CRX AND ITS REGULATORY NETWORK IN RETINAL DEGENERATIONS
CRX AND ITS REGULATORY NETWORK IN RETINAL DEGENERATIONS
批准号:
10330569
负责人:
SHIMING CHEN
金额:
$50.36万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
2000
资助国家:
美国
项目状态:
已结题
起止时间:
2000-03-01 至 2024-01-31
关键词:
AddressAffectBindingBiological AssayBiologyCRISPR/Cas technologyCRX proteinCatalogingCatalogsCell physiologyCellsChromatinCodeConeDNADevelopmentDiseaseDistalElementsEnhancersEpigenetic ProcessFutureGene ExpressionGene Expression RegulationGenesGeneticGenetic ScreeningGenetic studyGenomeGenomic approachGenomicsGrantGuide RNAHealthHumanHuman GenomeIndividualInstructionKnock-outKnockout MiceKnowledgeLinkLocationLocus Control RegionLogicMaintenanceMapsMeasuresMediatingModelingMusMutateMutationNucleic Acid Regulatory SequencesNucleotidesOrganOutcomePathogenicityPhenotypePhotoreceptorsPlayPositioning AttributeProteinsRed-Green Color BlindnessRegulationRegulator GenesRegulatory ElementReporterReporter GenesResearchRetinaRetinal DegenerationRetinal DiseasesRhodopsinRodRoleSpecificityTestingTissuesTranslatingUntranslated RNAVariantWorkblue cone monochromacycell typecellular developmentcombinatorialdisease phenotypedisease-causing mutationepigenomeexperiencefunctional genomicsgene regulatory networkgenetic resourcegenome-widehuman diseasein vivoinnovationinsightloss of functionnervous system disorderpromoterretinal rodstranscription factor
中文摘要
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方面的体内作用。
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。
英文摘要
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.
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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
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
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.
DOI:
10.1186/1471-2199-9-87
发表时间:
2008-10-14
期刊:
BMC molecular biology
影响因子:
--
作者:
[Mali RS, Peng GH, Zhang X, Dang L, Chen S, Mitton KP]
通讯作者:
Mitton KP
DOI:
10.3389/fnmol.2023.1134839
发表时间:
2023
期刊:
FRONTIERS IN MOLECULAR NEUROSCIENCE
影响因子:
4.8
作者:
[Sun, Chi, Chen, Shiming]
通讯作者:
Chen, Shiming
共 6 条
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海外基金