The Core Element of a CpG Island Protects Avian Sarcoma and Leukosis Virus-Derived Vectors from Transcriptional Silencing

Šenigl, Filip; Plachý, Jiřı́; Hejnar, Jiřı́

doi:10.1128/jvi.00419-08

Cited by 24 publications

(36 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…DNA methylation is a well-recognized silencing mechanism for retroviruses and other retrotransposons (22,61), frequently targeting the LTR promoter. As long-term ASV silencing in mammalian cells was known to be mediated by DNA methylation (22,47,62), an unexpected finding from our initial studies was that treatment with HDAC inhibitors was sufficient to reactivate epigenetically silent ASV proviruses in freshly infected or long-term-silent human cells (37). Furthermore, Daxx-HDAC complexes were found to have a prominent role in rapid silencing that we observed (17).…”

Section: Discussionmentioning

confidence: 75%

“…In these ASV-GFP viruses, the reporter genes are driven by alternative internal promoters, as well as the native viral long terminal repeat (LTR) promoter (18), with the GFP gene providing a readout for repressive epigenetic effects. The ASV LTR promoter region has been shown previously to be methylated in mammalian cells and functionally linked to ASV epigenetic silencing (22,47). As we found that viruses using diverse promoters to drive the GFP gene are silenced similarly in human cells (37), the GFP readout appears to serve as a general indicator of the epigenetic state of the provirus.…”

mentioning

confidence: 71%

See 1 more Smart Citation

Retroviral DNA Methylation and Epigenetic Repression Are Mediated by the Antiviral Host Protein Daxx

Shalginskikh

Poleshko

Skalka

et al. 2013

J Virol

View full text Add to dashboard Cite

Integrated retroviral DNA is subject to epigenetic transcriptional silencing at different frequencies. This process is mediated by repressive DNA methylation and histone modifications on viral chromatin. However, the detailed mechanisms by which retroviral silencing is initiated and maintained are not well understood. Using a model system in which avian sarcoma virus (ASV) DNA is epigenetically repressed in mammalian cells, we previously found that a cellular scaffolding protein, Daxx, acts as an antiretroviral factor that promotes epigenetic repression through recruitment of histone deacetylases (HDACs). Here we show that human Daxx protein levels are increased in response to retroviral infection and that Daxx acts at the time of infection to initiate epigenetic repression. Consistent with a rapid and active antiviral epigenetic response, we found that repressive histone marks and long terminal repeat (LTR) DNA methylation could be detected within 12 h to 3 days postinfection, respectively. Daxx was also found to be required for long-term ASV silencing maintenance and full viral DNA methylation, and it was physically associated with both viral DNA and DNA methyltransferases (DNMTs). These findings support a model in which incoming retroviral protein-DNA complexes are detected by Daxx, and the integrated provirus is rapidly chromatinized and repressed by DNA methylation and histone modification as part of an antiviral response. These results uncover a possible direct and active antiviral mechanism by which DNMTs can be recruited to retroviral DNA. Retroviruses are important agents of disease and serve as valuable vectors for gene delivery, and their study has provided seminal insights into cellular functions. A defining feature of retroviral replication is the integration of a DNA copy of the retroviral RNA genome into host chromatin, a process that establishes the DNA provirus. Integration provides a permanent association of viral DNA with the host cell and all of its progeny, and it also allows the provirus to efficiently mobilize the cellular transcriptional machinery for synthesis of viral mRNAs and viral RNA genomes.DNA integration is an essential step in retroviral replication, and it is catalyzed by the virus-encoded integrase (IN) protein. However, establishment of the provirus does not guarantee its expression; transcriptional repression by epigenetic mechanisms (epigenetic silencing) is often observed in both natural and interspecies retroviral infections. Examples include the silencing of retroviruses in embryonic stem cells (1, 2), the progressive silencing of expression of genes transduced by retroviral vectors during long-term cell propagation (3), and HIV latency (4). Epigenetic mechanisms also repress the expression of endogenous retroviruses (5-9).Retroviral epigenetic silencing is mediated by the enzymatic placement, and subsequent reading, of DNA methylation marks (addition of a methyl group to position 5 of the cytosine pyrimidine [5MeCpG]) and repressive nucleosomal histone modifications. Th...

show abstract

Section: Discussionmentioning

confidence: 75%

mentioning

confidence: 71%

Retroviral DNA Methylation and Epigenetic Repression Are Mediated by the Antiviral Host Protein Daxx

