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

• 批准号: 10502723
• 负责人: Mellissa Rae Wigle Mann
• 金额: $ 44.57万
• 依托单位: MAGEE-WOMEN'S RES INST AND FOUNDATION
• 依托单位国家: 美国
• 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/10502723
• 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; Genes; Genetic Transcription; Genome; Genomic Imprinting; Genomics; Growth; Hydatidiform Mole; Inherited; Investigation; Knowledge; Lead; Lysine; Maps; Medical; Metabolic; Methylation; Modeling; Mus; Neurologic; Nuclear; Nuclear Matrix; Nuclear Pore Complex Proteins; Patients; Phenotype; 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; chromatin remodeling; disease phenotype; fluorescence imaging; imprint; innovation; novel; nuclear pore complex protein p107; nuclease; recruit; stem cells; transcriptome sequencing; zygote; 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; Genes; Genetic Transcription; Genome; Genomic Imprinting; Genomics; Growth; Hydatidiform Mole; Inherited; Investigation; Knowledge; Lead; Lysine; Maps; Medical; Metabolic; Methylation; Modeling; Mus; Neurologic; Nuclear; Nuclear Matrix; Nuclear Pore Complex Proteins; Patients; Phenotype; 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; chromatin remodeling; disease phenotype; fluorescence imaging; imprint; innovation; novel; nuclear pore complex protein p107; nuclease; recruit; stem cells; transcriptome sequencing; zygote

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节过程，将特定基因的表达限制为一个 父母等位基因。了解早期发展过程中基因组烙印调节的医学重要，并且 社会含义，包括用于医学辅助的繁殖，这与罕见的烙印有关 疾病。但是，在调节基因组印记的调节中存在明显的知识差距 植入前的发展。而印迹控制区（ICR）处的DNA甲基化在 胚胎作为遗传的沉默标记，建立和/或保持活跃的特定机制 在未甲基化的ICR处的转录室，允许长的非编码RNA（LNCRNA）转录和 反过来，跨印迹域中的反射功能尚不清楚。为了解决这些差距，我们执行了 KCNQ1OT1印迹域的表观遗传调节剂的创新，RNA干扰屏幕（作为一个 小鼠外胚膜内胚层（XEN）干细胞中的模型域）。我们确定了41名候选人，包括 染色质重塑，激活剂和复制品。我们对一位候选人的投资，核动脉素（NUP）107， 导致发现了新型的NUP107和NUP153印迹域调节机制。在这里，我们 假设NUP107和NUP153是促进主动转录的核支架 通过募集染色质重塑剂和lncRNA转录激活剂，在iCRS中的隔室， 反过来，这导致了相邻基因的沉默。在AIM 1中，我们将检查有效力的角色 通过评估这些蛋白质之间的物理相互作用， KCNQ1OT1域及其lncRNA在1细胞晚期到胚泡胚胎。野生型胚胎将是 与具有NUP107或NUP153降解的胚胎相比，2个MATER基因组（Partthenotes）或2个父亲 例如基因组（母体DNMT3A/3B null）。接下来，我们将评估NUP107和NUP153的基因组定位， 以及染色质状态，使用这些胚胎中的超高输入切割和运行。进一步定义其功能 将在对照中进行印迹域，具有NUP107和NUP153抗体和RNA-seq的芯片seq和RNA-seq NUP107-和NUP153删除的XEN细胞。在AIM 2中，我们将确定染色质远方的功能， SMARCA5和SMARCC2，以及激活剂Kat2a和Taf6L，在产生主动转录时 ICRS的车厢。在Xen细胞中使用候选耗竭，我们将确定这些蛋白质的作用 KCNQ1OT1 lncRNA表达及其在域上的定位，开放/活性染色质形成，等位基因 抑制KCNQ1OT1域中相邻基因及其在其他印迹域中的功能。 接下来，我们将分析NUP107和NUP153的能力 在ICRS上执行其功能。最后，我们将评估SMARCA5，SMARCC2，TAF6L和KAT2A的作用 在植入前胚胎中在ICRS上建立主动转录。这项工作将导致发现 早期开发过程中印迹域调节的新型机制。