Regulatory mechanisms governing imprinted domains during early development

摘要 基因组印迹是一种表观遗传调控过程，它将特定基因的表达限制在一个 亲本等位基因。了解早期发育过程中的基因组印记调节具有重要的医学和 社会影响，包括医疗辅助生殖，这与罕见的印记有关 精神错乱。然而，在基因组印迹的调控方面存在着显著的知识空白。 植入前发育。而印迹控制区(ICR)上的DNA甲基化在 胚胎作为一种遗传沉默标志，建立和/或保持活跃的特定机制 位于未甲基化的ICR的转录间隔，允许长的非编码RNA(LncRNA)转录和 反过来，印迹结构域中的抑制功能仍不清楚。为了解决这些差距，我们执行了 针对Kcnq1ot1印迹结构域的表观遗传调控因子的创新的RNA干扰屏幕(作为 模型域)在小鼠胚胎外内胚层(XEN)干细胞中。我们确定了41名候选人，包括 染色质重构体、激活剂和抑制物。我们对候选核孔素(NUP)107的调查， 导致发现了一种新的印迹结构域调控机制NUP107和NUP153。在这里，我们 假设NUP107和NUP153作为促进活性转录的核支架发挥作用 通过招募染色质重构体和激活剂来转录合子中的ICR， 这反过来又会导致邻近基因的等位基因沉默。在目标1中，我们将研究受精后的作用 通过评估Kcnq1ot1 ICR上NUP107和NUP153之间的物理相互作用， Kcnq1ot1结构域及其在1-细胞晚期至囊胚期胚胎中的lncRNA。野生型胚胎将是 与NUP107或NUP153退化的胚胎相比，2个母体基因组(单倍体)或2个父本基因组- 类似基因组(母系DNMT3A/3b为空)。接下来，我们将评估NUP107和NUP153的基因组定位， 以及染色质状态，在这些胚胎中使用超低输入的Cut&Run。要进一步定义它们的功能，请访问 在对照上进行印迹结构域、NUP107和NUP153抗体的CHIP-SEQ和RNA-SEQ， Nup107和Nup153缺失的Xen细胞。在目标2中，我们将确定染色质重构体的功能， SMARCA5和SMARCC2，以及激活子KAT2A和TAF6L在产生激活转录中的作用 ICR的车厢。利用Xen细胞中的候选耗竭，我们将确定这些蛋白质在 Kcnq1ot1lncRNA的表达及其在结构域、开放/活性染色质形成、等位基因上的定位 抑制Kcnq1ot1结构域中的邻近基因，以及它们在其他印迹结构域中的功能。 接下来，我们将分析NUP107和NUP153的S招募这些染色质重构体和激活剂的能力 履行其在ICRS的职能。最后，我们将评估SMARCA5、SMARCC2、TAF6L和KAT2A的作用 在植入前胚胎中建立ICRs的主动转录。这项工作将导致发现 早期发育过程中印迹结构域调控的新机制。

ABSTRACT Genomic imprinting is an epigenetic regulatory process that restricts expression of specific genes to one parental allele. Understanding genomic imprint regulation during early development has significant medical and societal implications, including for medically assisted reproduction, which is associated with rare imprinting disorders. However, significant knowledge gaps exist for the regulation of genomic imprinting during preimplantation development. While DNA methylation at imprinting control regions (ICRs) plays a critical role in embryos as an inherited silencing mark, the specific mechanisms that establish and/or maintain an active transcription compartment at the unmethylated ICR, permitting long noncoding RNA (lncRNA) transcription and in turn, repressive function across an imprinted domain, remain unclear. To address these gaps, we performed an innovative, RNA interference screen for epigenetic regulators of the Kcnq1ot1 imprinted domain (as a model domain) in mouse extraembryonic endoderm (XEN) stem cells. We identified 41 candidates, including chromatin remodelers, activators and repressors. Our investigation of one candidate, nucleoporin (NUP) 107, led to the discovery of a novel NUP107 and NUP153 mechanism of imprinted domain regulation. Here, we hypothesize that NUP107 and NUP153 function as a nuclear scaffold for promoting an active transcription compartment at ICRs in zygotes by recruiting chromatin remodelers and activators for lncRNA transcription, which in turn, leads to allelic silencing of neighboring genes. In Aim 1, we will examine the postfertilization role of NUP107 and NUP153 at the Kcnq1ot1 ICR by assessing physical interactions between these proteins, the Kcnq1ot1 domain and its lncRNA in late 1-cell to blastocyst-stage embryos. Wildtype embryos will be compared to embryos with NUP107 or NUP153 degradation, 2 maternal genomes (parthenotes) or 2 paternal- like genomes (maternal Dnmt3a/3b null). Next, we will assess genomic localization of NUP107 and NUP153, as well as chromatin state, using ultralow-input CUT&RUN in these embryos. To further define their function at imprinted domains, ChIP-seq with NUP107 and NUP153 antibodies and RNA-seq will be conducted on control, Nup107- and Nup153-depleted XEN cells. In Aim 2, we will determine the function of chromatin remodelers, SMARCA5 and SMARCC2, and activators, KAT2A and TAF6L, in generating active transcription compartments at ICRs. Using candidate depletion in XEN cells, we will ascertain the role of these proteins on Kcnq1ot1 lncRNA expression and its localization at the domain, open/active chromatin formation, allelic repression of neighboring genes in the Kcnq1ot1 domain, as well as their function at other imprinted domains. Next, we will analyze NUP107 and NUP153’s ability to recruit these chromatin remodelers and activators to carry out their function at ICRs. Finally, we will assess the role of SMARCA5, SMARCC2, TAF6L and KAT2A in establishing active transcription at ICRs in preimplantation embryos. This work will lead to the discovery of novel mechanisms of imprinted domain regulation during early development.