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ARID1A influences HDAC1/BRD4 activity, intrinsic proliferative capacity and breast cancer treatment response

An Author Correction to this article was published on 31 January 2020

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Abstract

Using genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screens to understand endocrine drug resistance, we discovered ARID1A and other SWI/SNF complex components as the factors most critically required for response to two classes of estrogen receptor-alpha (ER) antagonists. In this context, SWI/SNF-specific gene deletion resulted in drug resistance. Unexpectedly, ARID1A was also the top candidate in regard to response to the bromodomain and extraterminal domain inhibitor JQ1, but in the opposite direction, with loss of ARID1A sensitizing breast cancer cells to bromodomain and extraterminal domain inhibition. We show that ARID1A is a repressor that binds chromatin at ER cis-regulatory elements. However, ARID1A elicits repressive activity in an enhancer-specific, but forkhead box A1-dependent and active, ER-independent manner. Deletion of ARID1A resulted in loss of histone deacetylase 1 binding, increased histone 4 lysine acetylation and subsequent BRD4-driven transcription and growth. ARID1A mutations are more frequent in treatment-resistant disease, and our findings provide mechanistic insight into this process while revealing rational treatment strategies for these patients.

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Fig. 1: CRISPR screens reveal ARID1A and BAF components as essential genes for treatment response.
Fig. 2: ARID1A knockout clonal cells show loss of response to ER antagonists but responsiveness to BET inhibitors.
Fig. 3: The SWI/SNF complex interacts with ER and is recruited to chromatin following drug treatment.
Fig. 4: FOXA1 promotes binding of ARID1A and BRG1 to a subset of potential enhancer elements.
Fig. 5: Loss of ARID1A results in decreased BRG1 and HDAC1 recruitment and increased histone H4 acetylation.
Fig. 6: Loss of ARID1A results in increased BRD4 recruitment and a gain in intrinsic proliferation.

Data availability

All CRISPR, ChIP-seq and RNA-seq data have been deposited at Gene Expression Omnibus and can be accessed at GSE123286. ATAC-seq data can be accessed at GSE134270. All proteomic data have been deposited at PRIDE and can be accessed at PXD011810. Source Data for Figs. 1 and 2 are available online.

Change history

  • 21 February 2020

    The Supplementary Information file initially published online was missing the Supplementary Figures and Note. The error has been corrected in the HTML version of the article.

  • 31 January 2020

    A Correction to this paper has been published: https://doi.org/10.1038/s41588-020-0582-9

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Acknowledgements

The authors thank Genomics, Proteomics, Bioinformatics, Preclinical genome editing, Histopathology and the Flow cytometry core facilities at CRUK Cambridge Institute. In particular, we thank C. D’Santos from Proteomics, A. Smith from Preclinical Genome Editing and M. Clayton, G. Cronshaw, R. Ellis and all the staff at CRUK. PX458 was a kind gift from F. Zhang and BRD4 antibody from C. M. Chiang. We acknowledge the suggestions from Z. Najafova in regard to mouse experiments, and A. Nicholson, M. Sen, X. Wang and S. A. Johnsen for reagents and antibodies. We thank R. Rony for his help in graphic design and models. We acknowledge the support of the University of Cambridge and Cancer Research UK. J.S.C. is supported by an ERC Consolidator award (no. 646876), a Susan G Komen leadership grant and CRUK core funding. S.N. is supported by a Komen grant. S.V.R. is funded by Innovation postdoc granted by the Norwegian Research Council. R.S. is funded by the Novo Nordisk Foundation (no. NNF15OC0014136).

