Molecular Mechanisms Employed by TRIM28 to Maintain Epigenetic Information through Different Stages of Embryonic Development

在胚胎发育期间，基因组中的信息是如何被解码的，这是人类最大的谜团之一。这个问题的核心是了解基因表达是如何调控的。某些基因是由受精前引入的化学修饰控制的。卵子和精子中DNA的这些标记(也称为染色质标记或基因组印记)是从父母传给孩子的，构成了控制基因世代表达的重要机制。尽管过去50年的研究揭示了染色质标记的复杂性，但关于识别和解释这些标记以激活或抑制印记基因的因素仍然缺乏信息。初步证据表明，一种特定蛋白质作为印记基因表达的调节器具有新的作用。通过进一步定义参与这种蛋白质的要求，这项研究计划将扩大对调节跨代基因表达控制的过程和因素的基础知识。除了加强对胚胎发育中一个基本问题的理解外，这个研究项目还促进了所有水平的学生的研究。此外，首席研究员和学生都参加了几个推广活动，以传播发育生物学的知识。受精后不久，哺乳动物胚胎经历了全基因组范围的去甲基化事件，涉及到酶促甲基胞嘧啶DNA残基的去除。这些表观遗传标记的去除允许合子基因组的重新编程，这是胚胎发育开始的关键过程。然而，许多基因组座位，包括印记基因，需要保存表观遗传信息，以便新有机体正常发育。转录抑制因子TRIM28最近成为印迹基因表达的关键调节因子。然而，该转录因子的作用机制仍不清楚。这项建议旨在解决TRIM28在不同发育阶段对基因组印记的要求，以及这种转录调控因子在小鼠胚胎发育中维持表观遗传信息所使用的分子机制。具体目标1的实验将通过分析Trim28在胚胎发育过程中不同时间条件耗尽的影响，分析TRIM28对印迹控制的阶段特异性要求。在具体目标2中，将通过上位性分析来研究TRIM28控制Dlk1-Gtl2基因座的二次DMR甲基化的机制。这些实验的结果不仅将为TRIM28的作用提供新的见解，还将为调控基因组印迹的机制提供新的见解。

How information in genomes is decoded during embryogenesis is one of the greatest mysteries of humankind. Central to this problem is an understanding of how gene expression is regulated. Certain genes are controlled by chemical modifications that are introduced before fertilization. These marks to DNA (also called chromatin marks or genomic imprints) in eggs and sperm are transmitted from parents to children and constitute an important mechanism for the control of gene expression across generations. While research in the last 50 years has uncovered the complexity of chromatin marks, information is still lacking about the factors that recognize and interpret these marks to activate or repress the imprinted genes. Preliminary evidence has revealed a novel role for a specific protein as a regulator of imprinted gene expression. By further defining the requirements for involvement of this protein, this research program will expand fundamental knowledge of the processes and factors that mediate control of gene expression across generations. In addition to enhancing understanding of a fundamental problem in embryonic development, this program of study fosters research by students of all levels. Additionally, both the lead researcher and students participate in several outreach activities to disseminate knowledge of developmental biology. Shortly after fertilization, mammalian embryos undergo a genome-wide demethylation event that involves the enzymatic removal of methyl groups from methyl-cytosine DNA residues. The removal of these epigenetic marks allows the reprogramming of the zygotic genome, an essential process for embryogenesis to begin. However, a number of genomic loci, including imprinted genes, need to preserve epigenetic information for the new organism to develop properly. The transcriptional repressor TRIM28 has recently emerged as a key regulator of imprinted gene expression. However, the mechanisms employed by this transcription factor are still unclear. This proposal aims to resolve the requirements of TRIM28 for genomic imprinting at different developmental stages and the molecular mechanisms used by this transcriptional regulator to maintain epigenetic information in developing mouse embryos. Experiments in Specific Aim 1 will analyze the stage-specific requirements of TRIM28 for imprinting control by analyzing the effects of conditional depletion of Trim28 at different times during embryonic development. In Specific Aim 2, the mechanisms by which TRIM28 controls secondary DMR methylation at the Dlk1-Gtl2 locus will be investigated through epistatic analysis. Results from these experiments will provide novel insight not only into the roles of TRIM28, but also into the mechanisms that regulate genomic imprinting.