Baculovirus-Mediated High-Level Expression of a Mammalian DNA Methyltransferase

Glickman, J. Fraser; Reich, Norbert O.

doi:10.1006/bbrc.1994.2562

Cited by 12 publications

(15 citation statements)

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“…Several reports have demonstrated the ability to express high levels of the truncated Dnmt cDNA in a transient fashion (12,13). In contrast, only a few reports have described high levels of MTase expression in stably transfected clones of transformed cells (14,33) or in a myoblast cell line (25).…”

Section: Discussionmentioning

confidence: 99%

“…Previous reports of high expression of functional MTase in transient transfections (12,13) contrasted with the inability to achieve high expression levels in stable transfection experiments. In fact, only one report described high levels of MTase expression in stably transfected clones that, however, displayed a transformed phenotype (14).…”

mentioning

confidence: 88%

“…RNA was prepared by extraction with guanidine thiocyanate and centrifugation in a cesium chloride step gradient, as described (24). RNA was passed once over an oligo(dT) [12][13][14][15][16][17][18] cellulose column (Pharmacia) as described (24). Poly(A) ϩ mRNA (12 g) was hybridized with 1 g of MMT2214AS (see Fig.…”

Section: Methodsmentioning

confidence: 99%

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Complementation of methylation deficiency in embryonic stem cells by a DNA methyltransferase minigene

Tucker

Talbot

Lee

et al. 1996

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

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Previous attempts to express functional DNA cytosine methyltransferase (EC 2.1.1.37) in cells transfected with the available Dnmt cDNAs have met with little or no success. We show that the published Dnmt sequence encodes an amino terminal-truncated protein that is tolerated only at very low levels when stably expressed in embryonic stem cells. Normal expression levels were, however, obtained with constructs containing a continuation of an ORF with a coding capacity of up to 171 amino acids upstream of the previously defined start site. The protein encoded by these constructs comigrated in SDS͞PAGE with the endogenous enzyme and restored methylation activity in transfected cells. This was shown by functional rescue of Dnmt mutant embryonic stem cells that contain highly demethylated genomic DNA and fail to differentiate normally. When transfected with the minigene construct, the genomic DNA became remethylated and the cells regained the capacity to form teratomas that displayed a wide variety of differentiated cell types. Our results define an amino-terminal domain of the mammalian MTase that is crucial for stable expression and function in vivo.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 88%

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Complementation of methylation deficiency in embryonic stem cells by a DNA methyltransferase minigene

Tucker

Talbot

Lee

et al. 1996

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

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“…These in vitro experiments need to be extended to other types of functional assays including modulation of inhibition by nucleic acids and interactions with other proteins. Our ability to express and purify recombinant DNA methyltransferase should facilitate in vitro studies of site-directed mutants (37,38). Other model systems, such as COS cell expression (39) or transgenic mice (1) might be useful in assessing the functional significance of phosphorylation of serine 514 in vivo.…”

Section: Post-translational Modification: Serine Phosphorylation Andmentioning

confidence: 99%

Peptide Mapping of the Murine DNA Methyltransferase Reveals a Major Phosphorylation Site and the Start of Translation

Glickman

Pavlovich

Reich

1997

Journal of Biological Chemistry

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The murine DNA methyltransferase catalyzes the transfer of methyl groups from S-adenosylmethionine to cytosines within d(CpG) dinucleotides. The enzyme is necessary for normal embryonic development and is implicated in a number of important processes, including the control of gene expression and cancer. Metabolic labeling and high pressure liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) were performed on DNA methyltransferase purified from murine erythroleukemia cells. Serine 514 was identified as a major phosphorylation site that lies in a domain required for targeting of the enzyme to the replication foci. These results present a potential mechanism for the regulation of DNA methylation. An essential mechanism for tissue-specific differentiation during embryonic development in mammals is the post-synthetic methylation of d (CpG) 1 dinucleotides in DNA (1). During embryogenesis, 70 -80% of d(CpG) dinucleotides become methylated in a developmentally regulated, tissue-specific fashion (2). The methylation of regulatory DNA elements frequently results in the transcriptional silencing of proximal genes (3). Although no general mechanism for this silencing has been identified, the methylation status of several DNA sequences is known to modulate binding by regulatory proteins (4, 5). HPLC-ESI-MS peptide mapping data demonstratedThe tissue-specific methylation patterns in mammalian cells presumably result from the action of the DNA methyltransferase in concert with other unidentified cellular factors (6), such as chromatin packaging proteins (7), active demethylation systems (8), or particular DNA structures (9) that dictate this enzyme's specificity for certain d(CpG)'s within the genome.The DNA methyltransferase enzyme interacts with p23, a known component of the progesterone receptor complex (10), and it is likely that control of genomic DNA methylation is coupled to receptor-mediated signaling systems in some, as yet unknown, way. A detailed knowledge of the factors involved in the regulation of the methyltransferase enzyme will be necessary to know how genomic methylation patterns are established.The murine DNA methyltransferase is an approximately 180 -190-kDa enzyme that transfers a methyl group from Sadenosylmethionine (AdoMet) onto the C-5 position of cytosine within the d(CpG) dinucleotides of double-stranded DNA. Targeted disruption of the gene for this enzyme is lethal to embryos at the middle stages of gestation (1). A number of functional domains of the enzyme have been identified and are presented in Fig. 1. The catalytic domain lies in the C-terminal third of the protein and contains regions of homology with the prokaryotic DNA methyltransferases, including a conserved AdoMet binding site and catalytic center. The N-terminal twothirds of the enzyme is separated from the catalytic domain by a flexible hinge region consisting of glycine-lysine repeats (11) and contains sequences that bind zinc and DNA independently (11,12). A minor groove DNA-binding motif (SPKK) shown to ...

