Perturbations to mammalian SWI/SNF (BAF) complexes contribute to over 20% of human cancers, with driving roles first identified in malignant rhabdoid tumor (MRT), an aggressive pediatric cancer characterized by biallelic inactivation of the core BAF complex subunit SMARCB1 (BAF47). However, the mechanism by which this alteration contributes to tumorigenesis remains poorly understood. We find that BAF47 loss destabilizes BAF complexes on chromatin, absent significant changes in intra-complex integrity. Rescue of BAF47 in BAF47-deficient sarcoma cell lines results in increased genome-wide BAF complex occupancy, facilitating widespread enhancer activation and opposition of polycomb-mediated repression at bivalent promoters. We demonstrate differential regulation by BAF and PBAF complexes at enhancers and promoters, respectively, suggesting distinct functions of each complex which are perturbed upon BAF47 loss. Our results demonstrate collaborative mechanisms of mSWI/SNF-mediated gene activation, identifying functions that are coopted or abated to drive human cancers and developmental disorders.

Trithorax-group genes and mammalian homologues, including BAF (mSWI/SNF) complexes, have been known for nearly 30 years to oppose Polycomb repressive activity1–5. This opposition underlies the tumor-suppression role of BAF3,5–7 and is expected to contribute to neurodevelopmental disorders, as evidenced by frequent driving mutations8,9. However, the mechanisms underlying opposition to Polycomb silencing are poorly understood. Here we report that recurrent disease mutations of BAF subunits induce genome-wide increases in Polycomb complex deposition and activity. We show that point mutations of the Smarca4 (Brg) ATPase domain cause loss of direct binding between BAF and PRC1 that occurs independently of chromatin. Release of this direct interaction occurs via an ATP-dependent mechanism, consistent with a role as a transient intermediate of eviction. Using a new in vivo assay, we find that BAF directly evicts Polycomb factors within minutes of its occupancy, together establishing a new mechanism for the widespread opposition underlying development and disease.

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 induced 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 SMARCA4-mutated disorders in a wide variety of tissues.

We report a single-cell chromatin immunocleavage sequencing (scChIC-seq) methodology for analyzing histone modifications, which involves targeting of the micrococcal nuclease (MNase) by tethering it to an antibody and selective PCR amplification of cleaved target sites. We show that the protocol reliably detects the H3K4me3 and H3K27me3 target sites in single human white blood cells (WBC), resulting data for successful identification of unique blood cell types based on clustering analysis.

Topoisomerase 3β (Top3β) is the only dual-activity topoisomerase in animals that can change topology for both DNA and RNA, and facilitate transcription on DNA and translation on mRNAs. Top3β mutations have been linked to schizophrenia, autism, epilepsy, and cognitive impairment. Here we show that Top3β knockout mice exhibit behavioural phenotypes related to psychiatric disorders and cognitive impairment. The mice also display impairments in hippocampal neurogenesis and synaptic plasticity. Notably, the brains of the mutant mice exhibit impaired global neuronal activity-dependent transcription in response to fear conditioning stress, and the affected genes include many with known neuronal functions. Our data suggest that Top3β is essential for normal brain function, and that defective neuronal activity-dependent transcription may be a mechanism by which Top3β deletion causes cognitive impairment and psychiatric disorders.

Modern next-generation sequencing-based methods have empowered researchers to assay the epigenetic states of individual cells. Existing techniques for profiling epigenetic marks in single cells often require the use and optimization of time-intensive procedures such as drop fluidics, chromatin fragmentation, and end repair. Here we describe ACT-seq, a novel and streamlined method for mapping genome-wide distributions of histone tail modifications, histone variants, and chromatin-binding proteins in a small number of or single cells. ACT-seq utilizes a fusion of Tn5 transposase to Protein A that is targeted to chromatin by a specific antibody, allowing chromatin fragmentation and sequence tag insertion specifically at genomic sites presenting the relevant antigen. The Tn5 transposase enables the use of an index multiplexing strategy (iACTseq), which enables construction of thousands of single-cell libraries in one day by a single researcher without the need for drop-based fluidics or visual sorting. We conclude that ACT-seq present an attractive alternative to existing techniques for mapping epigenetic marks in single cells. MAINTechniques for mapping epigenetic states in individual cells have enhanced our understanding of differentiation and cell-to-cell variation. Multiple single-cell approaches have been developed to map transcriptomes and chromatin organization, and these methods are proving invaluable in

Modern next-generation sequencing-based methods have empowered researchers to assay the epigenetic states of individual cells. Existing techniques for profiling epigenetic marks in single cells often require the use and optimization of time-intensive procedures such as drop fluidics, chromatin fragmentation, and end repair. Here we describe ACT-seq, a streamlined method for mapping genome-wide distributions of histone tail modifications, histone variants, and chromatin-binding proteins in a small number of or single cells. ACT-seq utilizes a fusion of Tn5 transposase to Protein A that is targeted to chromatin by a specific antibody, allowing chromatin fragmentation and sequence tag insertion specifically at genomic sites presenting the relevant antigen. The Tn5 transposase enables the use of an index multiplexing strategy (iACT-seq), which enables construction of thousands of single-cell libraries in one day by a single researcher without the need for drop-based fluidics or visual sorting. We conclude that ACT-seq present an attractive alternative to existing techniques for mapping epigenetic marks in single cells.

The molecular pathways underlying the development of innate lymphoid cells (ILCs) are mostly unknown. Here we show that TGF-β signaling programs the development of ILC2s from their progenitors. Specifically, the deficiency of TGF-β receptor II in bone marrow progenitors results in inefficient development of ILC2s, but not ILC1s or ILC3s. Mechanistically, TGF-β signaling is required for the generation and maintenance of ILC2 progenitors (ILC2p). In addition, TGF-β upregulates the expression of the IL-33 receptor gene Il1rl1 (encoding IL-1 receptor-like 1, also known as ST2) in ILC2p and common helper-like innate lymphoid progenitors (CHILP), at least partially through the MEK-dependent pathway. These findings identify a function of TGF-β in the development of ILC2s from their progenitors.