Chromatin remodeling and gene expression during preimplantation development

表观遗传信息的动态变化是配子染色质向胚胎染色质转变的基础。这些变化被认为是启动胚胎基因转录和获得多能性所必需的。这些转变的异常可能导致胚胎在早期植入前阶段的停滞。早期胚胎死亡是奶牛和人类不育的主要原因。我们和其他研究人员证实，着床前发育过程中组蛋白甲基化的变化是一个活跃的酶促过程。具体目标1将确定在植入前牛发育过程中组蛋白甲基化标记的位点特异性动力学及其与基因表达变化的关联。使用严格的实验设计，将生成整个植入前牛发育的ChIP-seq和RNA-seq数据集，以回答以下问题：1）抑制性组蛋白标记去除对EGA的作用是什么，2）与EGA相关的激活性组蛋白标记的沉积，3）哪些基因组特征逃避组蛋白修饰重塑，4）修饰的精子组蛋白对胚胎基因调控的作用是什么，5）不对称染色质状态持续多久以及它如何影响胚胎基因表达，以及6）全能状态的表观遗传景观是什么以及它如何随着早期谱系分化而变化。虽然人们普遍认为，配子染色质的显着重塑，由母体因素驱动，需要启动胚胎基因表达，转录和染色质重塑之间的关系知之甚少。具体目标2将确定胚胎基因表达和染色质重塑之间的关系，在牛植入前发育。将使用全长单分子RNA测序和使用ATAC-seq询问的全局染色质可及性景观来确定早期胚胎产生的转录物的性质。使用转录抑制和生物信息学分析的严格实验设计将确定胚胎发育过程中产生的转录本的特征以及转录与染色质重塑之间的关系。总之，这些实验将扩大我们对胚胎发育基本机制的理解。这些信息将有助于开发诊断工具和干预措施来治疗不孕症。此外，了解从分化的配子到多能卵裂球的转变将有助于阐明分化细胞的染色质重塑机制，这与克隆，干细胞和癌症生物学有关。因此，该项目的目标将有助于提高我们对人类发展和健康的理解。

Dynamic changes in epigenetic information underlie the transition of gametic chromatin to embryonic chromatin. These changes are thought to be required to initiate embryonic gene transcription and acquisition of pluripotency. Abnormalities in these transitions likely result in embryonic arrest at early preimplantation stages. Early embryonic mortality is a major cause of infertility in dairy cattle and humans. We and others established that histone methylation changes during preimplantation development is an active and enzymatically based process. Specific Aim 1 will determine the locus-specific dynamics of histone methylation marks during preimplantation bovine development and their association with changes in gene expression. Using a rigorous experimental design, ChIP-seq and RNA-seq datasets across preimplantation bovine development will be generated to answer the following questions: 1) what is the role of repressive histone mark removal on EGA, 2) is deposition of activating histone marks related to EGA, 3) which genomic features escape histone modification remodeling, 4) what is the role of modified sperm histones for embryonic gene regulation, 5) how long does the asymmetric chromatin state persist and how does it affect embryonic gene expression, and 6) what is the epigenetic landscape of the totipotent state and how does it change with early lineage differentiation. Although it is generally accepted that the dramatic remodeling of gametic chromatin, driven by maternal factors, is required to initiate embryonic gene expression, the relationship between transcription and chromatin remodeling is poorly understood. Specific Aim 2 will determine the relationship between embryonic gene expression and chromatin remodeling during bovine preimplantation development. The nature of transcripts produced by early embryos will be determined using full-length single-molecule RNA-sequencing and global chromatin accessibility landscapes interrogated using ATAC-seq. A rigorous experimental design using transcriptional inhibition and bioinformatics analysis will determine the characteristics of transcripts produced during embryo development and the relationship between transcription and chromatin remodeling. Together, these experiments will expand our understanding of basic mechanisms underlying embryonic development. This information will aid in developing diagnostic tools and interventions to treat infertility disorders. Moreover, understanding the transition from differentiated gametes to pluripotent blastomeres will shed light on mechanisms of chromatin remodeling of differentiated cells to pluripotency, which is relevant to cloning, stem cells, and cancer biology. Therefore, the aims of this project will contribute to improve our understanding of human development and health.