Author information

Authors and Affiliations

Authors

Contributions

S.N. contributed to conceptualization, methodology, experimental work, formal analysis and figure assembly, writing of text, reviewing and advising on the manuscript. S.V.R. contributed to experimental work, analysis and figure assembly of mouse experiments and advising on the manuscript. J.S. contributed to experimental work, analysis and figure assembly of mouse experiments and advising on the manuscript. D.C. and E.A.G. contributed to experimental work and advising on the manuscript. S.D., S.-E.G., A.R. and S.K. contributed to methodology and advising on the manuscript. E.K.P. contributed to experimental work, formal analysis and figure assembly and advising on the manuscript. J.-E.G.P. contributed to experimental work and advising on the manuscript. D.-L.C. contributed to statistical analyses, figure assembly, reviewing and advising on the manuscript. K.K. and C.S.R.C. contributed to bioinformatic analyses, figure assembly and advising on the manuscript. C.B. contributed to histopathological analyses and advising on the manuscript. N.G. and R.N. performed ARID1A immunohistochemistry and advising on the manuscript. A.B. contributed to methodology regarding PDX material and advising on the manuscript. C.C. contributed to methodology regarding PDX material, funding acquisition and advising on the manuscript. R.S. contributed to methodology, supervision and advising on the manuscript. K.Y. contributed to methodology, funding acquisition and advising on the manuscript. I.C. contributed to methodology, bioinformatic analyses and figure assembly, writing of text and advising on the manuscript. J.S.C. contributed to conceptualization, supervision, funding acquisition, writing of text, reviewing and advising on the manuscript.

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Correspondence to Jason S. Carroll.

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Competing interests

J.S.C. is the founder and chief scientific officer of Azeria Therapeutics.

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Extended data

Extended Data Fig. 1 Enrichment of BAF and P-BAF components in the CRISPR screen.

a. Scatterplot of CRISPR screening data, showing enrichment of BAF components following 26 days of different drug treatment, relative to DMSO treated control cells. n = 3 independent viral infections. b. Log2 fold changes showing gRNA enrichment/depletion against all BAF, P-BAF and ncBAF components in the CRISPR screen. Treatment conditions are compared to DMSO control. More proliferative changes represent enriched gRNA after treatment, indicating genes that contribute to drug resistance. c, e. Validation of ARID1A perturbation effect on proliferation and drug response using ARID1A siRNA on MCF7 (c) and ZR-75-1 (e), representative experiments shown from 2 similar independent experiments each cell line. p-values calculated by One way ANOVA test. * denote p < 0.05, *** denotes p < 0.001. Sample size mentioned in S4. Measure of centre represents mean ± SEM (c) and mean ± SD (e). d. Western blot of ARID1A protein levels after siRNA transfection in MCF7 cells. A representative image is shown from 3 similar independent experiments. Unprocessed Western blot in Source Data Fig. 2.

Extended Data Fig. 2 ARID1A co-binds ER and FOXA1-bound regulatory elements, but is depleted with estrogen treatment.

a-c. Single gene profiles showing the binding of ER, FOXA1 and ARID1A on overlapping sites in MCF7 cells. ChIP-seq was performed using three independent biological cell cultures. d. Overlap of binding sites for ER, FOXA1 and ARID1A binding sites in ZR-75-1 cells. e. Boxplots showing the normalized ChIP-seq tag density around 400 bp window around the center of ARID1A binding on DiffBind-defined estrogen independent (constant) and dependent (reduced with estrogen) sites in MCF7. Both classes show reduced ARID1A binding upon estrogen. p-values were calculated by Welch’s t-test, two-sided. Centre line shows the median values with bounds of box corresponding to the first and third quartiles and the upper and lower whiskers extend to the largest or the smallest value no further than 1.5 × IQR (inter-quartile range). Statistical test details are mentioned in Supplementary Table 5e.

