Aim 1: To determine the role of ATP-dependent remodeling in T cell differentiation.? ? During T helper cell differentiation, it is well know that dramatic changes occur in gene expression and chromatin structure. However, little is known about the role of ATP-dependent remodeling in these changes, or whether chromatin remodeling plays a causal role in gene regulation. We asked the role of BRG1, a SWI/SNF ATPase, in Th2 differentiation, a cell fate linked to fighting extracellular parasites and exacerbating allergy and asthma.? ? We found that BRG1 was required for Th2 cytokine expression. BRG1 was bound to genes that require BRG1 for expression, suggesting that the observed transcriptional regulation was direct. BRG1 was also required to establish and maintain chromatin structure at the target genes, suggesting that remodeling was responsible for the observed changes in gene expression. Together, these results indicate ATP-dependent remodeling plays an important role in immune function by regulating cell fate choice, by programming gene expression through chromatin remodeling. In the future, we will examine the role of other remodeling ATPases, and determine the role of BRG1 in regulating other genes in these cells.? ? Aim 2: To determine role of ATP-dependent remodeling in neuronal development.? ? ? During neuronal development, a specialized gene expression program is enacted, and errors in this program can lead to developmental and psychiatric disorders. We have begun to investigate the role of ATP-dependant remodeling in this process.? ? We identified changes in gene expression upon retinoic acid-induced differentiation of EC cells to neuronal precursors. We found that expression of several of these genes was dependent on remodeling enzymes. We are now asking whether the remodeling enzymes directly bind their targets, and whether they program the chromatin structure at these loci. In the future, we are interested in examining the transition from neuronal precursor to neurons.? ? Aim 3: To identify components of CHD5 and CHD7, putative remodeling enzyme complexes.? ? CHD5 has recently been identified as a tumor suppressor (neuroblastoma). CHD7 has recently been identified as the gene mutated in most cases of CHARGE syndrome, a rare disorder characterized by eye, ear, heart, neural, behavioral/cognitive and lymphocyte defects. CHD5 and CHD7 are homologous to ATPases that are known chromatin remodeling enzymes. However, neither putative remodeling enzyme has been characterized to date. We are investigating these enzymes using biochemical and molecular biology approaches.? ? First, we are identifying the components of CHD5 and CHD7 complexes. We produced antisera that can detect both proteins by immunoprecipitation and immunoblot. We characterized several tissues and cell lines for expression of these genes at the RNA and protein level. We determined by protein microsequencing that our antisera recognize the expected proteins. Next, we will attempt to identify other components of these enzymes by purifying protein complexes containing these proteins (using our antisera) and identifying associated proteins. Ultimately, we will determine the mechanism of remodeling for these proteins.? ? Second, we are determining the function of these ATPases in cells. We are developing loss-of-expression models for CHD7 to mimic the disease state, to ask what the role of CHD7 is in cells from the immune and neural systems. We are also developing reagents to add back CHD5 to neuroblastoma cells that have lost CHD5 expression, to ask how CHD5 contributes to normal and aberrant neuronal cells.
