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

概要/摘要（论文R 01）：胚胎干细胞（ESC）是转化细胞的基础， 治疗视网膜失明，影响全球超过3亿人。然而，无处不在的机制 染色质修饰物，如WDR 5，与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，a CHARGE和其他综合征的特征，通过不确定的途径触发视网膜缺陷。移植 的p53突变型ESC衍生的视网膜细胞继续进行临床试验，但尚不清楚p53是否调节ESC视网膜 命运决定一项耗资10亿美元的WDR 5抑制剂开发计划正在进行中。然而，对WDR 5知之甚少 除了作为MLL染色质修饰复合物的辅因子在促进转录中的作用外， 甲基化组蛋白H3上的赖氨酸4（H3 K4 me）。因此，预测这些医疗干预的结果 保持挑战性。在PI的K 08奖期间，我们发现WDR 5调节p53稳定性， 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。客观 拟议的研究是了解无处不在的染色质修饰剂和转录因子在关键时刻的相互作用。 发育窗口触发视网膜发生的最早事件。这一提议检验了中心假设 WDR 5与染色质上的p53和MAX以时间依赖性方式相互作用，通过以下方式促进视网膜发生： 激活视网膜特异性基因和抑制非视网膜的谱系特异性基因座。我们将测试这个 通过三个目标进行假设：（1）描述WDR 5、p53和共募集WDR 5基因座的功能， p53，在视网膜发生过程中;（2）定义抑制非视网膜命运的WDR 5和MAX的分子相互作用 （3）确定WDR 5-p53细胞命运途径在谱系分化过程中的作用 体内多能细胞的特化。我们的方法是重要的和创新的，因为它采用了国家的- 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，以获得有关视网膜发生最早期事件的基础见解。我们 研究将解决普遍研究的蛋白质的非规范功能，例如WDR 5的作用， 控制眼区TF活性，触发p53和PCGF 6的基因抑制和细胞命运功能， 肿瘤发生因此，收集到的见解将代表与传统观点的实质性偏离。我们 这些新的见解将垂直推进，并从根本上改变我们对无处不在的染色质 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。

SUMMARY/ABSTRACT (PARENT R01): 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 PCGF6 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.