ISWI Chromatin Remodeling Complexes in Eye Development

描述(申请人提供)：这项研究的长期目标是了解染色质重塑如何控制脊椎动物眼睛的发育，并剖析先天性白内障形成的分子基础。正如在所有发育过程中一样，眼睛的发育依赖于对基因表达的空间和时间模式的精确调控。转录激活和抑制发生在染色质的背景下，染色质是DNA和组蛋白的复合体，将DNA压缩到真核细胞的核中。染色质重塑酶和组蛋白修饰酶协同调节染色质中DNA的可及性，因此是基因表达的重要调节因子。我们特别感兴趣的是模仿开关(ISWL)染色质重塑酶如何控制早期胚胎中的眼睛发育，以及ISWL功能的丧失如何导致非洲爪蛙的白内障。为了实现这一目标，我们将阻止ISWL在非洲爪哇胚胎发育中的作用，特别是在发育中的眼睛中，并通过多种方法分析由此导致的眼睛畸形，包括显微镜检查、切片和精细结构分析以及基因表达分析。这些研究将为染色质重塑在眼睛发育中的作用提供关键的见解，并将提供一个新的实验系统来研究白内障的形成。这项提议有三个具体目标。目的1是描述缺乏正常ISWL蛋白水平的非洲爪哇胚胎眼睛的发育缺陷，解决发育中眼睛的形态和转录缺陷。目标2是通过对每个已知的非洲爪哇ISWI复合体所特有的亚基进行吗啡抑制，确定哪个含有ISWI的复合体对正常的眼睛发育负责。目的3是通过产生在伽马晶体蛋白启动子控制下表达显性-负性ISWI突变体的转基因非洲爪哇，来测试ISWL功能是否对发育中的眼睛正常发育和预防白内障是特定需要的。

DESCRIPTION (provided by applicant): The long-term objective of this research is to understand how chromatin remodeling controls the development of the vertebrate eye and to dissect the molecular basis of congenital cataract formation. As in all developmental pathways, eye development depends upon precise regulation of spatial and temporal patterns of gene expression. Transcription activation and repression occurs in the context of chromatin, a complex of DNA and histone proteins that compacts DNA into the eukaryotic nucleus. Chromatin remodeling enzymes and histone modifying enzymes cooperatively regulate the accessibility of DNA in chromatin, and are thereby essential regulators of gene expression. We are specifically interested in how the Imitation Switch (ISWl) chromatin remodeling enzyme controls eye development in the early embryo, and how the loss of ISWl function leads to cataracts in the frog Xenopus laevis. To accomplish this, we will, prevent ISWl function in developing Xenopus embryos, and specifically within the developing eye, and analyze the resulting eye malformations by a number of methods, including microscopic examination, sectioning and fine structure analysis, and gene expression analysis. These studies will provide critical insights into the role of chromatin remodeling in eye development and will provide a new experimental system in which to study cataract formation. This proposal has three specific aims. Aim 1 is to characterize the developmental defects in the eyes of Xenopus embryos lacking normal levels of ISWl protein, addressing both morphological and transcriptional defects in the developing eye. Aim 2 is to identify which ISWI-containing complex is responsible for normal eye development, using Morpholino inhibition of subunits unique to each of the known Xenopus ISWI complexes. Aim 3 is to test whether ISWl function is required specifically in the developing eye for normal development and to prevent cataracts, by generating transgenic Xenopus that express a dominant-negative ISWI mutant under control of the gamma-crystallin promoter.