Ramendra N. Saha - US grants

Very little is known about the role of epigenetic processes in maintenance or deregulation of mental health. Understanding these epigenetic processes is of paramount importance in light of the fact that incidence of many developmental and psychiatric disorders of the brain, such as the Autism Spectrum Disorders (ASDs), are on the rise at an alarming rate and are now major public health concerns. Recently two closely related H2A.Z hypervariants (H2A.z1 and H2A.z2) have been reported that differ from each other by only three amino acids. Despite such subtle differences, preliminary data using microarrays suggest largely independent roles of these hypervariants in neuronal gene transcription. Among the approximately 1000 genes affected by H2A.z1 or H2A.z2 knockdown, less than 5% were affected by lack of either isoform, strongly suggesting largely non-overlapping functions of these isoforms in neuronal gene regulation. One such gene, strongly regulated by H2A.z isoforms, is homerl, which, when mutated, is known to be a risk factor for schizophrenia and ASDs. Preliminary data show that mRNA levels of the short inducible homer1 isoform, homer1a, decrease after H2A.z1 depletion but increase when H2A.z2 is similarly depleted. Interestingly, gene ontology analysis of the microarray data reveals that several genes sensitive to H2A.z1, but not H2A.z2, depletion are known ASD and schizophrenia candidate genes, indicative of a possible hypervariant-specific role of H2A.Z in the etiology of these brain disorders. Thus, this proposal is designed to study these hypervariants in the context of neuronal function and synaptic plasticity. Three specific aims are proposed. First, the role of H2A.Z hypervariants will be studied in the rapid activity-induced induction of homer1a using RNA and chromatin immuno-precipitation techniques. Second, the DNA-binding sites of both H2A.Z isoforms across the entire genome and their roles in neuronal activity-dependent and -independent transcription will be studied by deep-sequencing of DNA and RNA from H2A.z1- or H2A.z2-depleted neurons. Third, the role of H2A.Z hypervariants in synaptic and non-synaptic neuronal function will be studied by electrophysiological and calcium imaging techniques.

Project summary  Neurodevelopmental disorders (NDDs) are becoming more prevalent among our children at an alarming rate.  Studies suggest that NDDs may be caused by inadvertent early-­life exposure to environmental toxins and  pollutants, especially the ones that are abundant indoors. We will study neurodevelopmental roles of one such  group of persistent environmental pollutants, polybrominated diphenyl ethers (PBDEs). This family of  organohalogenated flame-­retardants is used in several household products worldwide, with PBDE-­47 being the  most abundant in our environment. Our central hypothesis is that chronic exposure to PBDE-­47 and its  metabolites disrupts neurodevelopment by dysregulating epigenetic mechanisms that orchestrate  neurodevelopmental gene transcription. This proposal will test our central hypothesis via three specific aims. 1.  We will determine if chronic exposure to environmentally relevant concentrations of PBDE-­47 alters cortical  neurodevelopment. Experiments to test this possibility will be conducted in rat and human neuronal progenitor  cells (rNPCs and hNPCs) differentiating in vitro and in rats in vivo. Here, differentiating NPCs will be chronically  exposed to environmentally relevant doses of PBDE-­47 and its metabolites and neuronal maturation will be  subsequently assessed electro-­physiologically and functionally. 2. We will determine mechanisms of global  gene deregulation due to chronic exposure to PBDE47. Genome-­wide assays (RNA-­seq, ChIP-­seq, and CAP-­ seq) will be employed to test our hypothesis. 3. We will determine if chronic exposure to PBDE-­47 and its  metabolites alters the BAF (mammalian SWI/SNF) chromatin remodeling complex and thereby chromatin  permissiveness and gene transcription during neurodevelopment. Here, we will test the effects of chronic  PBDE exposure on functions of the BAF complex, a chromatin-­remodeling complex that is highly relevant for  neurodevelopment-­related gene transcription. We will mainly focus on a key BAF complex component,  BAF170 (SMARCC2). BAF170 is a candidate autism gene and is a ?hit? in our preliminary screening of PBDE-­ impacted genes. We will use RNAi and CRISPR-­based technology to understand the role of BAF170 in  neurodevelopmental gene expression, especially when challenged with PBDE-­47 exposure. Taken together,  this study will provide deeper insights into epigenetic mechanisms driving neurodevelopment and how  persistent environmental pollutants may modulate NDD risks by interfering with these mechanisms. 

Project summary (from the parent R01) Neurodevelopmental disorders (NDDs) are becoming more prevalent among our children at an alarming rate. Studies suggest that NDDs may be caused by inadvertent early-life exposure to environmental toxins and pollutants, especially the ones that are abundant indoors. We will study neurodevelopmental roles of one such group of persistent environmental pollutants, polybrominated diphenyl ethers (PBDEs). This family of organohalogenated flame-retardants is used in several household products worldwide, with PBDE-47 being the most abundant in our environment. Our central hypothesis is that chronic exposure to PBDE-47 and its metabolites disrupts neurodevelopment by dysregulating epigenetic mechanisms that orchestrate neurodevelopmental gene transcription. This proposal will test our central hypothesis via three specific aims. 1. We will determine if chronic exposure to environmentally relevant concentrations of PBDE-47 alters cortical neurodevelopment. Experiments to test this possibility will be conducted in rat and human neuronal progenitor cells (rNPCs and hNPCs) differentiating in vitro and in rats in vivo. Here, differentiating NPCs will be chronically exposed to environmentally relevant doses of PBDE-47 and its metabolites and neuronal maturation will be subsequently assessed electro-physiologically and functionally. 2. We will determine mechanisms of global gene deregulation due to chronic exposure to PBDE47. Genome-wide assays (RNA-seq, ChIP-seq, and CAP-seq) will be employed to test our hypothesis. 3. We will determine if chronic exposure to PBDE-47 and its metabolites alters the BAF (mammalian SWI/SNF) chromatin remodeling complex and thereby chromatin permissiveness and gene transcription during neurodevelopment. Here, we will test the effects of chronic PBDE exposure on functions of the BAF complex, a chromatin-remodeling complex that is highly relevant for neurodevelopment-related gene transcription. We will mainly focus on a key BAF complex component, BAF170 (SMARCC2). BAF170 is a candidate autism gene and is a ?hit? in our preliminary screening of PBDE-impacted genes. We will use RNAi and CRISPR-based technology to understand the role of BAF170 in neurodevelopmental gene expression, especially when challenged with PBDE-47 exposure. Taken together, this study will provide deeper insights into epigenetic mechanisms driving neurodevelopment and how persistent environmental pollutants may modulate NDD risks by interfering with these mechanisms. Note: Supplemental support is requested for the bold section.