Endogenous retroviral insertions drive non-canonical imprinting in extra-embryonic tissues [RNA-Seq]

Background: Genomic imprinting is an epigenetic phenomenon that allows a subset of genes to be expressed mono-allelically based on parent-of-origin, and is typically regulated by differential DNA methylation inherited from gametes. Imprinting is pervasive in murine extra-embryonic lineages and, uniquely, the imprinting of several genes has been found to be conferred non-canonically through maternally-inherited repressive histone modification H3K27me3. However, the underlying regulatory mechanisms of non-canonical imprinting in post-implantation development remain unexplored. Results: We identify imprinted regions in post-implantation epiblast and extra-embryonic ectoderm (ExE) by assaying allelic histone modifications (H3K4me3, H3K36me3, H3K27me3), gene expression and DNA methylation in reciprocal C57BL/6 and CAST hybrid embryos. We distinguish loci with DNA methylation- dependent (canonical) and independent (non-canonical) imprinting by assaying hybrid embryos with ablated maternally-inherited DNA methylation. We find that non-canonical imprints are localized to endogenous retrovirus-K (ERVK) long terminal repeats (LTRs), which act as imprinted promoters specifically in extra-embryonic lineages. Transcribed ERVK LTRs are CpG-rich and located in close proximity to gene promoters, and imprinting status is determined by their epigenetic patterning in the oocyte. Finally, we show that oocyte-derived H3K27me3 associates with non-canonical imprints is not maintained beyond pre-implantation development, and is replaced by secondary imprinted DNA methylation on the maternal allele in post-implantation ExE, while being completely silenced by bi-allelic DNA methylation in epiblast. Conclusions: This study reveals distinct epigenetic mechanisms regulating non-canonical imprinted gene expression between embryonic and extra-embryonic development, and identifies an integral role for ERVK LTR repetitive elements. Overall design: To evaluate the allelic regulation of histone modifications in the embryo, we assayed H3K4me3, H3K36me3 and H3K27me3 using ultra low-input ChIP-seq (Hanna et al. 2018) and post-bisulfite adaptor tagging (PBAT) in reciprocal hybrid (C57BL6/Babr x CAST/Ei) embryonic day (E) 6.5 epiblast and extra-embryonic ectoderm (ExE). We additionally profiled these epigenetic marks in E6.5 embryos derived from females with a double conditional knockout for Dnmt3a and Dnmt3b in oocytes (matDKO), driven by Zp3-cre. Consequently, these matDKO embryos will inherit no maternal DNA methylation, but are able to sufficiently establish DNA methylation post-fertilisation. Allelic gene expression was evaluated in E7.5 epiblast and ExE of all hybrid crosses.