Regulation of gene expression in the retina

视网膜基因表达的调控

• 批准号: 8149184
• 负责人: ANAND SWAROOP
• 金额: $ 245.05万
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8149184
• 关键词: Gene Expression Regulation; Retina

Background We have shown that NRL interacts with TATA-binding proteins (TBP), CRX, NR2E3, SP4 and other transcription factors to control gene expression. Deletion of Nrl in mouse leads to transformation of rods to cones, whereas expression of Nrl in committed cone precursors converts them into rods. A better understanding of the molecular networks that regulate NRL and/or are regulated by NRL and functional characterization of individual key components can provide fundamental insights into photoreceptor biology and dysfunction. 
 Results 1. Spatio-temporal gene profiling of rod photoreceptors We are using high throughput photoreceptor gene profiling to construct gene expression networks underlying rod functional maturation and homeostasis. We have taken advantage of a line of transgenic mice, which we generated to express green fluorescent protein (GFP) in newly born and mature rods under control of the Nrl promoter. RNA is extracted from flow-sorted mouse photoreceptor cells and used as template for exon array hybridization. A large amount of data is being analyzed to correlate gene expression changes to differentiation and maturation of photoreceptors, and to their aging and disease. The latter two sets of data will also be analyzed in the context of changes in photoreceptor function in aging and diseased photoreceptors (see project # EY00475-02). 2. Genes/pathways that guide rod differentiation and homeostasis (NRL targets) 2.1. We have employed in silico and chromatin immunoprecipitation (ChIP) followed by very high throughput sequencing (ChIP-seq) methods, in combination with Nrl-knockout gene profiles, to identify direct transcriptional targets of NRL in mature mouse rods. A number of candidates were confirmed by ChIP-qPCR and tested functionally by in vivo RNAi-mediated knockdown experiments in the developing mouse retina. Enhancer analysis was performed to identify candidate NRL co-activators/co-repressor partners on selected targets. 2.2. Among NRL targets is the rod-specific gene rhodopsin. Rhodopsin is a major phototransduction protein, and misregulation of its expression results in retinal diseases such as retinitis pigmentosa. We are elucidating transcription regulatory proteins that positively or negatively control rhodopsin expression. An analysis by mass spectrometry of individual components bound to different regions of the rhodopsin promoter has revealed the presence of several new proteins. Not surprisingly, a number of proteins are involved in splicing or chromatin remodeling. After validation by immunoblot analysis, we are characterizing three proteins that synergistically enhance NRL and CRX activity on the rhodopsin promoter. To delineate the mechanism of delay in rhodopsin expression during rod maturation, we are examining possible negative regulators of the rhodopsin promoter. We have identified evolutionarily conserved regions and performed in vivo electroporation and reporter gene assays to show that the most proximal conserved promoter region is sufficient to sustain reporter gene activity even at postnatal day (P)2. However, the region -174 to -1610 likely contains elements that negatively regulate rhodopsin expression in immature photoreceptor cells. In silico analysis combined with gene expression data identified putative transcription factors that bind in that region. Selected candidate negative transcription regulators of rhodopsin gene expression are being tested further. 