Deciphering the regulatory logic of rhodopsin expression

描述（由申请人提供）：基因表达由顺式调控元件（cre）控制，如包含转录激活因子和抑制因子结合位点的增强子和启动子。cre是一段非编码DNA，控制着基因何时、何地、以何种水平表达。本提案的总体目标是深入了解CRE功能背后鲜为人知的机制。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突变导致基因表达改变，这一建议也与深入了解疾病的遗传基础有关。在本提案中，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的不同组织中控制基因表达？相同的最小rh启动子区域（小于300个碱基对）在四种不同的发育和功能背景下控制rh的表达：幼虫光感受器、成体光感受器、昼夜节律“眼孔”光感受器和听觉神经元。我将利用我在导师的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，来破译这些不同背景下的顺式调控代码（指导阶段）。b)驱动特定细胞类型子集基因表达的cre与驱动同一细胞类型的所有细胞表达的cre有何区别？我将比较rh启动子，它控制光感受器亚型中高度受限的表达，与所有光感受器中表达的基因启动子。我将用生物信息学（指导阶段）和ChIP-seq技术（独立阶段）识别这种“泛pr”启动子。这将允许我测试这两种CRE类型共享一般激活子基序的假设，但rh基因具有额外的抑制子基序以实现亚型特异性。K99奖将使我获得实现这一目标所需的ChIP-seq技术的相关培训。c) CREs如何在特定细胞类型中实现稳健和均匀的基因表达水平？初步结果表明rh基因具有远端增强子，可确保高表达水平。我将识别和剖析控制rh表达定量方面的调控区域（独立期）。这将允许我将“表达级别”代码与“时空”CRE代码进行比较。d)功能非常不同但表达模式普遍且高度受限的基因是否使用相同的顺式调控代码？Desplan实验室先前的研究已经证实，肿瘤抑制因子疣和生长调节剂在双负反馈回路中被重复使用，介导蓝敏感Rhodopsin 5 （Rh5）或绿敏感Rhodopsin 6 （Rh6）表达的明确决定。由于熔化蛋白与rh5在相同的光感受器亚型中表达，疣蛋白与rh6共同表达，对于熔化蛋白/rh5或疣蛋白/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。我将解剖疣和融化的基因座来识别激活和抑制基元

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.