Summary Posttranslational histone modifications are important for gene regulation, yet the mode of propagation and the contribution to heritable gene expression states remains controversial. To address these questions, we developed a Chromatin in vivo (CiA) Assay system employing chemically-induced proximity to initiate and terminate chromatin modifications in living cells. We selectively recruited HP1α to induce H3K9me3-dependent gene silencing and describe the kinetics and extent of chromatin modifications at the Oct4 locus in fibroblasts and pluripotent cells. H3K9me3 propagated symmetrically and continuously at rates of ~0.18 nucleosomes/hr to produce domains of up to 10kb. After removal of the HP1α stimulus, heterochromatic domains were heritably transmitted, undiminished through multiple cell generations. Our data enabled quantitative modeling of reaction kinetics, which revealed that dynamic competition between histone marking and turnover determines the boundaries and stability of H3K9me3 domains. Applying this framework, we were able to predict the steady-state dynamics and spatial features of the majority of euchromatic H3K9me3 domains.

Signaling by the cytokine LIF and its downstream transcription factor, STAT3, prevents differentiation of pluripotent embryonic stem cells (ESCs) by opposing MAP kinase signaling. This contrasts with most cell types where STAT3signaling induces differentiation. We find that STAT3binding across the pluripotent genome is dependent upon Brg, the ATPase subunit of a specialized chromatin remodeling complex (esBAF) found in ESCs. Brg is required to establish chromatin accessibility at STAT3 binding targets, in essence preparing these sites to respond to LIF signaling. Moreover, Brg deletion leads to rapid Polycomb (PcG) binding and H3K27me3-mediated silencing of many Brg-activated targets genome-wide, including the target genes of the LIF signaling pathway. Hence, one crucial role of Brg in ESCs involves its ability to potentiate LIF signaling by opposing PcG. Contrary to expectations, Brg also facilitates PcG function at classical PcG target including all four Hox loci, reinforcing their repression in ESCs. These findings reveal that esBAF does not simply antagonize PcG, but rather, the two chromatin regulators act both antagonistically and synergistically with the common goal of supporting pluripotency.

The opposition between polycomb repressive complexes (PRC) and BAF (mSWI/SNF) complexes plays critical roles in development and disease. Mutations in the genes encoding BAF subunits contribute to over 20% of human malignancy, yet the underlying mechanisms remain unclear owing largely to a lack of assays to assess BAF function in vivo. To address this, we have developed a widely applicable recruitment assay system and find that BAF opposes PRC by rapid, ATP-dependent eviction, leading to the formation of accessible chromatin. Reversing this process results in reassembly of facultative heterochromatin. Surprisingly, BAF-mediated PRC eviction occurs in the absence of PolII occupancy, transcription, and replication. Further, we find that tumor suppressor and oncogenic BAF complex mutations result in differential effects on PRC eviction. These studies define a mechanistic sequence underlying the resolution and formation of facultative heterochromatin and demonstrate that BAF opposes polycomb complexes on a minute-by-minute basis to provide epigenetic plasticity.

Recent exon sequencing studies of human tumors have revealed that subunits of mSWI/SNF or BAF complexes are mutated in more than 20% of all human malignancies,1,2 yet the mechanisms involved in tumor suppression are unclear. BAF chromatin remodeling complexes are polymorphic assemblies that use energy provided by ATP hydrolysis to regulate transcription through the control of chromatin structure3 and the placement of Polycomb (PcG) across the genome4,5. Several proteins dedicated to this multi-subunit complex, including SMARCA4 (BRG1) and BAF250A (ARID1A), are mutated at frequencies similar to that of recognized tumor suppressors. In particular, the core ATPase BRG1 is mutated in 5-10% of childhood medulloblastoma6-9 and greater than 15% of Burkitt's Lymphoma.10,11 Here we find a novel function of BAF complexes in decatenating newly replicated sister chromatids, a requirement for proper chromosome segregation during mitosis. We find that deletion of Brg1, as well as the expression of Brg1 point mutants identified in human tumors leads to anaphase bridge formation (sister chromatids linked by catenated strands of DNA), and a G2/M phase block characteristic of the decatenation checkpoint. Endogenous BAF complexes directly interact with endogenous TopoIIα through BAF250a and are required for TopoIIα binding to about 12,000 sites over the genome. Our results demonstrate that TopoIIα's chromatin binding is dependent on the ATPase activity of Brg1, which is compromised in oncogenic Brg1 mutants. These studies indicate that the ability of TopoIIα to prevent DNA entanglement at mitosis requires BAF complexes and suggest that this activity contributes to the role of BAF subunits as tumor suppressors.

The molecular mechanisms of neurogenic fate determination are of particular importance in light of the need to regenerate neurons. Here we define the mechanisms of installing neurogenic fate by the transcription factor Pax6 acting together with the Brg1-containing BAF chromatin remodeling complex. We show that Pax6 physically interacts with Brg1-containing BAF complex and genetic deletion of either Pax6 or Brg1, in the neural stem cells in the adult mouse subependymal zone results in a strikingly similar fate conversion from neuronal progenitors to glia. The Pax6-BAF complex drives neurogenesis by directly activating transcription factors Sox11, Nfib and Pou3f4, which form a cross-regulatory network that maintains neurogenic fate downstream of the Pax6-BAF complex in neuroblasts. Our work identifies a novel concept of stratification in neural fate commitment with a strikingly specific role of the Pax6-BAF complex in initiating a cross-regulatory network essential for maintenance of the neurogenic lineage in the adult brain.

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.

Resolution and formation of facultative heterochromatin is essential to development, reprogramming, and oncogenesis. The mechanisms underlying these changes are poorly understood due to the inability to study heterochromatin dynamics and structure in vivo. We devised an in vivo approach to investigate these mechanisms and found that topoisomerase II (TOP2), but not TOP1, synergizes with BAF (mSWI/SNF) ATP-dependent chromatin remodeling complexes genome-wide to resolve facultative heterochromatin to accessible chromatin independent of transcription, indicating that changes in DNA topology through (de-)catenation rather than release of torsional stress through swiveling is necessary for heterochromatin resolution. In turn, TOP2 and BAF cooperate to recruit pluripotency factors, explaining some of the instructive roles of BAF complexes. Unexpectedly, we found that TOP2, also plays a role in the reformation of facultative heterochromatin, suggesting that facultative heterochromatin and accessible chromatin exist at different states of catenation or other topologies, which may be critical to their structures.

Aire mediates the expression of tissue-specific antigens in thymic epithelial cells to remove dangerous self-reactive T lymphocytes. However, the mechanism that allows expression of tissue-specific genes at levels that prevent harm is unknown. Here we show that Brg1 generates accessibility at tissue-specific loci to impose central tolerance. We found that Aire harbors an intrinsic repressive function that restricts chromatin accessibility and opposes Brg1 across the genome. Aire exerted this repressive influence within minutes upon recruitment to chromatin and restrained the amplitude of active transcription. Autoimmune mutations that impair Aire-induced activation also impair the its repression function, indicating dual roles for Aire. Together, Brg1 and Aire fine-tune the expression of tissue-specific genes at levels that prevent toxicity, yet promote immune tolerance.