Transcriptional Control of the Mouse aA-crystallin locus

摘要 这一研究计划的长期目标是阐明协调 调节晶状体发育和分化所需基因的表达。自.以来 晶状体是一个简单的组织，只由两种成熟的细胞类型组成，阐明了这些机制 提供对更复杂的组织分化所需的过程的洞察 为开发和设计前沿的生物新途径提供了基础 研究范围从干细胞替代疗法到有针对性的癌症治疗。中心前提 这一建议的核心是晶状体细胞的分化依赖于DNA-DNA的协调相互作用 转录因子与成纤维细胞生长因子和骨形态发生蛋白信号转导通路结合以协调晶状体特异性 形成成熟的眼晶状体所需的数百个基因的表达。哺乳动物晶状体的标志 纤维细胞分化是a-和b-/g-晶状体蛋白积聚的关键结构和保护因素 蛋白质、细胞伸长以及细胞核和其他细胞器的降解。在鉴别透镜中，特定的 一组基因在转录上被开启和关闭，这一过程的中心部分受到控制 通过染色质DNA与转录因子和染色质的可及性 重塑酶。在这些因子中，c-maf、Pax6、Prox1、c-jun、ETV5和Smads共同发挥作用 一个系统地调节单个晶体蛋白和其他蛋白的时空表达的单位 对眼睛晶状体的分化和功能至关重要的基因。具体地说，这项提议将：1) 阐明染色质在调节晶状体分化特异基因表达中的功能作用 在全基因组水平上，(2)定义DNA结合转录因子的特定作用及其作用 在晶状体分化过程中形成“开放的”染色质区域，以及(3)鉴定和表征 晶状体细胞核中不同的拓扑相关结构域(TADS)的作用和功能，包括 转录工厂，核仁和拼接斑点，作为晶状体分化的关键成分。 拟议的研究得到了强有力的初步数据的支持，这些数据表明“开放”的形成。 晶状体分化关键基因启动子和增强子中的染色质区域 必需转录因子的可及性、功能及离散TADS的鉴定 由来自不同染色体的晶体蛋白基因座组成，以协调它们在晶状体纤维细胞中的表达。 这一结果将首次确定晶状体特异基因表达所需的顺序事件 通过转录因子之间的相互作用和染色质构象的改变，将提供新的 对晶状体分化所需的晶状体纤维细胞核的3D组织的洞察，以及 发现驱动晶状体纤维细胞去核的新调节机制。

ABSTRACT The long-term goal of this research program is to elucidate the global mechanisms that coordinately regulate the expression of genes required for the development and differentiation of the ocular lens. Since the lens is a simple tissue composed of only two mature cell types, elucidation of these mechanisms provides insight into those processes required for the differentiation of far more complex tissues and provides the groundwork for the development and design of cutting-edge new avenues of biological research ranging from stem-cell replacement therapies to targeted cancer treatments. The central premise of this proposal is that differentiation of lens cells is dependent on the coordinated interactions of DNA- binding transcription factors with FGF and BMP signal transduction pathways to orchestrate lens-specific expression of hundreds of genes required to form the mature eye lens. The hallmark of mammalian lens fiber cell differentiation is accumulation of a- and b-/g-crystallins as key lens structural and protective proteins, cellular elongation, and degradation of nuclei and other organelles. In differentiating lens, specific groups of genes are transcriptionally turned on and off, and the central part of this process is controlled through the accessibility of chromatin DNA to associate with transcription factors and chromatin remodeling enzymes. Among these factors, c-Maf, Pax6, Prox1, c-Jun, Etv5, and Smads, act together as a unit to systematically regulate the spatial and temporal expression of individual crystallins and other genes essential for the differentiation and function of the eye lens. Specifically, this proposal will: 1) elucidate the functional role that chromatin plays in regulating lens differentiation-specific gene expression at the genome-wide level, (2) define the specific roles of DNA-binding transcription factors and their roles in formation of “open” chromatin regions during lens differentiation, and (3) identify and characterize the role and function of distinct topologically associating domains (TADs) in lens cell nuclei, including transcriptional factories, nucleoli and splicing speckles, as critical components of the lens differentiation. The proposed studies are supported by strong preliminary data demonstrating the formation of “open” regions of chromatin in the promoters and enhancers of key lens differentiation genes allowing accessibility and function of essential transcription factors and the identification of discrete TADs comprised of crystallin loci from different chromosomes to coordinate their expression in lens fiber cells. The results will define for the first time those sequential events required for lens-specific gene expression through the interplay between transcription factors and altered chromatin conformation, will provide novel insights into the 3D-organization of lens fiber cell nuclei that are required for lens differentiation, and uncover novel regulatory mechanisms that drive lens fiber cell denucleation.