Epigenetic Repression of Male Gametophyte-Specific Genes in the Arabidopsis Sporophyte

Molecular Plant  •  Volume 6  •  Number 4  •  Pages 1176–1186  •  July 2013

RESEARCH ARTICLE

Epigenetic Repression of Male GametophyteSpecific Genes in the Arabidopsis Sporophyte Robert D. Hoffmann1 and Michael G. Palmgren Centre for Membrane Pumps in Cells and Disease—PUMPKIN, Danish National Research Foundation, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark

ABSTRACT  Tissue formation, the identity of cells, and the functions they fulfill, are results of gene regulation. The male gametophyte of plants, pollen, is outstanding in this respect as several hundred genes expressed in pollen are not expressed in the sporophyte. How pollen-specific genes are down-regulated in the sporophyte has yet to be established. In this study, we have performed a bioinformatics analysis of publicly available genome-wide epigenetics data of several sporophytic tissues. By combining this analysis with DNase I footprinting data, we assessed means by which the repression of pollen-specific genes in the Arabidopsis sporophyte is conferred. Our findings show that, in seedlings, the majority of pollen-specific genes are associated with histone-3 marked by mono- or trimethylation of Lys-27 (H3K27me1/ H3K27me3), both of which are repressive markers for gene expression in the sporophyte. Analysis of DNase footprint profiles of pollen-specific genes in the sporophyte displayed closed chromatin proximal to the start codon. We describe a model of two-staged gene regulation in which a lack of nucleosome-free regions in promoters and histone modifications in open reading frames repress pollen-specific genes in the sporophyte. Key words: male gametophyte; epigenetics; gene repression; DNase I footprinting; transcription factors; bioinformatics.

Introduction Flowering plants undergo a life cycle in which the diploid sporophyte constitutes the predominant generation, and the haploid male and female gametophytes are reduced to only a few but highly specialized cells. Pollen, the male gametophyte, comprises in Arabidopsis thaliana three cells of which one drives vegetative growth of the pollen tube and is responsible for delivering the two sperm cells to the megaspore, the female gametophyte, where in an event called double fertilization the diploid embryo of the sporophytic generation is created (McCormick, 1993). In the last decade, genome arrays designed for Arabidopsis have been extensively used to create expression profiles of entire organs such as leaves or flowers, but also for single cell types, including pollen of different developmental stages and after penetrating the pistil in semi in vivo assays (Becker et al., 2003; Honys and Twell, 2004; Schmid et al., 2005; AlvesFerreira et al., 2007; Qin et al., 2009). From these experiments, we know quite well which genes are expressed in the individual stages of pollen development and, by comparing the expression of these genes with data for different sporophyte cell types, it has been shown that, in the Arabidopsis genome, several hundred genes are only expressed in pollen, but not or extremely restricted in the sporophyte (Honys and Twell, 2004). In pollen itself, the separation of sperm cells from

the vegetative nucleus has shed light on genes differentially expressed in these two cells (Borges et al., 2008, 2012) and, combined with next-generation sequencing techniques, have opened the door for identifying networks between individual pollen cells (Slotkin et al., 2009; Calarco et al., 2012). Moreover, high-throughput sequencing of gene transcripts from pollen and the sporophyte now allows for precise quantification of gene expression levels (Loraine et al., 2013). Recently, we have come a long way in understanding how gene expression is facilitated and gathered knowledge about the functions of proteins involved in this process. Transcription factors have been identified that direct expression of pollen-specific genes. These belong to the MADS family of transcription factors and form the subclass  MIKC*, which, by electrophoretic mobility shift assays, have been shown to recognize the motif CTA(A/T)4TAG and, in a less strict form,

1 To whom correspondence should be addressed. R.D.H. E-mail hoffmann@ life.ku.dk, tel. 0045-353-32577. M.G.P. E-mail [email protected], tel. 0045-353-32577.

© The Author 2013. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi:10.1093/mp/sst100, Advance Access publication 14 June 2013 Received 10 May 2013; accepted 5 June 2013