Extended Data Fig. 3 Enrichment of SWI/SNF factors with ER and FOXA1 in RIME.

a. ARID1A and BRG1 RIME were conducted on asynchronous MCF7 cells on two biological cell cultures. Label free quantification was performed to show the log 2 scaled normalized intensities of the BAF, P-BAF, ncBAF and common subunits of SWI/SNF complex. Rabbit polyclonal IgG is used as the negative control. b. ER qPLEX-RIME was performed on five primary tumours from ER + breast cancer patients and the ER interactors are shown as enrichment over IgG vs -log10 p-value, corrected by Benjamini and Hochberg multiplicity correction, two-sided. c, d. Boxplots illustrating the more enrichment of HDAC1 (c) and less enrichment of random factors (d) in ERα RIME in five patients compared to IgG negative control in human breast tumours. The values are scaled to the median of IgG and log2 transformed. e. Boxplots illustrating the enrichment of selected known ERα interactors from the RIME experiment in MCF7 cells at a representative timepoint (4-hydroxytamoxifen- 24 hrs) comparing to IgG negative control. The values are scaled to the median of IgG and log2 transformed. n = 5 independent biological cell cultures. For all boxplots, Centre line shows the median values with bounds of box corresponding to the first and third quartiles and the upper and lower whiskers extend to the largest or the smallest value no further than 1.5 × IQR (inter-quartile range).

Extended Data Fig. 4 Enrichment of SWI/SNF factors during Tamoxifen and Fulvestrant in ChIP-seq experiments.

a-d. Asynchronous MCF7 cells were treated with vehicle or Fulvestrant, an ER degrader and ChIP-seq was conducted for ARID1A (b), BRG1 (c) or SNF5 (d). Triplicate independent cell cultures were conducted. d. Single gene profile showing the induction of SWI/SNF complex binding during Fulvestrant treatment. e. Overlap of ARID1A lost sites during estrogen treatment with gained sites during Tamoxifen and Fulvestrant from three independent biological cell cultures. f. Overlap of ARID1A gained sites during Tamoxifen treatment with Fulvestrant and Tamoxifen downregulated genes.

Extended Data Fig. 5 FOXA1 promotes the binding of ARID1A and BRG1.

Hormone-deprived ZR-75-1 cells were transfected with control or FOXA1 siRNA and ChIP-seq was conducted for ARID1A (a) and BRG1 (b). n = 3 independent biological cell cultures. MA plots are shown with the average intensity of binding vs log2 fold change with FOXA1 siRNA relative to control siRNA. c. Scatterplot showing the association of the loss of ARID1A and BRG1 binding upon FOXA1 knockdown. PCC – Pearson Correlation coefficient, two-sided. d. Heatmaps shown on ARID1A and BRG1 FOXA1 independent (common) and dependent (lost sites with FOXA1 knockdown) sites in ZR-75-1 cells. e. Boxplots showing the normalized ChIP-seq tag density around 400 bp window of ARID1A and BRG1 on FOXA1 independent (constant, n = 70,429 sites) and dependent (lost sites with siFOXA1, n = 17,357 sites) sites in ZR-75-1. p-value calculated by Welch’s test, two-sided. n = 3 independent biological cell culture samples. Centre line shows the median values with bounds of box corresponding to the first and third quartiles and the upper and lower whiskers extend to the largest or the smallest value no further than 1.5 × IQR (inter-quartile range). Statistical test details are mentioned in Supplementary Table 5f.

Extended Data Fig. 6 FOXA1 promotes the binding of ARID1A and BRG1.

Hormone-deprived MCF7 and ZR-75-1 cells were transfected with control or FOXA1 siRNA and ChIP-seq was conducted for ARID1A and BRG1. n = 3 independent biological cell cultures. (a-b) Single gene profiles of CCND1 (a) and CDH1 (b) showing the effect on SWI/SNF complex binding with FOXA1 knockdown on MCF7 and ZR-75-1 cells. ER and FOXA1 binding overlap is shown. (c-d) ChIP-qPCR analyses on specific sites (CCND1 and CDH1 ER binding sites) showing ARID1A and BRG1 binding with FOXA1 knockdown in hormone-deprived MCF7 and ZR-75-1 cells (c) or ARID1A binding following Tamoxifen treatment in asynchronous MCF7 cells (d). n = 3 independent biological cell cultures. * denotes p ≤ 0.05, ** denotes p ≤ 0.01, *** denotes p ≤ 0.001. Precise p-values are mentioned in Supplementary Fig. 10. Mean is measured as centre shown with standard deviation. Details of the statistical tests are mentioned in Supplementary Fig. 10.

