ATRX binds to atypical chromatin domains at the 3' exons of zinc finger genes to preserve H3K9me3 enrichment

Abstract

ATRX is a SWI/SNF chromatin remodeler proposed to govern genomic stability through the regulation of repetitive sequences, such as rDNA, retrotransposons, and pericentromeric and telomeric repeats. However, few direct ATRX target genes have been identified and high-throughput genomic approaches are currently lacking for ATRX. Here we present a comprehensive ChIP-sequencing study of ATRX in multiple human cell lines, in which we identify the 3' exons of zinc finger genes (ZNFs) as a new class of ATRX targets. These 3' exonic regions encode the zinc finger motifs, which can range from 1-40 copies per ZNF gene and share large stretches of sequence similarity. These regions often contain an atypical chromatin signature: they are transcriptionally active, contain high levels of H3K36me3, and are paradoxically enriched in H3K9me3. We find that these ZNF 3' exons are co-occupied by SETDB1, TRIM28, and ZNF274, which form a complex with ATRX. CRISPR/Cas9-mediated loss-of-function studies demonstrate (i) a reduction of H3K9me3 at the ZNF 3' exons in the absence of ATRX and ZNF274 and, (ii) H3K9me3 levels at atypical chromatin regions are particularly sensitive to ATRX loss compared to other H3K9me3-occupied regions. As a consequence of ATRX or ZNF274 depletion, cells with reduced levels of H3K9me3 show increased levels of DNA damage, suggesting that ATRX binds to the 3' exons of ZNFs to maintain their genomic stability through preservation of H3K9me3.

Keywords: ATRX; Atypical chromatin; ESET; H3K9me3; KAP1; KRAB-ZNFs; SETDB1; TRIM28; ZNF274; zinc finger genes.

Figure 1.

ATRX binds to the 3′ regions of ZNF genes in K562 cells. (A) Observed over expected random distribution of significant ATRX peaks within HMM chromatin categories in K562. In (A) and (C) error bars represent standard deviation. Asterisks represent significantly overrepresented regions (*P-value < 0.05; ****P-value < 1 × 10⁻⁴) assessed by the hypergeometric test (see Tables S2 and S3 for details of statistical tests). (B) Gene Ontology analysis of genes that overlap with significant ATRX peaks in their gene bodies. (C) Observed over expected random distribution of significant ATRX peaks in ZNFs and non-ZNF (other) promoters and gene bodies in K562. The red line represents the expected value of a random distribution. (D) Hilbert curve plot of chromosome 19 showing the ZNF clusters (red, left), highly enriched ATRX regions in K562 (fold enrichment over input > 5, blue, middle), and overlap (right). The blue dot and the red arrow mark the start (5′) and end (3′) of the chromosome, respectively (see diagram on left). The green dots indicate the start and end of centromere, which is excluded from the analysis. (E) UCSC Genome browser screenshot of a typical ZNF cluster (genes in blue) on chromosome 19. The enrichment over input signal in K562 for ATRX, H3K9me3 and H3K36me3 ChIP-seq is shown. Significant peaks represented as bars below enrichment tracks. Normalized RPKM signal for RNA-seq is also shown. (F) Zoomed in snapshot of 2 ZNFs contained within the ZNF cluster shown in (E). (G) Average K562 enrichment ChIP-seq profiles of ATRX, H3K9me3 and H3K36me3 over all ZNF gene bodies ± 1 kb (n = 736).

Figure 2.

ATRX and the ZNF274/TRIM28/SETDB1 complex bind to ZNF genes with an atypical chromatin signature and distinctive genomic and epigenetic features. (A) Average K562 enrichment ChIP-seq profiles of ATRX, H3K9me3 and H3K36me3 at ZNFs classified by their ATRX content. Class I contains high levels of ATRX (n = 91), Class II contains medium to low levels of ATRX (n = 303) and Class III is devoid of ATRX enrichment (n = 342). (B) Spearman correlation heatmap of K562 ChIP-seq signal at ZNF Class I genes (left) and ZNF Class III genes (right). (C) Left: Distribution of genetic features among ZNF classes (sorted from high to low ATRX enrichment from top to bottom). Dashed lines show separation of the 3 classes. Colors represent presence of KRAB domains (black), number of zinc finger motifs (pink), G content at the C-terminal ZNF region (last 3 kb of the gene) (gray) and presence of sequences predicted to form G-quadruplexes (brown). RNA-seq bar shows the Z score of the normalized RPKM signal (log2(RPKM+1)) in K562; red: high expression signal and blue = low expression signal. For statistical tests between the classes see Table S3. Right: Box plots displaying the number of ZNF motifs, G-content at the ZNF region and RNA-seq values in K562 per ZNF Class. Asterisks show significant differences (P-value < 1 × 10⁻⁴). (D) Metagene analysis of ChIP-seq enrichment over input profiles at ZNF gene bodies ± 1 kb. (E) Spearman correlation heatmaps between ChIP-seq profiles genome-wide (left) and at ZNF genes (right). Black boxes indicate the significant correlations. (F) Immunoblots for endogenous ATRX Co-IP of chromatin bound proteins in K562 cells after pulldown with IgG or ATRX antibody. DAXX used as a positive control for the ATRX IP.

