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

项目总结/摘要 胚胎干细胞（ESCs）是视网膜失明的细胞疗法的基础， 全球超过300 M。然而，无处不在的染色质修饰剂，如WDR 5，与细胞核的相互作用机制， 广泛表达的胚胎转录因子（TF）如p53和MAX控制视网膜发生是未知的。 这种知识差距影响到关键领域。p53激活是CHARGE和其他综合征的特征， 通过不明途径导致视网膜缺陷。p53突变型ESC衍生的视网膜细胞的移植继续进行， 临床试验，但不知道是否p53调节ESC视网膜命运决定。投入10亿美元开发 WDR 5抑制剂治疗正在进行中。然而，除了WDR 5作为一种协同转录因子促进转录的作用外，对WDR 5的了解甚少。 MLL染色质修饰复合物的因子，其甲基化组蛋白H3上的赖氨酸4（H3 K4 me）。因此，在本发明中， 预测这些医疗干预的结果仍然具有挑战性。在PI的K 08奖励期间，我们 发现WDR 5调节p53稳定性以促进视网膜生成。此外，我们的初步数据显示， WDR 5直接与p53和MAX相互作用，调节非视网膜谱系特化、中胚层和生殖 细胞/减数分裂相关转录。拟议研究的目的是了解 普遍存在的染色质修饰剂和转录因子在一个关键的发育窗口触发最早的事件， 视网膜生成该提议验证了WDR 5与染色质上的p53和MAX相互作用的中心假设 以时间依赖性的方式通过激活视网膜特异性基因和通过抑制非特异性基因来促进视网膜发生。 视网膜的谱系特异性位点。我们将通过三个目标来验证这一假设：（1）描述WDR 5的功能， p53，以及在视网膜发生过程中共同招募WDR 5和p53的位点;（2）定义WDR 5的分子相互作用 （3）确定WDR 5-p53细胞在视网膜特化过程中的作用 体内多能细胞谱系特化过程中的命运途径。我们的方法是有意义和创新的 因为它采用了最先进的技术，如CUT&RUN，CRISPR-Cas9编辑，单细胞 转录组分析和ESC衍生的3D类器官平台，以获得关于最早的 视网膜发生的事件。我们的研究将解决非典型的功能，普遍研究的蛋白质，如 作为WDR 5的作用，其控制眼区TF活性，触发基因抑制和p53的细胞命运功能， MAX与肿瘤发生不同。因此，收集的见解将代表着对传统的实质性偏离， 意见.我们的洞察力将垂直推进，并从根本上改变我们对无处不在的染色质是如何 修饰剂和TF以时间方式相互作用以启动视网膜发生。我们的研究结果将进一步 与p53激活如何触发p53相关综合征中视网膜缺陷相关的关键概念、机制 ESC细胞系中现有的p53改变改变了正在进行的细胞治疗中的非视网膜谱系分化 试验，并预测目前正在开发的“治疗性”WDR 5抑制剂的脱靶效应。

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.