Methyltransferases Dnmt3b Research Articles

TAF12 Recruits Gadd45a and the Nucleotide Excision Repair Complex to the Promoter of rRNA Genes Leading to Active DNA Demethylation

Many studies have detailed the repressive effects of DNA methylation on gene expression. However, the mechanisms that promote active demethylation are just beginning to emerge. Here, we show that methylation of the rDNA promoter is a dynamic and reversible process. Demethylation of rDNA is initiated by recruitment of Gadd45a (growth arrest and DNA damage inducible protein 45 alpha) to the rDNA promoter by TAF12, a TBP-associated factor that is contained in Pol I- and Pol II-specific TBP-TAF complexes. Once targeted to rDNA, Gadd45a triggers demethylation of promoter-proximal DNA by recruiting the nucleotide excision repair (NER) machinery to remove methylated cytosines. Knockdown of Gadd45a, XPA, XPG, XPF, or TAF12 or treatment with drugs that inhibit NER causes hypermethylation of rDNA, establishes heterochromatic histone marks, and impairs transcription. The results reveal a mechanism that recruits the DNA repair machinery to the promoter of active genes, keeping them in a hypomethylated state.

Anti-AML Activity of Combined Epigenetic Therapy with Novel DNMT1 Inhibitors SGI-110 or SGI-1036 and Histone Deacetylase Inhibitor Panobinostat.

Lysine specific histone methylation and deacetylation and DNA hypermethylation are involved in the epigenetic silencing of tumor suppressor genes (TSG), e.g., p15 and p16. DNA methyltransferase (DNMT) inhibitors 5-azacytidine and 5-aza-2‘-deoxycytidine demethylate the CpG dinucleotide islands in or near gene promoters, leading to derepression of TSGs in AML. SGI-110 (S110) (Cancer Res. 2007; 67:6400) and SGI-1036 (SuperGen, Inc.) are novel, DNMT inhibitors, which also deplete DNMT1 levels. SGI-110 is a dinucleotide containing 5-aza-2‘-deoxycytidine and SGI-1036 is a non-nucleoside heterocycle. The multi-protein complex PRC (polycomb repressive complex) 2 that contains the three core proteins EZH2, SUZ12 and EED, has intrinsic histone methyltransferase (HMTase) activity. This is mediated by the SET domain of EZH2, which induces trimethylation of histone H3 on lysine (K)-27. We recently reported that treatment with the pan-HDAC inhibitor panobinostat (LBH589, Novartis Pharmaceutical Corp) acetylates and inhibits the ATP binding and chaperone function of hsp90, as well as depletes the levels of EZH2, Suz12 and EED in cultured and primary AML cells (Mol Cancer Ther. 2006; 5:3096). Within the PRC2 complex, EZH2 was shown to interact with and modulate the DNA methyltransferases DNMT1, DNMT3a and DNMT3b, which affects their binding to the EZH2-targeted gene promoters. In the present studies we determined the effects of SGI-110 or SGI-1036 and LBH589 on the PRC2 proteins EZH2 and SUZ12, and DNMT1, in the cultured (HL-60, OCI-AML3 and K562) and primary AML cells. Treatment with SGI-110 (0.5 to 2.0 μM) or SGI-1036 (0.5 and 1.0 μM) for 24 hours depleted protein levels of DNMT1 and EZH2 in the cultured and primary AML cells. SGI-110 and SGI-1036 promoted proteasomal degradation of DNMT1 and EZH2 since co-treatment with bortezomib significantly restored DNMT1 and EZH2 levels in the AML cells. Following treatment with SGI-110 or SGI-1036, bisulfite modification and methylation specific PCR demonstrated increase in unmethylated promoter DNA of p15 and JunB. This was associated with induction of the mRNA and protein levels of p15 and JunB, as well as caused inhibition of cell cycle progression (% of cells increased in G1 and increased in S phase) and colony growth in the soft agar. Treatment with 1.0 μM of SGI-110 or SGI-1036 also induced PARP cleavage activity of caspases and induced morphologic evidence of apoptosis in the AML cells. Co-treatment with 10 to 50 nM panobinostat enhanced SGI-110 or SGI-1036 mediated depletion of DNMT1 and EZH2, with more de-repression of the p15 and JunB and significant increase in apoptosis of AML cells. Collectively, these findings indicate that, SGI-110 and SGI-1036 deplete DNMT1 and EZH2 levels, as well as exert potent anti-AML activity. Additionally, combined epigenetic therapy consisting of SGI-110 or SGI-1036 in combination with panobinostat may represent a promising novel treatment of AML.

