Deciphering the regulatory logic of rhodopsin expression

破译视紫红质表达的调控逻辑

• 批准号: 8898819
• 负责人: Jens Rister
• 金额: $ 8.79万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8898819
• 关键词: Address; Adult; Architecture; Atherosclerosis; Auditory; Award; Base Pairing; Binding Sites; Bioinformatics; Biological Models; Biological Phenomena; Biology; Brain; Cells; ChIP-seq; Circadian Rhythms; Code; Collaborations; Collection; DNA; Data; Development; Disease; Dissection; Distal; Drosophila genus; Elements; Enhancers; Ensure; Equipment; Feedback; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic; Genomics; Goals; Growth; Health; Hemophilia A; Individual; Institution; Knowledge; Laboratories; Lead; Logic; Maintenance; Mediating; Medical; Mentors; Methods; Mitotic; Modeling; Mutation; Neurons; New York; Nucleic Acid Regulatory Sequences; Osteoporosis; Pattern; Phase; Photoreceptors; Phototransduction; Play; Polydactyly; Promoter Regions; Recording of previous events; Regulatory Element; Research; Retina; Rhodopsin; Role; Scientist; Specificity; Stretching; Structure; Systems Biology; Technology; Testing; Time; Tissue-Specific Gene Expression; Tissues; Training; Trans-Activators; Transcription Coactivator; Transcription Repressor/Corepressor; Transgenic Organisms; Tumor Suppressor Proteins; Universities; Untranslated RNA; base; blue cone monochromacy; career; cell type; design; developmental genetics; fly; genome-wide analysis; human disease; improved; in vivo; insight; light intensity; melting; mutant; novel; promoter; research study; spatiotemporal; transcription factor

DESCRIPTION (provided by applicant): Gene expression is controlled by cis-regulatory elements (CREs) such as enhancers and promoters that contain binding sites for transcriptional activators and repressors. CREs are stretches of non-coding DNA that control when, where, and at which levels genes are expressed. The overall goal of this proposal is to gain insights into the poorly understood mechanisms that underlie CRE function. Deciphering the rules that underlie their architecture will improve our understanding of the fundamental biological phenomenon of differential gene expression. As mutations in CREs lead to altered gene expression, this proposal is also relevant for gaining insights into the genetic basis of disease. n this proposal, I use the compact Drosophila rhodopsin (rh) promoters as a CRE model system to address the following questions: a) How does the same CRE control gene expression in different tissues at different developmental time points? The same minimal rh promoter regions (less than 300 base pairs) control rh expression in four different developmental and functional contexts: larval photoreceptors, adult photoreceptors, circadian 'eyelet' photoreceptors and auditory neurons. I will take advantage of a large collection of transgenic fly stocks that carry mutant rh promoters, which I have created in the mentor's lab, to decipher the cis-regulatory code in these different contexts (mentored phase). b) What distinguishes CREs that drive gene expression in a subset of a particular cell type from CREs that drive expression in all the cells o the same cell type? I will compare the rh promoters, which control highly restricted expression in subtypes of photoreceptors, to promoters of genes that are expressed in all photoreceptors. I will identify such 'pan-PR' promoters with bioinformatics (mentored phase) and ChIP-seq technology (independent phase). This will allow me to test the hypothesis that these two CRE types share general activator motifs, but rh genes have additional repressor motifs to achieve subtype specificity. The K99 Award will allow me to receive relevant training in ChIP-seq technology that is required for this goal. c) How do CREs achieve robust and uniform levels of gene expression within a particular cell type? Preliminary results suggest that rh genes have distal enhancers that ensure robust and high expression levels. I will identify and dissect the regulatory regions that control quantitative aspects of rh expression (independent phase). This will allow me to compare the 'expression level' code to the 'spatiotemporal' CRE code. d) Do genes with very different functions but common, highly restricted expression patterns use the same cis-regulatory code? Previous studies in the Desplan lab have established that the tumor suppressor warts and the growth regulator melted are re-used in a double-negative feedback loop to mediate an unambiguous decision for expression of either blue-sensitive Rhodopsin 5 (Rh5) or green-sensitive Rhodopsin 6 (Rh6). As melted is expressed in the same photoreceptor subtype as rh5 and warts is co- expressed with rh6, it is an intriguing question whether the same or a different cis-regulatory code is used for subtype-specific expression of melted/rh5 or warts/rh6. I will dissect the warts and melted loci to identify activator and repressor motifs that mediate their specific expression in two different photoreceptor subtypes (independent phase). I will also determine whether the same trans-acting factors are used for subtype-specific expression. This will allow me to compare the CRE code of two independent examples of highly restricted expression in the same cellular subtype. Using the insights gained from the experiments above, I will reconstruct the cis-regulatory logic of the rhodopsin promoters and will test the reconstructed promoters in mutant backgrounds to determine whether they depend on the same transcription factors. Moreover, I will assess whether they drive proper expression in other cellular contexts (see above). Hereby, I will test the completeness of our understanding of the cis- regulatory logic of rhodopsin expression. The training phase of this proposal will be performed in the lab of my mentor Dr. Claude Desplan in the Center for Developmental Genetics at New York University (NYU) in collaboration with the lab of my consultant Dr. Stephen Small. The NYU Center for Developmental Genetics and the nearby Center for Genomics and Systems Biology provide all essential equipment and facilities required for the proposed research. My long- term career goal is to establish an independent research group at an academic institution and to become a leading scientist in the field of gene regulation.

