Environmental chemicals impair epigenetic suppression of the endogenous retrovirus HML-2 in human primordial germ cells, predisposing the next generation to malignancies through HML-2 reactivation - Evidence supporting the epigenetic inheritance of diseases in mammals is accumulating, and the endogenous retrovirus (ERV) may play as a heritable epigenetic vehicle. HML-2, the ERV species most recently integrated into the human genome, is epigenetically silenced in normal cells, but our lab and others recently revealed that HML-2 is transiently activated in human primordial germ cells (PGCs) but silenced as PGCs differentiate to sex-specific germ cells. In contrast, HML-2 is often activated in common cancers (e.g., breast cancer, prostate cancer, or melanomas), and two HML-2 viral proteins are presumed oncogenic, supporting a growing paradigm that HML-2 reactivation may contribute to human carcinogenesis. We hypothesize that the epigenetic silencing of HML-2 during the post-PGC development of human germ cells may be impaired by environmental toxicants. The affected, incompletely silenced HML-2 loci may be vulnerable to reactivation, increasing the cancer risk in the next generation. Our mechanistic reasoning of this hypothesis is that assembly of protein complexes required for formation of heterochromatin – the main epigenetic machinery silencing ERVs – is known to be disrupted by transcriptional activation in its vicinity. Therefore, transactivation and/or DNA demethylation by toxic chemicals may impair epigenetic silencing of HML-2. HML-2 is strongly activated in pluripotent stem cell (PSC)-derived PGC-Like Cells (PGCLCs). In the xenogeneic reconstituted testis (xrTestis) organoid culture involving human PGCLCs and mouse embryonic testicular somatic cells, PGCLCs differentiate to gonocyte- like cells (GnCLCs), in which all HML-2 loci are efficiently silenced. Our Specific Aim 1 will expose xrTestes to toxicants (PFOA, dieldrin and benzo[a]pyrene), and the transcriptional and epigenetic states of all HML-2 loci in GnCLCs will be examined by bulk and single-cell deep sequencing. Degrees of relaxation in epigenetic silencing will be evaluated under pharmacologically induced, modest HML-2 reactivation using inhibitors of DNA methyltransferase 1 or H3K9 methyltransferases. Also using deep sequencing, Specific Aim 2 will determine whether impaired epigenetic silencing of HML-2 is repaired during the global epigenetic reprogramming in human PSCs and PGCLCs. Epigenetic silencing of selected HML-2 loci will be specifically relaxed by CRISPRa. Specific Aim 3 will create a humanized mouse model harboring the HML-2 locus associating the PRODH gene in the mouse genome safe-harbor locus Rosa26 and examine its epigenetic states and activation in mouse PGCs, gonocytes, and somatic cells muti-generationally after CRISPRa activation of the HML-2 locus or gestational exposures to toxicants. Specific Aim4 will perform computational analysis of RNA-seq data retrieved from databases to identify similarities and differences in the HML-2 loci activated in various human cancers and PGCLCs. The outcome of this project may create a novel paradigm focusing on the toxicological importance of HML-1 as a heritable epigenetic vehicle, linking exposure of pregnant women to toxicants and cancer risk in the subsequent generation.
