Deciphering the regulatory logic of rhodopsin expression

描述（由申请人提供）：基因表达受顺式调节元件（克雷斯）控制，如增强子和启动子，其含有转录激活子和阻遏子的结合位点。克雷斯是一段非编码DNA，它控制基因表达的时间、地点和水平。本提案的总体目标是深入了解CRE功能的基础机制。破译其架构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。由于克雷斯突变导致基因表达改变，因此该建议也与深入了解疾病的遗传基础有关。在这个提议中，我使用紧凑型果蝇视紫红质（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）控制四种不同发育和功能环境中的rh表达：幼虫光感受器、成虫光感受器、昼夜节律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型rh启动子的转基因果蝇种群，在这些不同的背景下（指导阶段）破译顺式调控密码。 B）在特定细胞类型的子集中驱动基因表达的克雷斯与在相同细胞类型的所有细胞中驱动表达的克雷斯有什么区别？我将比较控制光感受器亚型中高度限制性表达的rh启动子和在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）鉴定这种“泛PR”启动子。这将使我能够测试这两种CRE类型共享一般激活基序的假设，但rh基因有额外的阻遏基序，以实现亚型特异性。K99奖将使我能够接受这一目标所需的ChIP-seq技术的相关培训。 c）克雷斯如何在特定细胞类型内实现稳健和均匀的基因表达水平？初步结果表明，rh基因有远端增强子，以确保强大的和高的表达水平。我将确定和解剖控制rh表达定量方面的调控区域（独立阶段）。这将允许我比较“表达水平”代码和“时空”CRE代码。 d）功能差异很大但表达模式相同且高度受限的基因是否使用相同的顺式调控密码？Desplan实验室以前的研究已经确定，肿瘤抑制疣和生长调节剂融化被重新用于双负反馈回路，以介导表达蓝色敏感性视紫红质5（Rh 5）或绿色敏感性视紫红质6（Rh 6）的明确决定。由于melt在与rh 5相同的光感受器亚型中表达，并且疣与rh 6共表达，因此相同或不同的顺式调节密码是否用于melt/rh 5或疣/rh 6的亚型特异性表达是一个有趣的问题。我将解剖疣和熔化的位点，以确定激活和抑制基序， 介导它们在两种不同的光感受器亚型（独立期）中的特异性表达。我还将确定是否相同的反式作用因子用于亚型特异性表达。这将允许我比较在相同细胞亚型中高度限制表达的两个独立实例的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，并将在突变体背景下测试重建的启动子，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适当的表达（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理解的完整性. 本提案的培训阶段将在我的导师、纽约大学（NYU）发育遗传学中心的Claude Desplan博士的实验室进行，并与我的顾问Stephen Small博士的实验室合作。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了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。

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.