Silencing of proviruses in embryonic cells: efficiency, stability and chromatin modifications.

1] Department of Biochemistry and Molecular Biophysics, Columbia University [2] Department of Microbiology and Immunology, Columbia University.
EMBO Reports (Impact Factor: 7.86). 11/2012; DOI: 10.1038/embor.2012.182
Source: PubMed

ABSTRACT Embryonic stem cells repress retroviral infection through transcriptional silencing of proviral DNAs. We characterized two distinct mechanisms of silencing in embryonic mouse cells infected by Moloney murine leukaemia virus (MLV): a highly efficient one targeting the proline transfer RNA primer-binding site (PBSpro), and a less efficient one operating independently of the PBS. Rare virus-expressing populations were isolated, and the timing and efficiency of establishment of silencing were determined. Superinfection of the selected virus-expressing cells with a second virus carrying a distinguishable reporter revealed that the PBSpro-directed silencing was still largely intact, whereas the PBS-independent silencing was partially reduced. The timing and stability of silencing, and the associated chromatin modifications on newly established and endogenous proviruses were determined. The results indicate that epigenetic mechanisms with different specificity and efficiency are used to silence the exogenous retroviral sequences in embryonic cells.

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    ABSTRACT: Mammalian genomes are replete with retrotransposable elements, including endogenous retroviruses. DNA methyltransferase 3-like (DNMT3L) is an epigenetic regulator expressed in prospermatogonia, growing oocytes, and embryonic stem (ES) cells. Here, we demonstrate that DNMT3L enhances the interaction of repressive epigenetic modifiers, including histone deacetylase 1 (HDAC1), SET domain, bifurcated 1 (SETDB1), DNA methyltransfearse 3A (DNMT3A), and tripartite motif-containing protein 28 (TRIM28; also known as TIF1β and KAP1) in ES cells and orchestrates retroviral-silencing activity with TRIM28 through mechanisms including, but not limited to, de novo DNA methylation. Ectopic expression of DNMT3L in somatic cells causes methylation-independent retroviral silencing activity by recruitment of the TRIM28/HDAC1/SETDB1/DNMT3A/DNMT3L complex to newly integrated Moloney Murine Leukemia Virus (Mo-MuLV) proviral DNA. Concurrent with this recruitment, we also observed the accumulation of histone H3 lysine 9 trimethylation (H3K9me3) and heterochromatin protein 1 gamma (HP1γ), as well as reduced H3K9 and H3K27 acetylation at Mo-MuLV proviral sequences. Ectopic expression of DNMT3L in late passage mouse embryonic fibroblasts (MEFs) recruited cytoplasmically localized HDAC1 to the nucleus. The formation of this epigenetic modifying complex requires interaction of DNMT3L with DNMT3A as well as with histone H3. In fetal testes at embryonic day 17.5, endogenous DNMT3L also enhanced the binding among TRIM28, DNMT3A, SETDB1, and HDAC1. We propose that DNMT3L may be involved in initiating a cascade of repressive epigenetic modifications by assisting in the preparation of a chromatin context that further attracts DNMT3A-DNMT3L binding and installs longer-term DNA methylation marks at newly-integrated retroviruses.
    Journal of Virology 07/2014; · 4.65 Impact Factor
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    ABSTRACT: Retroviruses have evolved complex transcriptional enhancers and promoters that allow for their replication in a wide range of tissue and cell types. Embryonic stem (ES) cells, however, characteristically suppress transcription of proviruses formed after infection by exogenous retroviruses, and also of most members of the vast array of endogenous retroviruses in the genome. These cells have unusual profiles of transcribed genes and are poised to make rapid changes in those profiles upon induction of differentiation. Many of the transcription factors in ES cells control both host and retroviral genes coordinately, such that retroviral expression patterns can serve as markers of ES pluripotency. This overlap is not coincidental: retroviral-derived regulatory sequences are often used to control cellular genes important for pluripotency. These sequences specify the temporal control and perhaps "noisy" control of cellular genes that direct proper cell gene expression in primitive cells and their differentiating progeny. The evidence suggests that the viral elements have been domesticated for host needs, reflecting the wide-ranging exploitation of any and all available DNA sequences in assembling regulatory networks. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
    Molecular and Cellular Biology 12/2014; 35(5). · 5.04 Impact Factor
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    ABSTRACT: Retroviral DNAs are profoundly silenced at the transcriptional level in embryonic cell types. The transcriptional profile of pluripotent stem cells has been demonstrated to be extremely heterogeneous from cell to cell, and how the silencing of retroviral DNAs is achieved is not yet well characterized. In the current study, we investigated the transcriptional silencing dynamics in stem cells by independently monitoring the expression of two Moloney murine leukemia virus (MMLV) retroviral vectors newly introduced into embryonic carcinoma (EC) cells. Although MMLV is efficiently silenced by epigenetic mechanisms in most such cells, a small number of the doubly-transduced EC cells transiently show double-positive proviral expression. These cells were sorted and their expression patterns were studied over time as silencing is established. Our data suggest that retroviral silencing occurs stochastically, in an individual locus-specific fashion, and often without synchronous silencing of both viruses in the same cells. Surprisingly, the chromatin modifications that mark the silenced proviruses are unchanged even in cells that temporarily escape silencing. This local silencing effect is a feature of stem cell epigenomic regulation that has not previously been revealed.
    Retrovirology 04/2014; 11(1):31. · 4.77 Impact Factor

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