Extended Data Fig. 7 ATAC-seq analyses shows a negligible regulation of ARID1A on transcription-associated chromatin opening.

a. Heatmap showing ATAC-seq analysis in ARID1A KO clones 11 and 14 following Tamoxifen treatment. Common, gained and lost sites defined by DiffBind analysis. n = 4 independent biological cell cultures. FDR ≤ 0.05 corrected by Benjamini-Hochberg multiplicity correction, two-sided. b. Association of ARID1A KO upregulated and downregulated genes with ATAC-seq gained and lost sites.

Extended Data Fig. 8 ARID1A perturbation regulates ARID2 binding.

a. ARID2 ChIP-seq was conducted in wild type cells or the two ARID1A knock-out clonal cell lines and heatmaps are shown on ARID2 binding sites after Tamoxifen treatment. Also included was ARID1A ChIP-seq from wild type cells treated with vehicle or Tamoxifen. ARID2 binding overlapped with ARID1A binding and was dependent on ARID1A. n = 3 independent biological cell cultures. b. Signal intensity plot showing changes in ARID2 binding in wild type control cells or ARID1A knock-out cells at ARID2 binding sites. n = 3 independent biological cell cultures.

Extended Data Fig. 9 ARID1A promotes BRG1 and HDAC1 binding without affecting ER and H3K27ac occupancy.

a, b. BRG1, H3K27Ac, HDAC1 and ER (b) ChIP-seq were conducted in asynchronous wild type cells treated with vehicle or tamoxifen or in the two ARID1A knock-out clones (Clones 11 and 14) following tamoxifen treatment. The binding is shown on regions where HDAC1 is lost in ARID1A knockout cells relative to wild type cells. n = 3 independent biological cell cultures. c, d. Scatterplot showing the correlation of ER (c) or H3K27Ac (d) and HDAC1 binding in ARID1A knockout clone 11 versus wild type cells. n = 3 independent biological cell cultures. PCC – Pearson Correlation coefficient. p-values were calculated by Pearson correlation test, two-sided. e. Principal Component Analysis (PCA) of normalised peptide intensities of PDX tumours after ER qPLEX-RIME. n = 2 PDX each group. f. Details of ARID1A mutations observed within ER + PDX tumours used in ER qPLEX-RIME.

Extended Data Fig. 10 ARID1A regulates histone H4 acetylation.

Upregulation of histone H4 acetylation in ARID1A knock-out clone 11 and 14 in Vehicle (a) or Tamoxifen (b) treated cells comparing to wild type cells. Heatmap representing the changes in histone H4Ac marks upon ARID1A knockout with Vehicle or Tamoxifen treatment on ER binding sites close to ARID1A repressed genes. n = 3. (c) Empirical cumulative probability distribution plots of H4K8Ac and H4K12Ac ChIP-seq signals showing upregulation in intensity (y-axis) with ARID1A knockouts clones 11 and 14. Plots were made on ER sites close to ARID1A repressed genes (n = 686 sites) with more than 75% contribution to the variance in intensity. Window – 2 kb around the center of binding.

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Source Data Fig. 1

Unprocessed Western Blots for Extended Data Fig. 1d.

Source Data Fig. 2.

Unprocessed Western Blots for Fig. 2a.

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Nagarajan, S., Rao, S.V., Sutton, J. et al. ARID1A influences HDAC1/BRD4 activity, intrinsic proliferative capacity and breast cancer treatment response. Nat Genet 52, 187–197 (2020). https://doi.org/10.1038/s41588-019-0541-5

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