Epigenetic Control of Human Trophoblast Stem Cell Differentiation

胎盘形成需要将滋养层干细胞忠实地分配和分化为不同类型的细胞，创造出一个能够支持胎儿的器官。这一过程中的缺陷可能会给胎儿和母亲带来可怕的后果，并可能导致不孕不育、反复流产和死产。由胎盘缺陷引起的胎儿发育异常也可能在一生中产生慢性影响。这一提议将产生对控制人类滋养层细胞分化的表观遗传学事件的洞察，这对于理解胎盘缺陷的起源和病理至关重要。小鼠的遗传学研究已经确定了滋养层细胞分化的调节因素，但由于小鼠和人类之间的差异，它们的临床相关性尚不确定。人滋养层干细胞能够在体外概括早期滋养层细胞的分化，这代表着一项变革性的进展。该培养体系为研究基因突变对人滋养层细胞的影响提供了一种有力的手段。这些研究将使用HTS细胞来询问在小鼠中发现的调节因子是否对人类滋养层细胞分化至关重要。这项拟议工作的长期目标是识别促进人类正常胎盘发育的遗传和表观遗传因素。这项应用集中在BCOR，一个X连锁的基因，在小鼠的胎盘发育中起着至关重要的作用。BCOR在涉及低出生体重和宫内生长受限(IUGR)的人类综合征中发生突变，BCOR杂合子零突变的女性可能会经历反复流产。BCOR是表观遗传沉默复合体Polycomb抑制复合体1.1的保守成分。这一建议的中心假设是，BCOR是人类胎盘发育的关键表观遗传调节因子。拟议的工作有两个具体目标。第一个研究是研究BCOR是否通过将在人类患者中发现的BCOR零突变引入HTS细胞来调节人类滋养层细胞的分化，这可能是相关的IUGR和反复流产的原因。表型分析将确定这些突变是否影响滋养层干细胞分化为合体滋养层细胞和绒毛外滋养层细胞类型的能力，这两种细胞类型在胎盘形成中起着重要的功能作用。BCOR依赖的mRNA表达和表观遗传学变化将被用来识别影响滋养细胞分化的关键下游靶点，并可能编码有用的生物标志物，用于早期诊断因滋养细胞分化不当而引起的妊娠并发症。第二个目的是研究BCOR错义突变的功能重要性，询问它们如何影响蛋白质的功能。对BCOR蛋白错义突变的表征将提供对其如何影响滋养细胞分化的机械性见解，并将产生超出这些综合征患者的影响。

Placentation requires faithful allocation and differentiation of trophoblast stem cells into diverse cell types, creating an organ capable of sustaining the fetus. Defects in this process can have dire consequences for both the fetus and the mother and can contribute to infertility, recurrent miscarriages and stillbirth. Abnormal fetal growth caused by placental defects can also have chronic effects throughout life. This proposal will generate insights into the epigenetic events that control human trophoblast differentiation essential for understanding the origin and pathology of placental defects. Genetic studies in mice have identified regulators of trophoblast differentiation but their clinical relevance is uncertain due to differences between mice and humans. The derivation of human trophoblast stem (hTS) cells, which are able to recapitulate early trophoblast differentiation in vitro, represents a transformational advance. This culture system provides an incisive means to investigate the effects of genetic mutations in human trophoblasts. These studies will use hTS cells to ask whether regulators identified in the mouse are crucial for human trophoblast differentiation. The long-term goal of the proposed work is to identify genetic and epigenetic factors that promote normal placental development in humans. This application focuses on BCOR, an X-linked gene with essential roles in placental development in mice. BCOR is mutated in human syndromes involving low birth weight and intrauterine growth restriction (IUGR) and women with a heterozygous null mutation in BCOR can experience recurrent miscarriages. BCOR is a conserved component of the epigenetic silencing complex Polycomb Repressive Complex 1.1. The central hypothesis of this proposal is that BCOR is a key epigenetic regulator of human placental development. The proposed work has two specific aims. The first investigates whether BCOR regulates human trophoblast differentiation by introducing into hTS cells BCOR null mutations found in human patients that might be responsible for the associated IUGR and recurrent miscarriages. Phenotypic assays will determine whether these mutations affect the ability of trophoblast stem cells to differentiate into syncytiotrophoblast and extravillous trophoblasts cell types, which play important functional roles in placentation. BCOR-dependent mRNA expression and epigenetic changes will be used to identify key downstream targets that affect trophoblast differentiation and might encode useful biomarkers for early diagnosis of pregnancy complications arising from improper trophoblast differentiation. The second aim investigates the functional importance of BCOR missense mutations, asking how they affect function of the protein. Characterization of missense mutations in the BCOR protein will provide mechanistic insights into how it influences trophoblast differentiation and will have an impact that extends beyond the patients with these syndromes.