Hoffmann & Palmgren  •  Repression of Pollen-Specific Genes in the Sporophyte   

derivatives of this motif (Verelst et al., 2007; Adamczyk and Fernandez, 2009). Other promoter elements that direct or mediate enhanced gene expression in pollen have been identified by mutagenesis studies in, for example, tomato (POLLEN1LELAT52, AGAAA) and tobacco (GTGANTG10, GTGA) (Bate and Twell, 1998; Rogers et al., 2001). To form cell type identity, not only have certain genes to be actively expressed, but also have others to be repressed. Contrasting our knowledge about gene expression, we know little about how genes are repressed in certain cell types, so that a particular gene becomes cell type-specific. Few studies describe DNA motifs recognized by proteins repressing transcription in plants (Zhao et al., 2007; Leivar et al., 2008; Gao et  al., 2009). Early mutation studies of pollen-specific promoters did not identify DNA sequences mediating gene repression (Eyal et  al., 1995). Today, the only experimental evidence for a DNA motif conferring strict gene repression in every cell except the sperm cell in pollen is the 10-bp region GGCTGAATTT in the promoter of LGC1 in lily, which is recognized by the silencing transcription factor GERMLINE RESTRICTIVE SILENCING FACTOR (GRSF) in non-male germline cells (Haerizadeh et  al., 2006). RNA-directed DNA methylation (RdDM) is involved in repression of transposable elements in plants and mammals (Law and Jacobsen, 2010), but this repression is released in the vegetative nucleus of pollen. A recent study showed RdDM-targeted genes to be activated in pollen, suggesting a mechanism of repression of these genes in the sporophyte (Schmitz et al., 2013). In an international effort, the human ENCODE project has identified 45 million transcription factor occupancy events, all of which were identified by DNase I  footprinting (Neph et al., 2012; Thurman et al., 2012). With this method, genomic DNA is initially cleaved by DNase I. Resulting short nucleotide sequences are subsequently sequenced and aligned to the reference genome. When regulatory factors bind to genomic DNA, the underlying sequence is protected from cleavage by DNase I, leaving footprints at single nucleotide resolution level. This technique has also been employed to generate a genome-wide map of DNase I  activity in Arabidopsis leaves and flowers (Zhang et al., 2012b) and rice (Zhang et al., 2012a). DNase I-hypersensitive sites were identified upstream of expressed genes and, as in humans, an overlay of the data with experimentally confirmed transcription factor binding sites revealed distinct DNase footprints at these sites (Zhang et al., 2012b). We do not yet fully understand the different layers of gene regulation so that particular genes are transcribed or repressed, which eventually is the base for cell type identity. Besides proteins functioning as transcriptional regulators, epigenetic modifications of DNA and associated proteins have been identified as triggers for gene activity (Zhang et al., 2006; Jacob et al., 2009; Guo et al., 2010). In recent years, studies of epigenetic modifications in Arabidopsis combined with highthroughput sequencing showed that distinct histone modifications are associated with centromeric heterochromatin and

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euchromatin on chromosome arms (Zhang et al., 2006, 2007; Zilberman et al., 2007; Bernatavichute et al., 2008; Zilberman et al., 2008; Charron et al., 2009; Zhang et al., 2009; van Dijk et al., 2010; Zhou et al., 2010; Bouyer et al., 2011). Genes and transposable elements are selectively targeted and, within the group of genes, expressed and repressed genes carry different epigenetic marks. For example, trimethylation of Lys-4 on histone-3 (H3K4me3) has been shown to be associated with transcribed genes (Zhang et al., 2009), trimethylation of Lys-27 on histone-3 (H3K27me3) with repressed genes (Zhang et al., 2007; Roudier et al., 2011; Luo et al., 2013), and methylation of cytosine together with dimethylation of Lys-9 on histone-3 (H3K9me2) was found to silence transposable elements (Bernatavichute et  al., 2008; Cokus et  al., 2008). Disputes persist over the targets of histone-3 carrying a single methyl group on Lys-27 (H3K27me1). It was suggested that H3K27me1 is only associated with constitutive heterochromatin (Jacob et al., 2009; Luo et al., 2013). However, Roudier et al. (2011) also found genes with low expression signals or cell-specific expression to be associated with H3K27me1. Publicly available databases related to the model plant Arabidopsis store massive amounts of whole genome data from epigenetic experiments, sequence data for annotated genes, and DNase I  footprint data. In this work, we have data-mined such databases to obtain information that can shed light on how repression of pollen-specific genes in the sporophyte takes place. The results allow us to describe a model of a two-staged gene regulation in which histone positioning in promoters and epigenetic marks in gene bodies act separately to repress expression of pollen-specific genes in the sporophyte.

RESULTS Data Sets of Male Gametophyte- and Sporophyte-Specific Genes To analyze gene repression of male gametophyte-specific genes in the sporophyte, we first defined a set of genes being expressed in the male gametophyte but not in sporophytic tissues. Collection of this data set was based on available microarray data of 79 distinct Arabidopsis cell types or organs (Schmid et al., 2005). We eliminated entries of pseudogenes and transposable elements to have solely protein-coding genes, as defined by TAIR10, in our data set. We identified 541 genes that showed expression in mature pollen grains (log2 ≥ 7.0) and minimal expression (log2 < 3.5) in 8-day-old whole seedlings and green parts of 21-day-old seedlings (Supplemental Table  1). These genes were classified as male gametophyte-specific. As control groups of sporophyte-expressed genes, we selected the 541 genes with strongest expression signals in 8-day-old whole seedlings or green parts of 21-day-old seedlings (in the following named seedling-specific and leaf-specific, respectively), as these matched best the plant material used in studies we subsequently data-mined.

