The CHD3 remodeler PICKLE promotes trimethylation of histone H3 lysine 27 - PubMed (original) (raw)

The CHD3 remodeler PICKLE promotes trimethylation of histone H3 lysine 27

Heng Zhang et al. J Biol Chem. 2008.

Abstract

CHD3 proteins are ATP-dependent chromatin remodelers that contribute to repression of developmentally regulated genes in both animal and plant systems. In animals, this repression has been linked to a multiple subunit complex, Mi-2/NuRD, whose constituents include a CHD3 protein, a histone deacetylase, and a methyl-CpG-binding domain protein. In Arabidopsis, PICKLE (PKL) codes for a CHD3 protein that acts during germination to repress expression of seed-associated genes. Repression of seed-associated traits is promoted in pkl seedlings by the plant growth regulator gibberellin (GA). We undertook a microarray analysis to determine how PKL and GA act to promote the transition from seed to seedling. We found that PKL and GA act in separate pathways to repress expression of seed-specific genes. Comparison of genomic datasets revealed that PKL-dependent genes are enriched for trimethylation of histone H3 lysine 27 (H3K27me3), a repressive epigenetic mark. Chromatin immunoprecipitation studies demonstrate that PKL promotes H3K27me3 in both germinating seedlings and in adult plants but do not identify a connection between PKL-dependent expression and acetylation levels. Taken together, our analyses illuminate a new pathway by which CHD3 remodelers contribute to repression in eukaryotes.

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Figures

FIGURE 1.

FIGURE 1.

The presence of uniconazole-P results in increased LEC1 and_LEC2_ transcript levels in germinating pkl seeds. Quantitative RT-PCR was used to determine the relative transcript levels of_LEC1_ and LEC2 in germinating WT and pkl seeds in the absence or presence of 10-8

m

uniconazole-P (uni-P). Each sample was collected when 50% of the seeds had germinated under the indicated treatment. 18 S rRNA was used as a standardization control, and expression levels were normalized to wild-type seeds imbibed in the absence of uniconazole-P. Error bars represent the S.D. of the mean.

FIGURE 2.

FIGURE 2.

Intersection of genes that exhibit _PKL_- and uniconazole-dependent transcript levels. The Venn diagram indicates the number of loci for which the corresponding transcript is expressed at significantly different levels in the presence of the pkl mutation and/or uniconazole-P (uni-P). Expect indicates the number of genes that would be predicted to exhibit the indicated type of behavior if there was no interaction between genotype (wild type versus pkl) and treatment (± uniconazole-P). % indicates percentage of genes represented on array that are affected by treatment. For a complete list of_PKL_- and uniconazole-dependent genes identified by our analysis, please see supplemental Table 1.

FIGURE 3.

FIGURE 3.

Possible genetic models of how PKL and GA affect expression of target genes. Arrows denote activation; bars denote repression.

FIGURE 4.

FIGURE 4.

pkl and uniconazole-P act synergistically to increase gene expression. The sets of genes that exhibit PKL_- and/or uniconazole-dependent expression are depicted in a Venn diagram as in Fig. 2. The top number in each set indicates the percent of genes belonging to that set for which_pkl or uniconazole-P (uni-P) enhance the effect of each other, and the bottom number in each set indicates the percent of genes for they counteract each other. nd, denotes not determined.

FIGURE 5.

FIGURE 5.

Seed-specific genes are preferentially derepressed in the absence of_PKL_ and/or in the presence of uniconazole. χ2 analysis (see “Experimental Procedures”) was used to examine the intersection of genes that exhibit altered expression in response to_pkl_ and/or uniconazole-P (x axis) and genes preferentially expressed in a specific tissue (y axis). The χ2 value associated with each intersection is represented on the z axis. A_green bar_ denotes more genes observed in common between the two sets than expected (at p < 0.01), and a red bar denotes fewer genes observed than expected (at p < 0.01).

FIGURE 6.

FIGURE 6.

Genes that exhibit strongly elevated expression in the absence of_PKL_ are frequently targets of H3K27me3. χ2 analysis (see “Experimental Procedures”) was used to examine the intersection of genes linked to a remodeling pathway (x axis) and genes that exhibit increased expression in response to pkl according to distinct selection criteria (y axis). The percent of genes that exhibit increased expression in pkl plants that are associated with each intersection is represented on the z axis. The expected values category on the y axis indicates the percentage of_pkl_-dependent genes expected to found in the intersection of the compared sets of genes. A green bar denotes more genes observed in common between the two sets than expected (at p < 1 × 10-4), whereas a red bar denotes fewer genes observed than expected (at p < 1 × 10-4). In x axis categories, C denotes ChIP-chip data, and M denotes microarray-derived expression data. For further discussion of the chromatin remodeling pathways investigated, please see text.

FIGURE 7.

FIGURE 7.

H3K27me3 levels are decreased in pkl plants. ChIP was used to examine levels of H3K27me3, diacetylated H3 (H3Ac), and tetraacetylated H4 (H4Ac) at indicated loci in 50% germinated seedlings (white bars) or in 14-day-old plants (black bars). For each panel, a schematic of the gene of interest at the top left indicates the region that was assayed by ChIP. The graph on the_left_ indicates the level of H3K27me3 for the region of the GOI relative to MULE and ACT7 in wild-type plants. The graph in the middle indicates the level of H3K27me3 in WT relative to_pkl_ plants (thus a decrease in H3K27me3 in pkl relative to WT plants results in an increased signal). The graph on the_right_ indicates the level of H3Ac and H4Ac in pkl plants relative to WT plants (thus an increase in acetylation in pkl relative to WT plants results in an increased signal). The table in the right-hand corner of each panel indicates the expression of the gene as assayed by qRT-CR in pkl plants relative to WT plants in 50% germinated seedlings or in 14-day-old plants. ND denotes transcript not detected at indicated developmental stage. All data are the average of three biological replicates. Bars denote standard deviation.Asterisks in graphs depicting relative levels of H3K27me3, H3Ac, or H3Ac denote a fold change of at least 1.5 (95% confidence interval).

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