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

人类内源性逆转录病毒(HERV)是占人类基因组8%的逆转录病毒残留物。 HERV的转录通常被表观遗传机制抑制；但最近的研究，包括我们的研究， 已经提供了证据表明人类特有的HERV物种LTR5_HS在人类体内被特异性地重新激活 原始生殖细胞及其人多能干细胞来源的细胞培养模型 (人类原始生殖细胞样细胞)。目前从我们的初步研究中获得的证据 以及在最近的文献中提出的，支持将hPSC转换为 HPGCLCs和hPGCLCs(但不是hPSCs)从质膜释放病毒样颗粒(VLP)。 利用我们最近在长期培养hPGCLCs(LTC-hPGCLCs)方面取得的成功，这个项目将 检查(1)重新激活LTR5_HS是否会创建特定于hPGCLC的新边界 拓扑相关结构域(TADS)，可能支持hPSCs的胚系分化；以及(2) HPGCLCs释放的VLP是否能传递病毒和/或非病毒分子，包括逆转录病毒 逆转录酶，从LTC-hPGCLCs到其他人类细胞。我们的具体目标1将使用货物 (gRNA寡核苷酸嵌合阵列)-CRISPRi和piggyBac载体对697的全面抑制作用 LTR5_HS的已知拷贝和外源LTR5_HS从/向基因组的随机插入 HPSCs和LTC-hPGCLCs。工程细胞将接受TADS的深度测序分析(Hi- C SEQ)、转录本(RNA-SEQ)和表观基因组(ChIP-SEQ)。(A)的体外转化效率 HPSCs分化为hPGCLCs或代表三个生殖层的细胞，以及(B)雄性LTC-hPGCLC分化为 XrTestis异种重组睾丸器官培养系统中的生殖细胞将通过单个 细胞RNA-seq以及免疫荧光染色。具体目标2将尝试建立LTR5_HS 免疫金染色法检测LTC-hPGCLCs释放VLPs的来源及其富集物的研究 它们的成分，如来自病毒或宿主细胞的RNA种类、蛋白质和酶活性。 将进一步尝试用多肽抗原标签和/或荧光蛋白标记VLP以 确定VLP是否可以融合到周围的其他人类细胞中，并可能内化到其他细胞中 在微流控系统中产生的人类胚胎中的hPGCLCs以及其他类型的hPSC来源的hPGCLCs 胚胎细胞。如果拟议的实验成功完成，我们预计将获得重要的 对逆转录病毒衍生的人类特有基因组元件如何促进发育的机械性见解 生殖系细胞，潜在地创造了一种新的范式，突出了exexted的生理重要性 人类生殖中的HERV。我们的实验也可能支持VLP作为一种 病毒和/或宿主细胞分子的细胞间运输，潜在地革命性地改变了我们对 HERV在人类发育和健康中的生理作用。

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.