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. 2016 Aug 15;30(16):1866-80.
doi: 10.1101/gad.286278.116. Epub 2016 Aug 26.

Direct interrogation of the role of H3K9 in metazoan heterochromatin function

Taylor J R Penke  1 , Daniel J McKay  2 , Brian D Strahl  3 , A Gregory Matera  4 , Robert J Duronio  4
Affiliations

Direct interrogation of the role of H3K9 in metazoan heterochromatin function

Taylor J R Penke et al. Genes Dev. .

Abstract

A defining feature of heterochromatin is methylation of Lys9 of histone H3 (H3K9me), a binding site for heterochromatin protein 1 (HP1). Although H3K9 methyltransferases and HP1 are necessary for proper heterochromatin structure, the specific contribution of H3K9 to heterochromatin function and animal development is unknown. Using our recently developed platform to engineer histone genes in Drosophila, we generated H3K9R mutant flies, separating the functions of H3K9 and nonhistone substrates of H3K9 methyltransferases. Nucleosome occupancy and HP1a binding at pericentromeric heterochromatin are markedly decreased in H3K9R mutants. Despite these changes in chromosome architecture, a small percentage of H3K9R mutants complete development. Consistent with this result, expression of most protein-coding genes, including those within heterochromatin, is similar between H3K9R and controls. In contrast, H3K9R mutants exhibit increased open chromatin and transcription from piRNA clusters and transposons, resulting in transposon mobilization. Hence, transposon silencing is a major developmental function of H3K9.

Keywords: Drosophila; H3K9; genomics; heterochromatin; heterochromatin protein 1 (HP1); transposon.

