Molecular determinants of sex-specific DNA methylation signature acquisition in the mammalian germline

哺乳动物种系中性别特异性 DNA 甲基化特征获取的分子决定因素

• 批准号: 10723071
• 负责人: Rexxi Diptya Prasasya
• 金额: $ 12万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10723071
• 关键词: Aberrant DNA Methylation; Advisory Committees; Age; Binding; Biochemical; Bioinformatics; Cell Reprogramming; Cells; Coupled; Cues; DNA; DNA Methylation; DNA Modification Methylases; Deposition; Development; Dioxygenases; Embryo; Embryonic Development; Environment; Enzymes; Epiblast; Epigenetic Process; Event; Exclusion; Exhibits; Female; Fertility; Fertilization; Gametogenesis; Genetic; Genetic Diseases; Genetic Models; Genome; Genomic Imprinting; Genomics; Germ Cells; Gonadal structure; Growth; Histones; Human Genetics; Hypermethylation; Knowledge; Length; Mentors; Mentorship; Methylation; Methyltransferase; Modeling; Modification; Molecular; Mus; Mutation; Nature; Oocytes; Outcome; Ovarian aging; Parents; Pathway interactions; Pattern; Phase; Post-Translational Protein Processing; Process; Reproduction; Research Personnel; Resistance; Role; Signal Pathway; Signal Transduction; Site; Somatic Cell; Specific qualifier value; Spermatogonia; Structure of primordial sex cell; Supporting Cell; Testing; Time; Tissues; Training; WNT Signaling Pathway; bisulfite sequencing; candidate identification; demethylation; epigenome; epigenomics; experimental study; extracellular; follow-up; genotypic sex; granulosa cell; idiopathic infertility; in vivo; infertility treatment; male; methylation pattern; methylome; mouse model; multiple omics; mutant; novel; nuclease; offspring; overexpression; postnatal; prevent; public health relevance; recruit; reproductive; sertoli cell; sex; sexual dimorphism; sperm cell; transcription factor; transcriptomics; transdifferentiation; transmission process; whole genome

Project abstract Mammalian reproduction requires biparental genetic contributions due to the highly dimorphic nature of gamete epigenomes. DNA methylation (DNAme) is one of the most sexually dimorphic epigenetic marks in gametes, being hypermethylated in the sperm and alternatingly hypo- and hypermethylated in the oocyte. Aberrant DNAme in the germline can negatively impact fertility and offspring development. To prevent transmission of epimutations and establish the germline fate, primordial germ cells (PGCs) undergo global DNAme erasure following specification. While most of the genome achieves demethylation through replication-coupled passive dilution, the active demethylation pathway using the TET1 enzyme is required for methylation erasure of a subset of loci. I recently discovered that sperm-specific hypomethylated regions, while rare, require TET1 for reprogramming. Tissue-specific hypomethylation signatures often correlate with binding of developmentally relevant transcriptional factors, lending to the significance of these sperm-specific hypomethylated regions. Mechanisms of how sperm or oocytes acquire sex-specific DNAme remain a knowledge gap with relevance to fertility and development. While biochemically histone post-translational modifications (PTMs) have been shown to correlate with DNA methyltransferase (DNMT) accessibility, it remains unknown how these epigenetic marks become non-uniformly enriched within the germline genome. I hypothesize that histone PTMs enrichment and DNAme patterning in the germline are determined 1) intrinsically by the demethylation pathway used during PGC reprogramming and 2) extrinsically by the signaling milleu of the gonadal supporting cells. To test this hypothesis in vivo, genetic mouse models and multi-omics analyses will be used to elucidate what cellular signals are responsible for the acquisition of sex-specific DNAme signatures in the sperm and the oocyte. Aim 1 (K99) will test the catalytic and non-catalytic requirement for TET1 during PGC development for the establishment of the methylation signature of the oocyte genome. Aim 2 (R00) will employ genetic sex- reversal models of Dmrt1 overexpression in pre-granulosa cells (female-to-male) and constitutively active Wnt signaling in pre-Sertoli cells (male-to-female) to test the impact of altering the somatic signaling environment for DNAme acquisition in germ cells. In these models, I will integrate and identify correlations between changes in methylome and the relevant histone PTMs enrichment (methylation of H3K4 and H3K36). Single cell transcriptomics will be used to identify instructive cues for the establishment of sex-specific DNAme signatures in gametes. I will receive extensive training in advanced bioinformatics and single-cell genomics during the mentored phase of this proposal under the mentorship of Dr. Bartolomei, a pioneer in DNAme and genomic imprinting, within the UPenn Epigenetics Institute. With the additional guidance from my advisory committee, which includes leaders in the field of germ cell epigenetics and gonadal signaling pathways, I will be well prepared to become an independent investigator in the field reproductive epigenetics.

项目摘要 由于配子的高度二态性，哺乳动物的繁殖需要双亲遗传的贡献 表观基因组。 DNA甲基化（DNAme）是配子中性别二态性最强的表观遗传标记之一， 精子中高甲基化，卵母细胞中交替低甲基化和高甲基化。异常 种系中的 DNAme 会对生育力和后代发育产生负面影响。为了防止传播 表观突变并确定种系命运，原始生殖细胞 (PGC) 经历整体 DNAme 擦除 以下规格。虽然大多数基因组通过复制耦合被动实现去甲基化 稀释后，使用 TET1 酶的主动去甲基化途径是甲基化擦除所必需的 基因座的子集。我最近发现精子特异性低甲基化区域虽然很少见，但需要 TET1 来实现 重新编程。组织特异性低甲基化特征通常与发育性结合相关 相关转录因子，赋予这些精子特异性低甲基化区域的重要性。 精子或卵母细胞如何获得性别特异性 DNAme 的机制仍然是一个与相关知识差距 生育和发育。虽然生化组蛋白翻译后修饰（PTM）已被 虽然已被证明与 DNA 甲基转移酶 (DNMT) 可及性相关，但仍不清楚这些 表观遗传标记在种系基因组中变得不均匀富集。我假设组蛋白 种系中 PTM 富集和 DNAme 模式由 1) 本质上由去甲基化决定 PGC 重编程期间使用的途径和 2) 外在性腺支持的信号传导微系统 细胞。为了在体内检验这一假设，将使用遗传小鼠模型和多组学分析来阐明 哪些细胞信号负责在精子和精子中获取性别特异性 DNAme 特征 卵母细胞。目标 1 (K99) 将在 PGC 开发过程中测试 TET1 的催化和非催化要求 卵母细胞基因组甲基化特征的建立。目标2（R00）将采用遗传性- 前颗粒细胞（女性到男性）中 Dmrt1 过度表达和组成型活性 Wnt 的逆转模型 前支持细胞（男性到女性）中的信号传导，以测试改变体细胞信号传导环境的影响 用于在生殖细胞中获取 DNAme。在这些模型中，我将整合并识别变化之间的相关性 甲基化和相关组蛋白 PTM 富集（H3K4 和 H3K36 的甲基化）。单细胞 转录组学将用于识别建立性别特异性 DNAme 特征的指导性线索 在配子中。在此期间，我将接受高级生物信息学和单细胞基因组学方面的广泛培训 该提案的指导阶段由 DNAme 和基因组学先驱 Bartolomei 博士指导 宾夕法尼亚大学表观遗传学研究所内的印记。在我的咨询委员会的额外指导下， 其中包括生殖细胞表观遗传学和性腺信号通路领域的领导者，我会很好 准备成为生殖表观遗传学领域的独立研究者。