Shalginskikh

Poleshko

Skalka

et al. 2013

J Virol

View full text Add to dashboard Cite

show abstract

“…Epigenetic silencing is a common mechanism of host protection against retroviral infection; however, here we show that human cells are very inefficient in PERV silencing by DNA methylation. For example, the LTRs of Rous sarcoma virus or lentiviral vectors become silenced by methylation within weeks (69,70), in contrast to PERV LTRs, which are not substantially methylated even after 2 months (Fig. 6).…”

Section: Discussionmentioning

confidence: 99%

Role of DNA Methylation in Expression and Transmission of Porcine Endogenous Retroviruses

Matoušková

Veselý

Daniel

et al. 2013

J Virol

View full text Add to dashboard Cite

c Porcine endogenous retroviruses (PERV) represent a major safety concern in pig-to-human xenotransplantation. To date, no PERV infection of a xenograft recipient has been recorded; however, PERVs are transmissible to human cells in vitro. Some recombinants of the A and C PERV subgroups are particularly efficient in infection and replication in human cells. Transcription of PERVs has been described in most pig cells, but their sequence and insertion polymorphism in the pig genome impede identification of transcriptionally active or silenced proviral copies. Furthermore, little is known about the epigenetic regulation of PERV transcription. Here, we report on the transcriptional suppression of PERV by DNA methylation in vitro and describe heavy methylation in the majority of PERV 5= long terminal repeats (LTR) in porcine tissues. In contrast, we have detected sparsely methylated or nonmethylated proviruses in the porcine PK15 cells, which express human cell-tropic PERVs. We also demonstrate the resistance of PERV DNA methylation to inhibitors of methylation and deacetylation. Finally, we show that the high permissiveness of various human cell lines to PERV infection coincides with the inability to efficiently silence the PERV proviruses by 5=LTR methylation. In conclusion, we suggest that DNA methylation is involved in PERV regulation, and that only a minor fraction of proviruses are responsible for the PERV RNA expression and porcine cell infectivity.

show abstract

“…CpG islands isolated from the mouse and hamster adenine phosphoribosyltransferase (APRT) genes are alternative DNA elements which are shown to be effective at preventing DNA methylation . Deletion analysis of the hamster APRT island identified a 120 base‐pair core CpG island element (IE), making IE easier to use than the other DNA elements which can span several thousand base pairs. Inclusion of IE into the retroviral long terminal repeat (LTR) promoter led to efficient protection of the integrated viral vectors from silencing in NIL‐2, HEK293, and QT6 cells .…”

Section: Introductionmentioning

confidence: 99%

Insertion of core CpG island element into human CMV promoter for enhancing recombinant protein expression stability in CHO cells

Mariati

Yeo

Koh

et al. 2014

Biotechnology Progress

View full text Add to dashboard Cite

The human cytomegalovirus promoter (hCMV) is susceptible to gene silencing in CHO cells, most likely due to epigenetic events, such as DNA methylation and histone modifications. The core CpG island element (IE) from the hamster adenine phosphoribosyltransferase gene has been shown to prevent DNA methylation. A set of modified hCMV promoters was developed by inserting one or two copies of IE in either forward or reverse orientations either upstream of the hCMV enhancer, between the enhancer and core promoter (CP), or downstream of the CP. The modified hCMV with one copy of IE inserted between the enhancer and core promoter in reverse orientation (MR1) was most effective at enhancing expression stability without compromising expression level when compared with the wild-type (WT) hCMV. A third of 18 EGFP expressing clones generated using MR1 retained 70% of their starting expression level after 8 weeks of culture in the absence of selection pressure, while none of 18 WT hCMV generated clones had expression above 50%. MR1 also improved antibody expression stability of methotrexate (MTX) amplified CHO cell lines. Stably transfected pools generated using MR1 maintained 62% of their original monoclonal antibody titer after 8 weeks of culture in the absence of MTX, compared to only 37% for WT hCMV pools. Low levels of CpG methylation within both WT hCMV and MR1 were observed in all the analyzed cell lines and the methylation levels did not correlate to the expression stability, suggesting IE enhances expression stability by other mechanisms other than preventing methylation.

show abstract

The Core Element of a CpG Island Protects Avian Sarcoma and Leukosis Virus-Derived Vectors from Transcriptional Silencing

Cited by 24 publications

References 53 publications

Retroviral DNA Methylation and Epigenetic Repression Are Mediated by the Antiviral Host Protein Daxx

Retroviral DNA Methylation and Epigenetic Repression Are Mediated by the Antiviral Host Protein Daxx

Role of DNA Methylation in Expression and Transmission of Porcine Endogenous Retroviruses

Insertion of core CpG island element into human CMV promoter for enhancing recombinant protein expression stability in CHO cells

Contact Info

Product

Resources

About