Roles of the LTR5_Hs human-specific endogenous retroviruses in primordial germ cells

LTR5_Hs 人类特异性内源性逆转录病毒在原始生殖细胞中的作用

基本信息

批准号：
10573853
负责人：
TOSHIHIRO SHIODA
金额：
$ 25.05万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-03 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10573853
关键词：
Antigens CRISPR interference Cell Culture Techniques Cell Differentiation process Cell membrane Cells Cellular biology Characteristics Chimeric Proteins DNA Data Development Electron Microscopy Elements Embryo Endogenous Retroviruses Engineering Enhancers Epigenetic Process Ethics Excision Fluorescence Generations Genes Genetic Heterogeneity Genetic Materials Genetic Transcription Genome Genomics Germ Cells Germ Layers Guide RNA Health Hi-C Human Human Development Human Genome Human body Immunofluorescence Immunologic In Vitro Individual Infertility Inherited Knowledge Label Laboratories Life Cycle Stages Literature Macaca Microfluidic Microchips Microfluidics Monkeys Monoclonal Antibodies Oligonucleotides Organoids Outcome Peptides Physiological Play Pluripotent Stem Cells Population Pregnancy Preparation Production Proteins Protocols documentation RNA RNA-Directed DNA Polymerase Reproduction Reproductive Biology Retroviridae Rodent Role Serum Somatic Cell Stains Structure of primordial sex cell Surface System Testing Testis Vesicular stomatitis Indiana virus Viral Viral Envelope Proteins Virus-like particle access restrictions blastomere structure cell type deep sequencing demethylation egg embryo cell epigenome experimental study high risk population human migration human model human pluripotent stem cell induced pluripotent stem cell insight male novel particle pluripotency precursor cell reconstitution senescence single-cell RNA sequencing sperm cell success three dimensional structure transcriptome transcriptome sequencing vector viral DNA

项目摘要

Human endogenous retroviruses (HERVs) are remnants of retroviruses occupying 8% of human genome. Transcription of HERVs is generally suppressed by epigenetic mechanisms; but recent studies, including ours, have provided evidence that LTR5_Hs, the human-specific HERV species, is specifically reactivated in human Primordial Germ Cells as well as its human pluripotent stem cell (hPSC)-derived cell culture model hPGCLCs (human Primordial Germ Cell-Like Cells). Currently available evidence obtained from our preliminary studies as well as presented in very recent literature supports that LTR5_Hs is required for conversion of hPSCs to hPGCLCs and that hPGCLCs (but not hPSCs) release Virus-Like Particles (VLPs) from plasma membrane. Taking advantage of our recent success in Long-Term Culture hPGCLCs (LTC-hPGCLCs), this project will examine (1) whether the reactivation of LTR5_Hs creates new, hPGCLC-specific boundaries of the topologically associated domains (TADs), which may support germline differentiation of hPSCs; and (2) whether VLPs released from hPGCLCs can convey viral and/or non-viral molecules, including the retroviral reverse transcriptase, from LTC-hPGCLCs to other human cells. Our Specific Aim 1 will use the CARGO (chimeric array of gRNA oligos)-CRISPRi and the piggyBac vector for comprehensive suppression of the 697 known copies of LTR5_Hs and random insertion of exogenous copies of LTR5_Hs from/to the genome of hPSCs and LTC-hPGCLCs. The engineered cells will be subjected to deep sequencing analyses of TADs (Hi- C seq), transcriptomes (RNA-seq), and epigenomes (ChIP-seq). Efficiencies of (A) in vitro conversion of hPSCs to hPGCLCs or cells representing the three germ layers, and (B) male LTC-hPGCLC differentiation to gonocytes in the xrTestis xenogeneic reconstituted testis organoid culture system, will be evaluated by single cell RNA-seq as well as immunofluorescence staining. Specific Aim 2 will attempt to establish the LTR5_Hs origin of VLPs released from LTC-hPGCLCs by immunogold staining and enrich the VLPs for further analyses of their components such as RNA species, proteins, and enzymatic activities derived from viral or host cells. Attempts will be further made to label the VLPs with peptide antigen tags and/or fluorescence proteins to determine whether the VLPs can fuse into, and possibly internalized in, other human cells surrounding hPGCLCs in human embryoids generated in a microfluidics system as well as other types of hiPSC-derived embryonic cells. If the proposed experiments are successfully completed, we expect to obtain important mechanistic insights into how retrovirus-derived, human-specific genomic elements contribute to development of germline cells, potentially creating a novel paradigm that highlights the physiological importance of exapted HERVs in human reproduction. Our experiments may also support the novel concept that VLPs function as an intercellular conveyer of viral and/or host cell molecules, potentially revolutionize our knowledge about physiological roles of HERVs in human development and health.

人类内源性逆转录病毒（HERV）是占人类基因组8%的逆转录病毒的残余物。 HERV 的转录通常受到表观遗传机制的抑制；但最近的研究，包括我们的研究，已提供证据表明 LTR5_Hs（人类特异性 HERV 物种）在人类体内被特异性重新激活原始生殖细胞及其人类多能干细胞 (hPSC) 衍生的细胞培养模型 hPGCLC （人类原始生殖细胞样细胞）。从我们的初步研究中获得的现有证据以及最近文献中提出的观点支持 LTR5_Hs 是将 hPSC 转换为 hPGCLC 和 hPGCLC（但不是 hPSC）从质膜释放病毒样颗粒 (VLP)。利用我们最近在长期培养 hPGCLC (LTC-hPGCLC) 方面取得的成功，该项目将检查 (1) LTR5_Hs 的重新激活是否会创建新的、hPGCLC 特定的边界拓扑相关结构域 (TAD)，可能支持 hPSC 的种系分化；和（2） hPGCLC 释放的 VLP 是否可以传递病毒和/或非病毒分子，包括逆转录病毒逆转录酶，从 LTC-hPGCLC 到其他人类细胞。我们的具体目标 1 将使用 CARGO 第697章被人欺负了 LTR5_Hs 的已知拷贝以及 LTR5_Hs 的外源拷贝从/到基因组的随机插入 hPSC 和 LTC-hPGCLC。工程细胞将接受 TAD 的深度测序分析（Hi- C seq）、转录组（RNA-seq）和表观基因组（ChIP-seq）。 (A)体外转化的效率 hPSC 向 hPGCLC 或代表三个胚层的细胞，以及 (B) 雄性 LTC-hPGCLC 分化为 xrTestis 异种重建睾丸类器官培养系统中的生殖细胞将通过单次评估细胞 RNA-seq 以及免疫荧光染色。具体目标 2 将尝试建立 LTR5_Hs 通过免疫金染色确定 LTC-hPGCLC 释放的 VLP 的来源，并富集 VLP 以供进一步分析它们的成分，例如来自病毒或宿主细胞的 RNA 种类、蛋白质和酶活性。将进一步尝试用肽抗原标签和/或荧光蛋白来标记VLP，以确定 VLP 是否可以融合到周围的其他人类细胞中，并可能内化到其中微流体系统中产生的人类胚胎中的 hPGCLC 以及其他类型的 hiPSC 衍生的胚胎细胞。如果所提出的实验成功完成，我们期望获得重要的成果关于逆转录病毒衍生的人类特异性基因组元件如何促进发育的机制见解生殖细胞，有可能创造一种新的范式，强调外泌体的生理重要性人类生殖中的HERV。我们的实验也可能支持 VLP 作为病毒载体的新概念。病毒和/或宿主细胞分子的细胞间输送器，可能会彻底改变我们的知识 HERV 在人类发育和健康中的生理作用。