Regulatory mechanisms governing imprinted domains during early development

早期发育过程中管理印记域的调控机制

基本信息

批准号：
10697375
负责人：
Mellissa Rae Wigle Mann
金额：
$ 44.5万
依托单位：
MAGEE-WOMEN'S RES INST AND FOUNDATION
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-15 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10697375
关键词：
Acetylation Acetyltransferase Actins Address Alleles Antibodies Assisted Reproductive Technology Cells ChIP-seq Chromatin Clinical Management Complex DNA DNA Methylation DNMT3a Data Development Diploidy Disease Embryo Endoderm Epigenetic Process Fertilization Fishes Genes Genetic Transcription Genome Genomic Imprinting Genomics Growth Hydatidiform Mole Inherited Investigation Knowledge Lysine Maps Medical Metabolic Methylation Modeling Mus Neurologic Nuclear Nuclear Matrix Nuclear Pore Complex Proteins Patients Phenotype Physical assessment Play Pre-implantation Embryo Development Process Proteins RNA RNA interference screen Regulation Repression Reproduction Role SMARCA5 gene SMARCC2 gene TATA-Binding Protein Associated Factors TRIM Gene Teratoma Testing Untranslated RNA Work assisted reproduction blastocyst disease phenotype fluorescence imaging imprint innovation novel nuclear pore complex protein p107 nuclease recruit stem cells transcriptome sequencing zygote

项目摘要

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.

抽象的基因组印记是一种表观遗传调控过程，将特定基因的表达限制在一个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意义。社会影响，包括与罕见印记相关的医学辅助生殖然而，对于基因组印记的调控存在显着的知识差距。而印迹控制区 (ICR) 的 DNA 甲基化在植入前发育中起着关键作用。胚胎作为遗传性沉默标记，建立和/或维持活性的特定机制未甲基化 ICR 处的转录区室，允许长非编码 RNA (lncRNA) 转录和反过来，跨印记域的抑制功能仍不清楚。为了解决这些差距，我们进行了研究。针对 Kcnq1ot1 印迹域的表观遗传调节因子的创新 RNA 干扰筛选（作为模型域）在小鼠胚胎外内胚层（XEN）干细胞中我们鉴定了 41 个候选者，包括我们对一种候选物核孔蛋白 (NUP) 107 的研究。导致了一种新的印记域调控 NUP107 和 NUP153 机制的发现。研究发现 NUP107 和 NUP153 作为核支架促进活性转录通过招募用于 lncRNA 转录的染色质重塑剂和激活剂，在受精卵中的 ICR 区室中，这反过来又导致邻近基因的等位基因沉默。在目标 1 中，我们将检查受精后的作用。通过评估 NUP107 和 NUP153 在 Kcnq1ot1 ICR 之间的物理相互作用， 1 细胞晚期至囊胚期胚胎中的 Kcnq1ot1 结构域及其 lncRNA 将是野生型胚胎。与 NUP107 或 NUP153 降解的胚胎相比，2 个母本基因组（单性生殖）或 2 个父本基因组像基因组（母体 Dnmt3a/3b 无效）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 CUT&RUN 进一步定义它们的功能。将在对照上进行印迹结构域、使用 NUP107 和 NUP153 抗体的 ChIP-seq 以及 RNA-seq， Nup107 和 Nup153 耗尽的 XEN 细胞在目标 2 中，我们将确定染色质重塑剂的功能， SMARCA5 和 SMARCC2 以及激活剂 KAT2A 和 TAF6L 在生成活性转录中的作用使用 XEN 细胞中的候选物耗尽，我们将确定这些蛋白质对 ICR 的作用。 Kcnq1ot1 lncRNA 表达及其在域中的定位、开放/活性染色质形成、等位基因 Kcnq1ot1 结构域中邻近基因的抑制，以及它们在其他印记结构域中的功能。接下来，我们将分析 NUP107 和 NUP153 招募这些染色质重塑剂和激活剂的能力最后，我们将评估 SMARCA5、SMARCC2、TAF6L 和 KAT2A 的作用。在植入前胚胎中建立 ICR 的活性转录这项工作将导致发现。早期发育过程中印迹域调控的新机制。