Antitumor Activity of Cda-2, An Extract from Unine, in a High-Risk Myelodysplastic Syndrome Cells in Vitro

DNA methyltransferase inhibitors (DNMTI), including 5-azacytidine and 5-aza-2′- deoxycytidine, are a new class of epigenetic drug, which exhibit higher response rates in myelodysplastic syndrome (MDS) patients. Cell differentiation agent (CDA-2) is a kind of urine extracts, which contains several DNMTIs. A phase IV clinical trials for MDS showed total response rate is 69.22%. In the present study, we investigated the mechanism of CDA-2 on MDS using high-risk MDS cell line namely MuTz-1. MTT assay results showed that CDA-2 significantly inhibit the cell growth at a dose and time-dependent manner. Flow cytometer anlyasis showed that this growth inhibition was remarkblely associated with cycle arrest in G1-phase, but not associated with apoptosis. In addition, CDA-2 increased the expression of CD11b/CD14, a pair markers representing cell differentiation. we found the spectrum of hypermethylated tumor suppressor genes (TIMP3, CDKN2B, CHFR, CD44, RASSF1, TP73, IGSF4, CDH13 and DAPK) in MuTz-1 cells by Methylation-Specific Multiplex ligation-dependent Probe amplification (MS-MLPA), but the hypermethylation of these genes were remarkable decreased, as well as the expressions of DNA methyltransferase genes DNMT1 and DNMT3B at mRNA and protein level were downregulated in the treatment for 3 days with CDA-2. Also, we used CDA-2 for treatment of three MDS patients, whose several tumor suppressor genes are hypermethylated. After tour weeks of treatment, all the hypermethylation genes were undetected, part of this result was verified by methylation specific PCR (MSP) and bisulphite sequencing. In conclusion, our results demonstrated that CDA-2 may be an effective agent targeting hepermethylated tumor suppressor genes on MDS.

Neonatal Exposure to Diethylstilbestrol Alters Expression of DNA Methyltransferases and Methylation of Genomic DNA in the Mouse Uterus

Perinatal exposure to diethylstilbestrol (DES) can have numerous adverse effects on the reproductive organs later in life, such as vaginal clear-cell adenocarcinoma. Epigenetic processes including DNA methylation may be involved in the mechanisms. We subcutaneously injected DES to neonatal C57BL/6 mice. At days 5, 14, and 30, expressions of DNA methyltransferases (Dnmts) Dnmt1, Dnmt3a, and Dnmt3b, and transcription factors Sp1 and Sp3 were examined. We also performed restriction landmark genomic scanning (RLGS) to detect aberrant DNA methylation. Real-time RT-PCR revealed that expressions of Dnmt1, Dnmt3b, and Sp3 were decreased at day 5 in DES-treated mice, and that those of Dnmt1, Dnmt3a, and Sp1 were also decreased at day 14. RLGS analysis revealed that 5 genomic loci were demethylated, and 5 other loci were methylated by DES treatment. Two loci were cloned, and differential DNA methylation was quantified. Our results indicated that DES altered the expression levels of Dnmts and DNA methylation.

Epigenetic mechanisms in mammals

.DNA and histone methylation are linked and subjected to mitotic inheritance in mammals. Yet how methylation is propagated and maintained between successive cell divisions is not fully understood. A series of enzyme families that can add methylation marks to cytosine nucleobases, and lysine and arginine amino acid residues has been discovered. Apart from methyltransferases, there are also histone modification enzymes and accessory proteins, which can facilitate and/or target epigenetic marks. Several lysine and arginine demethylases have been discovered recently, and the presence of an active DNA demethylase is speculated in mammalian cells. A mammalian methyl DNA binding protein MBD2 and de novo DNA methyltransferase DNMT3A and DNMT3B are shown experimentally to possess DNA demethylase activity. Thus, complex mammalian epigenetic mechanisms appear to be dynamic yet reversible along with a well-choreographed set of events that take place during mammalian development.