描述（由申请人提供）：基因表达由顺式调控元件（CRE）控制，例如含有转录激活子和阻遏子结合位点的增强子和启动子。 CRE 是一段非编码 DNA，控制着基因表达的时间、地点和水平。该提案的总体目标是深入了解 CRE 功能背后的鲜为人知的机制。破译其结构背后的规则将提高我们对差异基因表达的基本生物学现象的理解。由于 CRE 的突变会导致基因表达的改变，因此该提议也有助于深入了解疾病的遗传基础。在这个提案中，我使用紧凑的果蝇视紫红质（rh）启动子作为CRE模型系统来解决以下问题：a）相同的CRE如何在不同发育时间点控制不同组织中的基因表达？相同的最小 rh 启动子区域（少于 300 个碱基对）在四种不同的发育和功能环境中控制 rh 表达：幼虫光感受器、成体光感受器、昼夜节律“孔眼”光感受器和听觉神经元。我将利用我在导师实验室中创建的大量携带突变 rh 启动子的转基因果蝇种群，破译这些不同环境下的顺式调控密码（指导阶段）。 b) 在特定细胞类型的子集中驱动基因表达的 CRE 与在同一细胞类型的所有细胞中驱动表达的 CRE 有何区别？我将比较控制光感受器亚型中高度受限表达的 rh 启动子与在所有光感受器中表达的基因启动子。我将通过生物信息学（指导阶段）和 ChIP-seq 技术（独立阶段）来识别此类“泛 PR”启动子。这将使我能够检验以下假设：这两种 CRE 类型共享一般激活子基序，但 rh 基因具有额外的阻遏基序以实现亚型特异性。 K99 奖将使我能够接受实现这一目标所需的 ChIP-seq 技术相关培训。 c) CRE 如何在特定细胞类型内实现稳定且一致的基因表达水平？初步结果表明 rh 基因具有远端增强子，可确保稳健和高表达水平。我将识别并剖析控制 rh 表达定量方面（独立相）的调控区域。这将使我能够将“表达级别”代码与“时空”CRE 代码进行比较。 d) 具有非常不同的功能但常见的、高度受限的表达模式的基因是否使用相同的顺式调控代码？ Desplan 实验室之前的研究已经证实，肿瘤抑制疣和融化的生长调节剂在双负反馈回路中重新使用，以介导表达蓝色敏感视紫红质 5 (Rh5) 或绿色敏感视紫红质的明确决定。视紫质 6 (Rh6)。由于melted与rh5在相同的光感受器亚型中表达，而warts与rh6共表达，因此对于melted/rh5或warts/rh6的亚型特异性表达是否使用相同或不同的顺式调控代码是一个有趣的问题。我将解剖疣和融化基因座，以确定激活子和抑制子基序 介导它们在两种不同光感受器亚型（独立相）中的特异性表达。我还将确定相同的反式作用因子是否用于亚型特异性表达。这将使我能够比较同一细胞亚型中高度受限表达的两个独立示例的 CRE 代码。 利用从上述实验中获得的见解，我将重建视紫红质启动子的顺式调控逻辑，并将在突变背景中测试重建的启动子，以确定它们是否依赖于相同的转录因子。此外，我将评估它们是否在其他细胞环境中驱动正确的表达（见上文）。在此，我将测试我们对视紫红质表达的顺式调控逻辑的理解的完整性。 该提案的培训阶段将在纽约大学 (NYU) 发育遗传学中心的我的导师 Claude Desplan 博士的实验室中与我的顾问 Stephen Small 博士的实验室合作进行。纽约大学发育遗传学中心和附近的基因组学和系统生物学中心提供拟议研究所需的所有必要设备和设施。我的长期职业目标是在学术机构建立一个独立的研究小组，并成为基因调控领域的领先科学家。