The process of transcription is essential for all living cells. Transcription is accomplished in four steps: promoter binding, RNA chain initiation, RNA transcript elongation, and RNA transcript termination. Regulation of transcription can occur at each of these steps. Recent studies suggest that transcript elongation by RNA polymerase II is one of the major targets of gene regulation. It has been established that regulation of gene expression at the level of transcript elongation involves factors interacting with histones. These factors facilitate transient displacement of histones from chromatin during transcript elongation and modify the rate of transcription through chromatin. The long-term research goal of this proposal is to understand the mechanism and the regulation of transcript elongation by eukaryotic RNA polymerases in chromatin. When eukaryotic genes are activated for transcription, their chromatin structure changes to accommodate transcription factors and to allow efficient transcription by RNA polymerases. However, for many genes there is evidence that transcribed regions are covered with nucleosomes. This raises the questions: 1. How do polymerases transcribe through the nucleosome barrier? 2. How do the factors interacting with histones change the rate of transcript elongation? These 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 biochemical, fluorescent, molecular genetic and single-molecule techniques. Our experiments will be focused on analysis of eukaryotic RNA polymerase II.
The specific aims are: 1. To test whether transient displacement of histones from nucleosome is the rate-limiting step during transcript elongation. 2. To determine whether elongation factors such as FACT facilitate transcription through chromatin by interacting with histones within polymerase-engaged nucleosomes. The discovery that some elongation factors play important roles in oncogenesis underscores the potential clinical significance of analysis of the mechanism of transcript elongation through chromatin.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058650-13
Application #
7807985
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
1999-01-01
Project End
2012-04-12
Budget Start
2010-05-01
Budget End
2012-04-12
Support Year
13
Fiscal Year
2010
Total Cost
$312,741
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Pharmacology
Type
Schools of Medicine
DUNS #
617022384
City
Piscataway
State
NJ
Country
United States
Zip Code
08854
Chang, Han-Wen; Studitsky, Vasily M (2017) Chromatin replication: TRANSmitting the histone code. J Nat Sci 3:
Valieva, Maria E; Gerasimova, Nadezhda S; Kudryashova, Kseniya S et al. (2017) Stabilization of Nucleosomes by Histone Tails and by FACT Revealed by spFRET Microscopy. Cancers (Basel) 9:
Sultanov, Daniel C; Gerasimova, Nadezhda S; Kudryashova, Kseniya S et al. (2017) Unfolding of core nucleosomes by PARP-1 revealed by spFRET microscopy. AIMS Genet 4:21-31
Valieva, Maria E; Armeev, Grigoriy A; Kudryashova, Kseniya S et al. (2016) Large-scale ATP-independent nucleosome unfolding by a histone chaperone. Nat Struct Mol Biol 23:1111-1116
Gerasimova, N S; Pestov, N A; Kulaeva, O I et al. (2016) Transcription-induced DNA supercoiling: New roles of intranucleosomal DNA loops in DNA repair and transcription. Transcription 7:91-5
Chang, Han-Wen; Pandey, Manjula; Kulaeva, Olga I et al. (2016) Overcoming a nucleosomal barrier to replication. Sci Adv 2:e1601865
Studitsky, Vasily M; Nizovtseva, Ekaterina V; Shaytan, Alexey K et al. (2016) Nucleosomal Barrier to Transcription: Structural Determinants and Changes in Chromatin Structure. Biochem Mol Biol J 2:
Kudryashova, Kseniya S; Chertkov, Oleg V; Nikitin, Dmitry V et al. (2015) Preparation of mononucleosomal templates for analysis of transcription with RNA polymerase using spFRET. Methods Mol Biol 1288:395-412
Gaykalova, Daria A; Kulaeva, Olga I; Volokh, Olesya et al. (2015) Structural analysis of nucleosomal barrier to transcription. Proc Natl Acad Sci U S A 112:E5787-95
Pestov, Nikolay A; Gerasimova, Nadezhda S; Kulaeva, Olga I et al. (2015) Structure of transcribed chromatin is a sensor of DNA damage. Sci Adv 1:e1500021

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