Vezf1 regulates genomic DNA methylation through its effects on expression of DNA methyltransferase Dnmt3b

The zinc finger protein vascular endothelial zinc finger 1 (Vezf1) has been implicated in the development of the blood vascular and lymphatic system in mice, and has been characterized as a transcriptional activator in some systems. The chicken homolog, BGP1, has binding sites in the beta-globin locus, including the upstream insulator element. We report that in a mouse embryonic stem cell line deletion of both copies of Vezf1 results in loss of DNA methylation at widespread sites in the genome, including Line1 elements and minor satellite repeats, some imprinted genes, and several CpG islands. Loss of methylation appears to arise from a substantial decrease in the abundance of the de novo DNA methyltransferase, Dnmt3b. These results suggest that naturally occurring mutations in Vezf1/BGP1 might have widespread effects on DNA methylation patterns and therefore on epigenetic regulation of gene expression.

Maternal and zygotic Dnmt1 are necessary and sufficient for the maintenance of DNA methylation imprints during preimplantation development

Parental origin-specific DNA methylation regulates the monoallelic expression of the mammalian imprinted genes. The methylation marks or imprints are established in the parental germline and maintained throughout embryonic development. However, it is unclear how the methylation imprints are maintained through extensive demethylation in cleavage-stage preimplantation embryos. Previous reports suggested that DNA methyltransferase(s) other than Dnmt1 is involved in the maintenance of the imprints during cleavage. Here we demonstrate, by using conditional knockout mice, that the other known DNA methyltransferases Dnmt3a and Dnmt3b are dispensable for the maintenance of the methylation marks at most imprinted loci. We further demonstrate that a lack of both maternal and zygotic Dnmt1 results in complete demethylation of all imprinted loci examined in blastocysts. Consistent with these results we find that zygotic Dnmt1 is expressed in the preimplantation embryo. Thus, contrary to the previous reports, Dnmt1 alone is sufficient to maintain the methylation marks of the imprinted genes.

Regulation of DNA methylation activity through Dnmt3L promoter methylation by Dnmt3 enzymes in embryonic development

The genomic DNA is methylated by de novo methyltransferases Dnmt3a and Dnmt3b during early embryonic development. The establishment of appropriate methylation patterns depends on a fine regulation of the methyltransferase activity. The activity of both enzymes increases in the presence of Dnmt3L, a Dnmt3a/3b-like protein. However, it is unclear how the function of Dnmt3L is regulated. We found here that the expression of Dnmt3L is controlled via its promoter methylation during embryonic development. Genetic studies showed that Dnmt3a, Dnmt3b and Dnmt3L are all involved in the methylation of the Dnmt3L promoter. Disruption of both Dnmt3a and Dnmt3b genes in mouse rendered the Dnmt3L promoter devoid of methylation, causing incomplete repression of the Dnmt3L transcription in embryonic stem cells and embryos. Disruption of either Dnmt3a or Dnmt3b led to reduced methylation and increased transcription of Dnmt3L, but severe hypomethylation occurred only when Dnmt3b was deficient. Consistent with the major contribution of Dnmt3b in the Dnmt3L promoter methylation, methylation of Dnmt3L was significantly reduced in mouse models of the human ICF syndrome carrying point mutations in Dnmt3b. Interestingly, Dnmt3L also contributes to the methylation of its own promoter in embryonic development. We thus propose an auto-regulatory mechanism for the control of DNA methylation activity whereby the activity of the Dnmt3L promoter is epigenetically modulated by the methylation machinery including Dnmt3L itself. Insufficient methylation of the DNMT3L promoter during embryonic development due to deficiency in DNMT3B might be implicated in the pathogenesis of the ICF syndrome.

The Colorful History of Active DNA Demethylation

Patterns of DNA cytosine methylation are subject to mitotic inheritance in both plants and vertebrates. Plants use 5-methylcytosine glycosylases and the base excision repair pathway to remove excess cytosine methylation. In mammals, active demethylation has been proposed to operate via several very different mechanisms. Two recent reports in Nature now claim that the demethylation process is initiated by the same enzymes that establish the methylation mark, the DNA methyltransferases DNMT3A and DNMT3B (Kangaspeska et al., 2008; Métivier et al., 2008).

