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CRX AND ITS REGULATORY NETWORK IN RETINAL DEGENERATIONS

CRX 及其在视网膜变性中的调节网络

基本信息

批准号：
10330569
负责人：
SHIMING CHEN
金额：
$ 50.36万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-03-01 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10330569
关键词：
Address Affect Binding Biological Assay Biology CRISPR/Cas technology CRX protein Cataloging Catalogs Cell physiology Cells Chromatin Code Cone DNA Development Disease Distal Elements Enhancers Epigenetic Process Future Gene Expression Gene Expression Regulation Genes Genetic Genetic Screening Genetic study Genome Genomic approach Genomics Grant Guide RNA Health Human Human Genome Individual Instruction Knock-out Knockout Mice Knowledge Link Location Locus Control Region Logic Maintenance Maps Measures Mediating Modeling Mus Mutate Mutation Nucleic Acid Regulatory Sequences Nucleotides Organ Outcome Pathogenicity Phenotype Photoreceptors Play Positioning Attribute Proteins Red-Green Color Blindness Regulation Regulator Genes Regulatory Element Reporter Reporter Genes Research Retina Retinal Degeneration Retinal Diseases Rhodopsin Rod Role Specificity Testing Tissues Translating Untranslated RNA Variant Work blue cone monochromacy cell type cellular development combinatorial disease phenotype disease-causing mutation epigenome experience functional genomics gene regulatory network genetic resource genome-wide human disease in vivo innovation insight loss of function nervous system disorder promoter retinal rods transcription factor

项目摘要

PROJECT SUMMARY Photoreceptor development and maintenance require precisely regulated gene expression. This regulation is controlled by the Gene Regulatory Network (GRN) of photoreceptor transcription factors (TFs) and their target non-coding cis-regulatory DNA elements (CREs). Mutations in either TFs or CREs can cause misregulation, leading to photoreceptor diseases. How mutations in coding sequences cause diseases, such as CRX-linked retinopathies, has been extensively studied, and many such mutations have been identified and can be tested for. However, only a few disease-associated mutations in non-coding DNA regulatory elements are known. One example is X-linked blue-cone monochromacy, a red/green color blindness associated with disruption of the Locus Control Region (LCR), a distal CRE of the Red/Green gene array. Little is known about the location or functional importance of other retinal CREs, presenting a bottleneck for identifying and evaluating disease-associated variants in the largely unexplored non-coding portion (99%) of the genome. Thus, there is an urgent need to catalog functional CREs and understand their sequence logic. To this end, we have identified the subset of CRX bound CREs that are Dependent on the activity of CRX to establish an active chromatin state and promote photoreceptor gene expression. We hypothesize that many of these CREs are essential for rod gene expression, cell fate, and survival, and their sequence features determine how they mediate the commands of CRX and/or other TFs. We propose to test this hypothesis in three specific aims using innovative high-throughput functional genomics approaches. Aim 1 will provide a deep understanding of two previously-identified CREs using loss-of-function studies in mice: By thoroughly characterizing the individual and combined deletions of the two rhodopsin (Rho) enhancers CBR and RER, we will decipher their in vivo roles in regulating endogenous Rho expression, rod cellular function, and health. Aims 2 and 3 will identify the subset of essential retinal CREs using unbiased high-throughput functional tests in mouse retinas: We will first identify activating CREs that are sufficient to enhance the expression of a reporter gene driven by a minimal rod gene promoter using massively parallel reporter assays (MPRA) (Aim 2); We will then identify active CREs that are necessary for rod gene expression and cell identity using a CRISPR-Cas9 genomic deletion screen (Aim 3). Finally, we will combine the information gained from these two complementary approaches to decipher the sequence logic mediating CRE regulatory function. Ultimately, we will identify the most functionally important photoreceptor CREs, gain a deep understanding of their genetic and epigenetic grammar, and be able to predict the effects of specific disruptions to these CREs on rod gene expression and cell fate. The findings will significantly advance our understanding of normal and pathogenic photoreceptor gene expression. It will also provide new resources for genetic screens and functional testing of disease mutations, and new insights into disease phenotype variability.

