ISWI Chromatin Remodeling Complexes in Eye Development

描述（由申请人提供）：本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。与所有发育途径一样，眼睛的发育取决于基因表达的空间和时间模式的精确调节。转录激活和抑制发生在染色质的背景下，染色质是将DNA压缩到真核细胞核中的DNA和组蛋白的复合物。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，从而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青蛙非洲爪蟾的白内障。为了实现这一点，我们将阻止ISW 1在非洲爪蟾胚胎发育中的功能，特别是在发育中的眼睛内，并通过许多方法分析所产生的眼睛畸形，包括显微镜检查、切片和精细结构分析以及基因表达分析。这些研究将提供关键的见解染色质重塑在眼睛发育中的作用，并将提供一个新的实验系统，在其中研究白内障的形成。这项建议有三个具体目标。目的1是表征缺乏正常水平的ISW 1蛋白的爪蟾胚胎的眼睛中的发育缺陷，解决发育中的眼睛中的形态和转录缺陷。目的2是确定的ISWI含有复杂的是负责正常的眼睛发育，使用吗啉抑制亚基独特的每个已知的非洲爪蟾ISWI复合物。目的3是通过产生在γ-晶状体蛋白启动子的控制下表达显性负性ISWI突变体的转基因非洲爪蟾来测试ISW 1功能是否在发育中的眼睛中特异性地需要以用于正常发育和预防白内障。

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.