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National Chung‐Cheng University
THESIS ADVISOR: VASILY M. STUDITSKY, PH.D.
GRADUATE PROGRAM IN CELLULAR AND MOLECULAR PHARMACOLOGY
PHARMACOLOGY DEPARTMENT CONFERENCE ROOM, 4TH FLOOR, RWJMS RESEARCH TOWER, PISCATAWAY
Friday, September 16, 2011
Nucleosome survival during transcription is essential for cell viability and aging, and nucleosomal barrier is involved in gene regulation at the level of transcript elongation. We have studied elements of nucleosomal structure and trans-acting factors affecting the rate of transcription through chromatin and the fate of nucleosomes during transcription.
Pol II proceeded much farther into the nucleosome after histone N-tail removal. Thus histone tails provide a significant part of the nucleosomal barrier to Pol II transcript elongation.
Nucleosomes having Sin mutations in histones H3 or H4 present a much weaker barrier to traversal by Pol II and less efficiently survive transcription. Sin mutations likely destabilize the critical survival intermediates, Ø-loop, and increase the nucleosome disassociation. Increases in traversal of Sin nucleosome and histones lacking H2A/H2B N-tails were mostly additive, indicating that traversal can be facilitated by distinct mechanisms.
Multiple transcribing Pol II complexes can efficiently overcome the high nucleosomal barrier and displace/evict the entire histone octamer and hexamer. Transcription by single Pol II complexes allows survival of the original H3/H4 histones, while multiple, closely spaced complexes of transcribing Pol II can induce displacement of all core histones.
Transcription factors IIS and FACT stimulated elongation and this effect was independent on the presence of tails. FACT is a histone chaperone that strongly facilitates transcription and nucleosome survival in vivo. FACT alternatively interacts with either proximal or distal H2A/H2B dimer, competing for DNA binding and facilitating DNA uncoiling from the octamer. In the uncoiled state, FACT stabilizes H2A/H2B binding to the nucleosome and prevents octamer displacement by Pol II. Thus, FACT-H2A/H2B interactions play a key role in overcoming the nucleosomal barrier and nucleosome survival during Pol II transcription.
In summary, we have identified the elements of nucleosomal structure (N-terminal histone “tails” and DNA-binding histone surface) and trans-acting factors (FACT and multiple complexes of Pol II) that are involved in formation of the nucleosomal barrier to transcribing Pol II and nucleosome survival. The mechanisms of their action have been described in terms of their effect on the structures of the critical intermediates and/or the rates of transitions between them.