Retrovirus vector silencing is de novo methylase independent and marked by a repressive histone code

Abstract

Retrovirus vectors are de novo methylated and transcriptionally silent in mammalian stem cells. Here, we identify epigenetic modifications that mark retrovirus-silenced transgenes. We show that murine stem cell virus (MSCV) and human immunodeficiency virus type 1 (HIV-1) vectors dominantly silence a linked locus control region (LCR) beta-globin reporter gene in transgenic mice. MSCV silencing blocks LCR hypersensitive site formation, and silent transgene chromatin is marked differentially by a histone code composed of abundant linker histone H1, deacetylated H3 and acetylated H4. Retrovirus-transduced embryonic stem (ES) cells are silenced predominantly 3 days post-infection, with a small subset expressing enhanced green fluorescent protein to low levels, and silencing is not relieved in de novo methylase-null [dnmt3a-/-;dnmt3b-/-] ES cells. MSCV and HIV-1 sequences also repress reporter transgene expression in Drosophila, demonstrating establishment of silencing in the absence of de novo and maintenance methylases. These findings provide mechanistic insight into a conserved gene silencing mechanism that is de novo methylase independent and that epigenetically marks retrovirus chromatin with a repressive histone code.

A retroviral vector suitable for ultrasound image-guided gene delivery to mouse brain

“…It has also been suggested that retroviral silencing may act through methylation-independent mechanisms, and methylation is only a secondary step in this pathway. [16][17][18][19] Consistent with this, retroviral silencing was reported in mouse embryonic stem cells that are de novo methyltransferase (dnmt3a and dnmt3b) null. 16,20 Up until now, when a UIGD technique is used, the only way to confirm whether embryos or adult mice have received the transgene has been to analyze brains taken from killed animals.…”

“…[16][17][18][19] Consistent with this, retroviral silencing was reported in mouse embryonic stem cells that are de novo methyltransferase (dnmt3a and dnmt3b) null. 16,20 Up until now, when a UIGD technique is used, the only way to confirm whether embryos or adult mice have received the transgene has been to analyze brains taken from killed animals. In order to construct adult mouse models using the UIGD technique, therefore, gene-transferred mice should be discriminated against other littermates without killing.…”

Sea urchin insulator protects lentiviral vector from silencing by maintaining active chromatin structure

“…2,5,6 Especially, acetylation status of lysine residues within histone tails are generally accepted as markers of chromatin activation. 27 For this purpose, we performed ChIP assay to detect histone acetylation levels of proviral ArsI and CMV promoter ( Figure 7).…”

“…The mechanism of transgene silencing remains to be elucidated; however, accumulating evidence suggest that epigenetic alterations of the transgene, such as DNA methylation and/or histone modifications, are likely components. 3,5,6 Engineering retroviral vectors to avoid epigenetic alterations is needed for successful application of gene therapy. Retroviral vectors currently in experimental and/or clinical practice are categorized into two groups: oncoretrovirus-based (eg Moloney-murine leukemia virus, MoMLV) and lentivirus-based (eg human immunodeficiency virus type-1, HIV-1) vectors.…”

Efficient transduction and long-term retroviral expression of the melanoma-associated tumor antigen tyrosinase in CD34+ cord blood-derived dendritic cells

“…28,29 Another report described a methylase-independent mechanism of proviral silencing through MoMuLV and immunodeficiency virus type 1 (HIV-1) cis-acting sequences. 30 Facing this problem we decided to transduce the CD34 + progenitor cells with titers ranging from 100 to 150 MOI, which resulted in a high tyrosinase mRNA and concomitant EGFP expression in the first 3 days after transduction. Nevertheless, we revealed a strong silencing of transgenes in the cells during the expansion phase of the progenitor cells.…”