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 退行性视网膜疾病是巨大的公共卫生负担，需要创新策略 理想的解决方案将克服与终末分化相关的先天障碍。 视网膜色素上皮（RPE）细胞的内源性再生有望实现这一目标。 应用，因为这些细胞可以重新编程以在胚胎羊膜动物中产生神经视网膜。 目前尚不清楚，RPE 细胞在早期羊膜发育过程中失去了神经能力。 提出全面绘制 RPE 在塑料发育阶段的基因调控图谱 探究 RPE 细胞分化时出现的重编程分子障碍。 鸡在胚胎第 4 天 (E4) 时具有可塑性，并且可以在视网膜切除术后重新编程为神经视网膜 然而，到胚胎第 5 天 (E5)，RPE 细胞命运受到限制并重新编程。 先前的研究表明，E4 RPE 细胞通过激活进行重新编程。 神经视网膜转录因子，如 VSX2、SIX6 和 PAX6，以及同时抑制 RPE 与此同时，表观遗传重编程事件重置了 DNA 甲基化和平衡。 染色质变成更活跃的构型，以促进随后的细胞身份变化，但事实并非如此。 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 我们的初步证据表明，亲神经基因在神经能力中仍然具有相对可诱导性。 E5 RPE，但早期的 RPE 分化程序是神经身份的固有障碍 这个阶段的分化程序是由 RPE 转录因子 MITF 和 OTX2 主导的，并且是不同的。 当前的提案将在染色质景观中观察到这些因素的可及性足迹。 基于这些发现，绘制与 RPE 能力相关的全基因组表观遗传模式 限制，包括先前已被证明可促进 RPE 的 DNA 和组蛋白修饰 此外，RPE 分化和重编程将在单细胞中进行分析。 分辨率，能够精确识别描述塑料和命运限制的转录组特征 RPE（目标 2）同时，关键效应转录因子（如 MITF 和 OTX2）的 DNA 结合活性， 将在分化或重编程的不同阶段对 RPE 进行分析，以及这些因素。 关键转录目标，将使用 CRISPR-Cas9 进行干扰，目的是恢复 RPE 神经 这些发现将共同提供更高级分化阶段的能力（目标 3）。 与 RPE 能力限制相关的染色质状态的广阔视野，同时探测 这些结果将提供驱动观察到的表型的染色质-转录因子相互作用。 了解 RPE 分化和这种细胞类型在神经元中使用的潜力的必要见解 替代策略。