Analysis of epigenetic regulation in early mammalian embryos via RNA interference

通过RNA干扰分析早期哺乳动物胚胎的表观遗传调控

• 批准号: 8097100
• 负责人: CHARLES R LONG
• 金额: $ 13.08万
• 依托单位: TEXAS A&M AGRILIFE RESEARCH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-07 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8097100
• 关键词: Adopted; Age; Animal Model; Assisted Reproductive Techniques; Assisted Reproductive Technology; Back; Biochemical; Biological; Biological Assay; Biological Phenomena; Birth; Cattle; Cell Differentiation process; Cells; Child; Chromatin; Clinic; Cloning; Conceptions; Congenital Abnormality; Couples; Cryopreservation; DNA Methylation; DNA Methyltransferase; DNA Modification Methylases; DNA Sequence; Data; Development; Disease; Embryo; Embryonic Development; Environment; Enzymes; Epigenetic Process; Ethics; Event; Failure; Family; Fertility; Fertilization; Fertilization in Vitro; Gene Expression; Gene Expression Regulation; Gene Silencing; Gene Targeting; Genes; Genetic Engineering; Genome; Goals; Health; Histones; Human; In Vitro; Individual; Infertility; Intracytoplasmic Sperm Injections; Laboratories; Lead; Link; Literature; Maintenance; Measures; Methylation; Methyltransferase Gene; Modeling; Molecular; Mus; Nature; Outcome; Pattern; Physicians; Predisposition; Procedures; Production; Property; Proteins; Publishing; RNA Interference; Regulation; Regulator Genes; Research; Risk; Role; Study models; Suggestion; System; Techniques; Technology; Testing; Time; United States; Woman; Work; adverse outcome; base; blastocyst; chromatin modification; critical period; demethylation; embryo culture; embryo stage 2; fitness; functional genomics; histone modification; improved; in vitro Model; innovation; knock-down; oocyte maturation; pluripotency; preimplantation; pressure; programs; public health relevance; research study; response; social

DESCRIPTION (provided by applicant): In the United States, it is estimated that about 1% of all babies will be born using assisted reproductive technologies and these numbers continue to grow at a steady rate. The promise of assisting infertile couples to have a family has been realized, but not without a growing concern over the effects of these revolutionary technologies on the proper development and ultimately the health and fitness of the child. Failed epigenetic programming is a primary suspect in the search for causes of unexplained infertility, congenital abnormalities and increased susceptibility to disease associated with both human assisted reproductive techniques and spontaneous conception. Epigenetic refers to differential patterns of gene expression based solely on the physical and biochemical properties of chromatin, without a change in DNA sequence. Two major mechanisms appear to be responsible for establishing and maintaining the epigenome, DNA methylation and histone modifications. Mammalian epigenetic marks are first erased and subsequently re-established during early embryonic development, concomitant with the period of embryo culture following IVF in fertility clinics. The long-term goal of our research is to develop a working model of epigenetic regulation during mammalian oocyte maturation, fertilization and pre-implantation embryonic development, so that in vitro embryo handling systems can be modified to improve the outcomes following ART. Our hypothesis states that subtle alterations of histone and DNA methyltransferases during the critical period of in vitro embryo culture manifest in aberrant embryo development. We will test our hypothesis by using RNA interference to study the functional genomics of epigenetic reprogramming in an established model of in vitro embryo development. The goal is to evaluate the molecular and biological effects of silencing a select group of epigenetic regulators on the establishment of epigenetic marks, maintenance of markers of pluripotency and initiation of differentiation. This Aim will investigate the role individual genes in epigenetic reprogramming and normal embryonic development by employing RNA interference (RNAi) techniques to silence the expression of genes regulating the epigenome during pre-implantation development. Global chromatin methylation patterns and quantitative gene expression during pre-implantation development will be assayed to observe the response to gene silencing. The successful completion of this innovative project will result in the identification of the proteins involved with maintaining and re-establishing the epigenetic program during the period of epigenetic reprogramming in the early embryo. These experiments will provide the first evidence of the functional genes controlling the embryonic epigenome and their effects on cell differentiation in a mammalian species other than the mouse. PUBLIC HEALTH RELEVANCE: Treatment of infertility using assisted reproductive technologies such as in vitro fertilization is at an all time high and growing rapidly in the United States. Numerous studies show a significantly increased risk of serious congenital abnormalities and disease associated with these procedures. Failed epigenetic abnormalities and disease associated with these procedures. Failed epigenetic programming is likely to blame and thus understanding the role of epigenetic gene regulation during embryonic development is critical for formulating infertility treatment options that reduce the possibility of adverse outcomes.

