Effects of dominant-negative Smarca4 mutations on the DNA accessibility landscape [ATAC-seq]
Ontology highlight
ABSTRACT: We generated a library of Smarca4 variants with mutations in conserved regions of the N-terminal ATPase domain based on mutants observed in primary tumors and cancer cell lines. Heterozygous expression of Smarca4 ATPase mutants led to decreased accessibility at active enhancers in mouse embryonic stem cells.
Project description:We investigated transcriptomic effects of a Smarca4 ATPase mutant observed in primary tumors and cancer cell lines. Heterozygous expression of G784E Smarca4 mutant led to transcriptional changes compared to cells expressing only wild-type Smarca4.
Project description:We investigated the chromatin effects of a Smarca4 ATPase mutant observed in primary tumors and cancer cell lines. Compared to cells expressing only wild-type Smarca4, heterozygous expression of G784E Smarca4 mutant led to reduction of H3K27ac and RNAP at a set of enhancer sites.
Project description:Mutation of SMARCA4 (BRG1), the ATPase of BAF (mSWI/SNF) and PBAF complexes, contributes to a range of malignancies and neurologic disorders. Unfortunately, the effects of SMARCA4 missense mutations have remained uncertain. Here we show that SMARCA4 cancer missense mutations target conserved ATPase surfaces and disrupt the mechanochemical cycle of remodeling. We find that heterozygous expression of mutants alters the open chromatin landscape at thousands of sites across the genome. Loss of DNA accessibility does not directly overlap with Polycomb accumulation, but is enriched in 'A compartments' at active enhancers, which lose H3K27ac but not H3K4me1. Affected positions include hundreds of sites identified as superenhancers in many tissues. Dominant-negative mutation induces pro-oncogenic expression changes, including increased expression of Myc and its target genes. Together, our data suggest that disruption of enhancer accessibility represents a key source of altered function in disorders with SMARCA4 mutations in a wide variety of tissues.
Project description:Mammalian SWI/SNF complexes are multi-subunit chromatin remodeling complexes associated with an ATPase, either SMARCA4 or SMARCA2. Heterozygous mutations in the SMARCA2 ATPase cause Nicolaides-Baraitser Syndrome (NCBRS), an intellectual disability syndrome associated with delayed speech onset. We engineered human embryonic stem cells (hESCs) to carry NCBRS-associated heterozygous SMARCA2 K755R or R1159Q mutations. While SMARCA2 mutant hESCs were phenotypically normal, differentiation to neural progenitors cells (NPCs) was severely impaired. We find that SMARCA2 mutations cause enhancer reorganization with loss of SOX3-dependent neural enhancers and prominent emergence of astrocyte-specific de novo enhancers. Changes in chromatin accessibility at enhancers were associated with an increase in SMARCA2 binding and retargeting of SMARCA4. We show that AP-1 family member FRA2 is aberrantly overexpressed in SMARCA2 mutant NPCs, where it functions as a pioneer factor at de novo enhancers. Together, our results demonstrate SMARCA2 mutations cause impaired differentiation through enhancer reprogramming via inappropriate targeting of SMARCA4.
Project description:We generated a library of Brg variants with mutations in conserved regions of the N-terminal ATPase domain based on mutants observed in primary tumors and cancer cell lines. Heterozygous expression of ATPase mutants leads to increased occupancy of Polycomb Repressive Complex 1 (PRC1) at bivalent CpG-island promoters. Increased PRC1 binding was accompanied by increases in H3K27me3, the mark left by the Polycomb Repressive Complex 2 (PRC2) ~2 kbp away.
Project description:Using ATAC-seq, we identified the accessibility changes in lung cancer cells (NCI-H1944) reconstituted with SMARCA4 WT or SMARCA4 mutants. We also determined the SMARCA2-regulated accessibility program in NCI-H1944 after SMARCA2 depletion. Lastly, we identified the SMARCA2 accessibility program that is rescued by SMARCA4 WT and mutants. The "SAMPLE_ID" sample characteristic is a sample identifier internal to Genentech.
Project description:SMARCA2 and SMARCA4 are two mutually exclusive ATPase subunits of SWI/SNF complex. SMARCA4 deficient lung cancer population selectively depend on SMARCA2 for cancer growth phenotype. Rescue experiments with ectopic expression of wild-type, bromodomain mutant and ATPase dead SMARCA2 and SMARCA4 highlight that ATPase domain is the drug target. In this study, we performed genome-wide microarray and differential gene expression profiling on isogenic lung cancer lines expressing cDNA rescue constructs for wild-type, bromodomain mutant and ATPase dead SMARCA2 and SMARCA4