ATRX Plays a Key Role in Maintaining Silencing at Interstitial Heterochromatic Loci and Imprinted Genes

Abstract

Histone H3.3 is a replication-independent histone variant, which replaces histones that are turned over throughout the entire cell cycle. H3.3 deposition at euchromatin is dependent on HIRA, whereas ATRX/Daxx deposits H3.3 at pericentric heterochromatin and telomeres. The role of H3.3 at heterochromatic regions is unknown, but mutations in the ATRX/Daxx/H3.3 pathway are linked to aberrant telomere lengthening in certain cancers. In this study, we show that ATRX-dependent deposition of H3.3 is not limited to pericentric heterochromatin and telomeres but also occurs at heterochromatic sites throughout the genome. Notably, ATRX/H3.3 specifically localizes to silenced imprinted alleles in mouse ESCs. ATRX KO cells failed to deposit H3.3 at these sites, leading to loss of the H3K9me3 heterochromatin modification, loss of repression, and aberrant allelic expression. We propose a model whereby ATRX-dependent deposition of H3.3 into heterochromatin is normally required to maintain the memory of silencing at imprinted loci.

Graphical abstract

Figure 1

ATRX Localizes to Genomic Regions that Are Enriched for Heterochromatic (H3K9me3/H4K20me3) Modifications and Histone H3.3 (A) ATRX ChIP-seq reads are mapped to genomic repeats and normalized for total read count compared to input. (B) (Left) H3K9me3 and (right) H4K20me3 are enriched under ATRX peaks compared to random genomic fragments. H3K9me3 and H4K20me3 ChIP-seq reads were quantitated under ATRX peaks and normalized for total read counts. Normalized read counts under random genomic fragments were used as a control. Boxes represent the 25^th, median, and 75^th percentiles; whiskers represent the 90^th and 10^th percentiles. (C) (Left) H3.3 ChIP-seq reads are mapped to genomic repeats and normalized for total read count compared to input. (Right) H3.3 ChIP-seq quantitation under ATRX peaks are normalized for total read count. (D) Overlap between heterochromatic H3K9me3 and H4K20me3 marks and histone H3.3 under ATRX peaks. Enrichment is defined as normalized read counts >75^th percentile of random genomic fragments. Overlap of H3K9me3/H4K20me3/H3.3 enrichments in random fragments of the genome is shown as a control. (E) ATRX is enriched at tandem repeats (TRs), ERVK elements, and CGIs (CGI) in the genome relative to random genomic fragments. (F) Proportion of ATRX-binding sites that overlie CGI, TRs, ERVK, and heterochromatin. See also Figure S1.

Figure 2

ATRX-Bound CGI Are Enriched for Marks of Heterochromatin (H3K9me3/H4K20me3 and DNA Methylation) and H3.3 (A) Normalized read counts of (left) H3K9me3, (middle) H4K20me3, and (right) H3.3 ChIP-seq at ATRX-bound CGI relative to genomic CGI are shown. Boxes represent the 25^th, median, and 75^th percentiles; whiskers represent the 90^th and 10^th percentiles. (B) Proportion of CGI that are methylated genome-wide in comparison to ATRX-bound CGI is shown. (C) Overlapping enrichment profiles of H3K9me3, H4K20me3, and H3.3 under ATRX CGI compared to genomic CGI are shown. (D) (Left) Proportion of CGI that are bound by ATRX following sequential filtering for DNA methylation, H3K9me3/H4K20me3 enrichment, H3.3 enrichment, and active transcription is shown. (Right) Proportion of CGI that are associated with imprinting and ATRX after sequential filtering is shown. (E) Screenshots of the (top) Spred2 locus, (bottom left) Zfp629 locus, and (bottom right) Zfp787 locus show a typical promoter CGI marked by high H3K4me3 and mid-level H3.3 compared to an ATRX-bound intragenic CGI, which is enriched with marks of heterochromatin (DNA methylation, H3K9me3, and H4K20me3) and H3.3. See also Figure S2.

