Identifying Protein Factors and Pathways Leading to the Active Chromatin State

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Acetylation, phosphorylation and methylation of the histones and ATP-dependent chromatin remodeling turned out to be universal components of this epigenetic gene regulation. Large protein ensembles known as chromatin remodeling complex RSC and histone chaperone FACT (Facilitates Chromatin Transcription) alter nucleosomes or their higher order arrays to make chromatin more accessible to transcription. Pol II itself and transcription elongation factors such as TFIIF, TFIIS, elongin, and pTEF-b may also work to establish and maintain the active state of the chromatin. Finally, our own results suggest that transcription elongation factors and chromatin modifiers may cooperate in the elimination of the chromatin barrier to transcription. Based on these findings, Pol II elongation factors and chromatin modifiers/re-modelers are considered promising targets for new anti-cancer drugs, which may help to re-gain transcription control of growth-promoting genes, as well as to overcome silencing of genes with the tumor suppressor functions. We establish an in vitro system for dissection of the chromatin remodeling mechanisms associated with transcription elongation and investigation of a crosstalk between Pol II elongation factors and chromatin re-modelers. I plan to dissect these mechanisms using mono and poly nucleosomal templates, yeast Pol II, and the purified transcription elongation factors and chromatin remodeling complexes. The utilization of yeast Pol II to understand gene regulation in higher eukaryotes is justified because: (i) yeast and mammalian RNA polymerases have the same subunit composition, and overall share more than 50% sequence identity and more than 80% sequence similarity; (ii) nucleosome organization is completely conserved in all eukaryotes, and (iii) the yeast allows an easy combination of biochemical and genetic approaches, which is practically impossible in mammalian systems. Thus, the future findings in this single cell organism have very high translational potential.