Supplemental Table 3 online). This is not to suggest that there is
no variation in the patterns of H3K27me3 between genotypes as
we do see examples of differences. However, the majority of
genes that exhibit differential expression between B73 and Mo17
do not show differences in H3K27me3 abundance.
We also characterized the conservation of H3K27me3 between
maize and other species and between the subgenomes derived
from the allotetraploid ancestry of maize (Gaut and Doebley, 1997).
It is somewhat difficult to compare the H3K27me3 patterns among
different species, as collecting similar tissues from species with
substantial morphological differences is complicated. However,
there was clear evidence for conservation of H3K27me3 targets
among species. In particular, when we looked at genes that were
marked with H3K27me3 in multiple tissues, we found that a large
proportion of maize H3K27me3 targets were marked by
H3K27me3 in both rice (He et al., 2010) and Arabidopsis (Lafos
et al., 2011). Given the conservation of H3K27me3 targets between species, we expected to find significant similarities in
H3K27me3 targets between the two subgenomes of maize. The
paralogs that were retained following the whole-genome duplication might be expected to exhibit similar patterns of H3K27me3.
However, the majority (72.2%) of pairs of syntenic genes had
H3K27me3 at only one of the two paralogs (Table 2). Even when
H3K27me3 was found at both paralogs, it frequently exhibited
differences in tissue-specific patterns. This divergence in tissuespecific H3K27me3 patterns for retained paralogs may reflect
subfunctionalization in gene expression patterns that has arisen
through differences in chromatin-based regulation of these paralogs. A recent analysis suggests that H3K27me3 may play a role in
functional divergence of Arabidopsis paralogs through contributing
to expression differences between paralogs (Berke et al., 2012).
Conservation of H3K27me3 Profiles among Tissues,
Genotypes, and Species
Targets of H3K27me3
Epigenetic regulation can contribute to developmental regulation
of gene expression or to (epi-)allelic variation among different
individuals. We profiled several distinct maize tissues from two
genotypes, B73 and Mo17, in order to assess the level of variation
for H3K27me3 distribution. There were many examples of tissuespecific differences in the H3K27me3 patterns. This is similar to
recent finding in Arabidopsis (Lafos et al., 2011). H3K27me3 is
often associated with repression of regulatory gene expression.
We found relatively few examples of genotypic differences in
H3K27me3. It is possible that the repetitive regions of the genome
or regions with structural variation between B73 and Mo17 exhibit
H3K27me3 variation among genotypes, but these regions were
not included in our analyses. Few studies have compared the
H3K27me3 profiles for multiple different genotypes of the same
species. Some differences were found between Arabidopsis ecotypes, and those primarily exhibit additive inheritance in F1 offspring (Moghaddam et al., 2011). When variation in H3K27me3
was assessed in two different rice subspecies, a small number of
examples of genes with differences in H3K27me3 levels were
In Arabidopsis, H3K27me3 is known to regulate genes that play
important roles in development, including AGAMOUS (AG)
(Schubert et al., 2006), SHOOT MERISTEMLESS (STM) (Schubert
et al., 2006), KNOX genes (Xu and Shen, 2008), and FLOWERING
LOCUS C (Bastow et al., 2004). A recent genome-wide profiling of
H3K27me3 in several tissues of Arabidopsis provides evidence
that H3K27me3 is present at genes coding for many transcription
factor families and at many of the genes involved in auxin biosynthesis, transport, and perception (Lafos et al., 2011). We found
that many developmentally important maize genes are targets of
H3K27me3 as well. The classical maize gene list (Schnable and
Freeling, 2011) includes genes that were identified by visible mutant phenotypes. Many (138 of 468) of these genes were targeted
by H3K27me3. This list includes a number of genes involved in
specific pathways (such as anthocyanin or starch synthesis) as
well as genes that contribute to development (such as kn1 and
tb1). H3K27me3 marks were present at many of the genes known
to be important for proper development. Maize transcription factors were also enriched for H3K27me3 targets. A family-specific
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analysis showed that certain families of maize transcription factors,
including YABBY, MADS, G2-like, homeobox, and NAC domain
genes, were particularly likely to be marked by H3K27me3. These
include the maize orthologs of AG, STM, and KNOX genes, which
are all targets of H3K27me3 in Arabidopsis. This study thus demonstrates that many genes with important regulatory roles are
targets of H3K27me3 and provides a catalog of maize genes
that are marked by H3K27me3.
