Barrier Insulators in Erythropoiesis

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DESCRIPTION (provided by applicant): Barrier insulators create a "barrier" to protect against heterochromatin-mediated gene silencing, critical for regulation of cell-type specific gene expression in normal development and differentiation. Perturbation of barrier insulator function, which frequently occurs in chromosomal translocations associated with sporadic and inherited genetic disease or cancerous states, contributes to alterations in gene expression characteristic of these conditions. The structure and function of vertebrate barrier elements are poorly understood. The overall aims of this project are to define and characterize barrier insulators controlling gene expression during erythropoiesis. The goal of aim one is to identify a common regulatory signature associated with functional barrier insulators in human erythroid cells. These studies address the hypothesis that there is a common regulatory signature for cell-type specific barrier insulators recognizable by chromatin architecture, binding of proteins with histone methyltransferase and acetyltransferase activity, ATP-dependent nucleosome remodeling activity, and other regulatory functions. This aim combines state of the art high throughput genomic technologies with functional studies. After barrier insulators have been identified and functionally validated, histone architecture, regulatory protein binding, and genomic organization will be integrated and analyzed. The goal of aim two is to address the hypothesis that USF (upstream stimulatory factor) proteins recruit enzymes and other proteins associated with activating histone modifications to block the mechanism(s) that lead to spreading of gene-silencing associated chromatin changes in barrier insulators. This hypothesis is based on studies of the chicken HS4 barrier insulator from the beta globin gene cluster and preliminary data from human erythroid cells. USF binding in hematopoietic stem and progenitor cells and erythroblasts will be assessed and integrated with genomic organization, histone architecture, and regulatory protein binding. The goal of the third aim is to characterize the multiprotein complexes associated with barrier insulators in erythroid cells, addressing the hypothesis that these complexes contain proteins of common function, including histone methyltransferases and acetyltransferases, nucleosomal remodeling proteins, and other critical regulatory proteins. Comprehensive analyses of multiprotein complexes regulating programs of gene expression have been hampered by low abundance, dynamic and context-dependent composition, and technologic difficulties in identifying complex constituents. To overcome these hurdles, characterization of the multiprotein complexes mediating barrier insulator function will be performed using state-of-the-art, quantitative proteomics techniques. This approach combines protein labeling by stable isotopes in vivo, integrated mass spectrometry and computational platforms, followed by validation studies. Together, these studies will provide novel insight into a critical process controlling gene expression and will ultimately lead to a comprehensive understanding of the regulatory interactions that control specific gene expression programs during cell growth and development. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to understand how a poorly understood, yet important genetic element that turns genes on and off at the right time in the right place works. When this element doesn't work, cells may not work, as occurs in children born with birth defects, or may grow out of control, as occurs in cancer. Understanding this element may provide us with important information on how some diseases occur and may provide ideas on how to treat them.