Identifying Protein Factors and Pathways Leading to the Active Chromatin State

组蛋白的乙酰化、磷酸化和甲基化以及依赖于ATP的染色质重塑被证明是这种表观遗传基因调控的普遍组成部分。大的蛋白质组合称为染色质重塑复合体RSC和组蛋白伴侣事实(促进染色质转录)改变核小体或其更高阶阵列，使染色质更容易被转录。POL II本身和转录延伸因子如TFIIF、TFIIS、拉伸素和pTEF-b也可能作用于建立和维持染色质的活性状态。最后，我们自己的结果表明，转录延长因子和染色质修饰剂可能在消除染色质对转录的障碍方面起到协同作用。基于这些发现，POL II延长因子和染色质修饰剂/重塑分子被认为是新的抗癌药物的有希望的靶点，这可能有助于重新获得促生长基因的转录控制，以及克服具有肿瘤抑制功能的基因的沉默。我们建立了一个体外系统，用于剖析与转录延长相关的染色质重塑机制，并研究Pol II延长因子和染色质重塑因子之间的串扰。我计划使用单核小体模板和多核小体模板、酵母POL II以及纯化的转录延伸因子和染色质重塑复合体来剖析这些机制。利用酵母Pol II来理解高等真核生物中的基因调控是合理的，因为：(I)酵母和哺乳动物的RNA聚合酶具有相同的亚基组成，总体上具有超过50%的序列一致性和超过80%的序列相似性；(Ii)核小体组织在所有真核生物中完全保守，以及(Iii)酵母允许生物化学和遗传方法的简单组合，这在哺乳动物系统中几乎是不可能的。因此，未来在这种单细胞生物体中的发现具有非常高的翻译潜力。

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.