3. Pathways upstream of NRL (photoreceptor cell fate specification) 3.1. To understand the regulation of Nrl expression during photoreceptor differentiation and in the mature retina, we are investigating cis-elements in the Nrl promoter and their cognate regulatory binding factors. We found that, upon in vivo electroporation, conserved sequences in the first and second proximal conserved regions (comprising a 900 bp promoter sequence) are responsible for sustained GFP expression specifically in rods. Furthermore, a minimal promoter sequence comprising the TATA like binding element and a second proximal conserved region are sufficient to drive specific GFP expression in rods. By electrophoretic mobility shift assay (EMSA), we have identified transcription factors that differentially bind to NRL conserved promoter regions in developing and mature mouse retina. Transcriptional activity of selected proteins on NRL minimal promoter is being tested by in vitro luciferase assays. 3.2. We have established that the transcriptional activity of NRL is modulated by di-sumoylation at the Lys-20 residue, a site that is conserved in NRL across species and in proteins of the MAF family (that includes NRL) (Ref. 3). NRL-K20R and NRL-K20R/K24R sumoylation mutants show reduced transcriptional activation of Nr2e3 and rhodopsin promoters (two direct targets of NRL) in reporter assays when compared with wild-type NRL. Consistent with this, in vivo electroporation of the NRL-K20R/K24R mutant into newborn Nrl-knockout mouse retina leads to reduced Nr2e3 activation and only partial rescue of the Nrl-knockout phenotype, in contrast to wild-type NRL that can convert cones to rods. Although PIAS3 (protein inhibitor of activated STAT3), an E3-SUMO ligase implicated in photoreceptor differentiation, can be immunoprecipitated with NRL, there appears to be redundancy in E3 ligases, and PIAS3 does not seem to be essential for NRL sumoylation. This differentiates NRL from NR2E3, which is modulated by PIAS3-mediated sumoylation. These data suggest that gene regulatory networks regulating cell fate in the retina not only include transcription factors and their target genes, but also an array of proteins that can modulate the activity of the transcription factors by post-translational modification, most likely in response to extracellular signalling pathways. To further investigate the role of PIAS3 in retinal development, PIAS3 floxed mice have been generated and are being bred with Rx-Cre (for deletion in all retinal cells) and with Crx-Cre (for photoreceptor specific deletion) mice to establish the role of SUMOylation in regulating NRL function. Preliminary data have also indicated that NRL stability is regulated by GSK3-mediated phosphorylation. GSK3 is a regulator of retinal progenitor differentiation and synaptogenesis. Deletion of GSK3a and/or GSK3b specifically in all retinal progenitor cells leads to a large increase of displaced ganglion cells (in the INL) and abnormal synaptogenesis. As compound GSK3a and GSK3b cKO with Rx-Cre is lethal, we are currently generating double KO mice using Six3-Cre and Crx-Cre. 4. Effects of NRL mutations on photoreceptor development and homeostasis The transgenic mice that we generated to express human NRL S50T and NRL P51S mutations in the Nrl-KO background underwent normal retinal development and rod