ISWI remodeling complexes in eye development

ISWI 重塑复合物在眼睛发育中的作用

• 批准号: 7366913
• 负责人: JOCELYN E KREBS
• 金额: $ 20.55万
• 依托单位: UNIVERSITY OF ALASKA ANCHORAGE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-01 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7366913
• 关键词: Address; Blindness; Brain; Bromodomain; CHRAC; Catalytic Domain; Cataract; Cell Nucleus; Child; Chromatin; Chromatin Remodeling Factor; Complex; DNA; Defect; Development; Dominant-Negative Mutation; Embryo; Embryonic Development; Enzymes; Event; Eye; Eye Development; Gene Expression; Genes; Genetic Transcription; Goals; Histones; ISWI; Individual; Injection of therapeutic agent; Microscopic; Neuraxis; PHD Finger; Pathway interactions; Pattern; Phenotype; Proteins; Rana; Regulation; Repression; Research; Role; Signal Transduction; Specific qualifier value; Staging; Structure; Study models; Switching Complex; Tissues; Transcriptional Activation; Transgenic Organisms; Work; Xenopus; Xenopus laevis; chromatin remodeling; congenital cataract; in vivo; insight; interest; knock-down; malformation; mutant; neurodevelopment; promoter; relating to nervous system; research study

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.

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和中枢神经系统（CNS）的发育。与所有发育途径一样，眼睛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中，染色质是 DNA 和组蛋白的复合物，可将 DNA 压缩到真核细胞核中。染色质重塑酶正向和负向调节染色质中 DNA 的可及性，因此是基因表达的重要调节因子。模仿开关（ISWI）染色质重塑酶是非洲爪蟾眼睛和中枢神经系统正常发育所必需的，缺乏 ISWI 的胚胎会出现先天性囊下白内障。然而，ISWI是体内多种重塑复合物的催化亚基。我们特别感兴趣的是不同的 ISWI 依赖性染色质重塑酶如何在胚胎发生过程中调节眼睛发育。为了实现这一目标，我们将通过抑制每个复合物特有的亚基来评估各个 ISWI 复合物的作用。我们将首先使用吗啉注射来抑制 WSTF（WICH 复合物的一个亚基）和 CHRAC-17（CHRAC 复合物的一个亚基）。我们将评估这些敲低对发育中的非洲爪蟾胚胎的影响，特别是在发育中的眼睛内，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产生的眼睛畸形。这些研究将为染色质重塑在眼睛发育中的作用提供重要的见解。该提案具有三个具体目标。目标 1 是利用吗啉抑制来表征缺乏功能性 WICH 或 CHRAC 复合物的非洲爪蟾胚胎的发育表型，解决形态学和转录缺陷。目标 2 是确定 WSTF 内的保守结构域（溴结构域、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。目标 3 是通过产生在诱导型和组织特异性启动子控制下表达显性失活 ISWI 突变体的转基因非洲爪蟾，确定眼睛发育所需 ISWI 活性的阶段。先天性白内障是儿童失明的主要原因。发育中的青蛙胚胎缺乏 ISWI 重塑活性，会导致大脑和眼睛发育出现许多缺陷，包括囊下白内障的形成。我们的工作将有助于理解正常眼睛发育的机制，并为研究先天性白内障形成提供新模型。