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Our approach of identifying pollen-specific genes was based on microarray experiments (Schmid et al., 2005). Microarrays are advantageous because they can be performed on single cell types; but, as hybridization properties of DNA microarray probes can vary from gene to gene, quantification of targets based on hybridization intensities alone may be inaccurate. In contrast, high-throughput cDNA sequencing provides direct counts of molecules (Mortazavi et  al., 2008; Loraine et  al., 2013) and whole transcriptome sequencing (RNA-Seq) data for Arabidopsis pollen and green parts of seedlings are available (Loraine et al., 2013). In the data of Loraine et al. (2013), we identified 788 pollen-specific genes (≥ 5 reads per million (RPM)), which were only marginally expressed in the sporophyte (≤ 1 RPM), and compared these with pollen-specific genes based on microarray data (Schmid et al., 2005). A total of 370 protein-coding genes were present in both data sets (Supplemental Figure 1). Among the 418 pollen-specific genes identified by RNA-Seq only, 204 were not represented on the ATH1 microarray. Among the 171 pollen-specific genes identified in the microarray data only, 131 had, in the RNA-Seq pollen data, absolute expression below the cut-off. In conclusion, RNA-Seq data confirmed to a high degree the data set of pollen-specific genes derived from microarray analysis.

Methylation of Lys-27 in Histone-3 Is a Dominant Repressive Epigenetic Mark for Pollen Genes in the Sporophyte To compare epigenetic modifications associated with male gametophyte-specific genes, we analyzed data provided by Roudier et  al. (2011). In the study, seven epigenetic modifications were mapped for the entire Arabidopsis genome with a 165-bp resolution using seedling tissue. Genes highly expressed in seedlings were typically marked by H3K4me2, H3K4me3, H3K36me3, and H2Bub (99%, 97%, 89%, and 72%, respectively; Figure  1). In seedlings, 67% and 35% of the pollen-specific genes were marked by H3K27me3 and H3K27me1, respectively. H3K27me1 and H3K27me3 were co-occurring at 16% of pollen-specific genes. Together with genes solely marked by one of the two histone modifications, 87% of all pollen genes were marked by H3K27me1 and/or H3K27me3 (Figure 1). To test the robustness of these results with respect to the selection of genes based on microarray data, we repeated our analysis with pollen-specific genes identified by highthroughput sequencing of Arabidopsis transcripts in pollen and aerial parts of seedlings (Loraine et  al., 2013). Among 788 pollen-specific genes in this data set, 64% and 32%

Figure 1.  Gametophyte- and Sporophyte-Specific Genes Have Distinct Histone Modifications in Seedlings. DNA methylation (5 mC) and six histone modifications were measured in seedlings (Roudier et al., 2011) and analyzed for their association with genes expressed in seedlings (gray) and pollen (black). (A) Pollen-specific genes were significantly more often associated with H3K27mono- and trimethylation, seedling-specific genes significantly more often with H2Bub, H3K36me3, H3K4me2, and H3K4me3; numbers to the right of bars indicate percentage of genes with an epigenetic modification in seedling tissue (Fisher’s exact test, * p < 0.0001, n = 541 for each seedling- and pollen-expressed gene). (B) Distribution of H3K27me1 and H3K27me3 in pollen- and seedling-specific genes. 87% of pollen-specific genes were marked by H3K27me1 and/ or H3K27me3, compared to 16% of seedling-specific genes (totals deviating from 100% due to rounding differences).