differentiation. No abnormal histological or functional (ERG) phenotype could be observed up to 12 months of age. However, upon short exposure (1 hr) to bright light, almost complete rod, but not cone, degeneration was observed even at young ages. The phenotype appeared to be modified by the genetic background. S50T and P51S mutations prevent NRL phosphorylation and increase its transcriptional activity on the rhodopsin promoter. Although it appears that NRL phosphorylation is dispensable for rod differentiation it is essential for rod maintenance upon tissue damage. 
 Significance 
As our studies identify novel genes and networks that control photoreceptor development and function, we are gaining new insights into photoreceptor biology and disease. Components of the regulatory networks can be modulated for treatment of retinal diseases. Additionally, our research will greatly assist in designing possible use of stem cells for therapies of degenerative diseases that involve photorecetpor dysfunction/death.

背景 我们已经表明，NRL与TATA结合蛋白（TBP），CRX，NR2E3，SP4和其他转录因子相互作用以控制基因表达。 NRL在小鼠中的缺失导致杆转化为锥体，而NRL在固定的锥前体中的表达将其转换为杆。更好地理解由NRL调节NRL和/或调节的分子网络，单个关键组件的功能表征可以为光感受器生物学和功能障碍提供基本见解。 
 结果 1。杆感光体的时空基因分析 我们使用高吞吐量的感光基因分析来构建杆功能成熟和稳态的基因表达网络。我们利用了一系列转基因小鼠，我们生成的是在NRL启动子控制的新出生和成熟棒中表达绿色荧光蛋白（GFP）。从流量的小鼠光感受器细胞中提取RNA，并用作外显子阵列杂交的模板。正在分析大量数据，以将基因表达的变化与感光体的分化和成熟以及其衰老和疾病相关。后两组数据也将在衰老和患病感受器中光感受器功能变化的背景下进行分析（请参阅项目＃EY 00475-02）。 2。引导杆分化和稳态的基因/途径（NRL目标） 2.1。 我们已经使用了硅和染色质免疫沉淀（CHIP），然后使用非常高的吞吐量测序（CHIP-SEQ）方法，并结合NRL-KNOCKOUT基因谱，以鉴定成熟小鼠棒中NRL的直接转录靶标。通过ChIP-QPCR证实了许多候选者，并通过体内RNAi介导的敲低实验在发育中的小鼠视网膜中进行了功能。进行增强子分析以识别候选NRL共激活因子/共抑制伙伴在选定目标上。 2.2。 NRL靶标在杆特异性基因视紫红质中。 Rhodopsin是主要的光转传蛋白，其表达的正调导致视网膜疾病（例如色素性视网膜炎）。我们正在阐明转录调节蛋白，它们积极或负面控制视紫红质的表达。通过质谱分析的分析，对呈播蛋白启动子不同区域的各个成分的分析揭示了几种新蛋白质的存在。毫不奇怪，许多蛋白质参与剪接或染色质重塑。通过免疫印迹分析验证后，我们表征了三种蛋白质，这些蛋白质可以协同增强Rhodopsin启动子上的NRL和CRX活性。为了描绘杆成熟过程中视紫红质表达延迟的机制，我们正在研究视紫红质启动子的阴性调节剂。我们已经确定了进化保守的区域，并在体内电穿孔和报告基因测定中进行了表明，最近端保守的启动子区域即使在产后（p）2也足以维持报告基因活性。然而，-174至-1610区域可能包含对未成熟感光细胞中阴视蛋白表达负调节的元素。在计算机分析中，结合基因表达数据确定了在该区域结合的推定转录因子。选定的候选候选阴性转录调节剂正在进一步测试。 3。NRL上游的途径（感光细胞命运规格） 3.1。 为了理解光感受器分化和成熟视网膜期间NRL表达的调节，我们正在研究NRL启动子中的顺式元素及其相关调节结合因子。我们发现，在体内电穿孔后，在第一和第二个近端保守区域（包括900 bp启动子序列）中保守的序列负责在杆中特别持续的GFP表达。此外，包括结合元件和第二个近端保守区域的最小启动子序列足以驱动杆中的特定GFP表达。通过电泳迁移率转移测定法（EMSA），我们确定了在发育和成熟小鼠视网膜中与NRL保守的启动子区域差异结合的转录因子。选定蛋白在NRL最小启动子上的转录活性正在通过体外荧光素酶测定进行测试。 3.2。我们已经确定，NRL的转录活性是通过Lys-20残基的二淀粉化来调节的，Lys-20残基，该位点在NRL中保守，跨物种和MAF家族的蛋白质（包括NRL）（包括NRL）（参考文献3）。与野生型NRL相比，NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R和NRL-K20R/K24R SUMOYLATION突变体的转录激活降低。与此相一致，与野生型NRL相反，可以转换为杆子的NRL NRL，NRL-K20R/K24R突变体中的NRL-K20R/K24R突变体中的NRL-K20R/K24R突变体导致NR2E3激活降低，并且仅部分营救NRL-KNOCKOUT表型。尽管PIAS3（激活STAT3的蛋白质抑制剂），但可以用NRL免疫沉淀与光感受器分化有关的E3-苏姆菌连接酶，但在E3连接酶中似乎有冗余，并且PIAS3似乎并不是NRL Sumoylation的必不可少的。 这将NRL与NR2E3区分开，该3由PIAS3介导的sumoylation调节。这些数据表明，调节视网膜中细胞命运的基因调节网络不仅包括转录因子及其靶基因，而且还包括一系列蛋白质，这些蛋白质可以通过翻译后修饰调节转录因子的活性，最有可能响应细胞外信号通路。为了进一步研究PIAS3在视网膜发育中的作用，PIAS3 Floxed小鼠已经产生，并用RX-CRE（用于所有视网膜细胞中的缺失）和CRX-CRE（用于光感受器特异性缺失）小鼠建立Sumoylation在调节NRL功能中的作用。初步数据还表明，NRL稳定性受GSK3介导的磷酸化调节。 GSK3是视网膜祖细胞分化和突触发生的调节剂。在所有视网膜祖细胞中，GSK3A和/或GSK3B的删除都会导致移位的神经节细胞（在INL）和突触异常。由于具有RX-CRE的复合GSK3A和GSK3B CKO是致命的，因此我们目前使用Six3-Cre和CRX-CRE生成双KO小鼠。 4。NRL突变对感光器发育和稳态的影响 我们生成的转基因小鼠在NRL-KO背景中表达人NRL S50T和NRL p51S突变进行了正常的视网膜发育和杆分化。在12个月大时，没有观察到异常的组织学或功能（ERG）表型。但是，在短暂暴露于明亮的光线（1小时）后，几乎完整的杆，但不是锥体，即使在年轻年龄也观察到变性。表型似乎是通过遗传背景修饰的。 S50T和p51S突变可防止NRL磷酸化并增加其在视紫红质启动子上的转录活性。尽管NRL磷酸化似乎可以用于杆分化，但对于组织损伤时的杆维持至关重要。 
 意义 
当我们的研究确定了控制感光体发育和功能的新型基因和网络时，我们正在获得对光感受器生物学和疾病的新见解。可以调节调节网络的组成部分，以治疗视网膜疾病。此外，我们的研究将大大有助于设计可能使用干细胞用于涉及光过多疾病功能障碍/死亡的退行性疾病的疗法。