Neuron-specific chromatin remodeling in social behavior and memory
Keywords: Developmental Biology.

Within the nucleus, DNA is packaged with histones and other proteins to form chromatin. This serves to protect and organize the genome, and to regulate processes like transcription. The BAF ATP-dependent chromatin remodeling complex controls the accessibility of DNA—and thus, transcription—by mobilizing histones and evicting Polycomb repressive complexes. Mutations in BAF subunits are enriched among individuals with neurodevelopmental disorders that feature intellectual disability and impaired social communication. Yet, how BAF mutations cause neurological symptoms, and more generally, how chromatin remodeling contributes to brain function, is poorly understood. This thesis work reveals BAF mechanisms in three neurodevelopmental disorders: autism, Down syndrome and Baraitser-Winter syndrome. Autism spectrum disorder (ASD) is a prevalent group of neurodevelopmental disorders that are defined by social deficits and restricted or repetitive behavior. Core BAF subunits including ARID1B are among the most frequently mutated genes in ASD, but the causal mechanisms are unknown. I hypothesized that BAF mutant ASD arises from impaired function of the neuronal BAF complex, which is defined by subunits ACTL6B, DPF1/3, and SS18L1. If true, mutations in neuronal-specific subunits would be expected to cause ASD. Whole exome sequencing revealed ACTL6B as the most significantly mutated gene in the Simons Recessive Autism Cohort, with loss-of-function mutations causing fully-penetrant Mendelian recessive ASD. Actl6b-/- mice on two genetic backgrounds exhibited patient-related social and memory impairments, stereotypies, hyperactivity, and corpus callosum hypogenesis, a clinical characteristic of BAF mutant ASD thought to arise from axonal targeting defects. I demonstrated a conserved role for ACTL6B in neural circuit formation by genetic complementation in fly olfactory projection neurons, where neurons expressing patient variants projected to the wrong glomerulus with 100% penetrance. These findings unveiled ACTL6B loss as a highly penetrant cause of Mendelian ASD, with conserved phenotypes in mouse and fly. Experiments using the Actl6b-/- mouse model have provided insight into molecular and cellular mechanisms. First, analysis of "resting state" gene expression and chromatin accessibility in network-silenced Actl6b-/- cortical neurons revealed signatures of activity like FOS expression, indicating a role for BAF in stabilizing resting state transcription. Second, genes involved in neurite targeting (e.g., semaphorins) and social behavior (e.g., serotonin receptor 1b; 5-HT1b) showed altered expression in Actl6b-/- neurons. Systemic administration of a 5-HT1b agonist fully rescued social impairments and hyperactivity in Actl6b-/- mice. Conditional deletion of the autism-related BAF subunit Arid1b in mouse serotonergic neurons caused social impairments that were rescued with the 5-HT1b agonist, indicating that serotonin-specific BAF chromatin remodeling is necessary for social behavior and facilitates pro-social neurotransmission. The 5-HT1b agonist rescued social behavior in 4 additional diverse mouse models for autism, indicating insufficient serotonin neurotransmission as a convergent and potentially causative mechanism for ASD. Through my studies of neuronal BAF, I identified the putative histone "reader" BRWD1 as a substoichiometric subunit and found that BRWD1 contributed to memory, transcriptional, and BAF targeting defects in a mouse model for Down syndrome. Down syndrome is the most common neurodevelopmental disorder in humans and is caused by the presence of an extra copy of chromosome 21 (trisomy 21). Down syndrome often presents with craniofacial features, organ malformations, intellectual disability; and it associated with increased risk for autism and Alzheimer's disease. BRWD1 is encoded within the Down syndrome-critical region of chromosome 21 and is expressed 1.5-fold in Down syndrome patient-derived neurons and in brain tissues of a mouse model for Down syndrome. I found that BRWD1 substoichiometrically associated with BAF in mouse embryonic stem cells, embryonic neurons, and in adult brain. Like BAF subunits, BRWD1 resisted dissociation from BAF in up to 4 M urea and co-migrated with the 2 MDa neuronal BAF complex on a glycerol gradient. BRWD1 was co-depleted from brain nuclear extracts with BAF antibodies to a similar degree as ACTL6B. In the Ts65Dn mouse model for Down syndrome, neuronal BAF complex mistargeting was rescued by selectively restoring BRWD1 to euploid copy number. Memory, synaptic gene expression, and Alzheimer's-associated APP expression were also rescued in the Down syndrome mouse model by selectively restoring Brwd1 to euploid copy number. These findings nominate BRWD1 as a substoichiometric targeting subunit of the neuronal BAF complex and implicate excess BRWD1 as a cause of Down syndrome-like phenotypes in mice. Finally, I examined the structure and function of actin in the BAF complex and uncovered a novel mechanism for BAF-Polycomb antagonism that is associated with Baraitser-Winter syndrome. Actin is best known as a cytoskeletal protein, but it also serves as a core subunit of the BAF complex, where its function is unclear. ACTB (β-actin) is haploinsufficient for ASD, while dominant mutations cause Baraitser-Winter syndrome. I found that the ability to polymerize was necessary for actin to assemble into BAF and that actin polymer stabilization with Jasplakinolide further enhanced its stoichiometry in the complex. Proteomic analysis of embryonic stem cell (es)BAF complexes with selective actin stabilization revealed that polymeric actin stabilizes intra-BAF interactions and regulates a subset of interactions with chromatin proteins including with RING1, TET1, and HP1α. esBAF showed enriched interactions with the non-canonical PRC1.6 complex and PRC2, particularly with substoichiometric targeting subunits JARID2, MTF2, and MGA that are necessary for de novo Polycomb domain formation—indicating a possible sequestration mechanism for BAF-Polycomb antagonism. β-actin deletion resulted in compensatory assembly of α-smooth muscle actin into BAF and expression of a dominant β-actin mutation that causes Baraitser-Winter syndrome reduced esBAF interactions with JARID2, TET1 and HP1α. These findings suggest that actin polymerization is necessary for assembly into BAF and may allosterically regulate BAF protein interactions. These studies lend insight into neurodevelopmental disease mechanisms and highlight a central role for the BAF complex in neuronal gene regulation and behavior