The goals of this proposal are to investigate fundamental mechanisms through which chromatin structure can regulate transcription and, when perturbed, creates conditions that lead to cancer. Studies will include the Swi/Snf and SAGA and complexes and the Set2/Rpd3S pathway in yeast, flies and human cells. The Swi/Snf nucleosome-remodeling complex functions as a tumor suppressor. Recurrent mutations in Swi/Snf are found in many cancers. To understand how loss of specific subunits affects Swi/Snf integrity and function, we will determine the Swi/Snf subunit interaction network in yeast and human cells. This information will uncover which subunits interact and form functional modules within the complex and how the functions of the complex change when subunits are missing or altered. We found that the Snf2 ATPase subunit of the yeast complex is modified by acetylation, and that this modification governs the dynamics of Swi/Snf occupancy genome wide. We will extend these studies to human Swi/Snf complexes to determine how they are regulated by acetylation. We will also purify Drosophila Swi/Snf complexes to determine its genomic binding sites and identify interacting proteins at different embryonic stages and in specific embryonic tissues. SAGA is a multisubunit histone acetyltransferase, histone ubiquitin protease and transcription co-activator. It contains several subunits with potential chromatin interacting domains and subunits that have been implicated in cancers and neurodegenerative disease. Major unresolved questions are which domains affect subunit functions and which localize SAGA to specific genes. We will address these questions by ChIP-Seq analysis after depleting specific subunits. We will explore novel proteins that interact with the ubiquitin protease module, which is no longer associated with SAGA in the absence of the Drosophila ataxin-7 subunit. We will pursue the function of this module and of intact SAGA in early zygotic transcription. The Set2 histone H3K36 methyltransferase, which functions as a tumor suppressor in human cells, is part of the Set2/Rpd3S pathway by which RNA polymerase II signals for histone deacetylation during transcription. We have found that acetylated histones accumulate in transcribed sequences because new acetylated histones replace the original histones in the absence of the Set2/Rpd3S pathway. Moreover, loss of Set2 leads to antisense transcription from within open reading frames. We will analyze the functions of Set2/Rpd3S pathway components in suppressing widespread antisense transcription. Conversely, we will analyze pathways by which antisense transcription represses coding gene transcripts, which also requires Set2, in mammalian cells. We will take both candidate and discovery approaches to identify novel Set2 targets. In addition, we will analyze the effects of cancer-associated mutations in Set2 with Set2 function in yeast and human cell lines. 1
This project will investigate the functions of several proteins that are often mutated in human cancers. These proteins modify the structures of chromosomes and affect gene expression. Their functions will be analyzed in yeast, flies and human cells by biochemistry, proteomics, genomics and genetics. 1
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