RESUMEN
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.
Asunto(s)
Silenciador del Gen , Vectores Genéticos , Retroviridae/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Evolución Biológica , Cromatina/genética , Metilasas de Modificación del ADN/metabolismo , Cartilla de ADN/genética , Drosophila/genética , Genes Reporteros , Globinas/genética , VIH-1/genética , Histonas/genética , Humanos , Lentivirus/genética , Ratones , Ratones TransgénicosRESUMEN
Retroviral vectors are transcriptionally silenced in hematopoietic stem cells, and this phenomenon must be overcome for effective gene therapy of blood diseases. The murine stem cell virus (MSCV) vector completely silences beta-globin reporter genes regulated by locus control region (LCR) elements 5'HS2 to 5'HS4 in seven of eight transgenic mice. Here, we show that no single known MSCV silencer element is sufficient for complete LCR beta-globin transgene silencing. However, partial silencing of high-copy transgenes is conveyed by the MSCV direct repeat and promoter elements. The CpG methylation pattern of silenced and expressed MSCV promoter transgenes is virtually identical, demonstrating that silencing does not absolutely correlate with methylation status. Combined mutations in all four MSCV silencer elements leads to expression of beta-globin in 6 of 10 transgenic mice. The same mutations incorporated into the HSC1 retrovirus vector direct neo gene expression in 71% of transduced F9 embryonic carcinoma cells. These studies demonstrate that combined mutation of four retroviral silencer elements relieves complete silencing in most transgenic mice and transduced F9 cells and suggests that novel silencer elements remain. Enhanced expression of the HSC1 vector in primitive stem cells is well suited for blood gene therapy applications.
