A Roadmap to Uncover RPE Plasticity

揭示 RPE 可塑性的路线图

• 批准号: 10639436
• 负责人: Katia Del Rio-Tsonis
• 金额: $ 36.13万
• 依托单位: MIAMI UNIVERSITY OXFORD
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10639436
• 关键词: ATAC-seq; Acute; Address; Atlases; Behavior; Binding; Binding Sites; Biological Assay; CRISPR/Cas technology; Cell Differentiation process; Cell Nucleus; Cells; Chick Embryo; Chicken Cells; Chickens; Chromatin; Chromosome Mapping; Competence; DNA; DNA Binding; DNA Methylation; DNA Modification Process; DNA mapping; Data Analyses; Data Set; Development; Embryo; Epigenetic Process; Event; Eye; FGF2 gene; Foundations; Gene Expression Regulation; Genes; Genetic Transcription; Genome Mappings; Goals; Intention; Maps; Methodology; Methods; Molecular; Molecular Probes; Natural regeneration; Nerve Regeneration; Neural Retina; Pattern; Phenotype; Public Health; Regulator Genes; Replacement Therapy; Repression; Resolution; Retina; Retinal Degeneration; Role; Series; Source; Structure of retinal pigment epithelium; Time; Vision; adult neurogenesis; blastomere structure; cell type; embryo cell; epithelial stem cell; genome-wide; genome-wide analysis; histone modification; innovation; insight; loss of function; multimodal data; multimodality; neural; neuronal replacement; novel; permissiveness; programs; regeneration potential; response; restoration; retinal neuron; retinal regeneration; single nucleus RNA-sequencing; transcription factor; transcriptome; transcriptome sequencing; transcriptomics

Abstract Degenerative retinal diseases represent an enormous public health burden and demand innovative strategies to replace retinal neurons. Ideal solutions will overcome innate barriers associated with terminal differentiation to endogenously regenerate retinal neurons. Retinal pigment epithelium (RPE) cells hold promise for this application, as these cells can reprogram to produce neural retina in embryonic amniotes. For reasons that are not well understood, RPE cells lose neural competence during early amniotic development. The present study proposes to comprehensively map the gene regulatory landscape of RPE at plastic stages of development and to probe the molecular barriers to reprogramming that emerge as these cells differentiate. RPE cells of the chicken are plastic at embryonic day 4 (E4) and can be reprogrammed to neural retina following retinectomy and treatment with FGF2. However, by embryonic day 5 (E5), RPE cell fate becomes restricted and reprogramming capacity is abrogated. Previous studies have demonstrated that E4 RPE cells reprogram through the activation of neural retina transcription factors, such as VSX2, SIX6, and PAX6, and the simultaneous repression of RPE differentiation programs. Concomitantly, an epigenetic reprogramming event resets DNA methylation and poises chromatin into a more active configuration to facilitate the ensuing change in cell identity. However, it is not understood how transcription factor networks interact with a dynamic chromatin landscape to determine RPE neural competence. Our preliminary evidence suggests that pro-neural genes remain comparably inducible in the E5 RPE, but that an incipient RPE differentiation program serves as an inherent barrier to neural identity at this stage. This differentiation program is led by the RPE transcription factors MITF and OTX2, and distinct accessibility footprints from these factors are observable in the chromatin landscape. The current proposal will build on these findings by mapping genome-wide epigenetic patterns associated with RPE competence restriction, including DNA and histone modifications that have been previously demonstrated to facilitate RPE reprogramming (Aim 1). Additionally, RPE differentiation and reprogramming will be profiled at a single cell resolution, enabling the precise identification of transcriptomic features that delineate plastic and fate restricted RPE (Aim 2). In parallel, the DNA binding activity of key effector transcription factors, such as MITF and OTX2, will be profiled across the RPE at different stages of differentiation or reprogramming. These factors, as well as key transcriptional targets, will be perturbed using CRISPR-Cas9 with the intention of recovering RPE neural competence at more advanced stages of differentiation (Aim 3). Together, these findings will provide an expansive view of chromatin states associated with RPE competence restriction, while simultaneously probing the chromatin – transcription factor interactions that drive the observed phenotypes. These results will provide imperative insights toward understanding RPE differentiation and the potentiality of this cell type for use in neuron replacement strategies.

抽象的 退化残留疾病代表了巨大的公共卫生伯恩伯恩（Burnen），要求创新的策略 更换残留神经元。理想的解决方案将克服与终端分化相关的先天障碍 内源性再生永久神经元。视网膜色素上皮（RPE）细胞对此有望 应用，因为这些细胞可以重新编程以在胚胎羊膜中产生神经视网膜。出于原因 不太了解的是，RPE细胞在早期羊膜发育期间失去神经能力。本研究 建议在发育的塑性阶段全面绘制RPE的基因调节景观 探测分子屏障以重新编程，因为这些细胞会分化。 RPE细胞 鸡在胚胎第4天（E4）是塑料，可以在视网膜切除术和 用FGF2治疗。但是，到胚胎第5天（E5），RPE细胞脂肪变得限制并重新编程 能力被废除。先前的研究表明，E4 RPE细胞通过激活重编程 神经视网膜转录因子（例如VSX2、66和PAX6）的简单表达 分化计划。同时，表观遗传重编程事件重置DNA甲基化和毒药 染色质成更活跃的构型，以促进随后的细胞身份变化。但是，不是 了解转录因子网络如何与动态染色质景观相互作用以确定RPE 神经能力。我们的初步证据表明，促神人基因在 E5 RPE，但初期的RPE差异化计划是神经身份的继承障碍 这个阶段。该差异化程序由RPE转录因子MITF和OTX2领导，并且不同 这些因素的可及性足迹在染色质景观中可观察到。当前的建议将 通过绘制与RPE能力相关的全基因组表观遗传模式来构建这些发现的基础 限制，包括先前已证明可以促进RPE的DNA和组蛋白修饰 重新编程（AIM 1）。此外，RPE分化和重编程将在单个单元格上进行分析 分辨率，可以精确地识别转录组特征，这些特征描绘了塑料和命运限制 RPE（AIM 2）。同时，关键效应转录因子（例如MITF和OTX2）的DNA结合活性， 将在分化或重新编程的不同阶段对RPE进行分析。这些因素以及 关键的转录目标将使用CRISPR-CAS9扰动，目的是恢复RPE中性 在更高级的差异阶段的能力（AIM 3）。这些发现将共同提供 与RPE能力限制相关的染色质状态的广阔视图，同时探测 驱动观察到的表型的染色质 - 转录因子相互作用。这些结果将提供 对理解RPE分化的势在必行见解和该细胞类型用于神经元的潜力 替代策略。