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Figures

Figure 1.
Figure 1.
H3K9R mutant cells proliferate with severely reduced H3K9me2 and me3 in pericentric heterochromatin. (A) Twin spot analysis of FLP–FRT-induced mitotic clones of HisC deletion wing disc cells rescued with an HWT transgene (top) or a K9R transgene (bottom). Wing discs were stained with DAPI to mark nuclei, anti-H3K9me2 to identify K9R cells, and anti-GFP to identify twin spots, (red lines). HisC deletion cells lack GFP, and control sister clones are homozygous HisC+ and express 2× GFP. Bar, 1000 µm. White boxes indicate magnified regions where the bar represents 20 µm. (B) Quantification of twin spot clone area. Each dot represents the area of the experimental (HWT or K9R) clone divided by the area of the control twin spot clone. (**) P < 0.005. (C) Western blot analysis of total cellular histone isolated from whole third instar larvae. (D) Polytene chromosome preparations from third instar larval salivary glands stained with DAPI and anti-H3K9me antibodies. Bar, 20 µm. Magnified images show H3K9me3 staining at the chromocenter (white box) and chromosome arms (yellow box). Bar, 10 µm. Note that H3K9me3 signal at the chromocenter is overexposed to reveal staining on chromosome arms.
Figure 2.
Figure 2.
H3K9 regulates chromatin organization at pericentromeric heterochromatin. (A) HWT and K9R salivary gland polytene chromosomes stained with DAPI and H3K9me2. Bar, 20 µm. The bottom images show a magnified view (white squares) of the chromocenter (dashed lines). Bar, 10 µm. (B) Quantification of chromocenter organization from yw (contains endogenous histones), HWT, and K9R. (C) K9R/HWT ratio of normalized FAIRE (formaldehyde-assisted isolation of regulatory elements) signal from third instar imaginal wing discs at FAIRE peaks called by MACS2 (see also Supplemental Fig. 2). (CPM) Counts per million. The X-axis indicates the average HWT and K9R signal at each peak. Darker colors in the heat map indicate a higher number of peaks. Red peaks are statistically significant as determined by edgeR. P < 0.01. Lines indicate a twofold change. (D) Genome browser shot of FAIRE peaks near the euchromatic gene engrailed and the heterochromatic gene concertina. (Map) Read mappability. (E) K9R/HWT ratio of normalized FAIRE signal plotted versus genome coordinates of FAIRE peaks on chromosomes 2 and 3. Green regions in the chromosome schematic indicate approximate locations of pericentromeric heterochromatin (Riddle et al. 2011; Hoskins et al. 2015). Blue indicates largely euchromatic regions. Loess regression line of modENCODE K9me3 chromatin immunoprecipitation (ChIP) signal is shown in red. (F) Box plot of average ratio of FAIRE signal for FAIRE peaks assigned to one of nine chromatin states (Kharchenko et al. 2010). See also Supplemental Figure 3, C and D.
Figure 3.
Figure 3.
HP1a relocalizes from pericentromeres to chromosome arms in the absence of H3K9. (A) HWT and K9R salivary gland polytene chromosomes stained with anti-H3K9me2 and anti-HP1a antibodies. Bar, 20 µm. The bottom panels show a magnified view of the chromocenter (white box) and a chromosome arm (yellow box) for each genotype. Arrows indicate telomeres. Bar, 5 µm. (B) α-HP1a Western blot of 3, 6, and 12 µg of whole-cell extract from HWT and K9R salivary glands. (C) K9R/HWT ratio of HP1a ChIP-seq (ChIP combined with high-throughput sequencing) signal from whole third instar larvae within 1-kb windows tiled across the five autosome arms. The top 20% of 1-kb windows with the highest counts are shown (see Supplemental Fig. 4 for all windows). Pie charts show the percentage of significantly altered windows on pericentromeres or chromosome arms as called by edgeR. P < 0.01. (D) Scatter plot of HP1a signal at FAIRE peaks with higher signal in K9R samples (top) or a random selection of FAIRE peaks that are not significantly different between HWT and K9R (bottom).
Figure 4.
Figure 4.
Differential expression of transcripts between HWT and K9R correlates with changes in FAIRE signal and HP1a localization. (A) K9R/HWT ratio of RNA-seq signal from third instar imaginal wing discs. Statistically different ratios identified using DESeq2 are indicated in red. P < 0.05. Blue lines indicate a twofold change. (B) Normalized FAIRE signal at all transcripts differentially expressed between HWT and K9R (red dots in A). Signal is expressed as counts per million (CPM). Black dots indicate transcripts annotated in the RefSeq reference transcriptome. Green dots here and in C indicate transcripts identified in Cufflinks transcriptome assembly but not in the reference transcriptome. (C) Scatter plot of normalized HP1a ChIP signal within transcripts differentially expressed between K9R and HWT (left) or a random selection of transcripts that are not significantly different between the two genotypes (right).
Figure 5.
Figure 5.
H3K9 represses transposon and piRNA cluster expression. (A) Scatter plot of FAIRE-seq signal ([RPKM] reads per kilobase per million) at individual transposon families (red) or piRNA clusters (blue). H3K4me2/me3-enriched promoter regions (grey) are shown for comparison. (B) Scatter plot of RNA-seq signal at transposons (red) and piRNA clusters (blue). A random selection of protein-coding RNAs was selected for comparison (gray). (C) Genome browser shot of FAIRE and RNA signal at the 42AB piRNA cluster. FAIRE signal is shown for both uniquely and multiple mapping reads. RNA signal contains uniquely mapping reads only. Highlighted areas indicate regions of increased FAIRE and RNA signal in K9R samples. (D,E) Scatter plot of HP1a ChIP-seq signal at transposon families (D) or piRNA clusters (E) comparing input and immunoprecipitated (IP) samples.
Figure 6.
Figure 6.
H3K9 represses gypsy transposon mobilization. (A) Schematic of the gypsy transposon mobilization assay. Ovo-binding site-dependent (green bars) gypsy insertion disrupts Gal80, resulting in Gal4-directed expression of yellow fluorescent protein (YFP) (Li et al. 2013). (B) Pupae showing one, two, or three or more YFP-positive clones representing gypsy mobilization events, which are limited to the mesoderm due to the twi-Gal4 driver and thus likely underestimate the total number of mobilization events in each animal. (C,D) Histogram of the average number of pupae (C; n = 200 for each genotype in six independent experiments) or larvae (D; n = 100 for each genotype in three independent experiments) with zero, one, two, and three or more mobilization events. Error bars represent standard deviation. (*) P < 0.05; (**) P < 0.005; (***) P < 0.0005.
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