Transcription factor function in chromatin

描述(申请人提供)：为了了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并促进特定细胞系形成的机制。通常，这涉及到转录沉默的基因的激活，这些基因很可能被整合到抑制性染色质结构中。有证据支持这样的观点，即分化、特异的转录调节因子和重塑染色质结构的酶相互作用，使基因组DNA更容易进入转录机制。SWI/SNF酶以一种依赖于ATP的方式改变核小体结构，并在体外和体内促进转录因子的功能。这些酶的成分对胚胎发育是必不可少的，其中一些起到了肿瘤抑制的作用。此外，SWI/SNF酶与其他已知的肿瘤抑制因子相互作用，并与细胞周期控制有关。因此，这些酶广泛地需要正常的细胞功能以及分化和发育，它们的错误调节与肿瘤的形成有关。长期以来，骨骼肌分化一直是研究组织分化基本原理的模型。我们最近的研究使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的生肌基因的激活。通过对现有方法的改进，我们现在也能够检测染色质结构的变化和调控蛋白的相互作用，从而在胚胎肌肉发生期间、成人肌肉发生期间和成人组织维持期间导致基因激活，从而使我们的观察具有前所未有的生物学意义。此外，我们可以使用最近发展的一种称为宫内电穿孔的新技术来评估特定调控蛋白在胚胎骨骼肌组织发育中的功能相关性。这一新的应用将集中在SWI/SNF(Aim 1)和协同染色质重塑酶(Aim 2)在单个肌源性基因座上的作用。我们还证明了SWI/SNF酶诱导高阶染色质结构的变化，从而导致分化过程中生肌基因的重排。我们将在肌肉发生过程中探讨这些变化的机制和后果(目标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的形成有重要影响。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.