Inil is a subunit of the mammalian, ATP-dependent, SWI/SNF chromatin remodeling complexes. These enzymes play a key role in cell growth by altering chromatin structure to render normally condensed DNA accessible to cellular machinery, an important preliminary step to facilitate gene expression, cell division, and development. Subunits of the SWI/SNF enzymes interact with Rb and BRCA-l, known tumor suppressor proteins. The recent identification of Ini1 mutations in familial malignant rhabdoid tumors has further heightened interest in the link between SWI/SNF complexes and tumor suppression. Previous work from our labs has identified and characterized mammalian SWI/SNF complexes as possessing ATP dependent chromatin remodeling activities and has identified a role for SWI/SNF chromatin remodeling in differentiation of muscle as well as other differentiation events. Recently, we have generated mice deficient for Ini and have found that Inil is required for proper early embryogenesis, and tumor formation in Inil-haploinsufficient mice has identified Inil as a bonajide tumor suppressor in mice. The research described in this proposal will draw upon the strengths of the two investigators, utilizing a combination of genetic, biochemical, and molecular approaches to elucidate the mechanistic role of Inil in transformation and differentiation. Additionally, these studies will examine the role of Ini chromatin remodeling enzymes. Mice heterozygous for Inil will be treated with DNA damaging agents, and will be bred to mice bear mg inactivated alleles of the p53 gene or the Rb gene to investigate the link between the Inil-based SWI/SNF activity and DNA damage repair pathways, and to explore the role of the two major tumor suppressor pathways in Inil-mediated tumor suppression. Mice bearing a conditional allele of Ini1 will also be generated via gene targeting experiments in ES cells. The specificity of the site of tumorigenesis in the Inil-deficient mice is striking; 100 percent of the tumors arise on the face, head, and neck of the mouse, whereas in humans bearing an inactivated Ini1 gene, most of the tumors arise in the kidney and/or brain. Interestingly, these structures are all derived from neural crest progenitor cells during development. In order to determine if tumorigenesis in the Inil mice is due to a defect in neural crest cell growth or differentiation, the conditional Inil mice will be bred with mice expressing Cre specifically in neural crest cells. The conditional Inil mice also will be crossed with mice expressing the Cre recombinase in cardiac and skeletal muscle in order to provide in vivo evidence for a link between SWI/SNF activity and muscle cell differentiation. Lastly, we will purify SWI/SNF complexes from Inil-deficient cells. Isolated purified protein will be characterized for subunit composition and the ability to interact with tumor suppressor proteins. Furthermore, we will assess the ability of purified complexes/subunits lacking Ini1 to remodel chromatin structure and induce specific gene activation events in vitro. These proposed experiments should serve not only to define the role of Inil in differentiation and chromatin remodeling, but should also provide much insight into the mechanisms of Inil-mediated tumor suppression.
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