项目摘要光感受器的发育和维持需要精确调控的基因表达。这调控由感光细胞转录因子（TF）的基因调控网络（GRN）控制和它们的靶向非编码顺式调节DNA元件（克雷斯）。TF或克雷斯中的突变可以导致失调，导致感光细胞疾病。编码序列的突变是如何导致疾病的，作为CRX连锁的视网膜病，已经被广泛研究，并且已经鉴定了许多这样的突变，可以测试。然而，只有少数与疾病相关的非编码DNA调控元件突变，是已知的。一个例子是X连锁的蓝锥单色性，一种红/绿色色盲，与基因座控制区（LCR）的破坏，LCR是红/绿色基因阵列的远端CRE。知之甚少其他视网膜克雷斯的位置或功能重要性，为识别和评估基因组中大部分未探索的非编码部分（99%）中的疾病相关变异。因此，迫切需要对功能性克雷斯进行编目并理解其序列逻辑。为此我们已经鉴定了CRX结合克雷斯的子集，其依赖于CRX的活性来建立CRX结合CREs的亚群。活跃染色质状态，促进感光基因表达。我们假设，克雷斯对视杆细胞基因表达、细胞命运和存活至关重要，确定它们如何调解CRX和/或其他TF的命令。我们建议检验这一假设在三个具体的目标，使用创新的高通量功能基因组学方法。目标1将提供使用小鼠功能丧失研究深入了解两种先前确定的克雷斯：通过彻底表征两个视紫红质（Rho）增强子CBR和RER的单独和组合缺失，我们将破译它们在调节内源性Rho表达、视杆细胞功能和健康中的体内作用。目的2和3将使用无偏高通量功能测试来识别基本视网膜克雷斯的子集在小鼠视网膜中：我们将首先鉴定激活克雷斯，其足以增强视网膜中的使用大规模平行报告基因测定（MPRA）由最小杆状基因启动子驱动的报告基因（Aim 2）;然后，我们将使用免疫组织化学方法鉴定对视杆细胞基因表达和细胞身份必需的活性克雷斯。 CRISPR-Cas9基因组缺失筛选（Aim 3）。最后，我们将联合收割机结合从这些两种互补的方法来破译介导CRE调节功能的序列逻辑。最后，我们将鉴定功能最重要的感光细胞克雷斯，深入了解它们的遗传特性，和表观遗传语法，并能够预测这些克雷斯的特定破坏对杆基因的影响表达和细胞命运。这一发现将大大促进我们对正常和致病性的理解。光感受器基因表达它还将为基因筛选和功能测试提供新的资源，疾病突变，以及对疾病表型变异性的新认识。

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Pias3-dependent SUMOylation controls mammalian cone photoreceptor differentiation.

DOI：
10.1038/nn.2618
发表时间：
2010-09
期刊：
NATURE NEUROSCIENCE
影响因子：
25
作者：
Onishi, Akishi;Peng, Guang-Hua;Chen, Shiming;Blackshaw, Seth
通讯作者：
Blackshaw, Seth

Photoreceptor degeneration changes magnetic resonance imaging features in a mouse model of retinitis pigmentosa.

DOI：
10.1002/mrm.22751
发表时间：
2011-06
期刊：
MAGNETIC RESONANCE IN MEDICINE
影响因子：
3.3
作者：
Wang, Qing;Song, Sheng-Kwei;Zhang, Huiying;Berkowitz, Bruce A.;Chen, Shiming;Wickline, Samuel A.;Chen, Junjie
通讯作者：
Chen, Junjie

Mutations in NR2E3 can cause dominant or recessive retinal degenerations in the same family.

DOI：
10.1002/humu.20858
发表时间：
2009-03
期刊：
HUMAN MUTATION
影响因子：
3.9
作者：
Escher, Pascal;Gouras, Peter;Roduit, Raphael;Tiab, Leila;Bolay, Sylvain;Delarive, Tania;Chen, Shiming;Tsai, Chih-Cheng;Hayashi, Masanori;Zernant, Jana;Merriam, Joanna E.;Mermod, Nicolas;Allikmets, Rando;Munier, Francis L.;Schorderet, Daniel F.
通讯作者：
Schorderet, Daniel F.

FIZ1 is part of the regulatory protein complex on active photoreceptor-specific gene promoters in vivo.