Analyzing DOT1L-dependent hippocampus development using single-cell high throughput sequencing

表观遗传过程涉及中枢神经系统的发育和功能。然而，对单个染色质修饰剂及其靶点在中枢神经系统生理和病理生理条件中的作用的了解仍然有限。染色质修饰如何影响海马体发育在很大程度上是未知的。组蛋白甲基转移酶干扰物端粒沉默1样（DOT1L）可使组蛋白H3赖氨酸79 （H3K79）甲基化。我们的初步数据表明，研究DOT1L的功能将为缩小这种表观遗传修饰物如何影响海马发育和功能的知识差距提供独特的机会。重要的是，我们的数据表明，DOT1L缺陷干扰了海马发育过程中基本过程的正确发挥。我们观察到齿状回（DG）和谷氨酰胺（CA）场的发育发生了变化。我们目前的见解表明，发育程序的脱轨反映在过早发生的神经元分化紊乱和赋予不正确的细胞身份。我们在此建议将胚胎日(E) 16.5对照和dot1l缺陷小鼠海马的初步单细胞RNA数据集扩展到进一步的发育时间点，从E12.5到出生。描述涵盖不同发育阶段的新兴数据集将使我们能够全面了解从不同但部分未知的干细胞群体到海马体神经元的分化轨迹。进一步了解DOT1L缺陷海马中详细转录程序的分子改变，需要研究H3K79甲基化和调控区域，特别是增强子的可及性。我们建议使用多样化的生物信息学分析来探索新兴的综合数据集，以1)破译H3K79甲基化对控制转录程序的影响，这些转录程序是指定异质神经元亚群所必需的，2)推断有助于海马裂和DG形成的转录程序，3)解决有助于DG和CA领域分化的不同神经干细胞亚群，4)确定在骨下和CA神经上皮边界需要和微调的基因活动。我们的目标是破译决定海马中不同神经元分化的主要调控因子，这些候选基因将通过功能获得或功能丧失实验在体内进行功能验证。

Epigenetic processes are implicated in central nervous system development and function. However, there is still limited insight into the role of individual chromatin modifiers and their targets in physiological and patho-physiological conditions in the central nervous system. It is largely unknown how chromatin modifications impact on hippocampus development. The histone methyltransferase Disruptor of Telomeric Silencing 1-Like (DOT1L) confers histone H3 lysine 79 (H3K79) methylations. Our preliminary data indicate that studying the function of DOT1L will give unique opportunity to narrow the gap of knowledge of how this epigenetic modifier affects hippocampal development and function. Importantly, our data indicate that DOT1L deficiency interferes with the correct exertion of fundamental processes during hippocampus development. We observe altered development of the dentate gyrus (DG) as well as the cornu ammonis (CA) fields. Our current insights suggest that the derailed developmental program is reflected by disturbed neuronal differentiation that occurs prematurely and confers incorrect cell identities.We here propose to extend a preliminary single-cell RNA data set from embryonic day (E) 16.5 control and DOT1L-deficient mouse hippocampus to further developmental time points, spanning from E12.5 until birth. Characterising the emerging data set covering various stages of development shall allow gaining comprehensive knowledge about differentiation trajectories from diverse, but partly unknown stem cell populations to the neurons of the hippocampus. Further insights into molecular alterations underlying derailed transcriptional programs in DOT1L deficient hippocampus shall come from studying H3K79 methylation and accessibility of regulatory regions, especially enhancers. We propose to use a diverse repertoire of bioinformatics analyses to explore the emerging comprehensive data set to 1) decipher the impact of H3K79 methylation in control of transcriptional programs necessary to specify the heterogeneous neuronal subpopulation, 2) infer transcriptional programs that contribute to hippocampal fissure and DG formation, 3) resolve different subsets of neural stem cells contributing to differentiation of DG and CA fields, and 4) determine gene activities needed and fine-tuned at the boundary of the subiculum and the CA neuroepithelium. We aim to decipher master regulators that determine differentiation of the diverse neurons in the hippocampus and these candidate genes shall be functionally validated in vivo through gain- or loss-of-function experiments.