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

项目摘要 哺乳动物的生殖需要双亲的遗传贡献，由于配子的高度二型性 表观基因组DNA甲基化（DNAme）是配子中最具性二态性的表观遗传标记之一， 在精子中是高甲基化的，在卵母细胞中是低甲基化和高甲基化的。异常 生殖系中的DNAme会对生育力和后代发育产生负面影响。为了防止传播 表位突变和建立生殖系的命运，原始生殖细胞（PGCs）经历全球DNAme擦除 以下规格。虽然大多数基因组通过复制偶联被动 稀释时，使用TET 1酶的主动去甲基化途径是消除DNA甲基化所必需的。 基因座的子集。我最近发现，精子特异性低甲基化区域虽然罕见，但需要TET 1才能用于 重新编程组织特异性低甲基化标记通常与发育中的 相关的转录因子，贷款的意义，这些精子特异性低甲基化区域。 精子或卵母细胞如何获得性别特异性DNA的机制仍然是一个知识空白， 生育力和发展。虽然在生物化学上组蛋白翻译后修饰（PTM）已经被发现， 显示与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内变得不均匀地富集。我假设组蛋白 生殖系中的PTM富集和DNAme模式化是由以下因素决定的：1）内在地通过去甲基化 PGC重编程过程中使用的信号通路和2）通过性腺支持的信号转导途径表达 细胞为了在体内验证这一假设，将使用遗传小鼠模型和多组学分析来阐明 什么样的细胞信号负责获得精子中的性别特异性DNAme签名， 卵母细胞目标1（K99）将测试PGC开发期间对TET 1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（R 00）将采用遗传性别- 前颗粒细胞中Dmrt 1过表达的逆转模型（女性对男性）和组成型活性Wnt 前Sertoli细胞（雄性到雌性）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DNA。在这些模型中，我将整合并识别变化之间的相关性 在甲基化组和相关组蛋白PTM富集（H3 K4和H3 K36的甲基化）中。单细胞 转录组学将被用来确定建立性别特异性DNA标签的指导性线索 在配子中。我将接受高级生物信息学和单细胞基因组学的广泛培训， 在DNA和基因组学领域的先驱Bartolomei博士的指导下， 在宾夕法尼亚大学表观遗传学研究所进行。在我的顾问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调查员。

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.