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

人内源性逆转录病毒（human endogenous retroviruses，HERVs）是逆转录病毒的残余物，约占人类基因组的8%。 HERVs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， 已经提供了证据表明，LTR5_H，人类特异性HERV种类，在人类中特异性再激活， 原始生殖细胞及其人多能干细胞（hPSC）衍生的细胞培养模型hPGCLC （人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 以及在最近的文献中提出的，支持LTR5_H是将hPSC转化为 hPGCLC和hPGCLC（而不是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）方面的成功，该项目将 检查（1）LTR5_Hs的重新激活是否产生了新的hPGCLC特异性边界， 拓扑相关结构域（TADs），其可支持hPSC的种系分化;和（2） 从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将使用货物 （gRNA寡核苷酸的嵌合阵列）-CRISPRi和piggyBac载体用于全面抑制697 LTR5_H的已知拷贝和LTR5_H的外源拷贝从/到LTR5_H的基因组的随机插入 hPSC和LTC-hPGCLC。工程化的细胞将进行TADs（Hi-TADs）的深度测序分析。 C seq）、转录组（RNA-seq）和表观基因组（ChIP-seq）。（A）体外转化的效果 （B）雄性LTC-hPGCLC分化为hPGCLC或代表三个胚层的细胞， 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 细胞RNA-seq以及免疫荧光染色。具体目标2将尝试建立LTR 5_HS 通过免疫金染色从LTC-hPGCLC释放的VLP的来源，并富集VLP用于进一步分析 它们的组分如RNA种类、蛋白质和来自病毒或宿主细胞的酶活性。 将进一步尝试用肽抗原标签和/或荧光蛋白标记VLP， 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 在微流体系统中产生的人胚样中的hPGCLC以及其他类型的hiPSC衍生的 胚胎细胞如果实验成功，我们将获得重要的成果。 对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性见解 生殖系细胞，可能创造一个新的范例，突出了exapted的生理重要性， 人类生殖中的HERV。我们的实验也可能支持新的概念，即VLP作为一种 病毒和/或宿主细胞分子细胞间输送器，潜在地革新了我们关于 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.