Role of H3K27me3 in Imprinting
There is evidence that H3K27me3 is critical for regulating imprinted gene expression in the endosperm of plants (Berger and
Chaudhury, 2009; Raissig et al., 2011). MEDEA, the first wellcharacterized imprinted gene in Arabidopsis (Grossniklaus et al.,
1998), encodes a putative H3K27memethyltransferase that is
required for endosperm development and is part of a specific
FIS-PRC2 complex that functions in endosperm development and
includes FIE and FIS2 (Kohler et al., 2003). This FIS-PRC2 complex
is necessary for proper imprinting at MEA (Gehring et al., 2006;
Jullien et al., 2006), at Pheres1 (Makarevich et al., 2006), and at
several other imprinted Arabidopsis genes (Wolff et al., 2011).
We found a striking enrichment for H3K27me3 at maize PEGs.
Nearly all of the PEGs (41/46) identified in maize (Waters et al.,
2011) had enrichment for H3K27me3 in endosperm tissue and, in
the three instances tested, we found that this H3K27me3 was
restricted to the silent maternal allele. A combined analysis of the
patterns of expression and H3K27me3 through development revealed two different types of pattern. Some of these PEGs were
expressed in numerous vegetative tissues. H3K27me3 was observed only in endosperm tissue for these constitutively expressed
PEGs. These might require specific recruitment of a FIS-PRC2
complex to mark the maternal allele with H3K27me3 during gametogenesis. The other group of PEGs had endosperm-specific
expression. These PEGs were marked by H3K27me3 throughout
development and might specifically lose H3K27me3 at the paternal allele. The remodeling of H3K27me3 during gametogenesis
could include specific recruitment of H3K27 methyltransferase
activity to some loci and recruitment of H3K27 demethylase activities to other loci. The H3K27me3 histone methyltransferase
activity is likely supplied by MEA or related genes (Kohler et al.,
2005), while the H3K27 demethylase activity could be provided by
REF6 or a related gene (Lu et al., 2011). It is worth noting that while
endosperm does have unique H3K27me3 patterns, there were
also many genomic regions that were marked similarly in endosperm and vegetative tissues. This suggests that the remodeling
of H3K27me3 is limited to specific genomic regions.
Mez2 and Mez3 Play Minimal Independent Roles in Gene
Regulation through H3K27me3
Most plant species have multiple genes encoding each of the
components of the PRC2 complex (reviewed in Hennig and
Derkacheva, 2009; Kohler and Hennig, 2010). The Arabidopsis
genome contains three orthologs to the Drosophila E(z) gene,
MEA, CLF, and SWN (Chanvivattana et al., 2004; Hennig and
Derkacheva, 2009; Zheng and Chen, 2011). MEA appears to
mainly function in gametes and endosperm development and
does not play a major role in vegetative development (Zheng and
Chen, 2011). The mutant phenotype of CLF (Goodrich et al.,
1997) provides evidence for a unique role of CLF in vegetative
tissues. No morphological abnormalities were observed in SWN
single mutants (Chanvivattana et al., 2004). However, swn clf
double mutants display more severe developmental abnormalities (Chanvivattana et al., 2004) and further reduction in genomic
levels of H3K27me3 (Lafos et al., 2011), providing evidence for
substantial redundant function of CLF and SWN. There is also
biochemical evidence that either CLF or SWN can participate in
PRC2 complexes found in vegetative tissues (Chanvivattana
et al., 2004; Makarevich et al., 2006).
Maize contains three E(z) orthologs (Springer et al., 2002). The
potential for an endosperm-specific function analogous to MEA is
less clear in maize. All three of the Mez genes were expressed in all
of the tissues that we studied, including endosperm. The imprinted
expression of only Mez1 (Haun et al., 2007) may suggest that Mez1
is critical for endosperm functions, but both Mez2 and Mez3 were
also detected in this tissue. Although no line with a Mutator
transposon inserted into an exon was identified for Mez1 (Haun and
Springer, 2008), we were able to identify exon insertion alleles for
both of the maize SWN orthologs, Mez2 and Mez3. Profiling the
distribution of H3K27me3 in the single and double mutants revealed a subset of H3K27me3-enriched loci that were dependent
upon these genes and provided evidence for specific contributions
of each of these genes as well as of both of the genes together
in genomic distribution of H3K27me3 marks. However, the
H3K27me3 that was Mez2/3 dependent often occurred in small
regions that were not conserved among species and did not appear to be correlated with gene expression levels or patterns. RNA
sequencing analysis of the mutant lines did not reveal evidence for
widespread gene expression differences. These results, together
with the lack of developmental abnormalities in the mez mutants,
suggest that the H3K27me3 that is controlled by Mez2/3 is largely
dispensable for proper developmental and gene regulation. It is
possible that these mez2/3-dependent H3K27me3 marks are important for precise regulation of gene expression and that loss of
these marks might relate to variation in a population of cells. The
conserved presence of SWN-like orthologs in many plants suggests that this gene must play an important role, but there is no
clear evidence for a specific unique function provided by SWN-like
genes in maize or Arabidopsis.
