The Tumor Suppressor ARID1A Controls Global Transcription via Pausing of RNA Polymerase II

Abstract

AT-rich interactive domain-containing proteins 1A and 1B (ARID1A and ARID1B) are mutually exclusive subunits of the chromatin remodeler SWI/SNF. ARID1A is the most frequently mutated chromatin regulator across all cancers, and ovarian clear cell carcinoma (OCCC) carries the highest prevalence of ARID1A mutations (∼57%). Despite evidence implicating ARID1A in tumorigenesis, the mechanism remains elusive. Here, we demonstrate that ARID1A binds active regulatory elements in OCCC. Depletion of ARID1A represses RNA polymerase II (RNAPII) transcription but results in modest changes to accessibility. Specifically, pausing of RNAPII is severely impaired after loss of ARID1A. Compromised pausing results in transcriptional dysregulation of active genes, which is compensated by upregulation of ARID1B. However, a subset of ARID1A-dependent genes is not rescued by ARID1B, including many p53 and estrogen receptor (ESR1) targets. Our results provide insight into ARID1A-mediated tumorigenesis and unveil functions of SWI/SNF in modulating RNAPII dynamics.

Keywords: ARID1A; ARID1B; RNAPII; SWI/SNF; ovarian clear cell carcinoma; pausing; transcription.

Figure 1 –. ARID1A binds active enhancers and promoters in OCCC cells.

(A) Genomic distribution of ARID1A ChIP-seq peaks. The pie chart shows that ARID1A is broadly distributed across TSS, intronic regions and intergenic regulatory elements. (B) Heatmap of H3K27ac and ARID1A ChIP-seq signal across the ARID1A peaks suggests that ARID1A occupancy is enriched across all active regulatory elements. (C) Correlation plot of H3K27ac and ARID1A ChIP-seq experiments reveals a strong overlap (Pearson correlation=0.86) between ARID1A and active transcription genome-wide. (D) Average profile of ATAC-seq and ARID1A ChIP-seq in OCCC suggests that ARID1A binds the +1 and −1 nucleosomes flanking the TSS. (E) Average profile of ATAC-seq and ARID1A ChIP-seq in OCCC reveals that ARID1A binds the nucleosome depleted region of enhancers. (F, G) Genome Browser screenshot of ATAC-seq and ChIP-seq (ARID1A, H3K27ac) at a representative promoter and enhancer, respectively.

Figure 2 –. ARID1A depletion causes transcriptional attenuation in OCCC cells with minor effects on chromatin accessibility.

(A) ARID1A is efficiently depleted from RMG-1 cells in 72 hours, using a doxycycline inducible shRNA. Whole cell and chromatin extracts were probed by immunoblot. (B) Average ATAC-seq profiles (ARID1A-WT, KD) show that accessibility at promoters is not affected by ARID1A depletion. (C) Average ATAC-seq profiles (ARID1A-WT, KD) show that accessibility at weak enhancers (3^rd and 4^th quartile of H3K27ac enrichment) is significantly reduced upon ARID1A depletion. (D) Average ATAC-seq profiles (ARID1A-WT, KD) show that accessibility at strong enhancers (1^st and 2^nd quartile of H3K27ac enrichment) is marginally affected by ARID1A depletion. (E) Comparison of ARID1A ChIP-seq profiles at weak and strong enhancers. (F) Correlation plot (spike-in normalized RNA-seq) of all expressed coding genes (GENCODE annotations) in RMG-1 cells, ARID1A-KD elicits broad transcriptional downregulation. (G) RNA-seq at select loci displays transcriptional attenuation upon ARID1A-KD. (H) Parental RMG-1 cells were infected with shRNAs against ARID1A and rescued with exogenously expressed full-length ARID1A. Quantitative RT-PCR at three candidate genes shows that downregulation of the mRNA is significantly reversed (n=3) by ARID1A overexpression (fold change compared to shLUC, normalized by 18s rRNA).

Figure 3 –. ARID1A depletion causes attenuation of nascent transcription.

(A) Box plots compare nascent transcription (GRO-seq) before and after ARID1A depletion. Normalized read depth were calculated for the entire set of active protein coding genes and enhancers (intergenic H3K27ac peaks). Additionally, transcription at all ribosomal genes (rRNA transcript type in v17 of GENCODE) and RNAPIII dependent genes is shown. (B) GRO-seq correlation plot for all transcribed coding genes reveals global transcriptional downregulation upon ARID1A-KD. (C) GRO-seq profiles at MYC and PEX13 loci. (D and E) GRO-seq and chromatin-enriched RNA-seq (CHROM-RNAseq) data were used to calculate Pausing Indexes of all expressed genes. Most genes show a decrease in their pausing index (proximal promoter reads/gene body reads) upon depletion of ARID1A (ARID1A-KD), suggesting a defect in pausing of RNAPII.

Figure 4 –. Accumulation of paused RNAPII in OCCC cells depends on ARID1A.

