ARID1A loss impairs enhancer-mediated gene regulation and drives colon cancer in mice

Abstract

Genes encoding subunits of SWI/SNF (BAF) chromatin-remodeling complexes are collectively mutated in ∼20% of all human cancers. Although ARID1A is the most frequent target of mutations, the mechanism by which its inactivation promotes tumorigenesis is unclear. Here we demonstrate that Arid1a functions as a tumor suppressor in the mouse colon, but not the small intestine, and that invasive ARID1A-deficient adenocarcinomas resemble human colorectal cancer (CRC). These tumors lack deregulation of APC/β-catenin signaling components, which are crucial gatekeepers in common forms of intestinal cancer. We find that ARID1A normally targets SWI/SNF complexes to enhancers, where they function in coordination with transcription factors to facilitate gene activation. ARID1B preserves SWI/SNF function in ARID1A-deficient cells, but defects in SWI/SNF targeting and control of enhancer activity cause extensive dysregulation of gene expression. These findings represent an advance in colon cancer modeling and implicate enhancer-mediated gene regulation as a principal tumor-suppressor function of ARID1A.

Figure 1. ARID1A loss drives invasive colon adenocarcinoma in mice

(a) Survival of MX1-Cre Arid1a^fl/fl mice (n=26) and control Arid1a^fl/fl mice (n=16) following injection of Poly I:C; (b) MX1-Cre Arid1a^fl/fl mouse with tumors in the (1) cecum, (2) mid-colon, and (3) rectum; (c) H&E staining on normal colon epithelium (left) and tumor (right) tissue sections from MX1-Cre Arid1a^fl/fl mouse; (d) ARID1A immunohistochemistry (IHC) on above tissue sections; (e) Alcian blue staining on tumor section from MX1-Cre Arid1a^fl/fl mouse; (f) ARID1A IHC on tumor section from MX1-Cre Arid1a^fl/fl mouse showing lymphocytic infiltrate.

Figure 2. ARID1A loss drives colon tumorigenesis independent of APC inactivation

(a) β-catenin IHC in wildtype mouse colon and tumor tissue sections from MX1-Cre Arid1a^fl/fl and Villin-Cre^ER-T2 Arid1a^fl/fl mice; (b) Total tumor count in the small intestine and colon of Apc^Min (n=12) and Apc^Min:Arid1a^KO mice (n=12), p-value shown from unpaired two-tailed T-test; (c) Colon adenoma in Apc^Min mouse with H&E staining, ARID1A IHC, and β-catenin IHC (β-catenin IHC magnification shown for marked tumor region); (d) Same as (c) for colon adenoma in Apc^Min:Arid1a^KO mouse.

Figure 3. ARID1A loss causes defects in SWI/SNF targeting to chromatin

a) Live cell morphology of HCT116 ARID1A WT, ARID1A^+/−, and Arid1a^−/− cells in culture; b) Proliferation measured by MTT assay, error bars show standard deviation of 3 measurements (technical replicates); c) Invasion measured by Matrigel-chamber based assay, error bars show standard deviation of 4 measurements (technical replicates); d) Protein levels of ARID1A, E-Cadherin, and β-actin; e) Protein levels of Vimentin and β-actin (Note: NIH 3T3 fibroblast cells included for positive control); f) Fold change (log₂) in SMARCA4 and SMARCC1 ChIP-Seq signals at SWI/SNF binding sites in Arid1a^−/− cells relative to WT; g) Immunoprecipitation of SWI/SNF complexes using antibodies targeting ARID1A and ARID1B; h) Protein levels of ARID1B and β-actin following ARID1B knockdown with 3 independent shRNAs; i) Proliferation following shRNA-mediated ARID1B knockdown measured by MTT assay; error bars show standard deviation of 3 measurements (technical replicates).

Figure 4. SWI/SNF complexes are targeted to enhancers and contribute to their activity

a) Distribution of SWI/SNF binding sites in HCT116 WT and Arid1a^−/− cells relative to histone modifications (Note: difference between WT and Arid1a^−/− cells is not significant in a paired t-test); b) ChIP-seq profiles of SMARCA4, SMARCC1, H3K4me3, H3K4me1, and H3K27ac in WT and ARID1A^−/− cells around all TSS-distal SWI/SNF binding sites (Notes: labels on the right of the figure indicate number of sites in each category; labels on top right corners indicate any alterations made in scaling of Y-axis); c) H3K27ac levels in WT and ARID1A^−/− cells at TSS-proximal (promoter) and TSS-distal (enhancer) enrichment regions (Note: numbers in the three corners denote numbers of activated (>2×), inactivated (<1/2×), and stable sites); d) Fold changes (log₂) of gene expression between WT and Arid1a^−/− cells for genes nearest to TSS-distal SWI/SNF binding sites split based on Arid1a^−/− / WT ChIP-Seq signal; e) ChIP-seq tracks of SMARCA4, SMARCC1, H3K4me3, H3K4me1, and H3K27ac in WT and ARID1A^−/− cells.

Figure 5. ARID1A loss impairs enhancer-mediated gene regulation in the colonic epithelium

a) Observed vs. expected TF motif instances at TSS-distal H3K27ac regions (enhancers) with reduced H3K27ac signal based on enrichment regions with stable H3K27ac signal. Motifs highly similar to AP1 and CTCF motifs are highlighted; b) Correlation between H3K27ac signal change (Arid1a^−/− / WT) and WT ChIP-Seq signal levels of different factors profiled in this work and by the ENCODE Project. c) ChIP-Seq profiles for SMARCA4, SMARCC1, JUND, FOSL1, and CTCF in WT HCT116 cells centered around TSS-distal H3K27ac regions (enhancers) that remain stable, show lost/weakened H3K27ac, or show gained/strengthened H3K27ac in Arid1a^−/− cells relative to WT; d) H3K27ac levels at TSS-proximal (promoter) and TSS-distal (enhancer) enrichment regions for colon epithelium from wildtype (WT) and Villin-Cre^ER-T2 Arid1a^fl/fl (Arid1a^−/−) mice (Note: numbers in the three corners denote numbers of activated (>2×), inactivated (<1/2×) and stable sites); e) Fold changes (log₂) of gene expression between WT and Arid1a^−/− mouse colon epithelium for genes nearest to TSS-distal H3K27ac regions (enhancers) split based on Arid1a^−/− / WT ChIP-Seq signal; f) Pearson's correlation among RNA-Seq samples based on FPKM values for mouse colon epithelium dissociated from individual wildtype mice (WT, n=3) and Villin-Cre^ER-T2 Arid1a^fl/fl mice (Arid1a^−/−, n=2); G) H3K7ac ChIP-Seq tracks, super-enhancer (SE) calls, and RNA-Seq tracks at Gsdmc locus in WT and Arid1a^−/− mouse colon epithelium. H) RNA-Seq FKPM values for individual WT and Arid1a^−/− mice for Gsdmc, Gsdmc2, Gsdmc3, and Gsdmc4.

Figure 6. Defective SWI/SNF targeting and control of enhancer activity in the ARID1A-deficient colonic epithelium (Model)!