Hoffmann & Palmgren  •  Repression of Pollen-Specific Genes in the Sporophyte   

were associated with H3K37me3 and H3K27me1, respectively (Supplemental Figure 1). With the exception of H2Bub, the epigenetic modifications associated with pollen-specific genes in both data sets (Schmid et  al., 2005; Loraine et  al., 2013) were similar and showed no significant differences (Fisher’s exact test, p < 0.05, n = 541 and n = 788). Thus, analysis of both data sets points to methylation of Lys-27 in histone-3 as a major repressive epigenetic mark for pollen genes in the sporophyte For Arabidopsis Chromosome 4, data of 11 histone modifications and DNA methylation are available, which were sampled in seedlings with a 900-bp resolution (Roudier et  al., 2011). These data include the histone modifications H4K20me1, H2Bub, H3K56Ac, H3K9me2, and H3K27me2, as well as the modifications assessed for the whole genome (see above). Of the 541 pollen- and seedling-specific genes in our data set, those localized on Chromosome 4 were included in the analysis (Supplemental Figure 2). Male gametophyte-specific genes were marked by significantly higher levels of histone-3 Lys-27 mono, di-, and trimethylation: 31%, 64%, and 52%, respectively (Supplemental Figure 2). As H3K27me2 and H3K27me3 are reported to co-occur (Roudier et al., 2011), we tested whether this applies to pollen-specific genes. Indeed, 43% of the pollen-specific genes on Chromosome 4 carried both H3K27me2 and H3K27me3 (data not shown). A recent study analyzed nine histone modifications in aerial parts of Arabidopsis seedlings, allowing assessment of histone modifications H3K9Ac, H3K18Ac, and H3K36me2 for their prevalence in gametophyte-specific genes (Luo et  al., 2013). We found these marks to occur only rarely at pollenspecific genes, which is in accordance with their proposed correlation with transcribed genes (Supplemental Figure  3). H3K27me3 was also in these data identified as a predominant mark of pollen-specific genes. However, in contrast to data from Roudier et al. (2011), H3K27me1 was only found at 1% of pollen-specific genes. We next wanted to test whether pollen-specific genes are consistently associated with H3K27me3. Several genomewide studies of the histone modification H3K27me3 have been performed using different plant material (Turck et al., 2007; Zhang et al., 2007; Charron et al., 2009; Bouyer et al., 2011; Lafos et al., 2011; Roudier et al., 2011; Luo et al., 2013). We analyzed epigenetic data for H3K27me3 (Lafos et  al., 2011) which were sampled in leaves of 9-week-old A. thaliana plants and compared it with the data for H3K27me3 based on seedling tissue (Roudier et al., 2011). In total, 68% of gametophyte-specific genes were marked by H3K27me3, of which 80% carried the modification in seedlings and mature leaves, 19% only in seedlings, and 2% in leaf tissue only (Figure 2), indicating that a large fraction of pollen-specific genes carries the modification H3K27me3 in both developmental stages of the sporophyte. Taken together, the epigenetic landscape of pollen-specific genes pointed towards H3K27me3 as the dominant repressive epigenetic mark and H3K27me1 as the minor.

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Figure 2.  Pollen-Specific Genes Are Marked by H3K27me3 in Seedlings and Leaves. Compared are pollen-specific genes for their association with H3K27me3 in seedling (Roudier et  al., 2011) and leaf (Lafos et  al., 2011) tissue. In total, 68% of the pollen-specific genes were marked by H3K27me3 in either seedlings or leaves. The bar chart shows the distribution of these genes into categories whether they are marked in both seedlings and leaves (80%), in seedlings only (19%), and in leaves only (2%) (totals deviating from 100% due to rounding differences; n = 541; n.d. = no H3K27me3 detected).

Highly Expressed Pollen-Specific Genes Are Not Targeted by RNA-Directed DNA Methylation As RdDM has been identified as a repressive mechanism of genes in the sporophyte (Schmitz et al., 2013), we analyzed whether pollen-specific genes identified in our analysis were among 283 Arabidopsis genes known to be targeted by RdDM (Schmitz et al., 2013). Of the 541 pollen-specific genes identified by us, only six (AT4G26730, AT4G08670, AT4G08400, AT3G26860, AT2G16040, AT2G10970) were reported as targeted by RdDM. Analyzing absolute expression levels for RdDM-targeted loci in pollen (Loraine et  al., 2013) showed that 94% of these had no or very few (≤ 1 RPM) transcripts in pollen (Figure  3). In contrast, transcripts in pollen were found (≥ 1 RPM) for 90% of pollen-specific genes identified in microarray data, of which 84% had more than 5 RPM and 55% more than 50 RPM. In seedlings, differences in epigenetic modifications associated with RdDM-targeted loci and pollen-specific genes were strong; 90% of RdDM-targeted loci had methylated DNA and 17% were decorated with H3K27me3, whereas, of pollen-specific genes analyzed in this study, 19% and 67% were associated with DNA methylation and histone modification H3K27me3, respectively (Roudier et al., 2011) (Figure 3). We conclude that, in the sporophyte, pollen-specific genes are not a preferential target of RdDM, but rather are associated with H3K27me3 decoration.

Promoters of Pollen-Specific Genes Are Not Accessible in the Sporophyte After having identified epigenetic modifications to be associated with pollen-specific genes (Figure  1), we were interested in knowing how accessible the promoter regions of leaf-expressed

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Figure 3.  RdDM-Targeted Loci Do Not Overlap with Highly Expressed Pollen-Specific Genes. Pollen-specific genes identified in this study are compared with RdDM-targeted loci (comprising transposable elements and genes) that show activation in pollen (Schmitz et al., 2013). (A) DNA methylation (5 mC) (black) and histone modification H3K27me3 (gray) were measured in seedlings (Roudier et al., 2011). (B) Absolute expression (reads per million, RPM) in pollen. x-axis indicates categories for groups of RPMs, y-axis the number of genes or loci on each category (Loraine et al., 2013).

