The two major goals of this project is to 1) provide the research community and public domain for the first time with a comprehensive genome-wide atlas of the histone methylation landscape in selected subpopulations of cortical GABAergic interneurons and other cells residing in mouse cerebral cortex;and 2) To gain first insights into chromatin remodeling mechanisms of GABAergic neurons during the transition from juvenile to mature age. Previous work on chromatin extracted from human and mouse cortex indicated that histone methylation at "GABAergic gene" promoters is dynamically regulated during the extended period of maturation at least until early adulthood, thereby linking chromatin remodeling mechanisms to the developmental clock. The rationale to focus on the epigenome of cortical interneurons goes beyond mere academic curiosity. Dysregulated gene expression in GABAergic interneurons is considered a hallmark of the molecular pathophysiology and a major factor for the synchronization deficits in neural networks that affect widespread areas of the cerebral cortex in subjects on the psychosis or autism spectrum. These GABA related gene expression deficits include distinct cell types such as the class of fast spiking interneurons commonly recognized by expression of the calcium binding protein, Parvalbumin. Therefore, in the context of this proposal, we plan to generate 4 isogenic lines of BAC (bacterial artificial chromosome) transgenic mice to express green fluorescent protein (GFP)-tagged histone H2B in GABAergic interneurons overall, and in 3 specific subpopulations defined by differential expression of calcium buffering proteins Parvalbumin (PARV) or Calbindin (CALB) or Calretinin (CALR). Recent methodological advances enable us to separate and sort with high efficiency GFP-tagged nuclei from brain tissue for the purposes of chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq). Focus will be on tri- and mono-methyl-histone H3-lysine 4 (H3K4me3, H3K4me1) which are enriched at transcription start sites (H3K4me3) and enhancer sequences including those further removed from proximal promoters (H3K4me1), and a mark associated with RNA polymerase II activity and transcriptional elongation across coding and non-coding regions (H3K36me3). We expect that the various interneuron subpopulations, which differ in terms of function and developmental history, will show cell type specific chromatin signatures in many portions of the genome. When analyzed in conjunction with cell-specific transcriptomes and other datasets, histone methylation mapping of specific interneuron types is likely to provide radically novel insights into the developmental history and (epi)genomic architecture of cells ascribed a key role in schizophrenia and related disease.
For the majority of patients diagnosed with schizophrenia, no straightforward genetic cause has been identified. One of the important theories about schizophrenia implies that in some brain regions, such as the prefrontal cortex, a number of genes are not switched on properly during normal development, as they are in healthy subjects. Many of these genes related to a type of cell called the 'GABA neuron'which comprise less than 10% of all cells (nerve cells and others) in the cortex, but are very powerful because in effect they regulate synchronization of large neural networks in the brain. To further understand the role of the GABA neurons in psychiatric disease, and to clarify why gene expression is abnormal in these cells, it will be important to explore their genomes and chromatin architectures at high resolution. This grant proposal is based on extremely innovative techniques that were recently developed in our laboratories. We will be able, for the first time, to selectively isolate chromosomal materials and chromatin from the GABA neurons of the mouse brain for the study of epigenetic markings (basically, chemical modifications that regulate gene expression and function without altering the genetic code) on a genome-wide level. We expect that the work resulting from this project will provide a valuable resource /chromatin atlas for the neuroscience research community, and will shed light on some of the developmental mechanisms that govern proper gene expression activity in mature GABA neurons.
|Mitchell, Amanda C; Jiang, Yan; Peter, Cyril et al. (2015) Transcriptional regulation of GAD1 GABA synthesis gene in the prefrontal cortex of subjects with schizophrenia. Schizophr Res 167:28-34|
|Houston, Isaac; Peter, Cyril J; Mitchell, Amanda et al. (2013) Epigenetics in the human brain. Neuropsychopharmacology 38:183-97|