SWI/SNF Mediates Polycomb Eviction and Epigenetic Reprogramming of the INK4b-ARF-INK4a Locus

Stable silencing of the INK4b-ARF-INK4a tumor suppressor locus occurs in a variety of human cancers, including malignant rhabdoid tumors (MRTs). MRTs are extremely aggressive cancers caused by the loss of the hSNF5 subunit of the SWI/SNF chromatin-remodeling complex. We found previously that, in MRT cells, hSNF5 is required for p16(INK4a) induction, mitotic checkpoint activation, and cellular senescence. Here, we investigated how the balance between Polycomb group (PcG) silencing and SWI/SNF activation affects epigenetic control of the INK4b-ARF-INK4a locus in MRT cells. hSNF5 reexpression in MRT cells caused SWI/SNF recruitment and activation of p15(INK4b) and p16(INK4a), but not of p14(ARF). Gene activation by hSNF5 is strictly dependent on the SWI/SNF motor subunit BRG1. SWI/SNF mediates eviction of the PRC1 and PRC2 PcG silencers and extensive chromatin reprogramming. Concomitant with PcG complex removal, the mixed lineage leukemia 1 (MLL1) protein is recruited and active histone marks supplant repressive ones. Strikingly, loss of PcG complexes is accompanied by DNA methyltransferase DNMT3B dissociation and reduced DNA methylation. Thus, various chromatin states can be modulated by SWI/SNF action. Collectively, these findings emphasize the close interconnectivity and dynamics of diverse chromatin modifications in cancer and gene control.

Mammalian DNA Methyltransferases: A Structural Perspective

The methylation of mammalian DNA, primarily at CpG dinucleotides, has long been recognized to play a major role in controlling gene expression, among other functions. Given their importance, it is surprising how many basic questions remain to be answered about the proteins responsible for this methylation and for coordination with the parallel chromatin-marking system that operates at the level of histone modification. This article reviews recent studies on, and discusses the resulting biochemical and structural insights into, the DNA nucleotide methyltransferase (Dnmt) proteins 1, 3a, 3a2, 3b, and 3L.

Characterization of Dnmt3b:Thymine-DNA Glycosylase Interaction and Stimulation of Thymine Glycosylase-Mediated Repair by DNA Methyltransferase(s) and RNA

Methylation of cytosine residues in CpG dinucleotides plays an important role in epigenetic regulation of gene expression and chromatin structure/stability in higher eukaryotes. DNA methylation patterns are established and maintained at CpG dinucleotides by DNA methyltransferases (Dnmt1, Dnmt3a, and Dnmt3b). In mammals and many other eukaryotes, the CpG dinucleotide is underrepresented in the genome. This loss is postulated to be the result of unrepaired deamination of cytosine and 5-methylcytosine to uracil and thymine, respectively. Two thymine glycosylases are believed to reduce the impact of 5-methylcytosine deamination. G/T mismatch-specific thymine-DNA glycosylase (Tdg) and methyl-CpG binding domain protein 4 can both excise uracil or thymine at U·G and T·G mismatches to initiate base excision repair. Here, we report the characterization of interactions between Dnmt3b and both Tdg and methyl-CpG binding domain protein 4. Our results demonstrate (1) that both Tdg and Dnmt3b are colocalized to heterochromatin and (2) reduction of T·G mismatch repair efficiency upon loss of DNA methyltransferase expression, as well as a requirement for an RNA component for correct T·G mismatch repair.

Rare Missense and Synonymous Variants in UBE1 Are Associated with X-Linked Infantile Spinal Muscular Atrophy

X-linked infantile spinal muscular atrophy (XL-SMA) is an X-linked disorder presenting with the clinical features hypotonia, areflexia, and multiple congenital contractures (arthrogryposis) associated with loss of anterior horn cells and infantile death. To identify the XL-SMA disease gene, we performed large-scale mutation analysis in genes located between markers DXS8080 and DXS7132 (Xp11.3-Xq11.1). This resulted in detection of three rare novel variants in exon 15 of UBE1 that segregate with disease: two missense mutations (c.1617 G-->T, p.Met539Ile; c.1639 A-->G, p.Ser547Gly) present each in one XL-SMA family, and one synonymous C-->T substitution (c.1731 C-->T, p.Asn577Asn) identified in another three unrelated families. Absence of the missense mutations was demonstrated for 3550 and absence of the synonymous mutation was shown in 7914 control X chromosomes; therefore, these results yielded statistical significant evidence for the association of the synonymous substitution and the two missense mutations with XL-SMA (p = 2.416 x 10(-10), p = 0.001815). We also demonstrated that the synonymous C-->T substitution leads to significant reduction of UBE1 expression and alters the methylation pattern of exon 15, implying a plausible role of this DNA element in developmental UBE1 expression in humans. Our observations indicate first that XL-SMA is part of a growing list of neurodegenerative disorders associated with defects in the ubiquitin-proteasome pathway and second that synonymous C-->T transitions might have the potential to affect gene expression.