描述（由申请人提供）：在美国，估计约有 1% 的婴儿将使用辅助生殖技术出生，并且这些数字继续以稳定的速度增长。帮助不孕夫妇组建家庭的承诺已经实现，但人们越来越担心这些革命性技术对孩子的正常发育以及最终的健康和健身的影响。在寻找无法解释的不孕症、先天性异常以及与人类辅助生殖技术和自然受孕相关的疾病易感性增加的原因时，失败的表观遗传编程是一个主要嫌疑点。表观遗传是指仅基于染色质的物理和生化特性而不改变DNA序列的基因表达的差异模式。两种主要机制似乎负责建立和维持表观基因组：DNA 甲基化和组蛋白修饰。哺乳动物的表观遗传标记在早期胚胎发育过程中首先被擦除，随后在生育诊所体外受精后的胚胎培养期间重新建立。我们研究的长期目标是开发哺乳动物卵母细胞成熟、受精和植入前胚胎发育过程中表观遗传调控的工作模型，以便可以修改体外胚胎处理系统以改善 ART 后的结果。我们的假设表明，在体外胚胎培养的关键时期，组蛋白和 DNA 甲基转移酶的细微变化会导致胚胎发育异常。我们将通过使用 RNA 干扰在已建立的体外胚胎发育模型中研究表观遗传重编程的功能基因组学来检验我们的假设。目的是评估沉默一组选定的表观遗传调节因子对表观遗传标记的建立、多能性标记的维持和分化起始的分子和生物学效应。该目标将通过采用 RNA 干扰 (RNAi) 技术来沉默在植入前发育过程中调节表观基因组的基因的表达，从而研究单个基因在表观遗传重编程和正常胚胎发育中的作用。将分析植入前发育期间的整体染色质甲基化模式和定量基因表达，以观察对基因沉默的反应。这一创新项目的成功完成将导致在早期胚胎表观遗传重编程期间与维持和重建表观遗传程序相关的蛋白质的鉴定。这些实验将为控制胚胎表观基因组的功能基因及其对小鼠以外的哺乳动物物种细胞分化的影响提供第一个证据。公共健康相关性：在美国，使用体外受精等辅助生殖技术治疗不孕症的比例空前高涨，并且增长迅速。大量研究表明，与这些手术相关的严重先天畸形和疾病的风险显着增加。失败的表观遗传异常和与这些手术相关的疾病。失败的表观遗传编程很可能是罪魁祸首，因此了解表观遗传基因调控在胚胎发育过程中的作用对于制定减少不良结果可能性的不孕症治疗方案至关重要。

项目成果

Oxygen-induced alterations in the expression of chromatin modifying enzymes and the transcriptional regulation of imprinted genes.

• DOI: 10.1016/j.gep.2018.01.001
• 发表时间: 2018-06
• 期刊: Gene expression patterns : GEP
• 作者: Skiles WM;Kester A;Pryor JH;Westhusin ME;Golding MC;Long CR
• 通讯作者: Long CR

Down-regulation of viral replication by lentiviral-mediated expression of short-hairpin RNAs against vesicular stomatitis virus ribonuclear complex genes.

通过慢病毒介导的针对水泡性口炎病毒核糖核复合体基因的短发夹 RNA 表达下调病毒复制。

• DOI: 10.1016/j.antiviral.2012.05.007
• 发表时间: 2012
• 期刊: Antiviral research
• 影响因子: 7.6
• 作者: Ramirez-Carvajal,Lisbeth;Long,CharlesR
• 通讯作者: Long,CharlesR

Examination of DNA methyltransferase expression in cloned embryos reveals an essential role for Dnmt1 in bovine development.

• DOI: 10.1002/mrd.21306
• 发表时间: 2011-05
• 期刊: MOLECULAR REPRODUCTION AND DEVELOPMENT
• 影响因子: 2.5
• 作者: Golding, Michael C.;Williamson, Gayle L.;Stroud, Todd K.;Westhusin, Mark E.;Long, Charles R.
• 通讯作者: Long, Charles R.

Reshaping the transcriptional frontier: epigenetics and somatic cell nuclear transfer.

• DOI: 10.1002/mrd.22271
• 发表时间: 2014-02
• 期刊: MOLECULAR REPRODUCTION AND DEVELOPMENT
• 影响因子: 2.5
• 作者: Long, Charles R.;Westhusin, Mark E.;Golding, Michael C.
• 通讯作者: Golding, Michael C.