Regulation of Transcriptional Elongation by HIV-1 TAT

描述(申请人提供)：人类转录延长因子P-TEFb由CDK9/细胞周期蛋白T异源二聚体组成，通过磷酸化RNA聚合酶II并克服负延长因子的停顿，促进一般和HIV特异性延长。它也是HIV-1 Tat蛋白的一个辅助因素，其P-TEFb的招募到新生病毒的mRNA对于HIV的复制是必不可少的。PI的实验室最近确定7SK SnRNA和六亚甲基双乙酰胺(HMBA)诱导蛋白1(HEXlM1)是两个P-TEFb相关核因子。它们与P-TEFb的相互作用需要P-TEFb在可能保守的CDK9 T环上磷酸化。当P-TEFb与7SK和HEX1M1结合时，失去了它的激酶和转录活性，形成了7SK SnRNP。通过7SK桥接HEXlM1：P-TEFb相互作用，HEXIM1抑制P-TEFb激酶。当细胞对全球转录抑制引起的压力做出反应时，7SK和HEXIM1都从P-TEFb中分离出来。与其对PTEFb的抑制一致，HEXlM1在体内强烈抑制HIV-1转录。我们的数据支持一个模型，在该模型中，磷酸化的P-TEFb通过HEXlM1和7SK的协同作用而被靶向抑制。 本研究试图通过对7SK SNRNP的结构和功能的分析，阐明HEXlM1/7SK抑制P-TEFb激酶的机制。实验还将研究7SK是否在其介导的HEXlM1：P-TEFb结合之外的抑制作用中发挥直接的、积极的作用。此外，还将分析RNAi降低HEXlM1表达对CTD磷酸化和HIV-1转录的影响。由于HEXlM1是由HMBA诱导的，HMBA是一种有效的细胞分化诱导剂，因此我们将研究HEXlM1和P-TEFb在控制这一关键过程中的作用。到目前为止的初步数据表明，蛋白磷酸酶-1(PP-1)在激活HIV-1转录和在体外将7SK/HEXIM1从P-TEFb中分离出来的过程中发挥了作用。主要的工作将致力于研究PP-1在体内调节应激诱导的7SK SnRNP的破坏和P-TEFb激活的可能作用。我们的最终目标是阐明在伸长阶段控制真核基因表达的分子机制。研究HEX1M1/7SK如何调节关键的延伸因子和TAT辅助因子P-TEFb的活性，以及这种调节如何影响HIV-1的转录和细胞基因表达，将在这方面提供非常有意义的信息。

DESCRIPTION (provided by applicant): The human transcription elongation factor P-TEFb, consisting of a CDK9/cyclin T heterodimer, promotes both general and HIV-specific elongation by phosphorylating RNA polymerase II and overcoming pausing by negative elongation factors. It is also a co-factor for the HIV-1 Tat protein, whose recruitment of P-TEFb to the nascent viral mRNA is essential for HIV replication. The PI's laboratory has recently identified the 7SK snRNA and hexamethylene bisacetamide (HMBA)-inducible protein 1 (HEXlM1) as two P-TEFb-associated nuclear factors. Their interactions with P-TEFb require the phosphorylation of P-TEFb on possibly the conserved CDK9 T-loop. P-TEFb loses its kinase and transcriptional activities when associated with 7SK and HEXlM1 to form the 7SK snRNP. With 7SK bridging the HEXlM1:P-TEFb interaction, HEXIM1 inhibits the P-TEFb kinase. Both 7SK and HEXIM1 are dissociated from P-TEFb when cells respond to stresses caused by a global inhibition of transcription. Consistent with its inhibition of PTEFb, HEXlM1 strongly suppresses HIV-1 transcription in vivo. Our data support a model in which the phosphorylated P-TEFb is targeted for inhibition by the coordinated actions of HEXlM1 and 7SK. This proposal seeks to elucidate the mechanism by which HEXlM1/7SK inhibit the P-TEFb kinase through structure-function analyses of the 7SK snRNP. Experiments will also be performed to investigate whether 7SK plays a direct, active role in the inhibitory action beyond its mediation of the HEXlM1:P-TEFb binding. In addition, the effect of reducing HEXlM1 expression by RNAi on CTD phosphorylation and HIV-1 transcription will be analyzed. Because HEXlM1 is induced by HMBA, a potent inducer of cell differentiation, the roles of HEXlM1 and P-TEFb in controlling this key process will be examined. The preliminary data so far implicate a role of protein phosphatase-1 (PP-1) in activating HIV-1 transcription and dissociating 7SK/HEXIM1 from P-TEFb in vitro. A major effort will be devoted to the investigation of a possible role of PP-1 in regulating the stress-induced disruption of the 7SK snRNP and activation of P-TEFb in vivo. Our ultimate goal is to elucidate the molecular mechanisms controlling eukaryotic gene expression at the stage of elongation. Investigation of how the activity of P-TEFb, a key elongation factor and Tat co-factor, is regulated by HEXlM1/7SK and how this regulation affects HIV-1 transcription and cellular gene expression will be very informative in this regard.