ISWI 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和中枢神经系统(CNS)的发育。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。染色质重塑酶对DNA在染色质中的可及性有正向和负向调节作用，因此是基因表达的重要调节因子。模仿开关(ISWI)染色质重塑酶是非洲爪哇眼睛和中枢神经系统正常发育所必需的，缺乏ISWI的胚胎会发生先天性囊下白内障。然而，ISWI是体内多种重塑复合体的催化亚单位。我们特别感兴趣的是不同的依赖ISWI的染色质重塑酶如何在胚胎发育过程中调节眼睛的发育。为了实现这一点，我们将通过抑制每个复合体特有的亚基来评估单个ISWI复合体的作用。我们将首先使用吗啡注射来抑制WSTF和CHRAC-17，WSTF是WICH复合体的一个亚单位，CHRAC-17是CHRAC复合体的一个亚单位。我们将评估这些基因敲除在非洲爪哇胚胎发育中的影响，特别是在发育中的眼睛，并通过显微镜检查、切片和精细结构分析以及基因表达分析来分析由此导致的眼睛畸形。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。这项提议有三个具体目标。目的1是用吗啡抑制法研究缺乏功能性WICE或CHRAC复合体的非洲爪哇胚胎的发育表型，解决形态和转录缺陷。目的2是确定WSTF中的保守结构域(溴域、PHD Finger和DDT结构域)对其发育功能的贡献。这一目标将包括注射WSTF的缺失突变体。目的3是通过在诱导启动子和组织特异性启动子的控制下产生表达显性-负性ISWI突变体的转基因非洲爪哇，来确定眼睛发育所需的ISWI活性的阶段(S)。先天性白内障是导致儿童失明的主要原因。发育中的青蛙胚胎缺乏ISWI重塑活性，导致大脑和眼睛发育中的许多缺陷，包括包膜下白内障的形成。我们的工作将有助于了解正常眼发育的机制，并为研究先天性白内障的形成提供一个新的模型。

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 central nervous system (CNS). 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 positively and negatively regulate the accessibility of DNA in chromatin, and are thereby essential regulators of gene expression. The Imitation Switch (ISWI) chromatin remodeling enzyme is required for normal development of the eye and CNS in Xenopus laevis, and embryos lacking ISWI develop congenital subcapsular cataracts. However, ISWI is the catalytic subunit of multiple remodeling complexes in vivo. We are specifically interested in how different ISWI-dependent chromatin remodeling enzymes regulate eye development during embryogenesis. To accomplish this, we will assess the roles of individual ISWI complexes by inhibiting subunits unique to each complex. We will begin by using morpholino injection to inhibit WSTF, a subunit of the WICH complex, and CHRAC-17, a subunit of the CHRAC complex. We will assess the effects of these knockdowns in the developing Xenopus embryo, specifically within the developing eye, and analyze the resulting eye malformations by 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. This proposal has three specific aims. Aim 1 is to characterize the developmental phenotypes of Xenopus embryos lacking functional WICH or CHRAC complexes, using morpholino inhibition, addressing both morphological and transcriptional defects. Aim 2 is to identify the contributions of the conserved domains within WSTF (bromodomain, PHD finger, and DDT domains) to its developmental functions. This aim will include injection of deletion mutants of WSTF. Aim 3 is to determine the stage(s) during which ISWI activity is required for eye development, by generating transgenic Xenopus that express a dominant-negative ISWI mutant under control of inducible and tissue-specific promoters. Congenital cataracts are the leading cause of blindness in children. Lack of ISWI remodeling activity in developing frog embryos results in numerous defects in brain and eye development, including the formation of subcapsular cataracts. Our work will contribute to the understanding of the mechanisms of normal eye development and provides a new model for studying congenital cataract formation.