Delayed de novo methylation in teratocarcinoma suggests additional tissue-specific mechanisms for controlling gene expression

Gautsch, James W.; Wilson, Michael C.

doi:10.1038/301032a0

Cited by 185 publications

(139 citation statements)

References 34 publications

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“…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.…”

Section: Discussionsupporting

confidence: 66%

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

et al. 2011

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Gene transfer to the early-stage embryonic brain using the ultrasound image-guided gene delivery (UIGD) technique has proven to be valuable for investigating brain development. Thus far, this technology has been restricted to the study of embryonic neurogenesis. When this technique is designed to be employed for the study in adult animals, a long-term stable gene expression will be required. We attempted to develop a retroviral vector suitable for expressing exogenous genes in the brains of postnatal and adult mice in the context of the UIGD technique. Retroviral vectors containing four different long terminal repeats (LTRs) (each from Moloney murine leukemia virus (MoMLV), murine stem cell virus (MSCV), myeloproliferative sarcoma virus (MPSV) and spleen focus-forming virus (SFFV)) were compared using the well-known CE vector having the EF1a internal promoter as a control. The MS vector containing MSCV LTR produced a higher viral titer and a higher level of gene expression than other vectors including CE. The MS vector drove the gene expression in cultured neural stem cells for 3 weeks. Furthermore, the MS vector could efficiently deliver the gene to the mouse central nervous system, as transgene expression was found in various regions of the brains and spinal cords as well as in all major neural cell types. The data from an in vivo luciferase imaging analysis showed that the gene expression from the MS vector was sustainable for almost 3 months. Our data suggested that the MS vector would be suitable to construct mice containing the transgene expressed in the brain or spinal cord in a quick and cost-effective manner.

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Section: Discussionsupporting

confidence: 66%

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

et al. 2011

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“…The retrotransposon Etn has also been shown to have inserted into the p53 gene in a mouse osteosarcoma (Mitreiter et al, 1994). Retroviruses are not transcribed in EC cells (Gautsch and Wilson, 1983) and are known to inactivate host genes into which they insert (Hartung et al, 1986). Thus it is not surprising that the MLV provirus in the p53 gene of P19 cells renders it transcriptionally silenced.…”

Section: Response To Radiationmentioning

confidence: 99%

Absence of p53-dependent cell cycle regulation in pluripotent mouse cell lines

et al. 1998

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We examined the expression of p53 in three lines of pluripotent embryonal carcinoma (EC) and ES cells. p53 mRNA and protein levels were constitutively high in two lines but absent from one. In the P19 line of EC cells neither p53 protein nor mRNA was detected. The ®rst intron of the p53 gene in these cells had been invaded by a murine leukemia virus and there was extensive hypermethylation of the p53 gene accompanying its inactivation. In all three cell lines, irradiation resulted in arrest of the cells in the G2 but not in the G1 phase of the cell cycle despite the induction of p21 cip1 in the cell lines expressing p53. Thus, the chromosomal stability of EC and ES cells appears to be not dependent on the p53 protein and we interpret our results to suggest that these cells may require the deletion of p53 dependent cell cycle regulation in order to become immortalized.

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“…Indeed, treatment of EC cells with the DNA methylation inhibitor 5-azacytidine leads to transcriptional reactivation of MLV (21). However, the kinetics of de novo methylation (20,23) and the persistence of proviral silencing in murine ES cells (mESCs) deficient in the de novo DNA methyltransferases Dnmt3a and Dnmt3b (7) indicate that DNA methylation-independent silencing pathways must exist as well.Intriguingly, genome-wide studies have shown that endogenous retroviral elements (ERVs) are marked by the repressive histone marks H3K9me2 and/or H3K9me3 in mESCs (24)(25)(26). Recently, we demonstrated that although ERVs exhibit reduced H3K9me2 (but not H3K9me3) and DNA methylation in mESCs lacking the "euchromatic" H3K9 KMTase G9a (27), these parasitic elements remain transcriptionally inert.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Lysine methyltransferase G9a is required for de novo DNA methylation and the establishment, but not the maintenance, of proviral silencing

Leung

Dong²,

Maksakova³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

112

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Methylation on lysine 9 of histone H3 (H3K9me) and DNA methylation play important roles in the transcriptional silencing of specific genes and repetitive elements. Both marks are detected on class I and II endogenous retroviruses (ERVs) in murine embryonic stem cells (mESCs). Recently, we reported that the H3K9-specific lysine methyltransferase (KMTase) Eset/Setdb1/KMT1E is required for H3K9me3 and the maintenance of silencing of ERVs in mESCs. In contrast, G9a/Ehmt2/KMT1C is dispensable, despite the fact that this KMTase is required for H3K9 dimethylation (H3K9me2) and efficient DNA methylation of these retroelements. Transcription of the exogenous retrovirus (XRV) Moloney murine leukemia virus is rapidly extinguished after integration in mESCs, concomitant with de novo DNA methylation. However, the role that H3K9 KMTases play in this process has not been addressed. Here, we demonstrate that G9a, but not Suv39h1 or Suv39h2, is required for silencing of newly integrated Moloney murine leukemia virus-based vectors in mESCs. The silencing defect in G9a −/− cells is accompanied by a reduction of H3K9me2 at the proviral LTR, indicating that XRVs are direct targets of G9a. Furthermore, de novo DNA methylation of newly integrated proviruses is impaired in the G9a −/− line, phenocopying proviral DNA methylation and silencing defects observed in Dnmt3a-deficient mESCs. Once established, however, maintenance of silencing of XRVs, like ERVs, is dependent exclusively on the KMTase Eset. Taken together, these observations reveal that in mESCs, the H3K9 KMTase G9a is required for the establishment, but not for the maintenance, of silencing of newly integrated proviruses.epigenetics | covalent histone modification | long terminal repeat R etroviruses have colonized all classes of vertebrates and are responsible for a range of pathologies in mammals, including cancer in distantly related species and acquired immunodeficiency syndrome in humans. Although productive retroviral infection by a subset of retroviruses is cytopathic, proviral elements of the acuteand slow-transforming classes induce tumorigenesis by expressing viral oncogenes and perturbing the expression of cellular genes, respectively. Given the potential deleterious effects of retroviral infection, a number of cell autonomous pathways that act at the transcriptional or post-transcriptional stages of the retroviral replicative cycle have evolved to inhibit retroviral expression (1, 2).Retroviral vectors based on the exogenous retrovirus (XRV) Moloney murine leukemia virus (MLV), such as the MFG vector (3), have been used extensively in the laboratory and in the clinic as vehicles for gene delivery. In both settings, transcriptional silencing has proven to be a major impediment to stable/long-term proviral expression (1, 4), due in part to the presence of negative regulatory elements in the proviral LTR that are bound by transcriptional repressors (5, 6) and the binding of chromatin proteins, including histone H1 (7, 8) and macroH2A1 (9). MLV is repressed with parti...

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Delayed de novo methylation in teratocarcinoma suggests additional tissue-specific mechanisms for controlling gene expression

Cited by 185 publications

References 34 publications

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

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

Absence of p53-dependent cell cycle regulation in pluripotent mouse cell lines

Lysine methyltransferase G9a is required for de novo DNA methylation and the establishment, but not the maintenance, of proviral silencing

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