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-近端暂停一直被认为是病毒基因表达的主要限速步骤。为了克服这一限制，HIV Tat蛋白招募人类超级延长复合体(SEC)，通过在TAR RNA上形成多亚单位复合体来暂停POL II，TAR RNA是一种发夹结构，位于所有病毒转录本的5‘端。SEC包含两个强大的转录延长因子P-TEFb和ELL2，它们的作用机制不同，但可以协同刺激POL II的延长，产生全长HIV转录本。值得注意的是，SEC是在没有TAR的情况下确定为TAT辅助因子的。目前尚不清楚这种RNA元件是仅仅作为一个平台来招募TAT-SEC到病毒启动子，还是像我们的初步数据所表明的那样，通过吸引其他尚未确定的因素来发挥额外的调节作用。为了更好地了解控制TAR特异性和TAT依赖的HIV转录的机制，我们建议使用体内和体外亲和纯化相结合的方法来分离和鉴定TAR RNPs的新成分，并研究它们在HIV基因表达中的功能意义和作用机制。除了存在于SEC中，P-TEFb还存在于另一个含有BRD4的催化活性复合体中，BRD4是一种著名的BET溴域蛋白，是表观遗传学读物，也是主要的癌症治疗靶点。虽然P-TEFb的BRD4募集是许多细胞尤其是癌症相关基因转录所必需的，但它对TAT的反式激活具有抑制作用，因为BRD4和TAT完全与P-TEFb结合。最近，我们发现了一种从中草药中提取的天然产物Rej，它可能通过激活特定的泛素-蛋白酶体途径选择性地降解BRD4，从而拮抗BRD4对TAT功能的抑制。基于这些数据，这一建议的另一个主要目标是研究REJ激活依赖TAT的HIV转录的机制，并阐明REJ诱导的降解BRD4的蛋白降解途径。该途径的关键成分，特别是BRD4特异的E3泛素连接酶的鉴定，将使我们能够选择性地控制细胞中BRD4的水平，为潜在的艾滋病毒/艾滋病和癌症的治疗干预。

 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.