Biological roles and Mediator-dependent transcription mechanisms of RNA polymerase II(G)

 描述(由申请人提供) RNA聚合酶II(Pol II)通过转录蛋白质编码和非编码的RNA，在真核生物基因组的读出中起着关键作用。正常的生理过程依赖于精确的转录调控，而转录失调是包括癌症在内的许多病理学的基础。Pol II与特定启动子的结合受多种(Co)因子的调控，其中包括多亚单位介体，它直接结合Pol II和转录激活子，从而促进基本上所有基因的激活。POL II(G)是最近发现的(最初在小牛胸腺和猪肝脏中)POL II的二联体，它含有高度相关的多肽Gdown 1和Pol II的正常12个亚基。体外转录研究表明，在没有介体的情况下，与POL II相关的Gdown 1通过阻止起始因子TFIIF与POL II的结合来抑制转录，从而抑制预起始复合体的形成。Mediator逆转POL II(G)被抑制的转录能力，尽管其潜在的机制尚不清楚。虽然Mediator在酵母和人类之间是保守的，但Gdown 1的保守只延伸到果蝇。因此，果蝇和高等后生动物的转录调控系统是通过获得一个额外的和功能上不同的POL II而进化的，尽管POL II(G)在高等后生动物转录调控中所扮演的特定角色(S)仍不清楚。为了了解POL II(G)的生物学作用(S)，初步研究表明，去除Gdown 1会导致果蝇和小鼠的早期胚胎死亡。这种致命性可能部分是由于严重抑制细胞增殖，如在Gdown-1缺陷的胚胎干细胞和癌细胞系中观察到的，以及通过基因表达的失调，如在Gdown-1缺陷的肝细胞中观察到的。然而，POL II(G)似乎不太可能主要对细胞生长起重要作用，因为Gdown 1在酵母和低等后生动物中不保守。为了了解Pol II(G)在体内的作用和介体依赖的激活机制，我们的具体目标是：(1)通过测定Pol II(G)直接调控的基因--通过Gdown 1基因缺失的囊胚、条件性Gdown 1基因敲除小鼠、小鼠胚胎干细胞和Gdown 1基因缺失的肝细胞的RNA-seq和ChIP-seq分析，研究Pol II(G)在胚胎和成人细胞中的生物学功能；(Ii)通过对Pol II、Gdown 1(wt和突变体)与介体之间接触位点的遗传和生化测定，以及通过CX-MS和相应复合物的低温EM结构分析，研究依赖介体的Pol II(G)转录的分子机制；(Iii)通过小鼠移植人癌细胞和流体动力注射致癌载体产生肝癌的模型，研究Gdown 1消融对肿瘤形成的影响。因此，除了揭示POL II(G)的生物学作用和Mediator对其调节的潜在机制外，这些研究还可能揭示癌症治疗的新靶点。

 DESCRIPTION (provided by applicant) RNA polymerase II (Pol II) plays a critical role in readout of the eukaryotic genome through its transcription of protein-coding and non-coding RNAs. Normal physiological processes depend upon precise transcriptional controls, whereas transcriptional dysregulation is the basis of numerous pathologies that include cancer. Pol II recruitment to specific promoters is regulated by multiple (co)factors that include the multi-subunit Mediator, which directly binds both Pol II and transcriptional activators to facilitate activation of essentially all genes. Pol II(G) is a ditinct, recently discovered (originally in calf thymus and pig liver) form of Pol II that contains the tighly associated polypeptide Gdown1 along with the normal 12 subunits of Pol II. In vitro transcription studies revealed that, in the absence of Mediator, the Pol II-associated Gdown1 represses transcription by preventing the association of initiation factor TFIIF with Pol II and thus inhibitng pre-initiation complex formation. Mediator reverses the repressed transcriptional capacity of Pol II(G), although the underlying mechanism remains unclear. While Mediator is conserved from yeast to human, the conservation of Gdown1 extends only to Drosophila melanogaster. Thus, the transcriptional regulatory system in fruit flies and higher metazoans has evolved through acquisition of an additional and functionally distinct Pol II, Pol II(G), although the specific rol(s) played by Pol II(G) in transcriptional regulation in higher metazoans remains unclear. Toward an understanding of the biological role(s) of Pol II(G), preliminary studies have demonstrated that Gdown1 ablation causes early embryonic lethality in both fruit flies and mice. This lethality may be explained in part by a severe inhibition of cell proliferation, as observed in Gdown1-deficient embryonic stem cell and cancer cell lines, and by dysregulation of gene expression, as observed in Gdown1-deficient hepatocytes. However, it seems unlikely that Pol II(G) is important primarily for cell growth since Gdown1 is not conserved in yeast and lower metazoans. With the general objective of understanding the in vivo roles and Mediator-dependent activation mechanisms for Pol II(G), our specific aims are: (i) to investigate the biological functions of Pol II(G) in embryonic and adult cells through determination of genes directly regulated by Pol II(G) -- by RNA-seq and ChIP-seq assays with Gdown1 null blastocysts, conditional Gdown1 knockout mouse, mouse embryonic stem cells, and Gdown1-null hepatocytes); (ii) to investigate the molecular mechanism of Mediator-dependent Pol II(G) transcription by genetic and biochemical determination of contact sites between Pol II, Gdown1 (wt and mutant) and Mediator and by CX-MS and cryoEM structural analyses of corresponding complexes; (iii) to examine the impact of Gdown1 ablation on tumorigenesis using mouse models that involve xenograft of human cancer cells and hydrodynamic injection of oncogenic vectors to generate liver cancer. Thus, in addition to revealing biological roles of Pol II(G) and the mechanism underlying its regulation by Mediator, these studies may reveal new targets for cancer therapeutics.