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）：基因表达受顺式调节元件（CRE）控制，例如增强子和启动子，这些增强子和启动子包含转录激活因子和阻遏物的结合位点。 CRE是控制基因何时，何处和以哪些水平表达的非编码DNA的伸展。该提案的总体目标是了解基于CRE功能的机制知之甚少。解释其建筑基础的规则将改善我们对差异基因表达的基本生物学现象的理解。由于CRE中的突变导致基因表达改变，该建议也与获得疾病遗传基础的见解有关。 n这个建议，我将紧凑型果蝇视紫红质（RH）启动子用作CRE模型系统来解决以下问题：a）在不同发育时间点，不同组织中相同的CRE控制基因表达如何？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，成人光感受器，昼夜节律“睫毛”光感受器和听觉神经元。我将利用大量的转基因飞股（我在导师实验室中创建的突变RH启动子）在这些不同的情况下（指导阶段）中解密顺式调节代码。 b）是什么区分了在特定细胞类型的子集中驱动基因表达的CRE与驱动所有相同细胞类型中所有细胞表达的CRE表达的CRE？我将比较RH启动子控制感光体亚型高度限制的表达与在所有感光体中表达的基因的启动子。我将使用生物信息学（指导阶段）和CHIP-SEQ技术（独立阶段）确定此类“ PAN-PR”启动子。这将使我能够测试这两种CRE类型共享通用激活因子基序的假设，但是RH基因具有额外的阻遏基序以实现亚型特异性。 K99奖将使我能够接受此目标所需的CHIP-SEQ技术培训。 c）CRE如何在特定细胞类型中实现稳健和均匀水平的基因表达？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。我将识别和剖析控制RH表达（独立阶段）定量方面的调节区域。这将使我可以将“表达式级别”代码与“时空” CRE代码进行比较。 d）具有截然不同的基因，但常见的，高度限制的表达模式使用相同的顺式调节代码？先前在Desplan Lab中的研究表明，抑制疣疣和融化的生长调节剂在双重反馈回路中重新使用，以调解表达蓝色敏感的Rhodopsin 5（RH5）或绿色敏感的Rhopopsin 6（Rh6）的明确决定。正如熔化在与RH5相同的光感受器亚型中表达的那样，与RH6共同表达，这是一个有趣的问题，是一个相同或另一个不同的顺式调节代码用于熔融/RH5或WARTS/RH6的亚型特异性表达。我将剖析疣并融化的基因座，以识别激活剂和阻遏图主题 在两个不同的光感受器亚型（独立阶段）中介导其特定表达。我还将确定是否将相同的反式作用因子用于亚型特异性表达。这将使我可以比较在同一细胞亚型中高度限制表达的两个独立示例的CRE代码。 使用从上面的实验中获得的见解，我将重建视紫红质启动子的顺式调节逻辑，并将在突变体背景下测试重建的启动子，以确定它们是否依赖相同的转录因子。此外，我将评估它们在其他蜂窝情况下是否适当表达（见上文）。在此，我将测试我们对视紫红质表达的顺式调节逻辑的理解的完整性。 该提案的培训阶段将在我的导师Claude Desplan博士的实验室中与我的顾问Stephen Small博士的实验室合作。纽约大学发展遗传学中心和附近的基因组学和系统生物学中心提供了拟议研究所需的所有必需设备和设施。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，并成为基因监管领域的主要科学家。