| Chromatin structure is an integral component of transcriptional control. Nucleosomes inhibit the ability of transcription factors to bind to their regulatory sequences and antagonize initiation by RNA polymerase. Chromatin structure is regulated by chromatin modifying factors that modify histones or alter histone-DNA interactions to either facilitate or impair transcription. In this thesis, I have characterized the function of the chromatin remodeling factor Chd1p in budding yeast. CHD proteins are well conserved, with members found from yeast to humans. However, their cellular roles are not known. To uncovered the function of Chd1p, I implemented genetic, genomic, and biochemical approaches. Through a synthetic lethal screen, SWI /SNF genes were determined to have genetic interactions with CHD1. These results implicated a role for Chd1p in chromatin structure and transcription. To examine the role of Chd1p in chromatin structure, Chd1p was purified from yeast and assayed for nucleosome remodeling activities. Purified Chd1p was shown to alter the structure of mononuclesomes in an ATP-dependent manner. Genome-wide expression profiling showed that the expression of 2–4% of yeast genes are affected in the absence of Chd1p. To further access the role of Chd1p in transcription, chromatin immunoprecipitation/microarray analyses was carried out to identify genes directly regulated by Chd1p. Genes encoding ribosomal proteins and translation components were determined to be preferentially precipitated by Chd1p. In addition, Chd1p was selectively localized to the coding regions of these genes and was enriched along the entire open reading frame. These findings indicate that Chd1p acts in transcription elongation. To better understand how Chd1p activity and recruitment may be regulated, an affinity purification of proteins that interact with the chromo domain of Chd1p was performed. Three proteins with links to transcription and chromatin structure have been identified. Genetic and genomic analyses of one Chd1p-interacting protein, Crp1p, has been carried out. Collectively, the work presented in this thesis has determined that Chd1p functions of as a chromatin modifying factor with a role in transcription elongation. |
