Molecular Mechanisms in Transcriptional Regulation

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DESCRIPTION (provided by applicant): Studies of the yeast HO gene have identified many important transcriptional regulators and also identified regulatory paradigms that are conserved in metazoans. Activation at HO involves the sequential recruitment of transcription factors, including sequence specific DMA-binding proteins, chromatin remodeling factors, histone acetyl transferase complexes, architectural transcription factors, and the Mediator complex. Although a great deal is known about these transcriptional activators, relatively little is understood about how mechanistically they activate transcription. This proposal combines genetic and biochemical approaches to understand the regulation of a complex promoter. Chromatin structure at HO is highly repressive, and that numerous transcription factors function in a complex relationship to overcome this repression. Our experiments suggest that TATA-Binding Protein (TBP) is the last factor recruited to the HO promoter. Thus the ultimate goal of the stepwise recruitment of factors to the HO promoter is to assemble various activators in the vicinity of the TATA element, so that they are poised to work in concert to promote TBP binding at the critical time. The HO TATA element is obscured by a nucleosome, and we suggest that this nucleosome must be moved for TBP to bind and for HO activation. Experiments are proposed to examine how activators, including Swi/Snf and the Nhp6 architectural factor, overcome nucleosomal repression to facilitate TBP binding at HO. Swi5, absolutely required for HO expression, binds first to the HO promoter, recruits chromatin modifying factors to bind, and then Swi5 is degraded. Remarkably, there is no Swi5 protein bound to the HO promoter at the time the gene is transcribed. Thus cells must have a "memory" that Swi5 was present, and this memory persists for a remarkably long time. We will determine whether persistent changes in chromatin structure represent this memory mark, and how such a chromatin mark is generated and maintained.