Epigenetic Regulation of Myometrial Contractility in Pregnancy and Labor

项目摘要/摘要 早产是世界范围内婴儿在出生后的前四周死亡的主要原因。这个 这项研究的首要目标是增进我们对遗传和表观遗传机制的理解。 在妊娠和分娩过程中调节子宫肌层静止和收缩及其失调 导致早产。我们认为孕酮(P4)/孕酮受体(PR)维持子宫肌层 通过抑制炎症(如IL-1b、IL-8、COX-2)的表达在大部分妊娠期间保持平静 和收缩(CAP)(如OXTR、Cx43)基因。我们认为这是通过两种基本机制实现的：(1) PR可以与转录因子(例如，核因子-kB、AP-1)捆绑到炎症基因的启动子上，并 招募辅阻遏子抑制基因表达；(2)PR也可能直接与编码基因的启动子结合 转录抑制因子(如ZEB1)激活它们的表达。ZEB1进而与CAP的启动子结合 基因和招募一个抑制复合体来抑制它们的表达。相比之下，在学期开始时， 早产，子宫肌层PR功能因其与核因子-kB的直接相互作用而受损，其增加 通过减少PR辅活化子的表达，将P4代谢成非活性产物的酶的表达 以及通过上调截短的PR亚型(例如PR-A)。截短的PR亚型可能已经减少 转录和反式抑制活性。我们推测炎症和CAP基因的这些变化 表达是通过染色质修饰和结构的改变来调节的。这项提议的目标是 明确P4/PR的抑制作用和PR的差异作用的基因和机制。 A和PR-B对炎症和CAP基因表达的影响，并表征染色质修饰 介导子宫肌层静止并伴随CAP和炎症基因表达增强导致 足月和早产。为了实现这些目标，我们将使用小鼠模型和人类子宫肌层细胞和 组织：(1)定义和表征转录因子/辅助调节因子复合体的成分，所述转录因子/辅助调节因子 与妊娠子宫肌层中的PR相互作用以调节其抗炎作用 ZEB1抑制CAP基因的表达；(2)分析这些PR相互作用的表达和启动子结合 孕期、足月和早产的因素及激素和MicroRNAs对胎儿的影响 (3)利用rna-seq和chip-seq发现与子宫肌瘤的维持密切相关的肌层基因。 怀孕和分娩的开始及其调节的潜在转录机制。RNA序列 将使人们能够发现与维持妊娠和分娩有关的基因。合并后的 CHIP-SEQ的使用将提供对转录和表观遗传机制的全球洞察 这些基因表达会发生变化。利用PR-B-KO小鼠进行的研究将阐明PR-A和PR-A的不同作用。 PR-B在调节子宫肌层转录组中的作用。总的来说，我们的发现将揭示新的基因和 可作为预防早产及其后果的治疗靶点的途径。

PROJECT SUMMARY/ABSTRACT Preterm birth is the leading cause of infant mortality during the first four weeks of life world-wide. The overarching goal of this research is to enhance our understanding of the genetic and epigenetic mechanisms that mediate myometrial quiescence and contractility during pregnancy and labor, and their dysregulation leading to preterm birth. We suggest that progesterone (P4)/progesterone receptor (PR) maintains myometrial quiescence throughout most of pregnancy by inhibiting expression of inflammatory (e.g. IL-1b, IL-8, COX-2) and contractile (CAP) (e.g. OXTR, CX43) genes. We propose that this occurs via two basic mechanisms: (1) PR may tether to transcription factors (e.g. NF-kB, AP-1) bound to promoters of inflammatory genes and recruit corepressors to inhibit gene expression; (2) PR also may bind directly to promoters of genes encoding transcriptional repressors (e.g. ZEB1) to activate their expression. ZEB1, in turn, binds to promoters of CAP genes and recruits a repressive complex to inhibit their expression. By contrast, during the initiation of term and preterm labor, PR function in myometrium is impaired by its direct interaction with NF-kB, by increased expression of enzymes that metabolize P4 to inactive products, by decreased expression of PR coactivators and by upregulation of truncated PR isoforms (e.g. PR-A). The truncated PR isoforms may have reduced transcriptional and transrepressive activity. We postulate that these changes in inflammatory and CAP gene expression are mediated by alterations in chromatin modifications and structure. The goals of this proposal are to define the genes and mechanisms that underlie the inhibitory actions of P4/PR and differential actions of PR- A and PR-B on inflammatory and CAP gene expression, and to characterize the chromatin modifications that mediate myometrial quiescence and accompany enhanced CAP and inflammatory gene expression leading to term and preterm labor. To achieve these goals, we will use mouse models and human myometrial cells and tissues to: (1) define and characterize components of the complex of transcription factors/coregulators that interact with PR in the pregnant myometrium to mediate its anti-inflammatory actions and that interact with ZEB1 to inhibit CAP gene expression; (2) analyze expression and promoter-binding of these PR-interacting factors during pregnancy and with term and preterm labor and the effects of hormones and microRNAs in their regulation; (3) use RNA-seq and ChIP-seq to discover myometrial genes that are critical for the maintenance of pregnancy and initiation of labor and the underlying transcriptional mechanisms for their regulation. RNA-seq will enable discovery of genes involved in maintenance of pregnancy and the initiation of labor. The combined use of ChIP-seq will provide global insight into the transcriptional and epigenetic mechanisms that underlie these gene expression changes. Studies using PR-B-KO mice will elucidate the differential roles of PR-A vs. PR-B in regulation of the myometrial transcriptome. Collectively, our findings will reveal novel genes and pathways that can serve as therapeutic targets to prevent preterm birth and its consequences.