Figure 3

ATRX Localizes Specifically to Methylated DMR in Imprinted Regions along with Histone H3.3 and ATRX and H3.3 Both Localize Specifically to the Methylated Allele (A) (Top) Screenshots show overlapping chromatin modifications at imprinted DMRs. Imprinted DMRs are simultaneously enriched for H3K4me3-activating marks and heterochromatic marks (DNA methylation, H3K9me3, and H4K20me3). ATRX and H3.3 also are enriched at these regions. (Bottom) Allelic distribution of H3.3 and H3K4me3 is shown. ChIP-seq reads of H3.3 and H3K4me3 in F1 Cast × 129 mouse ESCs with informative SNPs were assigned to either maternal or paternal alleles. (B) Allele specific localization of ATRX, H3K9me3, H3.3, and H3K4me3 at (top left) Peg13, (top right) Peg3, (bottom left) Nap1L5, and (bottom right) IG-DMR. ChIP products were PCR amplified and digested with allele-specific restriction enzymes. Products were separated by agarose gel electrophoresis. See also Figure S4.

Figure 4

ATRX Is Required for Correct Deposition of H3.3 and H3K9me3 at DMRs (A) Normalized read counts of H3.3 ChIP-seq in WT versus ATRX KO mouse ESCs at various genomic regions. ATRX-bound imprinted DMRs are highly enriched for H3.3 and enrichment is lost in the absence of ATRX. Boxes represent the 25^th, median, and 75^th percentiles; whiskers represent the 90^th and 10^th percentiles. (B) Screenshots show the (left) Igf2r/Airn, (middle) Trappc9/Peg13, and (right) Nap1L5/Herc3 DMRs that are enriched for H3.3 in WT cells and lose H3.3 in ATRX KO cells. (C) Screenshot shows the Trappc9 promoter that retains H3.3 in ATRX KO cells. (D) Screenshot of a genomic region (chr17:13,021,841-13,220,327) shows a number of promoters that are enriched for H3K4me3 and H3.3 in WT cells. H3.3 profiles are unchanged in the ATRX KO cells. (E and F) Relative enrichment of (E) H3.3 and (F) H3K9me3 at DMRs in WT and ATRX KO mouse ESCs. ChIP products were amplified by qRT-PCR and enrichment was calculated relative to GAPDH after normalizing for input. Results represent the mean and SD of three independent experiments. See also Figure S5.

Figure 5

Allelic Read Distribution at Imprinted Genes Is Disrupted in ATRX KO Cells (A and B) RNA-seq of WT and ATRX KO ESCs show skewed distribution of reads at a number of imprinted genes in the (A) PolyA− and (B) PolyA+ fractions in WT cells. Genes where allelic distribution differed by >10% in ATRX KO compared to WT cells are indicated in red. Allelic distribution of reads across randomly chosen genes in the genome are shown for comparison. Error bars represent SD of allelic skewing across the random dataset. (C) (Left) A schematic representation of the Zrsr1/Commd1 imprinted locus shows allelic expression and methylation patterns. (Right) Normalized read counts arise from the maternal SV129 Zrsr1 allele in WT and ATRX KO cells. See also Figure S6.

Figure 6

ATRX-Mediated Deposition of H3.3 Maintains Heterochromatic Profiles during Transcription (A) Schematic depiction of a gene containing a gene body CGI. Heterochromatic modifications such as H3K9me3 silence the internal CGI to prevent cryptic transcription. (B) Progression of RNA Pol II during transcription disrupts heterochromatin-modified nucleosomes at the internal CGI. (C) ATRX/Daxx mediate deposition of replacement H3.3 at the heterochromatic CGI and facilitate maintenance of chromatin modifications at this site. (D) Loss of ATRX leads to failure of H3.3 deposition, loss of chromatin modifications, and aberrant transcription.