Figure 3.

ATRX binding to ZNF 3′ exons is conserved across human cell lines. (A) Heatmap of ATRX ChIP-qPCR enrichment over ZNF genes in several human cell lines. The color represents the average enrichment of at least 2 independent biological replicates per cell line. ZNF class I genes show significant enrichment as compared to IgG for all assessed cell lines (for details see Table S3). (B) H3K9me3 (left) and ZNF274 (right) ChIP-seq enrichment over the same panel of ZNFs as in (A) for cell lines with available ENCODE data. (C) RNA-seq normalized expression values (log₂(RPKM+1)) from ENCODE for the panel of ZNF genes shown in (A) and (B). (D) Metagene profiles of ATRX and H3K9me3 ChIP-seq data over ZNFs gene bodies ± 1 kb in LAN6 neuroblastoma cell line. (E) Observed over expected random distribution of significant ATRX peaks in ZNFs and non-ZNF (other) promoters and gene bodies for LAN6. (F) UCSC Genome browser screenshot of a typical ZNF cluster (genes in blue) on chromosome 19. The enrichment over input signal for ATRX and H3K9me3 ChIP-seq in LAN6 is shown. Significant peaks represented as bars below enrichment tracks. (G) Zoomed in snapshot of 2 ZNFs contained within the ZNF cluster shown in (F).

Figure 4.

ATRX deficient cells have decreased levels of H3K9me3 at 3′ exons of ZNFs and other atypical chromatin regions. (A) Western blot of ATRX in chromatin preparations from control (V2) and 2 CRISPR ATRX KO K562 cell lines. Amido Black staining of histones is shown as a loading control. (B) ATRX ChIP-qPCR over ZNF genes in control and ATRX KO K562 cell lines. (C) Same as in (B) with H3K9me3 native ChIP-qPCR. In both (B) and (C), bars represent average of at least 3 biological replicates and the error bars represent the SEM. (D) Box plot of the H3K9me3 changes (represented in % with respect to the control) observed in the ZNF classes and non-ZNF H3K9me3-bound genes upon ATRX KO. Asterisks show significant changes with respect to the non-ZNF genes (*P-value< 0.05; **** P-value < 1 × 10⁻⁴). (E) ChIP-seq metagene profile of H3K9me3 in control (V2) and ATRX KO (KO2) K562 cell lines over ZNFs. (F) Distribution of reduced and unchanged H3K9me3 regions after ATRX KO in atypical chromatin and H3K9me3-only regions. (G) Observed over expected distribution of reduced and unchanged regions at atypical chromatin. Asterisks show significantly overrepresented regions (P-value < 1 × 10⁻⁴) (H) Genomic distribution of atypical chromatin regions.

Figure 5.

ZNF274 KO reduces ATRX and H3K9me3 levels at ZNFs. (A) ZNF274, (B) ATRX and (C) H3K9me3 ChIP-qPCR in K562 ZNF274 KO and K562 double ZNF274/ATRX KO at ZNF genes. Single ATRX KO2 cells are used for the H3K9me3 ChIP for comparison. In all graphs, the bars represent the average of at least 2 independent biological replicates. Error bars depict SEM. Results of statistical comparisons in Table S3. A non-specific sgRNA (random) used as control (see Table S7). (D) Chromatin immunoblot of γH2A.X in control (Rnd) and ZNF KO K562 cells. Histones used as loading control. (E) Representative K562 cell cycle profiles of control (random), ATRX and ZNF274 single and double KO assessed by BrdU/PI staining. n ≥ 6 biological replicates. (F) Graph depicting quantifications of (E). The bars show the average % of cells in each phase, error bars depict SEM. Asterisks show significant changes compared to the control (*P-value < 0.05; **P-value < 0.01).

Figure 6.

Model of ATRX regulation at ZNF 3′ exons. Left: ATRX forms a complex with ZNF274, TRIM28 and SETDB1 to facilitate the deposition and maintenance of H3K9me3 at ZNF 3′ exons. The presence of the mark establishes an atypical H3K9me3/H3K36me3 domain. Right: Upon ATRX depletion, H3K9me3 and the atypical chromatin domains at ZNF 3′ exons are lost. Loss of ATRX induces altered cell cycle, increased DNA damage and possibly recombination between ZNFs.