Transcription factor function in chromatin

描述（由申请人提供）：为了理解正常的发育和分化，有必要确定细胞启动新的基因表达程序和促进特定细胞系形成的机制。通常，这涉及到转录沉默基因的激活，这些基因很可能与抑制性染色质结构结合在一起。有证据支持分化特异性转录调控因子和改造染色质结构的酶合作，使基因组DNA更容易被转录机制获取的观点。SWI/SNF酶以ATP依赖的方式改变核小体结构，促进转录因子在体外和体内的功能。这些酶的组成部分是胚胎发育所必需的，有些还可以作为肿瘤抑制因子。此外，SWI/SNF酶与其他已知的肿瘤抑制因子相互作用，并参与细胞周期控制。因此，这些酶是正常细胞功能和分化发育所必需的，它们的错误调节与肿瘤的形成有关。骨骼肌分化一直是研究组织分化基本原理的模型。我们最近的研究利用细胞培养模型从机制上描述了染色质重塑酶如何促进特定肌生成基因的激活。通过对现有方法的修改，我们现在也能够检查染色质结构的变化和调节蛋白的相互作用，从而在胚胎肌发生、成人肌发生和成人组织维持过程中导致基因激活，从而使我们的观察结果具有前所未有的生物学相关性。此外，我们可以评估特定调节蛋白在胚胎骨骼肌组织发育中的功能相关性，使用最近开发的一种新技术，称为子宫内电穿孔。这项更新应用将集中在SWI/SNF （Aim 1）和协同染色质重塑酶（Aim 2）在单个肌源性位点上的贡献。我们还证明，SWI/SNF酶诱导高阶染色质结构的变化，导致分化过程中肌源性基因的重排。我们将探讨这些变化在肌发生过程中的机制和后果（目的3）。在分子水平上了解胚胎和成人骨骼肌的分化和维持将对肌肉再生和横纹肌肉瘤形成的研究产生重大影响，横纹肌肉瘤是肌源性肿瘤。

DESCRIPTION (provided by applicant): To understand normal development and differentiation, it is necessary to determine the mechanisms by which cells initiate new programs of gene expression and promote formation of specific cell lineages. Typically, this involves activation of genes that are transcriptionally silent and that are likely incorporated into repressive chromatin structure. Evidence supports the idea that differentiation specific transcriptional regulators and enzymes that remodel chromatin structure cooperate to render genomic DNA more accessible to the transcriptional machinery. SWI/SNF enzymes alter nucleosome structure in an ATP dependent manner and facilitate transcription factor function in vitro and in vivo. Components of these enzymes are essential for embryonic development and some act as tumor suppressors. Additionally, SWI/SNF enzymes interact with other known tumor suppressors and are implicated in cell cycle control. Thus these enzymes are broadly required for normal cell function and for differentiation and development, and their mis-regulation is implicated in tumor formation. Skeletal muscle differentiation has long been a model for studying fundamental principles of tissue differentiation. Our recent studies mechanistically describe how chromatin remodeling enzymes facilitate the activation of specific myogenic genes using cell culture models. Via modification of existing methodologies, we are now also capable of examining changes in chromatin structure and regulatory protein interactions leading to gene activation during embryonic myogenesis, during adult myogenesis, and during maintenance of adult tissue, thereby giving our observations unprecedented biological relevance. In addition, we can assess the functional relevance of specific regulatory proteins in developing embryonic skeletal muscle tissue using a novel adaptation of a recently developed technique called in utero electroporation. This renewal application will focus on the contributions of SWI/SNF (Aim 1) and cooperating chromatin remodeling enzymes (Aim 2) at individual myogenic loci. We also demonstrate that SWI/SNF enzymes induce changes in higher order chromatin structure that result in rearrangement of myogenic genes during differentiation. We will pursue the mechanisms and consequences of these changes during myogenesis (Aim 3). Understanding embryonic and adult skeletal muscle differentiation and maintenance at a molecular level will have significant impact on studies of muscle regeneration and on the formation of rhabdomyosarcomas, which are tumors of myogenic derivation.