Analysis of epigenetic regulation in early mammalian embryos via RNA interference

描述（由申请人提供）：在美国，估计约有1%的婴儿将通过辅助生殖技术出生，这些数字将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，但人们对这些革命性技术对儿童的适当发育并最终对儿童的健康和适宜性的影响日益感到关切。失败的表观遗传编程是在寻找原因不明的不孕症，先天性异常和增加易感性的疾病与人类辅助生殖技术和自然受孕相关的主要怀疑。表观遗传是指完全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。两个主要的机制似乎是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首先被清除，随后在胚胎早期发育期间重新建立，伴随着生育诊所体外受精后的胚胎培养时期。我们研究的长期目标是建立哺乳动物卵母细胞成熟、受精和着床前胚胎发育过程中表观遗传调控的工作模型，从而改进体外胚胎处理系统，以改善抗逆转录病毒治疗后的结果。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表现在胚胎的异常发育中。我们将通过在体外胚胎发育的既定模型中使用RNA干扰来研究表观遗传重编程的功能基因组学来验证我们的假设。目的是评估沉默一组表观遗传调控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。本研究将通过RNA干扰（RNAi）技术沉默胚胎着床前发育过程中调控表观基因组的基因的表达，探讨个体基因在表观遗传重编程和正常胚胎发育中的作用。在胚胎着床前的发育过程中，我们将检测整体染色质甲基化模式和定量基因表达，以观察基因沉默的反应。这一创新项目的成功完成将导致在胚胎早期表观遗传重编程期间维持和重建表观遗传程序所涉及的蛋白质的鉴定。这些实验将首次证明控制胚胎表观基因组的功能基因及其对小鼠以外哺乳动物细胞分化的影响。公共卫生相关性：在美国，使用辅助生殖技术（如体外受精）治疗不孕症的比例一直很高，而且增长迅速。大量研究表明，与这些手术相关的严重先天性异常和疾病的风险显著增加。失败的表观遗传异常和疾病与这些程序有关。失败的表观遗传编程可能是罪魁祸首，因此了解胚胎发育过程中表观遗传基因调控的作用对于制定减少不良后果可能性的不孕症治疗方案至关重要。

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.