TRANSCRIPTIONAL REGULATION BY EPIGENETIC FACTORS

描述（由申请人提供）：蛋白质的三胸蛋白群（trxG）和polycomb蛋白群（PcG）的基因调控机制仍然是当前发育和分子生物学中最积极追求的问题之一。这种持续的兴趣是基于认识到这些蛋白质家族在多细胞生物发育过程中被用于调节大量重要基因，包括早期发育的关键调节因子，同源（HOX）基因。TrxG和PcG蛋白通过改变靶基因的染色质结构发挥其活性。这是通过多种方式实现的，通过atp依赖性核小体重塑复合体改变核小体的包装，或通过酶修饰组蛋白尾部的一些残基。重要的是，无论TrxG和PcG蛋白以何种特定方式发挥其活性，它们都能够锁定特定的基因表达状态，然后在子细胞中以表观遗传方式遗传。对这两组蛋白质的研究具有重要的健康相关应用，因为其中一些蛋白质，如ALL1/HRX/MLL，被认为与许多癌症有关。我们最近的数据表明，TrxG蛋白复合物TAC1被招募到活跃转录热休克基因起始位点下游的区域。这种招募伴随着特定的TAG 1依赖性甲基化和组蛋白残基乙酰化，最终导致热休克基因转录的增加。本提案的目的是通过使用靶HOX基因Ultrabithorax （Ubx）作为模型，验证TAG 1的这种功能模式也适用于发育调节基因的假设。我们的初步结果表明，该复合物的一个关键成分Trithorax （TRX）位于Ubx起始位点的下游及其启动子处，这表明TRX参与了两个不同的转录步骤，即Poll转录的起始和延伸。基于这些和先前的研究，以及与同源原癌基因MLL的功能相似性，我们提出存在2个TRX复合物，可能参与这些不同的转录方面。两(2)个TRX复合物可能与转录起始和/或延伸所需的其他因子相互作用。为了验证这一假设并揭示TRX复合物在其他转录因子网络中的作用，我们开发了一种分选技术，可以获得Ubx被激活和沉默的细胞核。利用这种技术，我们提出解决以下问题：(i) TrxG和PcG蛋白与转录活性和沉默的Ubx基因的调控区域的关联有什么不同？（ii）新的类似mll的TRX复合物有什么特点？（iii） Ubx转录的哪些步骤需要不同的TRX蛋白复合物？（iv） TRX复合物在其他转录因子网络中的作用是什么？这些问题的答案不仅将为TAC1在转录调控过程中发挥作用的方式提供新的线索，还将揭示其他TrxG和PcG蛋白在表观遗传维持中的作用，以及这些表观遗传调节因子与RNA聚合酶起始和延伸相关因子的关系。鉴于TRX和MLL在结构和功能上的相似性，这些研究也将推进我们对真核生物转录调控的基本机制及其与癌症等疾病的相关性的认识。

DESCRIPTION (provided by applicant): The mechanisms of gene regulation by the trithorax-group (trxG) and Polycomb-group (PcG) of proteins remain 1 of the most actively pursued issues in the current developmental and molecular biology. This continuous interest is based on the realization that these protein families are employed in regulation of a large number of important genes during development of the multicellular organisms, including the key regulators of early development, the homeotic (HOX) genes. TrxG and PcG proteins exert their activities by altering the chromatin structure of their target genes. This is achieved in a variety of ways, by either changing the packaging of nucleosomes by the ATP-dependent nucleosome remodeling complexes, or by the enzymatic modifications of a number of residues in histone tails. Importantly, irrespective of the specific ways in which TrxG and PcG proteins exert their activities, they are capable of locking in a specific status of gene expression, which is then inherited in an epigenetic fashion in daughter cells. The studies of these 2 groups of proteins have an important health-related applications since some of these proteins, as for example ALL1/HRX/MLL, are believed to be involved in a number of cancers. Our recent data suggest that the TrxG protein complex TAC1 is recruited to the region downstream of the start site of the actively transcribed heat shock genes. This recruitment is accompanied by the specific TAG 1-dependent methylation and acetylation of histone residues and ultimately leads to an increase in heat shock gene transcription. The goal of this proposal is to test the hypothesis that this mode of functioning of TAG 1 is also true for the developmentally regulated genes, by using a target HOX gene Ultrabithorax (Ubx) as a model. Our preliminary results indicate that a key component of this complex, Trithorax (TRX), is found downstream of the start site of Ubx and at its promoter, suggesting that TRX is involved in 2 different steps of transcription, initiation and elongation of transcription by Poll. Based on these and previous studies, and on the functional similarities with the homologous proto-oncogene MLL, we propose the existence of 2 TRX complexes that may be involved in these different aspects of transcription. Two (2) TRX complexes may interact with other factors that are required for initiation and/or elongation of transcription. To test this hypothesis and to uncover the role of TRX complexes in the network of other transcription factors, we developed a sorting technique that allows to obtain nuclei in which Ubx is activated and silenced. Using this technique, we propose to address the following questions: (i) What is the difference in association of the TrxG and PcG proteins with the regulatory regions of the transcriptionally active and silenced Ubx gene? (ii) What are the features of the new MLL-like TRX complex? (iii) Which steps of Ubx transcription require different TRX protein complexes? (iv) What are the roles of TRX complexes in the network of other transcription factors? Answers to these questions will shed new light not only on the way TAC1 exerts its effects during transcriptional regulation, but will also reveal the roles of other TrxG and PcG proteins in epigenetic maintenance, as well as the relationship of these epigenetic regulators with factors that are involved in initiation and elongation of RNA polymerase. Given structural and functional similarities between TRX and MLL, these studies will also advance our knowledge of the basic mechanisms of transcriptional regulation in eukaryotes and their relevance to diseases like cancer.