METHODS
Plant Growth and Tissue Collection
The whole aboveground tissue was collected from 12-d-old deetiolated
seedlings that were grown in the dark at 30°C in 1:1 mix of autoclaved field
soil and MetroMix. For embryo, immature ear (;5 to 7 cm in length), tassel
(green soft tassels prior to shedding, ;15 to 20 cm in length), and endosperm collection, plants were grown to maturity in the field of the
University of Minnesota Saint Paul Agricultural Research station during
the summer of 2011. Endosperm and embryos were harvested from
multiple B73 and Mo17 ears 14 d after the self-pollination. The seeds for
each replicate came from a unique, single source (ear). Each biological
replicate was planted at a different time and represents the pooled
aboveground tissue from eight to 10 plants. Three replicate tissue collections were performed for all B73 and Mo17 tissues. For each replicate/
Genomic Profiling of H3K27me3 in Maize
tissue/genotype combination, 1 g of plant material was harvested on ice,
rinsed with water, and cross-linked with 1% formaldehyde for 10 min
under vacuum infiltration. Cross-linking was quenched by adding Gly
solution to a final concentration of 0.125 M under vacuum infiltration for 5
min. Treated tissue was frozen in liquid nitrogen and stored at 280°C until
chromatin extraction.
Isolation and Characterization of mez Mutations
The Pioneer Hi-Bred trait utility system population (Bensen et al., 1995) was
screened to identify Mutator transposon insertions in Mez1, Mez2, and Mez3.
Several putative loss-of-function mu insertion alleles were obtained for mez2
(-m4) and mez3 (-m1 and -m4). No loss-of-function alleles were identified for
Mez1 (Haun et al., 2009). The mez2-m4, mez3-m1, and mez3-m4 alleles were
backcrossed into B73 for at least seven generations. The double mutant
strain was created by crossing the backcross derived stocks to each other
and selecting homozygous double mutant progeny.
Immunoprecipitation of H3K27me3-Linked DNA, Labeling,
and Hybridization
Chromatin extractions were performed using the EpiQuik Plant ChIP kit
(Epigentek) according to the manufacturer’s recommendations. Extracted
chromatin was sheared in 600 mL EpiQuick buffer CP3F with five 10-s pulses
on a sonicator. To test and optimize sonication conditions, cross-linking was
reversed in a sample of sheared chromatin, and the resulting products were
analyzed on agarose gels. Sonication conditions were optimized to yield
predominantly 200- to 500-bp DNA samples. ChIP, reverse cross-linking,
and DNA cleanup were performed using the EpiQuik Plant ChIP kit according
to the manufacturer’s recommendations. Antibodies specific for H3K27me3
(#07-449) were purchased from Millipore. For each genotype, antibody, and
replicate, 50 to 100 ng of input and immunoprecipitated DNA was amplified
with a whole-genome amplification kit (WGA2; Sigma-Aldrich). The amplification of the no-antibody control (negative control) was always fivefold to
10-fold less efficient, confirming the specificity of immunoprecipitation. For
each amplified immunoprecipitated and input sample, 3 mg amplified DNA
was labeled using the Dual-Color Labeling Kit (Roche NimbleGen) according
to the array manufacturer’s protocol (Roche NimbleGen Methylation User
Guide v7.0). Each immunoprecipitated sample was labeled with Cy5, and
each input/control sonicated DNA was labeled with Cy3. Samples were
hybridized to the custom 1.4 or 2.1 M probe array (Gene Expression Omnibus [GEO] platforms GPL15621 and GPL13499) for 16 to 20 h at 42°C.
Slides were washed and scanned according to NimbleGen’s protocol for the
Gene-Pix4000B scanner. Images were aligned and quantified using NimbleScan software (Roche NimbleGen) producing raw data reports for each
probe on the array.
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model accounting for array, dye, and sample effects to the data using the
Limma package (Wettenhall and Smyth, 2004). Moderated t-statistics and
the log-odds score for differential MeDIP enrichment was computed by
empirical Bayes shrinkage of the standard errors with the false discovery rate
controlled to 0.05. For the analysis of H3K27me3 levels in Mo17, the probes
with substantial variation in CGH signal between B73 and Mo17 were
omitted. In addition, a linear model approach was used to control for minor
variation in hybridization due to genotype as determined from the CGH data.
