3/3 High-resolution mapping of cell type-specific DNA (hydroxy)methylation in the human brain during postnatal development and in psychiatric disease

3/3 出生后发育和精神疾病期间人脑中细胞类型特异性 DNA（羟基）甲基化的高分辨率图谱

• 批准号: 10356882
• 负责人: ERAN A MUKAMEL
• 金额: $ 12.98万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10356882
• 关键词: Aberrant DNA Methylation; Adolescence; Adult; Affect; Alleles; Attention; Autopsy; Base Pairing; Binding Sites; Bipolar Disorder; Brain; Brain region; Cell Nucleus; Cells; Childhood; Complement; DNA; DNA Methylation; Data; Data Set; Development; Disease; Enhancers; Epigenetic Process; Etiology; Fluorescence; Functional disorder; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genetic; Genetic Risk; Genetic Transcription; Genome; Glutamates; Histones; Human; Interneurons; Link; Maps; Measures; Mediating; Mental disorders; Methods; Methylation; Minor Lymphocyte Stimulatory Loci; Modification; Molecular; Neuroglia; Neurons; Nuclear; Nucleic Acid Regulatory Sequences; Nucleotides; Patients; Population; Prefrontal Cortex; Process; Quantitative Trait Loci; Regulator Genes; Regulatory Element; Reporting; Resolution; Role; Sampling; Schizophrenia; Sex Bias; Site; Sorting - Cell Movement; Specimen; Techniques; Testing; Time; Tissues; Untranslated RNA; Variant; Work; base; bisulfite sequencing; brain cell; case control; cell type; disorder control; disorder risk; epigenome; epigenomics; excitatory neuron; gamma-Aminobutyric Acid; genetic association; genetic variant; genome wide association study; histone modification; individual patient; inhibitory neuron; insertion/deletion mutation; methylome; neuropsychiatric disorder; postnatal; postnatal development; promoter; recruit; relating to nervous system; risk variant; sex; spatiotemporal; transcription factor; transcriptome; transcriptome sequencing

Project Summary: Most genetic variants associated with disease in genome-wide association studies (GWAS) lie in non-coding gene regulatory elements (GRE; e.g., promoters and enhancers). GREs are tissue- and cell type-specific and are identified through their epigenomic signatures, including low DNA methylation (DNAm), DNA accessibility and certain histone modifications. The PsychENCODE Consortium has characterized brain GREs across brain regions and developmental time points, as well as in the brains of psychiatric patients using mostly DNA accessibility and histone modification marks. These marks, however, identify large regions of enrichment (~300-2,000bp), providing only low resolution coverage of the important regulatory nucleotides, e.g., transcription factor binding sites. DNAm (especially cell type-specific DNAm) has received less attention, although it has been linked to psychiatric disorders, including schizophrenia (SZ) and bipolar disorder (BD). In the adult human brain, ~80% of CG and 1.5% of non-CG (CH) sites are methylated, and can be converted to hydroxymethylcytosine (hmC) and further demethylated. In postmitotic neuronal genomes, mCH and hmC accumulate to a significantly higher level than in other tissues--a distinct feature of the brain's epigenome. Bisulfite sequencing (BS)-based approaches that are used to measure (h)mC can pinpoint GREs with single base resolution, presenting a unique opportunity to refine the gene regulatory landscape of the brain cell types. Here we aim to create reference DNAm maps [mC and hmC, using BS and oxidative (ox)BS sequencing] and transcriptional profiles (using RNA-seq) in two major subtypes of neurons in the human dorsolateral prefrontal cortex (DLPFC), namely excitatory glutamatergic (Glu) and inhibitory GABA-ergic neurons. The proposed work is based on fluorescence-activated nuclear sorting (FANS) methods that we recently developed to separate nuclei of different cell types from autopsied human brain, and on our recent findings that showed unexpected relationships between DNA(h)m, GREs, and gene expression in the DLPFC Glu and GABA neurons. We will perform these studies at key time points of postnatal brain development and adulthood to map DNA(h)m within active neuron subtype-specific GREs that may be vulnerable to disruption during childhood and adolescence periods that coincide with the critical processes of brain maturation and circuit refinement (Aim 1). This work will be complemented with single nucleus mC profiling, which will allow us to define the developmental trajectories of mC in discrete subpopulations of Glu and GABA neurons (Aim2). Finally, we will profile Glu and GABA neurons in 150 autopsied DLPFC samples obtained from controls and cases of SZ and BD, and will then map neuron subtype-specific gene expression and (hydroxy)methylation quantitative trait loci (eQTL, mQTLS, hmQTLs) (Aim3). We will integrate QTL, transcriptome, and DNA(h)m data with the results of SZ and BP GWAS to reveal DNA(h)m and gene expression-mediated causal risk variants and genes, and to distinguish specific neuronal subtype(s) that are critical in the etiology of these disorders.

