Co-expression of de novo DNA methyltransferases Dnmt3a2 and Dnmt3L in gonocytes of mouse embryos

Sakai, Yasuhiro; Suetake, Isao; Shinozaki, Fuminori; Yamashina, Shohei; Tsuji, Shoji

doi:10.1016/j.modgep.2004.07.011

Cited by 110 publications

(120 citation statements)

References 25 publications

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“…Murine Dnmt3L expression is highly up-regulated at specific stages of germ cell development, coinciding with the acquisition of gametic methylation in the male and female germ lines (27,28). In males, Dnmt3a, particularly the Dnmt3a2 isoform, is also highly overexpressed at the same stage in gonocytes (28,29). Altogether, these data strongly indicate that DNMT3A2 is an important physiological target for stimulation by DNMT3L.…”

mentioning

confidence: 75%

Reconstitution and Mechanism of the Stimulation of de Novo Methylation by Human DNMT3L

Kareta

Botello

Ennis

et al. 2006

Journal of Biological Chemistry

173

156

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The DNMT3-like protein, DNMT3L, is required for germ line DNA methylation, although it is inactive as a DNA methyltransferase per se. Previous studies have shown that DNMT3L physically associates with the active de novo DNA methyltransferases, DNMT3A and DNMT3B, and stimulates their catalytic activities in a cell culture system. However, the mechanism by which DNMT3L stimulates de novo methylation remains unclear. Here, we have purified the full-length human DNMT3A2 and DNMT3L proteins and determined unique conditions that allow for the proper reconstitution of the stimulation of DNMT3A2 de novo methyltransferase activity by DNMT3L. These conditions include the use of buffers resembling physiological conditions and the preincubation of the two proteins. Under these conditions, maximal stimulation is reached at equimolar amounts of DNMT3L and DNMT3A2 proteins, and the catalytic efficiency of DNMT3A2 is increased up to 20-fold. Biochemical analysis revealed that whereas DNMT3L on its own does not significantly bind to the methyl group donor, S-adenosyl-L-methionine (SAM), it strongly increases the binding of SAM to DNMT3A2. DNA binding, on the contrary, was not appreciably improved. Analysis of DNA methyltransferase complexes in solution using size exclusion chromatography revealed that DNMT3A2 forms large structures of heterogeneous sizes, whereas DNMT3L appears as a monomer. Binding of DNMT3L to DNMT3A2 promotes a dramatic reorganization of DNMT3A2 subunits and leads to the formation of specific complexes with enhanced DNA methyltransferase activity and increased SAM binding.In mammals, the methylation of cytosines represents the only known form of covalent DNA modification that has a clear biological function, through its association with stable transcriptional silencing (1). Such silencing is critical for proper embryonic development, genome stability, X chromosome inactivation, genomic imprinting, and the silencing of retrotransposons (2-4). In addition, aberrant DNA methylation underlies many human diseases, including cancer, which is often associated with a genome-wide loss of DNA methylation and inappropriate silencing of tumor suppressor genes (5, 6). DNA methyltransferases (DNMTs) 2 are essential enzymes that catalyze the transfer of a methyl group from a donor molecule, S-adenosyl-L-methionine (SAM), to a cytosine ring, usually located within the context of a symmetrical CpG dinucleotide (7). Mammalian genomes carry three distinct active DNMT genes. Two de novo DNMTs, encoded by the DNMT3A and DNMT3B genes, have been identified (8). These proteins show a high similarity to each other and have several domains in common, including a highly conserved C-terminal domain containing catalytic DNMT motifs that are homologous to those originally defined for bacterial type II cytosine DNMTs (9). DNMT3A and DNMT3B are considered to be de novo DNMTs, since they are responsible for initiating DNA methylation during early embryonic development (10), and show a preference for unmethylated DNA in vitro (8,11). Specif...

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mentioning

confidence: 75%

Reconstitution and Mechanism of the Stimulation of de Novo Methylation by Human DNMT3L

Kareta

Botello

Ennis

et al. 2006

Journal of Biological Chemistry

173

156

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“…S9 A-C). For example, we found that the genes highly expressed only in male gonocytes, such as Dnmt3l, Tdrd1 and Miwi2/Piwi-like 4 (19,28,29), are down-regulated in the Nanos2 −/− male gonads, whereas Figla, Lhx8 and Nobox, which have been shown to be essential only for oogenesis and not for spermatogenesis (30)(31)(32), become accumulated in the Nanos2 −/− male gonads (6) (Fig. S9 D-I).…”

Section: Nanos2 Interacts With Specific Mrnas and May Promote Theirmentioning

confidence: 98%

NANOS2 interacts with the CCR4-NOT deadenylation complex and leads to suppression of specific RNAs

Suzuki

Igarashi

Aisaki

et al. 2010

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

135

154

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Nanos is one of the evolutionarily conserved proteins implicated in germ cell development. We have previously shown that NANOS2 plays an important role in both the maintenance and sexual development of germ cells. However, the molecular mechanisms underlying these events have remained elusive. In our present study, we found that NANOS2 localizes to the P-bodies, known centers of RNA degradation that are abundantly accumulated in male gonocytes. We further identified by immunoprecipitation that the components of the CCR4-NOT deadenylation complex are NANOS2-interacting proteins and found that NANOS2 promotes the localization of CNOT proteins to P-bodies in vivo. We also elucidated that the NANOS2/CCR4-NOT complex has deadenylase activity in vitro, and that some of the RNAs implicated in meiosis interact with NANOS2 and are accumulated in its absence. Our current data thus indicate that the expression of these RNA molecules is normally suppressed via a NANOS2-mediated mechanism. We propose from our current findings that NANOS2-interacting RNAs may be recruited to P-bodies and degraded by the enzymes contained therein through NANOS2-mediated deadenylation.germ cells | P-body | meiosis

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“…35 In addition, Dnmt3l, which is closely related to spermatogenesis and stimulates the DNA methylation catalytic activity of Dnmt3a, was shown to coexpress with Dnmt3a2 in mouse germ cells at different stages. 73 Evidence from Dnmt3a conditional knockout study revealed that both Dnmt3a-deficient male and female mice presented with impaired reproductive potential. Dnmt3a conditional knockout males were sterile due to azoospermia and lacked methylation at the H19 DMR (H19 differentially methylated region) and IG-DMR (intergenic-differentially methylated region) paternally imprinted loci in spermatogonia, indicating deficiency of Dnmt3a impaired spermatogenesis.…”

Section: Dnmt3a In Primordial Germ Cellsmentioning

confidence: 99%

The de novo DNA methyltransferase DNMT3A in development and cancer

Chen

Chan

2014

Epigenetics

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Co-expression of de novo DNA methyltransferases Dnmt3a2 and Dnmt3L in gonocytes of mouse embryos

Cited by 110 publications

References 25 publications

Reconstitution and Mechanism of the Stimulation of de Novo Methylation by Human DNMT3L

Reconstitution and Mechanism of the Stimulation of de Novo Methylation by Human DNMT3L

NANOS2 interacts with the CCR4-NOT deadenylation complex and leads to suppression of specific RNAs

The de novo DNA methyltransferase DNMT3A in development and cancer

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