Synergistic Function of DNA Methyltransferases Dnmt3a and Dnmt3b in the Methylation of Oct4 and Nanog

DNA methylation plays an important role in gene silencing in mammals. Two de novo methyltransferases, Dnmt3a and Dnmt3b, are required for the establishment of genomic methylation patterns in development. However, little is known about their coordinate function in the silencing of genes critical for embryonic development and how their activity is regulated. Here we show that Dnmt3a and Dnmt3b are the major components of a native complex purified from embryonic stem cells. The two enzymes directly interact and mutually stimulate each other both in vitro and in vivo. The stimulatory effect is independent of the catalytic activity of the enzyme. In differentiating embryonic carcinoma or embryonic stem cells and mouse postimplantation embryos, they function synergistically to methylate the promoters of the Oct4 and Nanog genes. Inadequate methylation caused by ablating Dnmt3a and Dnmt3b is associated with dysregulated expression of Oct4 and Nanog during the differentiation of pluripotent cells and mouse embryonic development. These results suggest that Dnmt3a and Dnmt3b form a complex through direct contact in living cells and cooperate in the methylation of the promoters of Oct4 and Nanog during cell differentiation. The physical and functional interaction between Dnmt3a and Dnmt3b represents a novel regulatory mechanism to ensure the proper establishment of genomic methylation patterns for gene silencing in development.

The Krüppel-associated Box Repressor Domain Can Trigger de Novo Promoter Methylation during Mouse Early Embryogenesis

The Krüppel-associated box (KRAB) domain is a transcriptional repression module responsible for the DNA binding-dependent gene silencing activity of hundreds of vertebrate zinc finger proteins. We previously exploited KRAB-mediated repression within the context of a tet repressor-KRAB fusion protein and of lentiviral vectors to create a method of external gene control. We demonstrated that with this system transcriptional silencing was fully reversible in cell culture as well as in vivo. Here we reveal that, in sharp contrast, KRAB-mediated repression results in irreversible gene silencing through promoter DNA methylation if it acts during the first few days of mouse development.

Cell cycle‐dependent transcription of cyclin B2 is influenced by DNA methylation but is independent of methylation in the CDE and CHR elements

DNA methylation is an important mechanism involved in embryogenesis and tumor development. Changing cytosines to 5-methylcytosines in CpG dinucleotides has been found to be responsible for the inactivation of tumor suppressor genes by repressing transcription. A central cell cycle regulator whose synthesis is controlled by transcription is cyclin B. In mammalian cells, cyclin B1 and B2 proteins are well characterized and often found to be overexpressed in cancer patients. Transcription from cyclin B1 and B2 promoters during the cell cycle is dependent upon a combination of two sites named 'cell cycle-dependent element' (CDE) and 'cell cycle genes homology region' (CHR), through repression in G(0) and G(1) followed by release in G(2)/M. Here we show that the cyclin B2 promoter contains a CpG island and that 5-aza-deoxycytidine treatment leads to deregulation of cell cycle-dependent mRNA expression from this gene via a loss of repression in G(0). Furthermore, deletion of the DNA methyltransferase genes DNMT1 and DNMT3b leads to an increase in transcription of cyclin B2. Additionally, DNA methylation in vitro prevents transcriptional activation of the cyclin B2 promoter in G(2)/M. Analysis in vivo of the cyclin B2 core promoter revealed that the CDE/CHR site is partially methylated. However, quantitative in vivo analysis of the CpG-methylation level of the CDE during cell division indicates that CpG methylation is independent of the cell cycle. We conclude that DNA methylation affects cell cycle-dependent transcription of cyclin B2 but that regulation through CDE/CHR is independent of cytosine methylation.