项目摘要：全基因组关联研究 (GWAS) 中与疾病相关的大多数遗传变异 存在于非编码基因调控元件（GRE；例如启动子和增强子）中。 GRE 是组织和细胞 类型特异性，并通过其表观基因组特征进行识别，包括低 DNA 甲基化 (DNAm)， DNA 可及性和某些组蛋白修饰。 PsychENCODE 联盟已对大脑进行了表征 跨大脑区域和发育时间点以及精神病患者大脑中的 GRE 主要是DNA可及性和组蛋白修饰标记。然而，这些标记识别了大片区域 富集（~300-2,000bp），仅提供重要调节核苷酸的低分辨率覆盖，例如， 转录因子结合位点。 DNAm（尤其是细胞类型特异性 DNAm）受到的关注较少， 尽管它与精神疾病有关，包括精神分裂症（SZ）和双相情感障碍（BD）。 在成人大脑中，约 80% 的 CG 和 1.5% 的非 CG (CH) 位点被甲基化，并且可以转化 转化为羟甲基胞嘧啶（hmC）并进一步去甲基化。在有丝分裂后神经元基因组中，mCH 和 hmC 积累的水平明显高于其他组织——这是大脑表观基因组的一个显着特征。 用于测量 (h)mC 的基于亚硫酸氢盐测序 (BS) 的方法可以通过单次精确定位 GRE 碱基分辨率，为完善脑细胞类型的基因调控景观提供了独特的机会。 在这里，我们的目标是创建参考 DNAm 图谱 [mC 和 hmC，使用 BS 和氧化 (ox)BS 测序] 人类背外侧神经元两种主要亚型的转录谱（使用 RNA-seq） 前额皮质 (DLPFC)，即兴奋性谷氨酸能 (Glu) 和抑制性 GABA 能神经元。这 拟议的工作基于我们最近开发的荧光激活核分选（FANS）方法 从尸检的人脑中分离不同细胞类型的细胞核，以及我们最近的发现表明 DNA(h)m、GRE 和 DLPFC Glu 和 GABA 神经元中基因表达之间的意外关系。 我们将在出生后大脑发育和成年期的关键时间点进行这些研究，以绘制地图 活跃神经元亚型特异性 GRE 中的 DNA(h)m 可能在儿童期和 青春期恰逢大脑成熟和回路细化的关键过程（目标 1）。 这项工作将得到单核 mC 分析的补充，这将使我们能够定义发育 Glu 和 GABA 神经元离散亚群中 mC 的轨迹（Aim2）。 最后，我们将分析从对照组和对照组获得的 150 个尸检 DLPFC 样本中的 Glu 和 GABA 神经元。 SZ 和 BD 病例，然后绘制神经元亚型特异性基因表达和（羟）甲基化图谱 数量性状基因座（eQTL、mQTLS、hmQTL）（Aim3）。我们将整合 QTL、转录​​组和 DNA(h)m 数据 结合 SZ 和 BP GWAS 的结果揭示 DNA(h)m 和基因表达介导的因果风险变异 基因，并区分在这些疾病的病因学中至关重要的特定神经元亚型。