ARID1A deficiency weakens BRG1-RAD21 interaction that jeopardizes chromatin compactness and drives liver cancer cell metastasis

Abstract

ARID1A, encoding a subunit of SWI/SNF chromatin remodeling complex, is widely recognized as a tumor suppressor gene in multiple tumor types including liver cancer. Previous studies have demonstrated that ARID1A deficiency can cause liver cancer metastasis, possibly due to the altered chromatin organization, however the underlying mechanisms remain poorly understood. To address the effect of Arid1a deficiency on chromatin organization, we generated chromatin interaction matrices, and exploited the conformation changes upon Arid1a depletion in hepatocytes. Our results demonstrated that Arid1a deficiency induced A/B compartment switching, topologically associated domain (TAD) remodeling, and decrease of chromatin loops. Further mechanism studies revealed that ATPase BRG1 of SWI/SNF complex could physically interact with RAD21, a structural subunit of chromatin architectural element cohesin; whereas ARID1A deficiency significantly diminished the coupled BRG1-RAD21. Interestingly, the tumor-associated genes within the switched compartments were differentially expressed depending upon Arid1a depletion or not. As a consequence of ARID1A deficiency-induced conformational alteration, the dysregulation of some genes such as PMP22 and GSC, promoted the invasion capacity of liver cancer cells. This study provides an insight into liver cancer tumorigenesis and progression related to ARID1A mutations.

Fig. 1. Arid1a deficiency alters chromatin conformation.

A Schematic flowchart of the immortalized primary hepatocytes termed AB17, isolated from the mouse with Arid1a^fl/fl genotype and infected by SV40LT and pLPC-H-Ras V12D viruses successively (Left). PCR and Western blotting to identify the knockout effect of AB17 cells post Ad-GFP or Ad-CRE infection (Right). B The comparison on chromatin 3D simulation of Chromosome 2 (Chr2), Chr11, and Chr12 between Arid1a WT (Ad-GFP) and KO (Ad-Cre) AB17 cells. C All chromatins at 1-Mb resolution (Top), a zoom-in of Chr11 at 250-kb resolution (Middle) and Chr11: 60-75 Mb at 25-kb resolution (Bottom) of the replicates and combined contact metrics in Arid1a WT (Ad-GFP) and KO (Ad-Cre) AB17 cells. D Hi-C contact maps of Chr2 in Arid1a WT (Ad-GFP) and KO (Ad-Cre) AB17 cells at 500-kb resolution (Top). Compartment A (red) and B (blue) are shown by PC1 eigenvectors (Bottom). Dashed boxes indicate the representative compartment switch in Chr2. E Percentages of compartment switching between Arid1a WT (Ad-GFP) and KO (Ad-Cre) AB17 cells. F The comparison on TAD number (Left) and average size in Mb (Right) between Arid1a WT (Ad-GFP) and KO (Ad-Cre) cells at 50-kb resolution. G Examples of TAD (encircled by dotted triangles) alteration in number and size. Shown in Hi-C contact maps, the larger domains were often observed in Arid1a KO (Ad-Cre) cells (Left, 15-35 Mb in Chr4); TADs were merged or vanished causing the visible reduction in number accompanied by the significantly larger sizes in Arid1a KO (Ad-Cre) cells (Right, 77-97 Mb in Chr14). H The statistics of changes in border strength induced by Arid1a deficiency from three categories: stronger, unchanged, and weaker. I The comparison on chromatin loop number between Arid1a WT and KO cells at 25-kb resolution.

Fig. 2. SWI/SNF complex associates with chromatin architecture via BRG1-RAD21 axis.

A-C. Endogenous co-IP assay with anti-ARID1A, CTCF, and RAD21 antibodies was performed in ARID1A WT and KO AB17 A, MEF cells (193-4), B and human HCC MHCC-97H cells C, and then blotting assay detected with the indicated antibodies. D Validation for the interaction between ARID1A/BRG1 and architectural elements CTCF/RAD21 in human liver cancer cell lines MHCC-97H, SK-HEP-1 and HepG2. E HEK293T cells transfected with the indicated constructs were subjected to co-IP assay with anti-T7 and -HA antibodies to verify the interaction between ARID1A and CTCF (Top) or RAD21 (Bottom). F The same as E but for detecting the interaction between BRG1 and CTCF (Top) or RAD21 (Bottom) with anti-Flag and -HA antibodies. G Validation for the interaction between BRG1 and RAD21 by co-IP assay in Flag-tagged BRG1-transfected HEK293T cells. H Glutathione S-transferase (GST) pull-down experiments using GST-RAD21 protein and in vitro translated BRG1 protein. BRG1 protein binding to GST or GST-RAD21 was detected by the anti-BRG1 antibody. I Immunofluorescent staining showing the colocalization of both BRG1 (green) and RAD21 (red) in mouse AB17, MEF cells (193-4), as well as human liver cancer MHCC-97H and SK-HEP-1 cells (Scale bar, 5 µm). J The ChIP-seq peaks of Ctcf, Rad21, Arid1a, and Brg1 were extensively overlapped in the genome distribution.

Fig. 3. ARID1A deficiency weakens BRG1-RAD21 axis via BRG1 suppression.

