Regulation of Transcriptional Elongation by HIV-1 Tat

 描述（由申请人提供）：RNA聚合酶II（Pol II）在整合的HIV-1前病毒DNA上的启动子近端暂停长期以来被认为是病毒基因表达的主要限速步骤。为了克服这种限制，HIV达特蛋白通过在TAR RNA上形成多亚基复合物（位于所有病毒转录物5'端的发夹结构）来募集人超延伸复合物（SEC）以暂停Pol II。SEC含有两种强大的转录延伸因子P-TEFb和ELL 2，它们通过不同的机制起作用，但可以协同刺激Pol II延伸以产生全长HIV转录物。值得注意的是，SEC作为达特辅因子的鉴定是在不存在TAR的情况下进行的。目前还不清楚这种RNA元件是否只是作为一个平台，将Tat-SEC招募到病毒启动子中，或者如我们的初步数据所示，可能通过吸引其他尚未确定的因子来发挥额外的调节作用。为了更好地理解控制TAR特异性和达特依赖性HIV转录的机制，我们建议使用体内和体外亲和纯化方法的组合来分离和鉴定TAR RNP的新组分，并研究其在HIV基因表达中的功能意义和作用机制。 除了存在于SEC中之外，P-TEFb还存在于另一种含有BRD 4的催化活性复合物中，BRD 4是一种众所周知的BET布罗莫结构域蛋白，表观遗传阅读器和主要的癌症治疗靶标。尽管P-TEFb的BRD 4募集对于许多细胞尤其是癌症相关基因的转录是必需的，但是它抑制Tat反式激活，因为BRD 4和达特完成与P-TEFb的结合。最近，我们已经鉴定了REJ，一种来自传统中草药的天然产物，其可以拮抗BRD 4对达特功能的抑制，可能通过激活特异性泛素-蛋白酶体途径来选择性地降解BRD 4。基于这些数据，本提案的另一个主要目标是研究REJ激活Tat依赖性HIV转录的机制，并阐明REJ诱导降解BRD 4的蛋白水解途径。该途径的关键组分的鉴定，特别是BRD 4特异性E3泛素连接酶，将使我们能够选择性地控制细胞BRD 4水平，用于HIV/AIDS和癌症的潜在治疗干预。

 DESCRIPTION (provided by applicant): Promoter-proximal pausing of RNA polymerase II (Pol II) on integrated HIV-1 proviral DNA has long been recognized as a major rate-limiting step in viral gene expression. To overcome this restriction, the HIV Tat protein recruits the human Super Elongation Complex (SEC) to paused Pol II through forming a multi-subunit complex on the TAR RNA, a hairpin structure located at the 5' end of all viral transcripts. SEC contains two powerful transcription elongation factors P-TEFb and ELL2 that act by different mechanisms but can synergize in their stimulation of Pol II elongation to generate full-length HIV transcripts. Notably, the identification of the SEC as a Tat cofactor was performed in the absence of TAR. It is unclear whether this RNA element simply serves as a platform to recruit Tat-SEC to the viral promoter, or as suggested by our preliminary data, may play additional regulatory roles by attracting other yet-to-be identified factors. To better understand the mechanism controlling TAR-specific and Tat- dependent HIV transcription, we propose to use a combination of in vivo and in vitro affinity-purification approaches to isolate and identify new components of the TAR RNPs and investigate their functional significance and mechanism of action in HIV gene expression. In addition to existing in the SEC, P-TEFb is also found in another catalytically active complex containing BRD4, a well-known BET bromodomain protein, epigenetic reader and major cancer therapeutic target. Although the BRD4 recruitment of P-TEFb is required for transcription of many cellular especially cancer-related genes, it is inhibitory to Tat-transactivation because BRD4 and Tat complete for binding to P-TEFb. Recently, we have identified REJ, a natural product derived from a traditional Chinese medicinal herb, which can antagonize BRD4 inhibition of Tat function through possibly activating a specific ubiquitin-proteasome pathway to selectively degrade BRD4. Based on these data, another major objective of this proposal is to investigate the mechanism by which REJ activates Tat-dependent HIV transcription and also to elucidate the proteolytic pathway that is induced by REJ to degrade BRD4. The identification of key components of this pathway especially the BRD4-specific E3 ubiquitin ligase will allow us to selectively control the cellular BRD4 level for potential therapeutic intervention in HIV/AIDS and cancer.