Regulation of Transcriptional Elongation by HIV-1 TAT

描述（由申请人提供）：人转录延伸因子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和克服负延伸因子的暂停来促进一般和HIV特异性延伸。它也是HIV-1达特蛋白的辅因子，其将P-TEFb募集至新生病毒mRNA对于HIV复制是必需的。PI的实验室最近鉴定了7SK snRNA和双乙酰胺（HMBA）诱导蛋白1（HEXlMl）作为两种P-TEFb相关的核因子。它们与P-TEFb的相互作用需要P-TEFb在可能保守的CDK9 T环上的磷酸化。P-TEFb在与7SK和HEXlMl结合形成7SK snRNP时失去其激酶和转录活性。在7SK桥接HEXIM 1：P-TEFb相互作用的情况下，HEXIM 1抑制P-TEFb激酶。7SK和HEXIM 1都是从P-TEFb解离时，细胞响应于由转录的整体抑制引起的应激。与其对PTEFb的抑制一致，HEXlMl强烈抑制体内HIV-1转录。我们的数据支持一种模型，其中磷酸化的P-TEFb被HEXlMl和7SK的协调作用靶向抑制。 该建议试图通过7SK snRNP的结构-功能分析来阐明HEXlM1/7SK抑制P-TEFb激酶的机制。还将进行实验以研究7SK是否在其介导HEXlMl：P-TEFb结合之外的抑制作用中发挥直接的、积极的作用。此外，将分析通过RNAi降低HEXlMl表达对CTD磷酸化和HIV-1转录的影响。因为HEXlMl由HMBA（细胞分化的有效诱导剂）诱导，所以将检查HEXlMl和P-TEFb在控制该关键过程中的作用。到目前为止的初步数据暗示蛋白磷酸酶-1（PP-1）在体外激活HIV-1转录和从P-TEFb解离7SK/HEXIM 1中的作用。一个主要的努力将致力于研究PP-1在调节应激诱导的7SK snRNP的破坏和体内P-TEFb的激活中的可能作用。我们的最终目标是阐明真核生物基因在延伸阶段表达的分子机制。研究HEXIM 1/7 SK如何调节P-TEFb（一种关键的延伸因子和达特辅因子）的活性以及这种调节如何影响HIV-1转录和细胞基因表达在这方面将是非常有益的。

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.