Expression changes in EZH2, but not in BMI-1, SIRT1, DNMT1 or DNMT3B are associated with DNA methylation changes in prostate cancer

The polycomb proteins BMI-1, EZH2, and SIRT1 are characteristic components of the PRC1, PRC2, and PRC4 repressor complexes, respectively, that modify chromatin. Moreover, EZH2 may influence DNA methylation by direct interaction with DNA methyltransferases. EZH2 expression increases during prostate cancer progression, whereas BMI-1 and SIRT1 are not well investigated. Like EZH2 expression, DNA methylation alterations escalate in higher stage prostate cancers, raising the question whether these epigenetic changes are related. Expression of EZH2, BMI-1, SIRT1, and the DNA methyltransferases DNMT1 and DNMT3B measured by qRT-PCR in 47 primary prostate cancers was compared to APC, ASC, GSTP1, RARB2, and RASSF1A hypermethylation and LINE-1 hypomethylation. SIRT1 and DNMT3B were overexpressed in cancerous over benign tissues, whereas BMI-1 was rather downregulated and DNMT1 significantly diminished. Nevertheless, cancers with higher DNMT1 and BMI-1 expression had worse clinical characteristics, as did those with elevated EZH2. In particular, above median DNMT1 expression predicted a worse prognosis. EZH2 and SIRT1 overexpression were well correlated with increased MKI67. Immunohistochemistry confirmed limited EZH2 and heterogeneous DNMT3B overexpression and explained the decrease in BMI-1 by pronounced heterogeneity among tumor cells. EZH2 overexpression, specifically among all factors investigated, was associated with more frequent hypermethylation, in particular of GSTP1 and RARB2, and also with LINE-1 hypomethylation. Our data reveal complex changes in the composition of polycomb repressor complexes in prostate cancer. Heterogeneously expressed BMI-1 and slightly increased EZH2 may characterize less malignant cancers, whereas more aggressive cases express both at higher levels. SIRT1 appears to be generally increased in prostate cancers. Intriguingly, our data suggest a direct influence of increased EZH2 on altered DNA methylation patterns in prostate cancer.

DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA

Mammals use DNA methylation for the heritable silencing of retrotransposons and imprinted genes and for the inactivation of the X chromosome in females. The establishment of patterns of DNA methylation during gametogenesis depends in part on DNMT3L, an enzymatically inactive regulatory factor that is related in sequence to the DNA methyltransferases DNMT3A and DNMT3B. The main proteins that interact in vivo with the product of an epitope-tagged allele of the endogenous Dnmt3L gene were identified by mass spectrometry as DNMT3A2, DNMT3B and the four core histones. Peptide interaction assays showed that DNMT3L specifically interacts with the extreme amino terminus of histone H3; this interaction was strongly inhibited by methylation at lysine 4 of histone H3 but was insensitive to modifications at other positions. Crystallographic studies of human DNMT3L showed that the protein has a carboxy-terminal methyltransferase-like domain and an N-terminal cysteine-rich domain. Cocrystallization of DNMT3L with the tail of histone H3 revealed that the tail bound to the cysteine-rich domain of DNMT3L, and substitution of key residues in the binding site eliminated the H3 tail-DNMT3L interaction. These data indicate that DNMT3L recognizes histone H3 tails that are unmethylated at lysine 4 and induces de novo DNA methylation by recruitment or activation of DNMT3A2.

Role of the Dnmt3 family in de novo methylation of imprinted and repetitive sequences during male germ cell development in the mouse

DNA methylation is an important epigenetic modification regulating various biological phenomena, including genomic imprinting and transposon silencing. It is known that methylation of the differentially methylated regions (DMRs) associated with paternally imprinted genes and of some repetitive elements occurs during male germ cell development in the mouse. We have performed a detailed methylation analysis of the paternally methylated DMRs (H19, Dlk1/Gtl2 and Rasgrf1), interspersed repeats [SineB1, intracisternal A particle (IAP) and Line1] and satellite repeats (major and minor) to determine the timing of this de novo methylation in male germ cells. Furthermore, we have examined the roles of the de novo methyltransferases (Dnmt3a and Dnmt3b) and related protein (Dnmt3L) in this process. We found that methylation of all DMRs and repeats occurred progressively in fetal prospermatogonia and was completed by the newborn stage. Analysis of newborn prospermatogonia from germline-specific Dnmt3a and Dnmt3b knockout mice revealed that Dnmt3a mainly methylates the H19 and Dlk1/Gtl2 DMRs and a short interspersed repeat SineB1. Both Dnmt3a and Dnmt3b were involved in the methylation of Rasgrf1 DMR and long interspersed repeats IAP and Line1. Only Dnmt3b was required for the methylation of the satellite repeats. These results indicate both common and differential target specificities of Dnmt3a and Dnmt3b in vivo. Finally, all these sequences showed moderate to severe hypomethylation in Dnmt3L-deficient prospermatogonia, indicating the critical function and broad specificity of this factor in de novo methylation.

The A, B, Gs of silencing: Figure 1.