Results were visualized using Integrated Genomics Viewer (IGV) (Robinson
et al., 2011). Microarray results were deposited with the National Center for
Biotechnology Information (NCBI) GEO under accession number GSE39456,
and data tracks formatted for IGV are available at http://genomics.tacc.
utexas.edu/data/h3k27_epigenetic_variation/.
Analysis of H3K27me3 Distribution
Normalized H3K27me3 data, represented as log2(H3K27me3 IP/input), for
all B73 tissue samples were segmented into discrete regions using the
DNAcopy algorithm (Venkatraman and Olshen, 2007). The resulting
segments were filtered to identify H3K27me3-enriched or -depleted
segments using the expectation maximization algorithm (Dempster et al.,
1977) assuming two subdistributions. Filtered segments required a 95%
probability of falling into the higher subdistribution and also required an
H3K27me3 segment average [log2(H3K27me3 IP/input)] of >0.8 to be
identified as H3K27me3 enriched. The relative levels of H3K27me3 in
Mo17 or mez mutant tissues was determined calculating average log2
(H3K27me3 IP/input) values in Mo17 or mez mutant samples for the filtered segments defined in their respective tissue in B73. Expectation
maximization was applied to the relative levels of H3K27me3 in mez
mutant samples compared with the wild type to identify significant differences in H3K27me3 levels in mez mutants compared with the wild type.
Tissue Specificity of Gene Expression
We estimated tissue specificity by calculating Shannon entropy (Schug
et al., 2005; Zhang et al., 2007) for genes in a maize gene expression atlas
(Sekhon et al., 2011). The data were averaged across biological and
technical replicates. Genes that had 0 expression level in all tissues were
excluded from the analysis. For the remaining 31,764 genes, entropy was
calculated as HðpÞ ¼ 2 ∑ni¼1 pi log pi , where pi is a relative abundance of
the gene’s transcript in tissue i. We also tried two additional data filters.
First, we ignored genes if the sum of their expression levels across all
tissues was below a certain threshold (g ∈ [2, 4, 6, 8, 10]). Second, we set
expression level in a tissue to 0 whenever it was below a certain cutoff (t ∈
[1, 2, 4, 6, 8]). However, those thresholds didn’t produce any significant
impact on the value distribution (data not shown.)
Array Design and Annotation
RNA Sequencing
Two array platforms were used in this experiment. All seedling tissues were
assayed using a NimbleGen 2.1M feature oligonucleotide array (GEO
platform GPL13499) as described (Eichten et al., 2011). All other tissues were
assayed using a NimbleGen 3 3 1.4M feature oligonucleotide array (GEO
platform GPL15621) containing a subset of 1.4 million probes from the larger
array design that are single copy in the B73 genome. Only the subset of
probes that were present on both microarray platforms were used in the
analyses. Adjacent probes were spaced every 56 bp for chromosome 9 and
every 200 bp for the other maize (Zea mays) chromosomes.
RNA sequencing analysis of all tissues described above was performed
as described (Eichten et al., 2012). Three biological replicates of five B73
and Mo17 tissues and one biological replicate of B73, mez3-m1, mez3m4, mez2-m4, and mez3-m4 mez2-m4 seedlings were prepared, with
eight plants pooled for each of the replicates. All RNA samples were
prepared by the University of Minnesota BioMedical Genomics Center in
accordance with the TruSeq library creation protocol (Illumina). Samples
were sequenced on the HiSequation 2000 developing six to 17 million
reads per replicate. Raw reads were filtered to eliminate poor quality reads
using CASAVA (Illumina). Transcript abundance was calculated by mapping
reads to the maize reference genome (AGPv2) using TopHat (Trapnell et al.,
2009). A high degree of correlation between replicates was observed (r >
0.98). RPKM values were developed using “BAM to Counts” across the
exon space of the maize genome reference WGS (ZmB73_5a) within the
iPlant Discovery Environment (www.iplantcollaborative.org).
Normalization and Linear Modeling
XYS files exported from NimbleScan were imported into the Bioconductor
statistical environment (http://bioconductor.org/). Sample-dependent H3K27me3
enrichments were estimated for each probe by fitting a fixed linear
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cDNA Synthesis and ChIP-qPCR Validations of H3K27me3 Levels
cDNA synthesis and qRT-PCR analysis for mez mutants were performed as described (Makarevitch et al., 2012) with the following
primers: mez3forward (59-CCCTATCCCGTTTGCCAGGTCTGACC-39)
and mez3reverse (59-CCAAGGGGGCCGTCGCAGAAC-39); mez2forward
(59-AAACTTTCCATTCCGGAGATTCGTG-39) and mez2reverse (59-GTTTGCCACTTCTATGCAGGTCTTAAG-39). H3K27me3 levels were validated
for 11 genes in two biological replicates of four B73 tissues and for five
genes in seedlings of B73 and in mez2/3 mutants (see Supplemental Data
Set 1 online for details). For all samples, three technical replicates were
performed. The cycle threshold results were averaged for technical
replicates, and percentage of input DNA was calculated for H3K27me3immunoprecipitated samples as well as for immunoglobulin control samples according to Haring et al. (2007).
