Genetic and molecular studies of Drosophila chromatin remodeling factors

描述（由申请人提供）：核小体和染色质的其他组分可通过阻断转录因子和其他调节蛋白进入DNA来抑制转录。核小体和连接体组蛋白之间的相互作用促进30 nm纤维的形成和更高级染色质结构的越来越紧凑的形式，进一步限制了调控蛋白对DNA的访问。染色质结构的改变导致多种人类疾病，包括癌症和出生缺陷。虽然在确定染色质重塑因子和组蛋白修饰酶的作用机制方面已经取得了巨大的进展，但关于它们如何通过改变核小体的结构和间距来调节转录，仍有许多东西有待了解。更少的是知道如何更高层次的染色质结构的调节和用于控制基因表达在真核细胞。为了解决这些重要问题，我们实验室使用果蝇作为模式生物，研究染色质重塑和修饰酶的功能。我们的工作主要集中在Polycomb和trithorax基团在转录和发育中的作用。多梳族蛋白通过甲基化组蛋白H3的赖氨酸27在广泛的染色质结构域上调节细胞的多能性和分化中发挥重要作用。这种染色质修饰在从苍蝇到人类的生物体中的表观遗传基因沉默中起着关键作用。一个关键问题是如何克服Polycomb组抑制，以允许分化所需的基因表达。我们实验室最近的研究表明，Kismet（KIS），一种ATP依赖的染色质重塑因子，通过阻止H3K27甲基化扩散到活性基因中，在这一过程中起着重要作用。为了验证这一假设，我们将比较KIS和H3K27甲基化的全基因组分布，并确定KIS是否作为抑制性修饰扩散的障碍。我们实验室的其他研究表明，另一种染色质重塑因子，模仿SWI（ISWI），通过促进连接体组蛋白H1与染色质的结合，在染色质致密化和转录抑制中起着全球性的作用。为了验证这一假设，我们将比较ISWI和组蛋白H1功能丧失所导致的基因表达变化。我们还将使用互补的遗传学，生物化学和细胞生物学方法来确定ISWI如何促进组蛋白H1与染色质的结合，并确定参与这一过程的其他因素。通过在单一模式生物中研究多种染色质重塑因子，我们将更好地了解它们在转录、发育和疾病中的作用。

DESCRIPTION (provided by applicant): Nucleosomes and other components of chromatin can repress transcription by blocking the access of transcription factors and other regulatory proteins to DNA. Interactions between nucleosomes and linker histones promote the formation of 30 nm fibers and increasingly compact forms of higher-order chromatin structure, further limiting the access of regulatory proteins to DNA. Alterations in chromatin structure lead to a variety of human diseases, including cancer and birth defects. Although tremendous progress has been made toward determining the mechanism of action of chromatin-remodeling factors and histone-modifying enzymes, much remains to be learned about how they regulate transcription by altering the structure and spacing of nucleosomes. Even less is known about how higher-order chromatin structure is regulated and used to control gene expression in eukaryotic cells. To address these important issues, our laboratory uses Drosophila melanogaster as a model organism to study the function of chromatin-remodeling and modifying enzymes. Much of our work has been focused on the roles of Polycomb and trithorax group in transcription and development. Polycomb group proteins play important roles in the regulation of cellular pluripotency and differentiation by methylating lysine 27 of histone H3 over broad chromatin domains. This chromatin modification plays a key role in epigenetic gene silencing in organisms ranging from flies to humans. A key issue concerns how Polycomb group repression is overcome to permit the expression of genes required for differentiation. Recent studies in our lab have suggested that Kismet (KIS), an ATP-dependent chromatin-remodeling factor, plays an important role in this process by preventing the spread of H3K27 methylation into active genes. To test this hypothesis, we will compare the genome-wide distributions of KIS and H3K27 methylation and determine if KIS acts as a barrier to the spread of the repressive modification. Other studies in our laboratory have suggested that another chromatin-remodeling factor, Imitation-SWI (ISWI), plays a global role in chromatin compaction and transcriptional repression by promoting the association of the linker histone H1 with chromatin. To test this hypothesis, we will compare changes in gene expression resulting from the loss of ISWI and histone H1 function in vivo. We will also use complementary genetic, biochemical and cell biological approaches to determine how ISWI promotes the association of histone H1 with chromatin and identify other factors involved in this process. By studying multiple chromatin- remodeling factors in a single model organism, we will gain a much better understanding of their roles in transcription, development and disease.