Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET

Abstract

Endogenous retroviruses (ERVs), retrovirus-like elements with long terminal repeats, are widely dispersed in the euchromatic compartment in mammalian cells, comprising approximately 10% of the mouse genome. These parasitic elements are responsible for >10% of spontaneous mutations. Whereas DNA methylation has an important role in proviral silencing in somatic and germ-lineage cells, 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). 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 fibroblasts. 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) 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.

Transposable elements in the mammalian germline: a comfortable niche or a deadly trap?

“…TRIM28 induces the heterochromatinization of this sequence by recruiting H3K9 di-and trimethylation, HP1 and the NuRD histone deacetylase complex (Sripathy et al, 2006). It was recently shown that TRIM28 also binds to the 5 0 sequence of endogenous LTR-retrotransposons in ES cells and early embryos, and induces their repression in conjunction with ESET-dependent H3K9 trimethylation (Matsui et al, 2010;Rowe et al, 2010). Interestingly, although reactivated IAP sequences lose H3K9 trimethylation and gain H4 acetylation, they still harbor a normal level of DNA methylation in TRIM28-deficient cells.…”

“…As a result of the diversity and redundancy of histone-modifying enzymes, the importance of these marks is difficult to assess functionally. Decreases in H3K9 methylation levels through inactivation of H3K9 methyltransferases have variable outcomes on TE silencing in mouse ES cells: inactivation of ESET leads to a robust reactivation of a range of LTR-retrotransposons (Matsui et al, 2010), Suv39h deficiency induces a modest IAP reactivation (Martens et al, 2005), while inactivation of G9a has no effect (Dong et al, 2008). Reduction in H4K20 methylation through Suv420h1 and Suv420h2 deficiency does not affect TE expression in ES cells (Matsui et al, 2010).…”

“…Decreases in H3K9 methylation levels through inactivation of H3K9 methyltransferases have variable outcomes on TE silencing in mouse ES cells: inactivation of ESET leads to a robust reactivation of a range of LTR-retrotransposons (Matsui et al, 2010), Suv39h deficiency induces a modest IAP reactivation (Martens et al, 2005), while inactivation of G9a has no effect (Dong et al, 2008). Reduction in H4K20 methylation through Suv420h1 and Suv420h2 deficiency does not affect TE expression in ES cells (Matsui et al, 2010). Combined loss of H3K9 and H4K20 trimethylation can induce the reactivation of L1 elements, as was observed in mouse fibroblasts lacking the heterochromatin-associated retinoblastoma protein (Montoya-Durango et al, 2009).…”

Factors affecting the persistence of drug-induced reprogramming of the cancer methylome

“…A similar phenomenon may be occurring with H3K9me3. While studies have indicated that H3K9me2/3 is required for the maintenance of DNA methylation 44 and participates in the direction of DNMT1 during replication, 48 recent work has demonstrated that H3K9me3 and DNA methylation generally repress distinct sets of genes. 49 That DAC-induced loss of H3K9me3 from repressed areas often leads to its replacement by H3K27me3 9 suggests that the relationship between H3K9me3 and remethylation rate may be indirect, and a function of the accumulation of H3K27me3 in hypomethylated regions.…”

Overcoming barriers to the clinical utilization of iPSCs: reprogramming efficiency, safety and quality

“…The first reports at direct reprogramming employed Moloney-based retroviral vectors that are known to undergo silencing in the ESC state (Takahashi and Yamanaka, 2006;Takahashi et al, 2007;Yu et al, 2007); this self-silencing property provided an advantage for initial attempts as the temporal requirement of factor expression, which was defined that proviral silencing in embryonic stem cells requires the histone methyltransferase Eset (Maherali and Hochedlinger, 2008;Matsui et al, 2010). There are several drawbacks for the genome integration system for example their infectivity is limited to dividing cells, thus restricting the range of cell types that can be reprogrammed; silencing occurs gradually during the course of iPSC induction, resulting in a lowered efficiency of conversion compared to nonsilencing viral methods and iPSCs made with retroviruses often maintain viral gene ex- Yamanaka, 2006 Blelloch et al, 2007;Yu et al, 2007;Brambrink et al, 2008Brambrink et al, 2008Stadtfeld et al, 2008b Free of genetic modification No genetic modification, still requires at least one factor to be transduced Okita et al, 2008Chang et al, 2009Soldner et al, 2009Stadtfeld et al, 2008cFusaki et al, 2009;Gonzalez et al, 2009Kaji et al, 2009Woltjen et al, 2009Bosnali and Edenhofer, 2008Huangfu et al, 2008aShi et al, 2008b;Ichida et al, 2009;Lyssiotis et al, 2009 pression thus limiting their utility (Maherali and Hochedlinger, 2008).…”