(A) ARID1A depletion (ARID1A-KD) affects RNAPII levels at the majority of active TSS. The heatmap shows 5,914 TSS targeted by SWI/SNF, ranked by mean intensity of ARID1A. (B) Average profiles of RNAPII ChIP-seq (ARID1A-WT, KD) around the TSS of 5,914 genes presented in panel (A). (C) UCSC Genome Browser screenshot of RNAPII ChIP-seq profiles (ARID1A-WT, KD) at PEX13 and MYC loci. There is a significant reduction of RNAPII occupancy at the promoter proximal regions. (D) Box plots showing read depth of RNAPII ChIP-seq at the TSS of ARID1A target genes. RNAPII occupancy is significantly reduced (p<2.2×10⁻¹⁶) after depletion of ARID1A, in two independent replicates. (E) Immunoblot analysis of the chromatin fraction of OCCC cells, before and after ARID1A depletion. While total RNAPII levels (assayed with two different antibodies, see methods) and phosphorylation of Ser2 remain unchanged, phosphorylation of Ser5 is reduced. Lamin C is shown as loading control. Quantitative immunoblots are shown in Supplemental Fig. S3B. (F) Travelling ratio analysis reveals changes in RNAPII occupancy at proximal promoter vs. gene body. Incremental plots demonstrate that ARID1A depletion elicits a reduction of pausing genome-wide. (G) Parental RMG-1 cells were infected with shRNAs against ARID1A and rescued with exogenously expressed full-length ARID1A. Quantitative PCR at three candidate genes shows that downregulation of RNAPII at TSS is significantly reversed (n=3) by ARID1A overexpression.

Figure 5 –. ARID1A depletion impairs RNAPII pausing without affecting transcriptional initiation in OCCC cells.

(A) UCSC Genome Browser screenshot of RNAPII at the FOS locus. ARID1A-KD reduces paused RNAPII at the 5’ of FOS, treatment with flavopiridol (2h) inhibits elongation and fully restores accumulation of RNAPII at the TSSs, similar to ARID1A-WT. (B) Box plot of RNAPII ChIP-seq binding at the TSS of 6,281 transcribed genes. We compare ARID1A-WT and ARID1A-KD conditions, with or without flavopiridol (FP). Upon elongation inhibition elicited by FP, accumulation of RNAPII in ARID1A-KD+FP is comparable or even higher than that of ARID1A-WT+FP, suggesting that ARID1A loss does not affect integrity of the pre-initiation complex but hinders accumulation of RNAPII at proximal promoters. (C) Comparison of RNAPII ChIP-seq at 6,281 TSS of flavopiridol treated wild type RMG-1 (ARID1A-WT + FP) and flavopiridol treated ARID1A depleted RMG-1 (ARID1A-KD + FP). The heatmap suggests similar accumulation of RNAPII at the proximal promoter in ARID1A-WT+FP and ARID1A-KD+FP cells. (D) Travelling ratio measures changes in RNAPII occupancy at proximal promoter vs. gene body (Fig. 4). Flavopiridol treated wild type RMG-1 (ARID1A-WT + FP) are compared to flavopiridol treated ARID1A depleted RMG-1 (ARID1A-KD + FP). We observe minor differences between the two conditions, further suggesting that flavopiridol fully restores physiological levels of pausing.

Figure 6 –. Upregulation of ARID1B restores RNAPII pausing and transcription at most genes.

(A) Immunoblot analysis of ARID1A and ARID1B during a time course of doxycycline (days 3, 5, 7). ARID1B is increasingly upregulated in response to ARID1A depletion, peaking at day 7. Quantitative immunoblots are shown in Supplemental Fig. S4H. (B) ChIP-qPCR analysis of RNAPII and ARID1B binding for a panel of ARID1A target genes at 0 hours (CTRL), 3 days, and 7 days of treatment with doxycycline. ARID1B gradually replaces ARID1A at the TSS, whilst RNAPII proximal-promoter occupancy is restored. Similarly, qRT-PCR for the same genes (fold enrichment compared to CTRL, normalized to 18S rRNA) suggests that transcription resumes its physiological level at day 7, concurrent with highest occupancy of ARID1B. (C) Parental RMG-1 cells were transduced with shRNAs against ARID1A and a full-length ARID1B vector. ChIP-qPCR for RNAPII, and qRT-PCR at three candidate loci, show that ARID1A-dependent transcriptional regulation is complemented (p<0.0111) by ARID1B (qRT-PCR fold change compared to shLUC, normalized by 18s rRNA). (D) Correlation plot (spike-in normalized RNA-seq) for all expressed coding genes shows that global transcriptional downregulation provoked by ARID1A depletion (day 3 of DOX induction, green) is largely rescued by ARID1B upregulation (day 7 of DOX induction, purple). Genes that are persistently dysregulated at day 7 have been functionally evaluated (IPA) and may represent ARID1A-specific targets.