A Glycerol sedimentation assay with 5 to 35% gradients for detecting the co-segregation of Brg1 and Rad21 in nuclear fractions in Arid1a WT and KO AB17 cells. The dotted lines enclose the co-density stratifications of Brg1 and Rad21. B Sucrose sedimentation (5 to 50%) assay in Arid1a WT and KO AB17 cells. The co-fractionation of Brg1 and Rad21 circled with dotted lines. C, D The same as A and B respectively but in HCC cell line MHCC-97H. E HEK293T cells transfected with the T7-ARID1A construct exhibited no significant impact on CTCF and RAD21 (Left). Similarly, no impact on ARID1A in CTCF (Medium) and RAD21 over-expressed cells (Right). F Neither CTCF nor RAD21 was effected by ARID1A deficiency in AB17 (Left) and MHCC-97H cells (Right). G Brg1 mRNA expression levels in Arid1a WT and KO AB17 cells, Q506 cells, primary hepatocytes, MEFs (193-4 and 102-2 cells), and HCC cell line MHCC-97H with three biological replicates. Error bars were painted by SEM. p value was calculated by a two-tailed Student’s t-test. mRNA expression of Gapdh was employed as an internal control.

Fig. 4. Compartment switching dysregulates cancer-related genes.

A Volcano plot showing the DEGs (fold change >2, p value <0.05) between Arid1a WT (Ad-GFP) and KO (Ad-Cre) AB17 cells. The green and red dots represent down- and up-regulated genes post Arid1a deficiency, respectively. B The enrichment analysis on down- (Left) and up-regulated (Right) genes. The top ten significantly enriched terms were shown (p value <0.01). The color-coded circles symbolize the enriched GO terms and pathways. C Fold change (Ad-Cre/Ad-GFP log2) of mRNA expression (FPKM) of the genes residing at regions with compartment switching. D Venn diagrams displaying the overlap of DEGs and A/B compartment switch-related genes (Left). The heatmap showing the expression profiles of the switching compartments controlled-genes with triplicate datasets, of which down- and up-regulated cancer-related genes labelled by green and red pentagrams, respectively (Right). E qRT-PCR for the compartment switching-controlled genes in Arid1a WT (Ad-GFP) and KO (Ad-Cre) AB17 and Q506 hepatocytes as well as MEFs (193-4 and 102-2 cells) with three biological replicates. Error bars were painted by SEM. p value was calculated by a two-tailed Student’s t-test. mRNA expression of Gapdh was employed as an internal control.

Fig. 5. Weakened chromatin loops and remodeled TADs cause aberrant transcription.

A Hi-C contact maps of Pmp22 and Gsc, which were dysregulated in the switched compartments caused by Arid1a deficiency. TADs (Contact domains) are visible as triangle-shaped regions with a high frequency of interactions, and the position information are labelled in black font. Locally enriched peaks discovered via HiCCUPS are referred to as chromatin loops, and location coordinates are shown in green (the target gene) and yellow (the potential enhancers) font in line with the colors of graphic symbols. B Expanded views of the potential enhancer locus and proximal promoter of Pmp22, with H3K4me1 and H3K27ac ChIP-seq tracks for recognition on the potential enhancers, and Ctcf and Rad21 ChIP-seq tracks for pointing the anchored loops in AB17 cells. Vertical black bars label the enhancers and promoter primers examined in the 3C assay. C Identification on enhancers of E-P loops in Pmp22. The position of primer for the enhancer is labelled by the vertical red bar. Histone marks H3K27ac and H3K4me1 were significantly enriched at enhancer locus of Pmp22 in AB17 with IgG as control. D Curve graphs indicate the significantly reduced ligation frequencies at E-P anchored loops of Pmp22 in Arid1a-deficient cells. E Validation on a regulatory pattern of the up-regulated gene Gsc within switched A compartment. H3K27ac and H3K4me3 ChIP-seq tracks were obtained from AB17 cells and ENCODE database (H3K27ac: ENCSR000CDH; H3K4me3: ENCSR000CAP), and the location of enhancer boxed with the yellow rectangle. F A significantly increased the H3K27ac signal of the Gsc gene detected in Arid1a knockout cells with IgG as control.

Fig. 6. ARID1A deficiency promotes liver cancer cell metastasis via dysregulation on PMP22 and GSC.

A Immunoblotting on Pmp22 and Gsc in liver tissues of 3 and 14 month old Arid1a^fl/fl mice and Arid1a^LKO littermates. B Representative IHC staining (Left) and quantification of positive staining (Right) for Pmp22 and Gsc in liver tissues of 3-month-old Arid1a^fl/fl mice and Arid1a^LKO littermates. Scale bar = 50 μm. Data are presented as the mean ± SEM (n = 5 per group). *p < 0.05, **p < 0.01, calculated by a two-tailed Student’s t-test. C Representative images of invasive MHCC-97H cells transfected with Pcdh-PMP22 construct; lentiviral shARID1A-1, and ARID1A-knockdown cells co-transfected with Pcdh-PMP22 in transwell assays (Scale bar, 100 µm) (Left) and quantification of the invasive cells (Right). Data are shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, calculated by a two-tailed Student’s t-test. D Representative images and quantification of the invasive MHCC-97H cells treated with the indicated lentiviral shRNA plasmids for ARID1A (shARID1A-1) and GSC (shGSC-1) in transwell assays. E The relapse-free survival and the overall survival in HCC patients with high or low expression of PMP22 (Left) and GSC (Right), examined by Kaplan-Meier analysis. F Schematic diagram of the dysregulated mechanism related to PMP22 and GSC loci upon ARID1A deficiency. Based on our data, we proposed the working model of the SWI/SNF complex in maintaining chromatin organization mediated by the BRG1-RAD21 axis in hepatocytes. Once ARID1A is deficient, the chromatin loop tethering promoter to the enhancer of Pmp22 might be weakened due to the attenuated anchorage force arising from insufficient BRG1 (Top); the unaided boundaries lead to TAD remodeling surrounding Gsc locus to some extent (Bottom).