TRIM28-Regulated Transposon Repression Is Required for Human Germline Competency and Not Primed or Naive Human Pluripotency

摘要

class="head no_bottom_margin" id="sec1title">IntroductionMost studies on the mechanisms that regulate human pluripotency have focused on protein-coding genes, which constitute less than 5% of the human genome. In contrast, TEs, which account for nearly half the human genome, have received significantly less attention. Recent reports have shown that TEs are dynamically expressed in human germline cells, the naive and primed states of human pluripotency, human pre-implantation embryos, and human germ cell tumors (, , , , , ). Yet the mechanisms that regulate the dynamic expression of TEs in human pluripotency and human germline development are not well understood.One of the most dynamically expressed families of TEs in human pre-implantation embryos are long terminal repeat (LTR) retrotransposons, which constitute about 8% of the human genome (). Notably, full-length human-specific LTR5 (LTR5_HS) human endogenous retroviral K (HERVK) TEs are expressed exclusively in 8-cell, morula, and pre-implantation human epiblast cells as well as in germ cell tumors (, ). In contrast, the primate-specific LTR7-HERVHs are expressed throughout human pre-implantation embryo development as well as in primed human embryonic stem cells (hESCs), but are repressed when primed hESCs are converted to the naive state (). LTR5_HS TEs regulate viral infection in human pluripotent stem cells, whereas LTR7-HERVHs regulate primed pluripotent stem cell self-renewal (, , , ).Dynamic TE expression is not restricted to human embryos. In the mouse embryo, murine endogenous retrovirus L (MuERV-L) is expressed at the 2-cell and 8-cell stage of mouse embryo development, whereas intracisternal A particles (IAPs) are expressed in mouse oocytes, cleavage embryos, and blastocysts (). Long interspersed nuclear element 1 (LINE1), a non-LTR TE, is expressed during mouse zygotic genome activation where it functions to enable chromatin accessibility (href="#bib7" rid="bib7" class=" bibr popnode">Fadloun et al., 2013, href="#bib13" rid="bib13" class=" bibr popnode">Jachowicz et al., 2017, href="#bib23" rid="bib23" class=" bibr popnode">Rowe et al., 2010). Therefore TE expression in germline competent pluripotent cells in both mouse and human is a fundamental requirement for pluripotent cell biology.Although regulation of TE expression in human embryos and pluripotent stem cells is not well known, one of the major mechanisms responsible for regulating TE expression in mouse embryos is Trim28 (also named Kap1 and Tif1b) (href="#bib26" rid="bib26" class=" bibr popnode">Schultz et al., 2001, href="#bib27" rid="bib27" class=" bibr popnode">Schultz et al., 2002, href="#bib35" rid="bib35" class=" bibr popnode">Wolf and Goff, 2007, href="#bib37" rid="bib37" class=" bibr popnode">Zuo et al., 2012). In mouse, a zygotic knockout of Trim28 causes embryonic lethality shortly after implantation (href="#bib2" rid="bib2" class=" bibr popnode">Cammas et al., 2000), whereas a maternal mouse knockout causes variable epigenetic instability at imprinting control regions (ICRs), and no live births (href="#bib18" rid="bib18" class=" bibr popnode">Messerschmidt et al., 2012). In naive mouse ESCs, Trim28 is essential for repression of IAPs, as well as mouse ESC self-renewal and survival (href="#bib23" rid="bib23" class=" bibr popnode">Rowe et al., 2010), whereas in primed hESCs, a short-term knockdown of TRIM28 leads to HERV derepression (href="#bib32" rid="bib32" class=" bibr popnode">Turelli et al., 2014); however, the role of TRIM28 in the basic properties of human primed or naive pluripotency is not known.In the current study we report the generation of TRIM28 null mutations in primed and naive hESCs using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein nuclease (Cas9) technology (href="#bib3" rid="bib3" class=" bibr popnode">Cong et al., 2013). We show that a null mutation in TRIM28 is compatible with both primed and naive human self-renewal, despite massive derepression of TEs. We demonstrate that a null mutation in TRIM28 leads to loss of human germline competency from primed hESCs, indicating that it is the ability to differentiate into the germline, not pluripotent self-renewal per se, that is particularly sensitive to loss of TRIM28 in humans.