TLS蛋白参与调控正性转录延伸因子b乙酰化/去乙酰化修饰的作用及机制

RNA polymerase Ⅱ-dependent transcription can be subdivided into multiple stages, of which elongation stage is considered to be the major rate-limiting step. P-TEFb, positive transcriptional elongation factor b, as the name suggested, plays a key role in the transition of abortive elongation to productive elongation. Cellular P-TEFb presents in two distinct forms, the active and inactive form. The normal function of P-TEFb depends on the dynamic equilibrium of the two forms. Cooperating with HEXIM1, 7SK-RNA takes part in regulating the equilibrium of p-TEFb. Evidence shows that the acetylation/deacetylation of P-TEFb plays an important role in the equilibrium, but the mechanism remains largely unknown.. We screened the RNAs that bind to TLS, a RNA binding protein with transcriptional acitvity, and identified the 7SK-RNA as a new partner of TLS. Our prelimilary results and other's published data suport the idea that TLS, facilitated by 7SK-RNA, modulates the acetyltransferase/deacetyltransferase activities of HEXIM1 complex,and consequently affects the acetylation/deacetylation of P-TEFb.. The current proposal is aimed to testify the above idea by focucing on the following four poits. Firstly, the role amd mechanism of 7SK-RNA on facilitating the interaction of TLS and HEXIM1. Secondly, the effect of TLS or TLS/7SK-RNA on the binding of HEXIM1 and acetyltransferase/deacetyltransferase. Thirdly, the effect of TLS or TLS/7SK-RNA on the acetyltransferase activity of HEXIM1 complex. Lastly, the effect of TLS on the acetylation of P-TEFb.. This is a story about a detailed mechanism of transcriptional regulation. The research would benefit the understanding of the relevant cellular events.

正性转录延伸因子b是真核细胞基因转录过程中延伸阶段的关键调控蛋白,其正常功能依赖于它失活/活化两种形式的动态平衡。7SK-RNA和HEXIM1参与调控这个平衡。研究表明正性转录延伸因子b的乙酰化/去乙酰化修饰在此调控中起重要作用，但具体机制仍需探讨。根据已有文献和初步试验结果我们推测，在7SK-RNA的协助下TLS通过抑制HEXIM1/乙酰化酶/去乙酰化酶复合体的乙酰化酶活性参与调控正性转录延伸因子b的乙酰化修饰状态。为证实上述推测，本项目拟阐明如下四个问题⑴7SK-RNA协助TLS和HEXIM1结合⑵TLS及TLS/7SK-RNA对HEXIM1和乙酰化酶或去乙酰化酶结合的影响及机制⑶TLS及TLS/7SK-RNA对HEXIM1复合体乙酰化酶活性的影响⑷TLS对正性转录延伸因子b乙酰化状态的影响。本项目是对基因转录调控中一个细节机制的探索，有助于更好地了解上述各分子所参与的生理病理过程。

正性转录延伸因子 b（P-TEFb）是真核细胞基因转录过程中延伸阶段的关键调控蛋白。本项目假设这因子中包括由P-TEFb、EP300、TLS、7SK-RNA组成的RNA-蛋白质复合物，并且TLS在触发P-TEFb释放/回收的过程中起关键作用。我们研究了TLS和EP300、TLS和7SK-RNA的相互作用，证明了TLS在7SK-RNA蛋白复合物中的结合。..在本项目的进展中，我们了解到另外有课题组已经发表了相关研究（Cell. 2013 May 9;153(4):855-68），证明不是TLS，而是另外的SRSF2蛋白，控制7SK-RNA蛋白复合物中P-TEFb的释放和回收。..相应的，我们调整了研究内容。.一方面，我们继续关注长链非编码RNA的功能，特别是非编码RNA在肿瘤中的作用。我们发现了另一个非编码RNA，GAS5，在前列腺癌中起重要作用。前列腺癌经手术去势和抗雄激素治疗后大多会进展为去势抵抗性前列腺癌（CRPC）。研究表明CRPC阶段虽癌细胞对去势和抗雄不再敏感，但雄激素受体信号仍是其最重要的通路，且持续增强，具体机制不明。我们发现一个与雄激素受体功能相关的长链非编码 RNA（lncRNA）GAS5。进一步研究证实：GAS5 的表达受雄激素受体负调控；过表达GAS5抑制雄激素受体的转录活性；GAS5的量与CRPC进展负相关；敲减 GAS5 能促进CRPC细胞增殖。基于以上数据，GAS5 和雄激素受体组成负反馈环路，且在CRPC进展中起重要作用。..另一方面，围绕TLS的结合蛋白EP300，我们研究了EP300突变在膀胱癌发生发展中的作用。课题组前期的大样本高通量测序证明，膀胱移行细胞癌中EP300发生热点突变，具体机制不明。我们克隆了9个EP300的突变，证明EP300-R1627W 突变通过改变EP300蛋白的底物结合中心，从而改变其转录活性，促进癌细胞的生长和侵袭，是膀胱移行细胞癌的“driver”突变。