and pollen-specific genes are. For this, we analyzed the genomic region 900 bp upstream of the start codon by mining DNase I footprint data (Zhang et al., 2012b) (Figure 4). The accessibility for DNase I in the promoter regions of pollen-specific genes was low, exhibiting lowest levels in proximity to the start codon. The control group of leaf-expressed genes, however, was found to have open chromatin with a peak about 170 bp upstream of the start codon. We grouped pollen-specific genes into bins of the 200 genes with lowest and the 200 with highest expression signals in leaves, and did not find changes in promoter accessibility. Also, no altered DNase I accessibility was observed in relation to whether the pollen-specific genes were associated with H3K27me1 or H3K27me3 (Figure 4).

Transcription Factor Binding Sites of Pollen-Specific Genes Are Occupied by Proteins in the Sporophyte Besides epigenetic modifications, repression of gametophytespecific genes in the sporophyte might be induced by binding of proteins that promote heterochromatin formation (Kotake et  al., 2003). We reasoned that, if pollen-specific genes are to some extent negatively regulated by proteins in the sporophyte, these proteins would be likely to recognize DNA motifs in promoter regions of pollen-expressed genes and, accordingly, these motifs would likely be overrepresented in pollen-specific genes. We first tested the DNase I sensitivity at CArG-box motifs, namely CN(A/T)6NG (N = every nucleotide) which are recognized by members of the MADS transcription

factor family in the sporophyte as well as pollen (Huang et al., 1995; Alvarez-Buylla et  al., 2000; Verelst et  al., 2007). For this motif-footprint analysis, we included all motifs located within 500 bp upstream of the 5′ UTR (TAIR 10) of the genes in question and averaged DNase I  cuts for every nucleotide 50 bp upstream and downstream of the motif. DNase I data were data-mined (Zhang et al., 2012b). In the sporophyte, promoter regions of both pollen-specific and sporophyte-expressed genes having the CN(A/T)6NG motif showed a decreased accessibility for DNase I (Figure 5), which indicates that DNA binding proteins were protecting the genomic DNA at this motif from DNase I cleavage. We additionally tested the motifs AGAAA (POLLEN1LELAT52) and GTGA (GTGANTG10), identified to enhance expression in tomato and tobacco pollen, respectively (Bate and Twell, 1998; Rogers et al., 2001). The AGAAA motif showed a decreased accessibility for DNase I similar to the CN(A/T)6NG motif, and low cleavage values extended three bases upstream of the motif. Surprisingly, for the motif GTGA, we identified a positive DNase I cleavage peak, suggesting that this motif is particularly less bound by DNA binding proteins (Figure 5). For all motifs, positive or negative peaks were more distinct for sporophyte-expressed genes. To identify more DNA motifs overrepresented in promoters of pollen-specific genes, we used SCOPE (Carlson et al., 2007) and performed de novo motif identification in the region 400 bp upstream of the translation start site. Of the identified 31 motifs, most were derivatives of the well-characterized

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Figure 4.  DNase I Cleavage in Promoter Regions in the Sporophyte. DNase I cleavage data measured in leaves for the region 900 bp upstream of the start codon (position +1) of pollen-expressed genes were datamined (Zhang et al., 2012b). (A, B) Genes identified as marked by an epigenetic modification (gray curves) were compared for their DNase I accessibility with genes not being marked (black curves). (A) H3K27me3, (B) H3K27me1. (C) The 541 pollen-specific genes were grouped in bins of the 200 genes with lowest (gray curve) and highest (black curve) expression signals in leaves. (D) Pollen- (black curve) and leaf- (gray curve) specific genes were analyzed (n = 541 for each leaf- and pollen-expressed genes). x-axes represent genomic sequence 900 bp upstream of the start codon of analyzed genes, y-axes DNase I cut per nucleotide (mean).

Figure 5.  DNA Motifs in Promoters Are Bound by Proteins or Hypersensitive to DNase I. DNA motifs located in the region 500 bp upstream of the transcription start site were identified for pollen-specific genes (black curves) and genes expressed in leaves (gray curves). DNase I cleavage data of the 100 bp surrounding each motif were data-mined (Zhang et al., 2012b) and averaged for all motifs found in the respective group of genes. Gray boxes indicate position of the motifs and the motifs’ sequences are stated above. DNase I footprints indicating binding of proteins to the motif were found for CN(A/T)6NG and AGAAA. Hypersensitive to DNase I were GTGA and GCCCA. Profiles for leaf-expressed genes and pollen-specific genes were similar, but the average DNase I cleavage values for leaf-expressed genes were higher in all plots (n of each motif stated in the plots; note that scales of y-axes change). x-axes represent genomic sequence surrounding the motifs, y-axes DNase I cut per nucleotide (mean).

