The dynamic regulation of chromatin structure, essential for the expression of eukaryotic genes, is achieved in part by the combined activity of histone modifying and chromatin remodeling complexes. Our long term goal is to understand how these complexes act coordinately to regulate transcription. RSC (Remodels the Structure of Chromatin) is a chromatin remodeling complex conserved from yeast to human that is required for viability. Although RSC is known to regulate the transcription of genes involved in important biological processes such as cell division and the responses to DNA damage and stress, how RSC is recruited to its target genes as well as how it functions to regulate their expression remains to be defined. The RSC complex possesses multiple subunits that can recognize and bind acetylated histones, which supports the prevailing view that the recognition of acetylated histone residues is important for RSC recruitment and function. We recently showed that acetylation in coding sequences is inversely correlated with histone occupancy, which further suggests that RSC recognizes and binds acetylated histones to remove them. Interestingly, RSC also interacts with RNA Polymerase II (Pol II), which suggests that the polymerase may target RSC to transcribed genes. In support of this hypothesis, our preliminary data shows that the interaction of RSC with Pol II is lost in a kin28-ts (C-terminal domain (CTD) kinase) mutant, indicating that phosphorylation of the Pol II CTD by Kin28 may promote the recruitment of RSC to coding sequences during transcription elongation. We thus propose a two-step model in which RSC is initially recruited to coding sequences by elongating polymerases, and subsequently recognizes particular patterns of histone acetylation to target nucleosomes for remodeling or eviction. To understand the mechanism involved in targeting RSC to transcribed coding regions, we will perform genome-wide localization assays in wild type cells and cells mutant for Pol II CTD kinases and histone acetyltransferases. Using a novel assay that we have recently developed, we will identify and characterize critical histone lysine residues important for the interaction of RSC with chromatin. We will then analyze the effect of depleting RSC on transcription elongation to define the mechanism by which RSC is able to facilitate Pol II movement through coding regions. These contributions will be significant in that they are expected to lend insight into the mechanism of how RSC is recruited to chromatin, as well as how it functions to regulate the transcription of its target genes in a healthy organism. This information will be valuable to our understanding of how mutations in the RSC complex lead to the initiation and progression of diseases such as cancer, and could potentially aid in identifying new therapeutic targets.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM095514-04
Application #
8696870
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Carter, Anthony D
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Oakland University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
Rochester
State
MI
Country
United States
Zip Code
48309
Spain, Marla M; Ansari, Suraiya A; Pathak, Rakesh et al. (2014) The RSC complex localizes to coding sequences to regulate Pol II and histone occupancy. Mol Cell 56:653-66
Burugula, Bala Bharathi; Jeronimo, CĂ©lia; Pathak, Rakesh et al. (2014) Histone deacetylases and phosphorylated polymerase II C-terminal domain recruit Spt6 for cotranscriptional histone reassembly. Mol Cell Biol 34:4115-29
Govind, Chhabi K; Ginsburg, Daniel; Hinnebusch, Alan G (2012) Measuring dynamic changes in histone modifications and nucleosome density during activated transcription in budding yeast. Methods Mol Biol 833:15-27