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Mechanisms controlling stochastic gene expression during eye development

眼睛发育过程中控制随机基因表达的机制

基本信息

批准号：
9116841
负责人：
Robert John Johnston
金额：
$ 36.45万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-01 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9116841
关键词：
Address Affect Anosmia Architecture Autistic Disorder Binding Biological Assay Biological Process Boxing CRISPR/Cas technology Cell Nucleus Cells Chromatin Chromatin Loop Color Color Visions DNA Development Disease Distant Drosophila genus Elements Enhancers Epigenetic Process Eye Eye Development Feedback Figs - dietary Gene Expression Gene Expression Regulation Genes Genetic Transcription Genome Goals Health Human Immune Immunologic Deficiency Syndromes Indium Individual Light Lymphoma Mediating Modeling Modification Molecular Motor Motor Neurons Mutate Mutation Neurons Organism Photoreceptors Polycomb Proteins Regulation Regulator Genes Response Elements Retinal Cone Role Stem cells System Technology Testing Tissues Trans-Activators Transgenes Variant Vision Vision Disorders Work cell fate specification cell type fly histone modification mutant olfactory receptor promoter stem stem cell differentiation transcription factor visual photoreceptor

项目摘要

DESCRIPTION (provided by applicant): During development, cell fate specification depends on both reproducible and stochastic regulatory mechanisms. Very little is known about stochastic mechanisms that turn gene expression on or off randomly in individual cells to diversify fates. Random cell fate choices are important for the diversification of visual and olfactory receptors, specification of motor neuron subtypes, dendritic self-avoidance in neurons, determination of immune cell fates, and differentiation of stem cells. Studying stochastic gene expression will enhance our understanding of vision disorders, anosmia, autism, immunodeficiencies, and lymphoma. The main goal of our work is to address how gene expression is regulated in a stochastic on/off manner during development using specification of Drosophila color photoreceptors as a model. The color vision system of the fly is a random mosaic of two photoreceptor subtypes determined by the stochastic on/off expression of the transcription factor Spineless. We recently discovered that each copy of the spineless gene makes an intrinsic, random expression decision within a single nucleus exposed to the same milieu of trans-acting factors. Our studies suggest that stochastic spineless expression requires regulation by specific transcription factors in R7 photoreceptors, insulator DNA elements that mediate DNA looping and Polycomb Response Elements/Trithorax Response Elements (PREs/TREs) that regulate expression. We hypothesize that a unique combination of transcription factors expressed in all R7s is required for stochastic ss expression (Aim 1). We propose that stochastic on/off expression is dictated by the ss locus randomly assuming one of two DNA looping configurations mediated by insulators (Aim 2). These configurations determine the proximity of a repressive PRE/TRE to the ss promoter. In on cells, the repressive PRE/TRE remains distant from the promoter to allow activation; in off cells, the repressive PRE/TRE is proximal to the promoter to repress expression (Aim 3). We will test these hypotheses by assessing transcription factor binding and combinatoriality, determining functionality of insulators and PRE/TRE DNA elements by generating deletions using CRISPR/Cas9 and conducting transgene assays, and examining the DNA looping conditions and histone modifications in the Spineless on and Spineless off cells using the recently developed cgChIP technology. Achieving these aims will characterize how regulation by trans factors, insulator-mediated DNA looping, and chromatin state control stochastic gene expression. Our findings will have a profound impact on our understanding of how the 3D architecture of the nucleus and epigenetic modifications control gene expression.