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CArG-box motifs (Riechmann and Meyerowitz, 1997) which we assessed in the form of CN(A/T)6NG. However, one motif, GCCCA, which is reported to be associated with stress response (Welchen and Gonzalez, 2006), was outstanding because of its GC-rich sequence. We analyzed DNase I footprints for this motif and, to our surprise, found three positive DNase I cleavage peaks (Figure 5): one in the center of the motif, one 20 bp downstream, and the third 20 bp upstream. The overall accessibility for DNase I  was high, for the pollen-specific genes between 1.0 and 1.5, peaking at about 2.2. Also, when we assessed the DNase I footprint in the 900-bp region upstream of the start codon, we found up to 100% more DNase I cuts per nucleotide as for the average of all 541 pollen-specific genes. Genes expressed in leaves exhibited the same pattern of open chromatin with three peaks in 20-bp intervals for the GCCCA motif (Figure 5). We looked at the positions of the motif in promoter regions of the pollen and leaf group of genes and found it frequently occurring twice or three times at a short distance (10–25 bp) from each other in pollen genes, and repeats of two, three, or four motifs in leaf-expressed genes. Taken together, promoter regions carrying the well-characterized CArG-boxes, which include the motif CTA(A/T)4TAG overrepresented in pollen gene promoters (Verelst et al., 2007), and AGAAA appeared to be bound by proteins in the sporophyte. Other DNA motifs, such as GTGA (Rogers et al., 2001) and GCCCA (Welchen and Gonzalez, 2006), did not show such a correlation.

Discussion Definition of Pollen-Specific Genes In this study, two data sets for pollen-specific genes were created, based on microarray (Schmid et al., 2005) and RNA sequencing data (Loraine et al., 2013), respectively. We found that 63% of the genes defined as pollen-specific in microarray data were also identified as pollen-specific in RNA-Seq data. A higher number of pollen-specific genes was identified in RNA-Seq data. Two reasons account for that difference: of all 788 pollen-specific genes identified in RNA-Seq data, only 584 (74%) are present on the ATH1 microarray. Moreover, 197 genes had, in microarray data, sporophytic expression values higher than the chosen cut-off level (log2 ≥ 3.5). Together, this suggests that chosen cut-off levels were conservative and confirms the robustness of the data sets for pollen-specific genes. As, for Arabidopsis, transcripts of many organs and cell types have been measured with microarrays, a comparison of relative expression between these samples is well suited for the identification of pollen-specific genes.

Methylation of Lys-27 in Histone-3 at Pollen-Specific Genes in the Sporophyte Correlates with Repression of Gene Expression In this study, we data-mined publicly available databases to identify genetic and epigenetic factors associated with repression of pollen-specific genes in the Arabidopsis sporophyte.

In the sporophyte, we observed for both data sets analyzed a highly statistically significant association of the epigenetic modifications H3K27me1 and H3K27me3 with repressed pollen genes. This points to the presence of a novel independent mechanism for sporophytic repression of genes highly expressed in pollen. Data from three independent studies (Lafos et  al., 2011; Roudier et  al., 2011; Luo et  al., 2013) found pollen-specific genes to be associated with the histone modification H3K27me3. In contrast, we found in only one study employed in our analysis that H3K27me1 marks pollen-specific genes (Roudier et al., 2011), whereas, in the other study employed (Luo et  al., 2013), H3K27me1 is nearly absent from pollenspecific genes. Compared to Roudier et al. (2011), Luo et al. (2013) generally find fewer genes associated with epigenetic modifications, which may explain the discrepancy between the two results. We observed that, in the sporophyte, a number of pollenspecific genes were marked by histone modifications associated with transcribed genes (H3K4me3, H3K9me3, H3K9Ac, H3K36me2, H3K36me3, H3K56AC, and H2Bub) (Roudier et al., 2011; Luo et al., 2013). Expression of the pollen-specific genes which is restricted to certain cell types in the sporophyte might be masked in microarray and RNA-Seq experiments. But genes with this cell type-specific expression are likely to have altered histone modifications in these cells and ChIP experiments will detect them as decorated with histone modifications generally associated with expressed genes. Arabidopsis lines carrying loss-of-function alleles for genes involved in H3K27me3 deposition (FIE within the Polycomb Repressive Complex 2, PRC2) or stabilization of repression (EMF1, TFL2/LHP1) show early flowering phenotypes along with up-regulation of key regulators in flower development (Turck et al., 2007; Bouyer et al., 2011; Kim et al., 2012). The overall number of H3K27me3 marked genes that are up-regulated in these plants, however, is low. Similarly, we identified only a small percentage of pollen-specific genes marked by H3K27me3 to be up-regulated in these mutant lines. This indicates that PRC2 is not the only repressive system or that, in addition, activators such as H3K4me3 are required for gene expression (Bouyer et al., 2011). As we discuss below, the inaccessibility of promoter regions might confer the maintenance of gene repression. Schmitz et al. (2013) found RdDM-targeted loci to be activated in pollen based on microarray analysis. However, no minimum expression cut-off level was applied and RNA-Seq data revealed that almost all loci are not transcribed in pollen. This is in line with the assumption that the loci targeted by RdDM have acquired this regulation by nearby transposons, which first were repressed by RdDM, and subsequently the mechanism spread to genes (Schmitz et al., 2013). Our approach of identifying pollen-specific genes was also based on microarray data. However, the minimum expression signal in pollen we defined as cut-off eliminated most genes with no or few transcripts in pollen. Thus, our data suggest that pollen-specific

