Regulatory mechanisms governing imprinted domains during early development

摘要 基因组印记是一种表观遗传调控过程， 亲本等位基因了解早期发育过程中的基因组印记调控具有重要的医学和生物学意义。 社会影响，包括医学辅助生殖，这与罕见的印记有关 紊乱然而，在基因组印迹的调控方面存在着重大的知识空白。 着床前发育虽然DNA甲基化在印迹控制区（ICR）中起着关键作用， 胚胎作为遗传沉默标记，建立和/或维持活性的特定机制， 在未甲基化的ICR处的转录区室，允许长非编码RNA（lncRNA）转录， 反过来，通过印记域抑制功能仍然不清楚。为了弥补这些差距，我们 一个创新的，RNA干扰筛选Kcnq 1 ot 1印迹结构域的表观遗传调节因子（作为一种 模型结构域）。我们确定了41名候选人，包括 染色质重塑剂、激活剂和抑制剂。我们对一种候选物核孔蛋白（NUP）107的研究， 导致发现了NUP 107和NUP 153印迹结构域调节的新机制。这里我们 假设NUP 107和NUP 153作为促进活性转录核支架起作用 通过募集染色质重塑剂和lncRNA转录激活剂， 这又导致相邻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 NUP 107和NUP 153在Kcnq 1 ot 1 ICR的表达，通过评估这些蛋白质之间的物理相互作用， Kcnq 1 ot 1结构域及其lncRNA在1-细胞晚期至囊胚期胚胎中的表达野生型胚胎将 与NUP 107或NUP 153降解的胚胎相比，2个母本基因组（单性生殖）或2个父本基因组- 类似基因组（母本Dnmt 3a/3b无效）。接下来，我们将评估NUP 107和NUP 153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CUT&Run。为了进一步确定其功能， 印迹结构域，使用NUP 107和NUP 153抗体的ChIP-seq和RNA-seq将在对照上进行， Nup 107和Nup 153耗尽的XEN细胞。在目标2中，我们将确定染色质重塑的功能， SMARCA 5和SMARCC 2以及激活剂KAT 2A和TAF 6L在产生活性转录中的作用 在ICRs的隔间。利用XEN细胞中的候选物耗竭，我们将确定这些蛋白质在 Kcnq 1 ot 1 lncRNA表达及其在结构域的定位，开放/活性染色质形成，等位基因 在Kcnq 1 ot 1结构域的相邻基因的抑制，以及它们在其他印迹结构域的功能。 接下来，我们将分析NUP 107和NUP 153招募这些染色质重塑剂和激活剂的能力， 在国际民事代表处履行其职能。最后，我们将评估SMARCA 5、SMARCC 2、TAF 6L和KAT 2A在肿瘤发生中的作用。 在植入前胚胎的ICR处建立活跃的转录。这项工作将导致发现 早期发育过程中印记域调控的新机制。

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.