Epigenetic Mechanisms Underlying Trophoblast Syncytialization

摘要 胎盘作为母子之间的唯一通道，是胎儿发育所必需的。 真兽类哺乳动物。胎盘发育中一个独特的过程是滋养层细胞融合形成 多核极化合体滋养层(STB)，作为母胎的主要部位 交换。虽然胎盘发育的许多方面已经得到了很好的研究，但一个综合性的全球 合胞化的分析和对协调合胞体融合的表观遗传过程的理解 缺席了。我们的初步研究利用了多种全基因组方法(RNA-seq、基因组- Scale DNA甲基化和CHIP-SEQ)表明，激活组蛋白乙酰化的收益是 合胞化BeWo细胞的主要表观基因组位移。基于这些发现，我们假设 CBP和p300组蛋白乙酰转移酶在H3K27ac/H3K9ac上的组蛋白乙酰化 (HATS)代表合胞化所需的一个从根本上保守的表观遗传过程。这个 拟议的探索性研究将解决与这一假设有关的两个主要问题。利用 滋养层细胞特异性条件性基因敲除，特异性目标1将研究CBP/p300信号转导的作用 在胎盘里。实验将评估胎盘、迷路和血管系统的发育，标志 CBPfl/fl：Cre+(CBP-CKO)和p300fl：Cre+(p300-CKO)小鼠的合胞化和融合 相对于各自的Flox控制(fl/fl：Cre-)凋落物。这些研究将提供体内功能验证 寻找BeWo细胞的发现。对特定目标2的研究将推断基因的协调变化 表达、DNA甲基化(5-MC和5-HMC)、组蛋白修饰和三维基因组 足月胎盘组织中原代人绒毛细胞滋养层细胞的组织。通过合并表达式 侧写和表观遗传学分析，这些研究将推断潜在的染色质相关过程 合胞化并将研究结果从BeWo细胞扩展到原代滋养层细胞。总的来说，这些研究 将提供组蛋白乙酰转移酶CBP/p300和胎盘发育之间的机制联系 并在理解关键转录和表观基因组网络方面产生重要的新资源 在合胞化过程中。

ABSTRACT As the sole conduit between mother and offspring, the placenta is essential fetal development in eutherian mammals. A unique process in placental development is the fusion of trophoblasts to form the multinucleated polarized syncytiotrophoblast (STB) layer that serves as the primary site for maternofetal exchange. While many aspects of placental development have been well studied, an integrative global analysis of syncytialization and understanding of the epigenetic processes orchestrating syncytial fusion is absent. Our preliminary studies leveraging multiple genome-wide approaches (RNA-seq, genome- scale DNA methylation and ChIP-seq) suggest that gain in activating histone acetylation is the predominant epigenomic shift in syncytializing BeWo cells. Based on these findings, we hypothesize that histone acetylation at H3K27ac/ H3K9ac mediated via CBP and p300 histone acetyltransferases (HATs) represents a fundamentally conserved epigenetic process required for syncytialization. The proposed exploratory studies will address two main questions relating to this postulate. Utilizing trophoblast-specific conditional knockouts, Specific Aim 1 will examine the role of CBP/ p300 signaling in the placenta. Experiments will evaluate placental, labyrinth and vasculature development, markers of syncytialization and syncytial fusion in CBPfl/fl:Cre+ (CBP-CKO) and p300fl/fl:Cre+ (p300-CKO) mice, relative to respective flox-control (fl/fl:Cre-) littermates. The studies will provide in vivo functional validation for findings from BeWo cells. Studies in Specific Aim 2 will deduce coordinated changes in gene expression, DNA methylation (5-mc and 5-hmc), histone modifications and three-dimensional genome organization in primary human villous cytotrophoblasts from term placenta tissue. By merging expression profiling and epigenetic analysis, these studies will deduce underlying chromatin associated processes in syncytialization and extend findings from BeWo cells to primary trophoblasts. Collectively, these studies will provide a mechanistic link between histone acetyltransferases CBP/p300 and placental development and generate an important new resource in understanding key transcriptional and epigenomic networks in the syncytialization process.