Molecular Determinants for WDR5-Driven Transcriptional Regulation at Lineage-Specifying Genes During Retinogenesis

项目摘要/摘要 胚胎干细胞(ESCs)形成了视网膜失明的变革性细胞疗法的基础，这影响了 全球超过3亿人。然而，无处不在的染色质修饰剂，如WDR5，与之合作的机制 广泛表达的胚胎转录因子(TF)，如P53和MAX，控制视网膜的发生尚不清楚。 这一知识鸿沟影响着关键领域。P53激活，这是Charge和其他综合征的一个特征，触发 视网膜缺陷通过未确定的途径。P53突变型胚胎干细胞来源的视网膜细胞移植仍在继续 临床试验，但尚不清楚P53是否调节ESC视网膜命运。耗资10亿美元开发 WDR5抑制剂正在进行中。然而，除了WDR5作为一种联合转录因子促进转录外，人们对它知之甚少。 MLL染色质修饰复合体的因子，它使组蛋白H3(H3K4me)上的赖氨酸4甲基化。因此， 预测这些医疗干预的结果仍然具有挑战性。在PI的K08颁奖期间，我们 发现WDR5调节P53的稳定性以促进视网膜生成。此外，我们的初步数据显示 WDR5直接与P53和MAX相互作用，调节非视网膜谱系、中胚层和胚层 细胞/减数分裂相关转录。拟议研究的目标是了解 在关键的发育窗口期普遍存在的染色质修饰物和TF会触发 视网膜发生。这一提议检验了WDR5与P53和MAX在染色质上相互作用的中心假设 以时间依赖的方式通过激活视网膜特异基因和抑制非 视网膜，血统特定的基因座。我们将通过三个目标来检验这一假设：(1)描述WDR5的功能， P53，以及在视网膜形成过程中共招募WDR5和P53的基因座；(2)定义WDR5的分子相互作用 和MAX在视网膜发育过程中抑制非视网膜命运；(3)确定WDR5-P53细胞的作用 体内多能细胞谱系分化过程中的命运通路。我们的方法是重要的和创新的 因为它采用了最先进的技术，如剪切和运行、CRISPR-Cas9编辑、单元格 转录组分析和ESC衍生的3D有机类平台，以获得关于最早的 视网膜发生的事件。我们的研究将解决普遍研究的蛋白质的非正则功能，如 作为WDR5的作用，控制眼场转录因子活性，触发基因抑制和细胞命运功能的P53和 MAX不同于肿瘤的发生。因此，收集到的见解将代表着与常规的重大背离。 视图。我们的洞察力将垂直推进，并从根本上改变我们对染色质普遍存在的理解 修饰剂和TF以一种暂时的方式相互作用，以启动视网膜发生。我们的研究结果将会进步 与P53激活如何触发P53相关综合征中的视网膜缺陷相关的关键概念、机制 ESC细胞系中现有的P53改变如何改变正在进行的细胞治疗中的非视网膜谱系分化 目前正在开发的“治疗性”WDR5抑制剂的非靶向效应的试验和预测。

Project Summary/Abstract Embryonic stem cells (ESCs) form the basis for transformative cell therapies for retinal blindness, which affects over 300M worldwide. Yet, the mechanisms by which ubiquitous chromatin modifiers, like WDR5, cooperate with broadly expressed, embryonic transcription factors (TFs), like p53 and MAX, control retinogenesis are unknown. This knowledge gap affects critical fields. p53 activation, a feature of CHARGE and other syndromes, triggers retinal defects via undetermined pathways. Transplantation of p53-mutant ESC-derived retinal cells continue in clinical trials but it is not known if p53 regulates ESC retinal fate determination. A $1 billion effort to develop WDR5 inhibitors is ongoing. Yet, little is known about WDR5 beyond its role in promoting transcription as a co- factor of the MLL chromatin modifying complex, which methylates lysine 4 on histone H3 (H3K4me). Thus, predicting the outcome of these medical interventions remain challenging. During the PI’s K08 award period, we discovered that WDR5 regulates p53 stability to promote retinogenesis. Further, our preliminary data reveals that WDR5 directly interacts with p53 and MAX to regulate non-retinal lineage specification, mesoderm and germ cell/meiosis-related transcription. The objective of the proposed research is to understand how interplay of ubiquitous chromatin modifiers and TFs at a critical developmental window trigger the earliest events of retinogenesis. This proposal tests the central hypothesis that WDR5 interacts with p53 and MAX on chromatin in a time-dependent manner to promote retinogenesis by activating retinal-specific genes and by repressing non- retinal, lineage-specifying loci. We will test this hypothesis through three aims: (1) Delineate functions of WDR5, p53, and of loci that co-recruit WDR5 and p53, during retinogenesis; (2) Define molecular interactions of WDR5 and MAX that inhibit non-retinal fates during retinal specification; (3) Determine the role of the WDR5-p53 cell fate pathway during lineage specification of pluripotent cells in vivo. Our approach is significant and innovative because it employs state-of-the-art technologies, such as CUT&RUN, CRISPR-Cas9 editing, single cell transcriptome profiling, and ESC-derived 3D organoid platforms, to obtain foundational insights about the earliest events of retinogenesis. Our research will address non-canonical functions of popularly-studied proteins, such as roles for WDR5 that control eye field TF activity, trigger gene repression and cell fate functions of p53 and MAX distinct from tumorigenesis. Thus, gleaned insights will represent substantial departures from conventional views. Our insights will vertically advance and fundamentally alter our understanding of how ubiquitous chromatin modifiers and TFs interact in a temporal manner to initiate retinogenesis. Results from our studies will advance key concepts related to how p53 activation triggers retinal defects in p53-associated syndromes, mechanisms by which existing p53 alterations in ESC lines alter non-retinal lineage differentiation in ongoing cell therapy trials, and prediction of off-target effects of ‘therapeutic’ WDR5 inhibitors that are currently in development.