Transcriptional silencing of mammalian genes is mediated by at least two modes of methylation: (1) cytosine methylation of DNA, and (2) methylation of the histones tails—both of which play an important role in transcriptional silencing from chromosomal DNA. Although there is some evidence that these pathways are interdependent, experimentally, they have largely been treated as two distinct processes. In this issue of Genes & Development, Smallwood et al. (2007) have taken a pioneering step toward defining the molecular mechanism by which these two types of methylations can cooperate to silence a euchromatic gene, and demonstrate that the DNA methyltransferase 1 (DNMT1) cytosine methyltransferase physically and functionally interacts with three Heterochromatin Protein 1 (HP1) family members ( , , and ) to implement gene silencing. DNA methylation is required for normal development in mammals. It is brought about by the de novo methyltransferases DNMT3a and DNMT3b and is propagated by the maintenance methyltransferase DNMT1 (for review, see Goll and Bestor 2005). DNA methylation is associated with silencing repetitive elements in the genome, X-chromosome inactivation in female mammals, and silencing of individual genes during development (for review, see Li and Bird 2007). Furthermore, DNA methylation is misregulated in cancer cells such that tumor suppressor genes are silenced to favor the growth of the tumor while repetitive regions of the genome are desilenced, which can contribute to genomic instability (for review, see Baylin and Jones 2007). However, the exact molecular mechanism of DNA methylation in transcriptional regulation and during the pathogenesis of cancer remains unclear. The core histone proteins are wrapped by 147 base pairs of DNA forming the intact nucleosome. The histone N termini, or tails, extend away from the core of the nucleosome and are available for interactions with the DNA, histone-modifying enzymes, and other proteins, which alter the nucleosome structure (Shilatifard 2006). There are multiple modification sites on each histone tail, and some amino acids in the histone tail can be modified in two or more ways. Histone methylation at Lys 9 of H3 is associated with silencing at both heterochromatin and euchromatic sequences in a variety of systems (for review, see Ebert et al. 2006; Shilatifard 2006). Recent evidence demonstrates that it is also associated with transcribed regions of euchromatic genes, indicating that this modification can have distinct meanings in different contexts (Vakoc et al. 2005; Eissenberg and Shilatifard 2006). Not only can the lysine residues within histones be modified by methylation, this modification can occur in three different states. Lysine residues can accept up to three methyl groups and therefore be mono-, di-, or trimethylated. For H3K9, the pattern of mono-, di-, and trimethylation is mediated by the catalytic properties of different enzymes. For example, the G9a methyltransferase mediates dimethylation of H3K9 and is associated with silencing of euchromatic genes (Tachibana et al. 2002; Peters et al. 2003). Not only can G9a regulate the pattern of H3K9 methylation, it also functions in some way in regulating DNA methylation at multiple sites (Xin et al. 2003; Feldman et al. 2006). Furthermore, it has been demonstrated in Arabidopsis that loss of DNA methyltransferase activity can result in alteration in H3K9 methylation patterns (Soppe et al. 2002). The attachment of methyl groups to histone proteins occurs predominantly at specific lysine or arginine residues, and each site requires the enzymatic activity of a specific class of enzymes. Furthermore, methylated lysine residues within histones can serve as a specific binding site for different proteins. For example, HP1 can recognize and bind to diand trimethylated H3K9 via its chromodomain (Bannister et al. 2001; Lachner et al. 2001). HP1 was first characterized in Drosophila as a nonhistone chromosomal protein that localized to heterochromatin and was required for gene silencing (Eissenberg and Elgin 2000). Both flies and mammals have more than one HP1-like protein, with each having distinct localizations (for review, see Hediger and Gasser 2006). Mammals have three HP1 isoforms: HP1 , HP1 , and HP1 . HP1 localizes primarily to pericentric heterochromatin, HP1 localizes primarily to promoters of silent euchromatic genes, and HP1 localizes primarily to coding regions of transcribed genes (Hediger and Gasser 2006). The basis for this localization is unknown, but in Drosophila it appears to depend, in part, on the hinge region and chromo shadow domain of each protein (Smothers and Henikoff 2001). Diversification of function of different HP1s could explain the recent finding of Blobel and colleagues (Vakoc et al. 2005) that H3K9 methylation is found in the coding regions of actively Corrresponding author. E-MAIL ASH@Stowers-Institute.org; FAX (816) 926-4112. Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1559407.