Conditional knockout effects of SMCHD1 in oocytes and embryos

卵母细胞和胚胎中 SMCHD1 的条件性敲除效应

• 批准号: 10228093
• 负责人: Keith E Latham
• 金额: $ 7.83万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10228093
• 关键词: Ablation; Alleles; Cell Nucleus; Cells; Chromatin; Chromosomes; DNA Methylation; DNA Transposable Elements; Data; Development; Embryo; Embryonic Development; Enhancers; Epigenetic Process; Event; Failure; Fertilization; Foundations; Gametogenesis; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Histone H3; Inner Cell Mass; Inner Supporting Cell; Knock-out; Life; LoxP-flanked allele; Lysine; Maintenance; Mammals; Mediating; Meiosis; Messenger RNA; Modeling; Modification; Mus; Oocytes; Oogenesis; Ovulation; Phenotype; Pilot Projects; Pregnancy; Process; Proteins; Quantitative Reverse Transcriptase PCR; Regulation; Role; Small Interfering RNA; Source; Structure; Transgenic Mice; conditional knockout; egg; embryo cell; flexibility; gene product; gene repression; innovation; knockout animal; novel; preimplantation; programs; sperm cell; zona pellucida glycoprotein; zygote

In mammals, the ability to regulate transcription is absent at the start of life. This fundamental ability is acquired during early cleavage stages through the formation of a transcriptionally repressive chromatin state (TRCS), wherein transcriptional enhancers first become necessary. Establishing the TRCS soon after fertilization is vital for two reasons: 1) it is important to suppress activation of endogenous transposable elements, which if activated can be mutagenic, and 2) it is essential to correctly execute the correct transcriptional program for embryo viability. This includes activating and repressing thousands of genes during four successive waves of embryonic genome activation (EGA1 to 4). Failure to establish the TRCS and to regulate EGA waves correctly kills embryos. We discovered that Structural maintenance of chromosomes flexible hinge domain containing protein one (SMCHD1) is a maternal effect gene that potentially orchestrates all of these events. SMCHD1 1) promotes EGA1 termination, 2) is implicated in repressing genes that are up-regulated during EGA2 to 4, and 3) supports inner cell mass (ICM) formation and embryo viability revealing long-term impacts of early SMCHD1 actions. Our overall model is that oocyte-expressed SMCHD1 terminates EGA1 and helps to establish the TRCS to allow correct gene regulation during EGA2. Embryo-expressed SMCHD1 then maintains and extends gene repression and enables optimum control of EGA3 & EGA4 necessary for embryo viability. The study of SMCHD1 mechanisms of action thus provides an important new entry for discovering fundamental mechanisms regulating embryonic genome function and viability. We created a novel floxed Smchd1 allele, which can be used to achieve oocyte-specific ablation of SMCHD1 function, and thus create embryos that lack maternal, embryonic or both sources of SMCHD1, as needed to dissect SMCHD1 earl functions. This proposal will provide essential preliminary data on the phenotype of these knockout animals to allow more in-depth mechanistic studies to be pursued. In Aim 1 we will determine the effects of oocyte-specific knockout on oogenesis and early embryo viability. In Aim 2 we will assess SMCHD1’s role in controlling genes that are activated during the first two waves of gene expression during the 2-cell stage. Overall, this project seeks to solve long-standing fundamental mysteries of how mammalian embryos become competent for life.

在哺乳动物中，在生命之初就缺乏调节转录的能力。这个基本能力就是 在早期裂解阶段通过形成转录抑制染色质状态获得 （TRCS），其中转录增强子首先变得必要。不久后成立TRCS 受精至关重要有两个原因：1）抑制内源转座子的激活很重要 元素，如果被激活可能会产生诱变，并且 2）正确执行正确的操作至关重要 胚胎活力的转录程序。这包括在过程中激活和抑制数千个基因 胚胎基因组激活的四个连续波（EGA1 至 4）。未能建立 TRCS 并 正确调节 EGA 波可杀死胚胎。 我们发现，含有蛋白质一的染色体柔性铰链结构域的结构维持 (SMCHD1) 是一种母体效应基因，可能协调所有这些事件。 SMCHD1 1) 促进 EGA1 终止，2) 涉及抑制在 EGA2 至 4 期间上调的基因，以及 3) 支持内细胞团 (ICM) 形成和胚胎活力，揭示早期 SMCHD1 的长期影响 行动。我们的总体模型是卵母细胞表达的 SMCHD1 终止 EGA1 并帮助建立 TRCS 允许在 EGA2 期间进行正确的基因调控。胚胎表达的 SMCHD1 然后维持并 延长基因抑制并实现对胚胎活力所需的 EGA3 和 EGA4 的最佳控制。这 因此，SMCHD1作用机制的研究为发现基本机制提供了一个重要的新入口。 调节胚胎基因组功能和活力的机制。 我们创建了一种新型 floxed Smchd1 等位基因，可用于实现卵母细胞特异性消融 SMCHD1 发挥作用，从而产生缺乏母体、胚胎或两者来源的 SMCHD1 的胚胎，如 需要剖析 SMCHD1 Earl 函数。该提案将提供重要的初步数据 这些基因敲除动物的表型，以便进行更深入的机制研究。在目标 1 中，我们 将确定卵母细胞特异性敲除对卵子发生和早期胚胎活力的影响。在目标 2 中，我们将 评估 SMCHD1 在控制前两波基因表达期间激活的基因中的作用 在2细胞阶段。总体而言，该项目旨在解决长期存在的基本谜团： 哺乳动物胚胎具有生命能力。