Transcriptional activation is generally accompanied by the """"""""remodeling"""""""" of promoter chromatin state. However, the molecular mechanisms by which chromatin remodeling is regulated and linked to changes in gene expression are poorly understood. The promoter of the yeast acid phosphatase gene PHO5 has served as a classical model to address the relationship between chromatin structure and transcription, providing major insights into gene regulation and promoter activity. Upon phosphate starvation, PHO5 is activated by Pho4p, a transcription factor that mediates promoter nucleosome disassembly. While PHO5 regulation has been widely investigated, little is known about the rate-limiting step of activation, the precise regulatory role of nucleosome disassembly, and the chromatin remodelers involved in nucleosome removal. This proposal outlines a research plan to employ a combination of biochemical, genetic, and imaging techniques to study the PHO5 promoter chromatin transition. We will use electron microscopy to determine the probabilities of promoter nucleosome configurations at PHO5 throughout the activation process. These studies will quantitatively test a recently published mathematical model describing chromatin stochasticity at the PHO5 promoter. We will also generate and characterize informative Pho4p activation-domain mutants. These mutations will be be used in a variety of assays to probe the role of Pho4p in chromatin remodeling and transcription. Finally, we will systematically identify genes involved in PHO5 activation by performing a suppressor screen, using a Pho4p activation-domain mutant. Suppressors will be extensively characterized, with future experiments planned to probe their effects on the PHO5 chromatin transition. The proposed research is expected to broaden our understanding of chromatin remodeling as it relates to transcription and will offer valuable information regarding the fundamental role of transcriptional activators. Furthermore, this work is expected to reveal novel factors that regulate promoter chromatin transitions. Aberrant gene regulation is ubiquitous in dozens of human diseases including several types of cancer. This proposal aims to shed light on the impact of nucleosome-mediated transcriptional regulation that may be applicable to several genes, of clinical importance. Gene expression is inextricably linked to the packaging and subsequent accessibility of genomic DNA. By studying factors that regulate DNA accessibility, we increase our understanding of the mechanisms underlying development, differentiation, and disease.
| Brown, Christopher R; Eskin, Julian A; Hamperl, Stephan et al. (2015) Chromatin structure analysis of single gene molecules by psoralen cross-linking and electron microscopy. Methods Mol Biol 1228:93-121 |
| Brown, Christopher R; Mao, Changhui; Falkovskaia, Elena et al. (2013) Linking stochastic fluctuations in chromatin structure and gene expression. PLoS Biol 11:e1001621 |
| Brown, Christopher R; Mao, Changhui; Falkovskaia, Elena et al. (2011) In vivo role for the chromatin-remodeling enzyme SWI/SNF in the removal of promoter nucleosomes by disassembly rather than sliding. J Biol Chem 286:40556-65 |
| Mao, Changhui; Brown, Christopher R; Griesenbeck, Joachim et al. (2011) Occlusion of regulatory sequences by promoter nucleosomes in vivo. PLoS One 6:e17521 |
| Mao, Changhui; Brown, Christopher R; Falkovskaia, Elena et al. (2010) Quantitative analysis of the transcription control mechanism. Mol Syst Biol 6:431 |
