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，生命开始时没有调节转录的能力。这种基本能力是 通过形成转录反射性染色质状态，在早期裂解阶段获得 （TRC），其中首先需要转录增强器。不久之后建立TRC 受精至关重 元素，如果激活可能是诱变的，那么2）必须正确执行正确执行正确的元素 胚胎生存能力的转录程序。这包括激活和反映数千个基因 四个成功的胚胎基因组激活波（EGA1至4）。未能建立TRC和 调节EGA波正确杀死胚胎。 我们发现染色体的结构维护柔性铰链结构含有蛋白质 （SMCHD1）是一个母体效应基因，它可能会协调所有这些事件。 SMCHD1 1）晋升 EGA1终止，2）涉及反映在EGA2至4和3期间上调的基因，3） 支持内部细胞质量（ICM）形成和胚胎生存能力，揭示了早期SMCHD1的长期影响 动作。我们的总体模型是卵母细胞表达的SMCHD1终止EGA1，并有助于建立 TRC允许在EGA2期间进行正确的基因调节。然后，胚胎表达的SMCHD1维护和 扩展基因表达，并实现对EGA3和EGA4胚胎生存能力的最佳控制。这 因此，SMCHD1作用机制的研究为发现基础提供了重要的新作品 调节胚胎基因组功能和生存力的机制。 我们创建了一个新颖的smchd1等位基因，可用于实现卵母细胞特异性的消融 SMCHD1功能，从而创建缺乏Mater，Embryonic或SMCHD1来源的胚胎， 需要解剖SMCHD1早期功能。该建议将提供有关该建议的基本初步数据 这些基因敲除动物的表型，以允许进行更多深入的机械研究。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生存能力的影响。在目标2中，我们将 评估SMCHD1在控制前两个基因表达中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以来的基本谜团 哺乳动物的胚胎能够胜任生命。