Hoffmann & Palmgren  •  Repression of Pollen-Specific Genes in the Sporophyte   

genes are associated with repressive histone modifications but generally are not targets of RdDM in the sporophyte.

Promoter Regions of Pollen-Specific Genes Have a Compact Structure in the Sporophyte Independently of Mono- and Trimethylation of Lys-27 in Histone-3 Our data show that, in leaf tissue, promoters of pollenspecific genes are more or less resistant to degradation by DNase I, which strongly indicates that the promoter regions have a compact structure—a consequence of nucleosomes being present in them (Tirosh and Barkai, 2008; Hartley and Madhani, 2009). This observation was independent of whether the genes were marked by histone modifications H3K27me1 or H3K27me3. In an Arabidopsis mutant line having highly decreased H3K27me3 levels, expression of genes marked by H3K27me3 in a wild-type remains unchanged for most genes (Bouyer et al., 2011). These findings suggest that what represses pollen-specific genes in the Arabidopsis sporophyte is the closed chromatin in their promoters. The factors controlling this state of closed chromatin, however, have not yet been identified. Leaf-expressed genes, in contrast, had open chromatin which is connected to nucleosome-free regions that allow access of transcription factors to the DNA (Tirosh and Barkai, 2008).

DNA Motifs Recognized by MADS Transcription Factors Are Occupied by Proteins in the Sporophyte In our motif-footprint analysis, the DNA motif CN(A/T)6NG (CArG-box), which is recognized by members of the MADS

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family of transcription factors (Huang et  al., 1995), showed strong protection from DNase I cleavage in sporophyte- and gametophyte-specific genes. Indeed, all motifs we analyzed had similar footprints in both groups; peaks were more distinct for sporophyte-expressed genes, which is due to their generally more open chromatin, increasing the visibility of positive and negative deflections (Boyle et al., 2011). The DNase footprint peak we found for AGAAA was extended to the left of the motif, which suggests that proteins binding it depend for proper recognition of the motif on the nucleotides immediately upstream. The two MADSdomain transcription factors SOC1 and SVP bind CArG-box motifs with an AAA extension at the 3′ end; for SVP, this motif is CCAAAAATAGAAA, which includes AGAAA (Tao et  al., 2012). Our data thus indicate that the POLLEN1LELAT52 element might in certain cases be an extension of less conserved CArG-box motifs.

Nucleosome-Free Regions Might Be Established by DNA Motif GCCCA We discovered that the DNA motifs GTGA and GCCCA in promoter regions were hypersensitive to DNase I cleavage, meaning they were bound by proteins to a lesser degree. It is possible that repetitive DNA stretches lead to errors in the alignment of DNase I reads and that such a bias in the data causes the enrichment of reads at a certain motif as we see it in our data; however, as all motifs we analyzed are located within 500 bp upstream of transcribed genes, it is unlikely that they were within repetitive DNA. Also,

Figure 6.  A Model for Two-Staged Gene Repression in Arabidopsis. (A) DNase I footprint profiles measured in Arabidopsis leaves. Repressed pollen-specific genes have closed promoter regions with lowest levels of DNase I accessibility in proximity to the start codon (left, orange). The promoter regions of expressed genes (right, green) are especially open in the region about 150 bp upstream of the start codon (ATG) (Zhang et al., 2012b), falling together with repeats of the DNA motif GCCCA (n = 541 for each pollen- and seedling-expressed genes). (B) In the sporophyte, repressive histone modifications (e.g. H3K27me3) are present in the gene body of pollen-specific genes (left), whereas expressed genes are marked by histone modifications promoting gene expression (e.g. H3K4me3) (Roudier et al., 2011). (C) In a combined model, pollen-specific genes are repressed in the sporophyte by mobile histones that occupy their promoters, causing the chromatin to be inaccessible, and by epigenetic marks in their gene bodies conferring additional repression. Expressed genes’ histones are removed from core promoter regions and the DNA is accessible for transcription factors in these nucleosome-free regions (gray ovals, histones; red boxes, DNA motif; red flags, repressive histone modifications; green flags, activating histone modifications).

