Alterations in gene expression cause numerous cancers including ovarian, bladder, breast and colorectal tumors, as well as neurodegenerative conditions, such as Alzheimer's disease. The dynamic organization of the human genome into chromatin regulates transcription initiation and elongation, while defects in chromatin modifications, assembly, disassembly and remodeling results in the misregulation of many oncogenes. This has led to chromatin modifying complexes becoming one of the main chemotherapeutic drug targets. Numerous studies have identified and correlated the components involved in chromatin transcriptional regulation (CTR), which include histone variants, histone post-translational modifications (PTMs), histone chaperone assembly factors and chromatin remodeling factors. Remarkably, genetic, biochemical, structural and deep sequencing studies have not fully revealed the mechanisms of CTR. We hypothesize that nucleosome unwrapping, histone PTMs, histone variants, histone chaperones and chromatin remodelers function together to significantly influence TF occupancy and H2A-H2B heterodimer exchange to regulate transcription. We have developed precise chemical tools and single molecule measures capable of determining the mechanisms and functions of CTR. Using these methodologies, we will determine the mechanisms by which histone variants, PTMs, chaperones and chromatin remodeling complexes function together to cooperatively (additively and multiplicatively), anti-cooperatively and redundantly regulate transcription. We propose in the renewal application the following Specific Aims: (1) Determine the mechanisms by which nucleosomes influence transcription factor dynamics;(2) Determine the transcriptional regulatory mechanisms of H2A.Z, H3.3 and nucleosome entry-exit PTMs. (3) Determine the transcriptional regulator mechanisms behind H2A-H2B heterodimer exchange and removal. These studies will provide a foundation for understanding the mechanisms and functional interactions behind chromatin transcriptional regulation and the misregulation of numerous oncogenes.
Regulation of gene expression and genomic stability by epigenetic factors such as histone modifications, histone variants and nucleosome dynamics have become a dominant field in cancer epigenetics because of the reversible nature of epigenetic alterations. Recent advances in understanding epigenetic factors have led to the emergence of epigenetic therapy and the recent FDA approval of multiple epigenetic drugs for cancer treatment. This proposal uses a combination of advanced protein chemistry and single molecule approaches to reveal the molecular mechanisms behind the function of chromatin epigenetic regulators. These studies will provide molecular information for understanding drug resistance, tumorigenesis, and for designing new cancer drugs and therapies.
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