Endogenous retroviruses (ERVs), retrovirus-like elements with long terminal repeats, are widely dispersed in the euchromatic compartment in mammalian cells, comprising ∼10% of the mouse genome1. These parasitic elements are responsible for >10% of spontaneous mutations2. Whereas DNA methylation has an important role in proviral silencing in somatic and germ-lineage cells3,4,5, an additional DNA-methylation-independent pathway also functions in embryonal carcinoma and embryonic stem (ES) cells to inhibit transcription of the exogenous gammaretrovirus murine leukaemia virus (MLV)6,7,8. Notably, a recent genome-wide study revealed that ERVs are also marked by histone H3 lysine 9 trimethylation (H3K9me3) and H4K20me3 in ES cells but not in mouse embryonic fibroblasts9. However, the role that these marks have in proviral silencing remains unexplored. Here we show that the H3K9 methyltransferase ESET (also called SETDB1 or KMT1E) and the Krüppel-associated box (KRAB)-associated protein 1 (KAP1, also called TRIM28)10,11 are required for H3K9me3 and silencing of endogenous and introduced retroviruses specifically in mouse ES cells. Furthermore, whereas ESET enzymatic activity is crucial for HP1 binding and efficient proviral silencing, the H4K20 methyltransferases Suv420h1 and Suv420h2 are dispensable for silencing. Notably, in DNA methyltransferase triple knockout (Dnmt1-/-Dnmt3a-/-Dnmt3b-/-) mouse ES cells, ESET and KAP1 binding and ESET-mediated H3K9me3 are maintained and ERVs are minimally derepressed. We propose that a DNA-methylation-independent pathway involving KAP1 and ESET/ESET-mediated H3K9me3 is required for proviral silencing during the period early in embryogenesis when DNA methylation is dynamically reprogrammed.
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We thank M. Okano for Dnmt1-/-Dnmt3a-/-Dnmt3b-/- ES cells; T. Jenuwein and P. Singh for Suv39h1-/-Suv39h2-/- ES cells; T. Jenuwein and G. Schotta for Suv420h1-/-Suv420h2-/- ES cells; K. Hata for Dnmt3l knockout mice testes; M. Hijikata for the large-T antigen expression vector; S. Kuramochi-Miyagawa for IAP and LINE-1 probes; S. Smale for HP1α antiserum; and L. Gaudreau for H2AZ antiserum. We are also grateful to P. Goyal and members of the Shinkai laboratory for technical support and D. Mager for critically reading the manuscript. This work was supported in part by the Genome Network Project from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, Grants-in-aid from MEXT to Y.S. and CIHR grants 77805 and 92090 to M.C.L. M.C.L. is a Scholar of the Michael Smith Foundation for Health Research.
Author Contributions T.M. and D.L. are equally contributing first authors; M.C.L. and Y.S. are equally contributing senior authors. Y.S., M.C.L., D.L. and T.M. planned studies and interpreted the data. T.M. validated the Eset CKO lines and performed most of the ERV-related studies. D.L. performed the siRNA and exogenous retrovirus-related experiments. H. Miyashita generated Eset CKO ES cells and mice. I.A.M. conducted the MusD sequencing analysis. H. Miyachi injected Eset CKO ES cells into blastocysts to make chimaeric mice. H.K. provided antibodies against methylated histones. M.T. cloned mouse Eset cDNA and provided his expertise in most of the studies conducted in this work. Y.S., M.C.L., D.L. and T.M. wrote the paper.
The authors declare no competing financial interests.
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Matsui, T., Leung, D., Miyashita, H. et al. Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET. Nature 464, 927–931 (2010). https://doi.org/10.1038/nature08858
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