Regulation of Pet1/FEV binding and chromatin accessibility during serotonergic neuron development

项目摘要/摘要 神经递质5-羟色胺(5-羟色胺)与许多精神疾病和 神经发育障碍，包括焦虑，抑郁，自闭症，精神分裂症，注意力- 缺陷/多动障碍和强迫症。5-羟色胺神经元如何成熟和获得 传播者的身份和成虫的特征仍然知之甚少。在胚胎和早期 出生后，5-羟色胺神经元的谱系特异性基因表达模式由一个网络编排 包括ETS家族转录因子Pet1(人 对于5-羟色胺神经传递的建立是必不可少的。有趣的是，我们最近 发现Pet1在胎儿向出生后早期过渡期间切换靶点，从控制5-羟色胺上调 胎儿时期的合成基因到出生后突触兴奋性所需的激活基因。 这项研究调查了一种假设，即顺式调控染色质可及性的变化 元件决定了PET1调控过程中可用的转录靶点的谱系 发展。此外，我假设Pet1结合也塑造了染色质结构，以引导 发育中5-羟色胺神经元的基因表达轨迹。为了测试这些假设，在目标1中，我将映射 转座酶可及染色质分析显示5-羟色胺神经元的全球开放染色质图谱 在多个胚胎和出生后早期发育时进行高通量测序(ATAC-SEQ) 并探讨开放染色质与5-羟色胺神经元基因表达的关系。在目标2中，我将分析 在5-羟色胺神经元发育的同一阶段，与Aim-1相同的Pet1 DNA占有率的变化 用Pet1染色质免疫沉淀结合高通量测序(CHIP-SEQ)，并测定 通过ATAC，Pet1在发育关键染色质重塑或维持中的重要性 Pet1基因敲除小鼠的SEQ。通过阐明Pet1如何动态调节对细胞生长至关重要的基因表达 5-羟色胺神经元的成熟，我将提供对神经精神病学的病理生理学的洞察 5-羟色胺基因表达被认为受到干扰的情况。此外，这项研究还提供了 为我提供宝贵的培训机会，让我精通小鼠遗传学和基因转录 表观遗传学，分子神经科学的广泛概念知识，以及实验经验 设计、数据解释以及口头和书面交流，这些对我作为一名 正在接受培训的内科医生-科学家。

PROJECT SUMMARY/ABSTRACT The neurotransmitter serotonin (5-HT) is implicated in the pathophysiology of many psychiatric and neurodevelopmental disorders, including anxiety, depression, autism, schizophrenia, attention- deficit/hyperactivity disorder, and compulsive disorders. How serotonin neurons mature and acquire their transmitter identity and adult characteristics remain poorly understood. During the embryonic and early postnatal period, lineage specific gene expression patterns of serotonin neurons are orchestrated by a network of developmentally critical transcription factors, including the ETS family transcription factor Pet1 (human ortholog FEV) that is essential for the establishment of serotonin neurotransmission. Intriguingly, we recently found Pet1 switches targets during fetal to early postnatal transition, from controlling the upregulation of 5-HT synthesis genes during fetal life to activating gene required for synaptic excitability during the postnatal period. This study investigates the hypothesis that the changes in the chromatin accessibility of cis-regulatory elements dictate the repertoire of transcriptional targets that are available for Pet1 regulation during development. Furthermore, I hypothesize that Pet1 binding also shapes chromatin architecture to direct the gene expression trajectories of developing serotonin neurons. To test these hypotheses, in Aim 1, I will map the global open chromatin landscape of serotonin neurons using Assay for Transposase Accessible Chromatin with high throughput sequencing (ATAC-seq) at multiple embryonic and early postnatal developmental time points, and investigate the relation of open chromatin to 5-HT neuron gene expression. In Aim 2, I will analyze the changes in Pet1 DNA occupancy during the same stages of serotonin neuron development as in Aim 1 using Pet1 chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-seq), and determine the importance of Pet1 for developmentally critical chromatin remodeling or maintenance by performing ATAC- seq in Pet1 knockout mice. By elucidating how Pet1 dynamically regulates gene expression critical for the maturation of serotonin neurons, I will provide insight into the pathophysiology of the neuropsychiatric conditions in which serotonin gene expression is thought to be perturbed. Additionally, this study provides valuable training opportunity for me to gain technical proficiency in mouse genetics and transcriptomic and epigenetic profiling, broad conceptual knowledge in molecular neuroscience, and experience with experimental design, data interpretation, and oral and written communication that are crucial for my own growth as a physician-scientist in training.