The Role of H3K79 Methylation and Dot1L in Neuronal Function and Neurodevelopmental Disorders

项目总结 本研究旨在确认组蛋白赖氨酸甲基转移酶DOT1L在神经功能中的作用及其机制。 对神经发育障碍(NDDS)的贡献。NDD包括一系列高度流行的疾病 在发育过程中表现出来，可能导致智力残疾、发育迟缓和自闭症 谱系障碍。最近的研究表明，许多染色质调节因子在NDDS中发生突变，包括 组蛋白甲基转移酶DOT1L。DOT1L甲基化赖氨酸79的组蛋白3(H3K79me) 具有活跃的转录功能。我们发现H3K79me在有丝分裂后高度丰富并动态调节 神经元。我们的初步数据还表明，H3K79me对神经元功能至关重要。我们找到了那个病人 突变导致DOT1L甲基转移酶活性丧失，表明H3K79me耗尽可导致 NDDS。进一步，我们发现DOT1L缺失改变了突触基因的转录，并双向调节 GluA2，AMPA受体亚单位。最后，我们发现在DOT1L条件性基因敲除中存在长期记忆缺陷 (CKO)小鼠。然而，DOT1L在神经元功能和认知中的作用仍不清楚。我假设 DOT1L调节突触基因的表达，DOT1L的部分缺失扰乱了这一调控，导致NDDS。 在目标1中，我将使用杂合子来定义部分DOT1L缺失引起的染色质和转录中断 DOT1L CKO小鼠模型结合H3K79me2切割靶标(Cut&Tag)和 RNA测序。在目标2中，我将通过以下方式研究部分DOT1L缺失对神经元功能和认知的影响 使用杂合子DOT1L CKO小鼠模型和对照进行电生理学和行为学研究 实验。总而言之，这项工作将确立DOT1L在神经元功能和NDDS等方面的作用 这将有助于理解染色质调节因子在大脑功能中的作用。

PROJECT SUMMARY This proposal aims to identify the role of the histone lysine methyltransferase Dot1L in neuronal function and its contribution to neurodevelopmental disorders (NDDs). NDDs include a spectrum of highly prevalent conditions that manifest during development that can cause intellectual disability, developmental delays, and autism spectrum disorder. Recent work demonstrated that many chromatin regulators are mutated in NDDs, including the histone methyltransferase Dot1L. Dot1L methylates histone 3 of lysine 79 (H3K79me) which is associated with active transcription. We found that H3K79me is highly abundant and dynamically regulated in postmitotic neurons. Our preliminary data also indicate that H3K79me is critical for neuronal function. We found that patient mutations result in a loss of Dot1L methyltransferase activity indicating that depletion of H3K79me can cause NDDs. Further, we found that Dot1L depletion alters transcription of synaptic genes and bidirectionally regulates GluA2, an AMPA receptor subunit. Finally, we found long-term memory deficits in Dot1L conditional knockout (cKO) mice. However, the role of Dot1L in neuronal function and cognition remain unclear. I hypothesize that Dot1L regulates synaptic gene expression and that partial Dot1L loss disrupts this regulation leading to NDDs. In Aim 1, I will define chromatin and transcriptional disruptions caused by partial Dot1L loss using a heterozygous Dot1L cKO mouse model coupled with H3K79me2 cleavage under targets and tagmentation (CUT&Tag) and RNA-sequencing. In Aim 2, I will examine the impact of partial Dot1L loss on neuronal function and cognition by using the heterozygous Dot1L cKO mouse model and controls to perform electrophysiology and behavioral experiments. Cumulatively, this work will establish a role for Dot1L in neuronal function and NDDs and more broadly will contribute to understanding of the role of chromatin regulators in brain function.