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

Abstract

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...

Epigenetic dysregulation by nickel through repressive chromatin domain disruption

“…However, the alternate possibility of gene silencing resulting in spreading of H3K9me2 in a subset of the spreading domains cannot be ruled out. Nevertheless, H3K9me2 spreading into active regions remains a significant event because this could play a role in stably silencing the genes, given that H3K9me2 could be a precursor to DNA methylation and long-term gene silencing (40,41). Although spreading of H3K9me2 into promoters was associated with reduction in gene expression, we detected a subset of genes whose expression was not affected by H3K9me2 spreading.…”

S -adenosyl methionine is necessary for inhibition of the methyltransferase G9a by the lysine 9 to methionine mutation on histone H3

“…Unmodified H3 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] peptide (23 μM) was incubated with 25 nM G9a-SET in the presence of 50 μM SAM (radioactive: nonradioactive molar ratio = 0.016) in a buffer containing 50 mM Hepes (pH 7.9), 0.5 mM DTT, 0.25 mM PMSF, and 2 mM MgCl 2 . Where applicable, different H3 1-20 K9X peptides (Tufts University Peptide Synthesis Core) were present in the reaction.…”

“…G9a is an ∼120-kDa protein and contains an SET domain at its C terminus that catalyzes monoand dimethylation of histones at lysine 9 (11), and we previously showed that H3K9M peptides are capable of inhibiting activity of SUV39H1 and G9a, two H3K9-directed methyltransferases, in vitro (7). Recent studies identified a role for G9a in heterochromatin maintenance and transcriptional repression, such as the silencing of repeat elements, including long interspersed nuclear elements and endogenous retroviruses (12)(13)(14). Up-regulation of both G9a and G9a like protein (GLP) has implications in a variety of human cancers, including solid tumors as well as acute myeloid leukemia (15)(16)(17).…”

A Histone Methyltransferase ESET Is Critical for T Cell Development

“…Histone methylation is also crucial in thymocyte development, as reported in Trim28-deficient mice, which showed defective trimethylation of lysine 4 in histone 3 (5,6). In contrast to transcriptional activating epigenetic marks such as histone 3 lysine 4 methylation (7), the importance of repressive epigenetic marks such as histone 3 lysine 9 (H3K9) methylation (8) on T cell development is still obscure because deletion of Suv39h1, the first identified H3K9 histone methyltransferase (HMT), did not result in an apparent defect in thymocyte differentiation (9). Similarly, G9a, another methyltransferase regulating mono-and dimethylation of H3K9 (10,11), was also shown to be dispensable because G9a 2/2 mice had normal T cell differentiation (12).…”