Supplemental Data Set 2B. Segments Targeted by H3K27me3 in
B73 Tassel.
Supplemental Data Set 2C. Segments Targeted by H3K27me3 in
B73 Ear.
Supplemental Data Set 2D. Segments Targeted by H3K27me3 in B73
Embryo.
Supplemental Data Set 2E. Segments Targeted by H3K27me3 in B73
Endosperm.
Supplemental Data Set 3. Genes Targeted by H3K27me3 in B73 in at
Least One of Five Tissues Profiled.
ACKNOWLEDGMENTS
Accession Numbers
H3K27me3 profile data were deposited with NCBI GEO under accession
number GSE39456 and data tracks formatted for IGV are available at
http://genomics.tacc.utexas.edu/data/h3k27_epigenetic_variation/. RNA
sequencing data are available at NCBI under numbers SRP013432 and
SRP009313.
Supplemental Data
We thank Peter Hermanson for assistance in growing and crossing
plants and performing ChIP hybridizations and Jennifer Rundquist for
performing qPCR validations. The Texas Advanced Computing Center at
the University of Texas at Austin provided high performance computing
and storage resources. The Minnesota Supercomputing Institute provided access to software and user support for data analyses. The
research was supported by a grant from the National Science Foundation
(IOS-0922095) to M.V.W. and N.M.S. and by a grant from the National
Science Foundation (0957312) to I.M.
The following materials are available in the online version of this article.
Supplemental Figure 1. Validation of Altered H3K27me3 Patterns
Using qPCR.
Supplemental Figure 2. Comparison of ChIP-chip H3K27me3 Profiles
with ChIP-seq Profiles from Wang et al. (2009).
Supplemental Figure 3. Distribution of Segments Relative to the Number
of FGS Genes per Segment.
Supplemental Figure 4. H3K27me3 Negatively Correlates with Gene
Expression Levels.
Supplemental Figure 5. GO Annotation of Genes Targeted by
H3K27me3.
Supplemental Figure 6. H3K27me3 Modifications Are Enriched at
Some Families of Maize Transcription Factors.
Supplemental Figure 7. Correlation between H3K27me3 Levels and
Expression Levels for Several Classical Genes.
Supplemental Figure 8. Allele-Specific H3K27me3 Modifications in
Maize Endosperm for Imprinted Genes with Parental Expression.
Supplemental Figure 9. Altered Levels of H3K27me3 in mez Mutants.
Supplemental Figure 10. GO Annotation of Gene Targets for
H3K27me3.
Supplemental Figure 11. Mez2/3-Dependent H3K27me3 Plays a Minimal Role in Regulation of Gene Expression.
Supplemental Table 1. Localization of H3K27me3 Marks in the
Genome of Maize B73 Seedlings.
Supplemental Table 2. Relationships between Gene Expression and
H3K27me3 in B73 Tissues.
Supplemental Table 3. Variation in H3K27me3 between B73 and Mo17
Seedlings.
Supplemental Data Set 1. Results of ChIP-qPCR Validations of
H3K27me3 Levels.
Supplemental Data Set 2A. Segments Targeted by H3K27me3 in B73
Seedlings.
AUTHOR CONTRIBUTIONS
I.M., M.W.V., and N.M.S. designed the research. I.M., S.R.E., R.B., and
A.J.W. performed research. O.N.D., R.B.M., and C.L.M. contributed new
analytic/computational/etc. tools. I.M., S.R.E., R.B., C.L.M., and M.W.V.
analyzed data. I.M. and N.M.S. wrote the article.
Received October 17, 2012; revised December 18, 2012; accepted
February 19, 2013; published March 5, 2013.
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Genomic Distribution of Maize Facultative Heterochromatin Marked by Trimethylation of H3K27
Irina Makarevitch, Steven R. Eichten, Roman Briskine, Amanda J. Waters, Olga N. Danilevskaya,
Robert B. Meeley, Chad L. Myers, Matthew W. Vaughn and Nathan M. Springer
Plant Cell; originally published online March 5, 2013;
DOI 10.1105/tpc.112.106427
This information is current as of June 18, 2017
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