The vital process of transcription by RNA polymerase II (pol II) occurs in chromatin environment in eukaryotic cells;in fact, moderately transcribed genes retain nucleosomal structure. Recent studies suggest that chromatin structure presents a strong barrier for transcribing pol II in vitro, and that DNA-histone interactions are only partialy and transiently disrupted during transcript elongation on moderately active genes. Furthermore, elongating pol II complex is one of the major targets during gene regulation. The nucleosomal barrier and protein factors interacting with histones and pol II participate in this regulation. These studies raise the following questions: (1) How do eukaryotic polymerases overcome the nucleosome barrier? (2) What are the mechanisms of histone recovery during progression of pol II through chromatin? (3) How do the factors interacting with chromatin and with the target enzymes change the rate of pol II progression through chromatin? The long-term research goal of this proposal is to understand the mechanism and the regulation of transcript elongation by pol II in chromatin. The specific questions will be addressed in a highly purified transcription system in vitro. We will analyze transcription of homogeneous and well-defined mono- and polynucleosomal chromatin templates using a combination of biochemical, fluorescent, molecular genetic and single-molecule techniques. Our experiments will be focused on analysis of eukaryotic pol II. Our preliminary studies have shown that transcription through chromatin is accompanied by transient DNA uncoiling from histones and unfolding of histone octamer. The structures of these intermediates and the rates of interconversion between them largely determine the outcome of the process of transcription through chromatin. Accordingly, the specific aims are: 1. To provide molecular description of the changes in DNA-histone interactions during transcription through chromatin by pol II and the rates of interconversion between the intermediates. 2. To identify protein-protein interactions perturbed during transcription-dependent unfolding of the histone octamer, and factors involved in this process. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page

Public Health Relevance

Analysis of transcript elongation is important for human health because: (a) expression of at least some human proto-oncogenes (e.g. c-myc and c-fos) is regulated at the level of transcript elongation, (b) elongation factors play important roles in numerous human diseases, including HIV and leukemia, (c) histone modifications associated with transcribed chromatin are changed in human cancers. (d) Chromatin recovery during transcript elongation is essential for normal aging.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058650-17
Application #
8606464
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
1999-01-01
Project End
2016-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
17
Fiscal Year
2014
Total Cost
$365,925
Indirect Cost
$160,925
Name
Research Institute of Fox Chase Cancer Center
Department
Type
DUNS #
064367329
City
Philadelphia
State
PA
Country
United States
Zip Code
19111
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Hsieh, Fu-Kai; Kulaeva, Olga I; Patel, Smita S et al. (2013) Histone chaperone FACT action during transcription through chromatin by RNA polymerase II. Proc Natl Acad Sci U S A 110:7654-9
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Hsieh, Fu-Kai; Fisher, Michael; Ujvari, Andrea et al. (2010) Histone Sin mutations promote nucleosome traversal and histone displacement by RNA polymerase II. EMBO Rep 11:705-10
Kulaeva, Olga I; Hsieh, Fu-Kai; Studitsky, Vasily M (2010) RNA polymerase complexes cooperate to relieve the nucleosomal barrier and evict histones. Proc Natl Acad Sci U S A 107:11325-30
Morozov, Alexandre V; Fortney, Karissa; Gaykalova, Daria A et al. (2009) Using DNA mechanics to predict in vitro nucleosome positions and formation energies. Nucleic Acids Res 37:4707-22
Studitskii, V M (2009) [Mechanisms of distant enhancer action on DNA and in chromatin] Mol Biol (Mosk) 43:204-14

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