Bryan L. Betz, Ph.D.

Dissertation research performed under the guidence of Dr. Bernard E. Weissman

ABSTRACT
    The organization of genomic DNA into nucleosomes regulates gene expression by limiting access of transcription factors to binding domains. SWI/SNF chromatin remodeling complexes counteract this by manipulating nucleosome structure. Disruption of components of these complexes is implicated in the development of human neoplasia. In particular, truncating mutations and homozygous deletions in the hSNF5/INI1/BAF47 subunit occur in almost all malignant rhabdoid tumors. Whether hSNF5 mutations occur in other malignancies remains unknown. To investigate this question, a panel of various human tumors was screened for hSNF5 aberrations. We found that hSNF5 loss is restricted to only a limited subset of malignancies. This includes both renal and extrarenal rhabdoid tumors and possibly rhabdomyosarcomas. Furthermore, how loss of hSNF5 contributes to tumorigenesis remains unknown. The SWI/SNF subunit BRG1 is required for RB-mediated cell cycle arrest. Thus, we hypothesized that hSNF5 deficiency also disrupts RB signaling. However, we found that unlike BRG1 deficient cells, hSNF5 deficient cells retain functional RB, suggesting this mechanism does not account for hSNF5's tumor suppressor abilities. To further characterize how hSNF5 loss contributes to tumorigenesis, it was introduced into multiple deficient cell lines. In all cases, reexpression inhibited colony formation and induced cell cycle arrest characterized by a flattened morphology. Cells accumulated in G0/G1 and exhibited strong induction of p16ink4a, hypophosphorylated RB, and downregulation of cyclin A. These data suggest hSNF5 acts upstream of RB to induce growth arrest. In support of this, SV40 T/t abolished hSNF5-induced G1 arrest and activation of RB. Likewise, HPV-16 E7 was sufficient to partially overcome cell cycle arrest. Our results demonstrate that hSNF5 loss is not functionally equivalent to BRG1 loss in human tumor cell lines. Furthermore, hSNF5-induced cell cycle arrest of deficient cells is mediated in part through activation of p16ink4a expression. These findings provide insight into mechanisms of hSNF5-mediated tumor suppression. They also indicate that hSNF5 loss is a useful marker for the differential diagnosis of certain malignancies.