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particularly GCCCA was surrounded by highly accessible chromatin, lowering the risk for a bias in the data, and we therefore believe that the DNase I  hypersensitivity of the motifs was true. The motif GCCCA was particular DNase I-hypersensitive, which generally indicates absence of protein binding. A similar motif has been identified in yeast, where it is involved in the formation of nucleosome-free regions (Hartley and Madhani, 2009).

Epigenetic Data Data from genome-wide analyses of genes marked by epigenetic modifications in the Arabidopsis sporophyte were downloaded from supplemental material accompanying the publications (Lafos et al., 2011; Roudier et al., 2011; Luo et al., 2013) or kindly provided by the authors (Schmitz et al., 2013). Microsoft Excel was used to identify pollen-specific and sporophyte-expressed genes within these data.

Arabidopsis Genome Data A Model for Repression of Pollen-Specific Genes in the Sporophyte The data presented in this work allow us to describe a twostaged model for the repression of pollen-specific genes in the sporophyte (Figure 6). First, promoters have inaccessible chromatin which disfavors transcription factor binding to DNA and/or initiation of transcription, resulting in repression of genes. Second, open reading frames are decorated by the histone modifications H3K27mono-, di-, and trimethylation, which strengthen the repression. As evidenced by this study, data of large-scale genomic experiments are very powerful when assessing information that applies to dozens or hundreds of genes. Plant material used in genome-wide studies often consists of whole organs or entire plants, and thus creates an averaged picture of the genome in which a multitude of cell types are mixed. Pollen, however, is released from plants and the vegetative nucleus of Arabidopsis pollen can further be isolated by FACS (Borges et al., 2012), resulting in a single cell type that offers unique genetic discoveries (Calarco et  al., 2012). In the future, we therefore imagine pollen to be a useful model for epigenomic studies.

METHODS Selection of Gametophyte- and Sporophyte-Specific Genes Samples were retrieved from microarray data (Schmid et al., 2005); ATGE_73 (mature pollen), ATGE_100 (green parts of seedlings, 21 d old) and the average of ATGE_94 and ATGE_96 (root and green parts of seedlings, respectively, 8 d old). Entries classified as pseudogenes or transposable elements (TAIR10) were deleted. Genes were arbitrarily defined as pollen-specific when they had log2 expression signals ≥ 7.0 in ATGE_73 and < 3.5 in ATGE_100 and ATGE_94_96. Selected as comparison groups for sporophyte-expressed genes were the 541 genes with highest log2 values in ATGE_94_96 (seedlingexpressed) and ATGE_100 (leaf-expressed). A comparative set of pollen-specific genes was obtained from high-throughput sequencing data for Arabidopsis pollen and aerial parts of seedlings (Loraine et  al., 2013). Protein-coding loci were considered as pollen-specific when they had normalized expression values of at least 5  RPM in pollen and < 1 RPM in seedlings.

Annotation and chromosomal positioning information of Arabidopsis genes and 5′ UTRs was downloaded from TAIR database (ftp://ftp.Arabidopsis.org/home/tair/Sequences/ blast_datasets/TAIR10_blastsets/). Arabidopsis promoter regions (–500-bp upstream gene loci) were scanned for DNA motif occurrences using the Patmatch tool on the TAIR website (www.Arabidopsis.org/cgi-bin/patmatch/nph-patmatch.pl).

Identification of DNA Motifs We performed DNA motif identification using SCOPE (Carlson et al., 2007). SCOPE supports analysis of promoter regions of 100, 200, 400, 800, or more base pairs, of which we identified the motif GCCCA within the 400-bp promoter region of the 541 genes we identified as pollen-specific.

DNase I Footprinting Data for DNase I  cleavage in the genome of Arabidopsis leaves were downloaded from http://128.104.239.218/cgi-bin/ gb2/gbrowse/Tair10 (Zhang et  al., 2012b). We uploaded the data to Google BigQuery at https://developers.google.com/ bigquery/ and queried DNase I cut per nucleotide values of the genomic locations corresponding to the genes we wanted to analyze. DNase I footprint profiles for DNA motifs were created by defining the motif’s nucleotide furthest away from the transcription start site of its downstream gene as base of the motif. DNase I  cuts at each nucleotide of the motif’s base ± 50 bp surrounding it were counted and averaged for all motifs found in the region –500 bp upstream of gene loci (TAIR 10) using Microsoft Excel.

SUPPLEMENTARY DATA Supplementary Data are available at Molecular Plant Online.

FUNDING We acknowledge the support of the Danish National Research Foundation through the PUMPkin Center of Excellence. No conflict of interest declared.

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