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“…The diverse role played by DNA methylation in mammals has motivated several groups to express mammalian m 5 C MTases in Escherichia coli (26), baculovirus (23,27), and mammalian (COS) cells (28). In the E. coli and COS cell expression systems, the amount of protein produced was low, whereas protein expression was high using the baculovirus system.…”

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confidence: 99%

Recombinant Human DNA (Cytosine-5) Methyltransferase

Pradhan¹,

Bacolla²,

Wells³

et al. 1999

Journal of Biological Chemistry

551

197

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A method is described to express and purify human DNA (cytosine-5) methyltransferase (human DNMT1) using a protein splicing (intein) fusion partner in a baculovirus expression vector. The system produces ϳ1 mg of intact recombinant enzyme >95% pure per 1.5 ؋ 10 9 insect cells. The protein lacks any affinity tag and is identical to the native enzyme except for the two Cterminal amino acids, proline and glycine, that were substituted for lysine and aspartic acid for optimal cleavage from the intein affinity tag. Human DNMT1 was used for steady-state kinetic analysis with poly(dIdC)⅐poly(dI-dC) and unmethylated and hemimethylated 36-and 75-mer oligonucleotides. The turnover number (k cat ) was 131-237 h ؊1 on poly(dI-dC)⅐poly(dI-dC), 1.2-2.3 h ؊1 on unmethylated DNA, and 8.3-49 h ؊1 on hemimethylated DNA. The Michaelis constants for DNA (K m CG ) and S-adenosyl-L-methionine (AdoMet) (K m AdoMet ) ranged from 0.33-1.32 and 2.6 -7.2 M, respectively, whereas the ratio of k cat /K m CG ranged from 3.9 to 44 (237-336 for poly(dI-dC)⅐poly(dI-dC)) ؋ 10 6 M ؊1 h ؊1 . The preference of the enzyme for hemimethylated, over unmethylated, DNA was 7-21-fold. The values of k cat on hemimethylated DNAs showed a 2-3-fold difference, depending upon which strand was pre-methylated. Furthermore, human DNMT1 formed covalent complexes with substrates containing 5-fluoro-CNG, indicating that substrate specificity extended beyond the canonical CG dinucleotide. These results show that, in addition to maintenance methylation, human DNMT1 may also carry out de novo and non-CG methyltransferase activities in vivo.Methylated cytosine is found in the genome of organisms ranging from prokaryotes to mammals (1). Methylation of DNA in eukaryotes is implicated in various biological and developmental processes, such as gene regulation (2), DNA replication (3), genomic imprinting (4), embryonic development (5), carcinogenesis (6), and genetic diseases (7). The bulk of the methylation takes place during DNA replication in the S-phase of the cell cycle (8). The maintenance methylation ensures the propagation of tissue-specific methylation patterns established during mammalian development. The methyl transfer reaction proceeds via nonspecific binding of the enzyme to DNA, recognition of the specific DNA target site, and recruitment of the methyl group donor S-adenosyl-L-methionine (AdoMet) 1 to the active site of the enzyme. DNA (cytosine-5) methyltransferases (m 5 C MTase) introduce a methyl group onto carbon 5 of the target cytosine through a covalent intermediate between the protein and the target cytosine (9). During this process, the cytosine is flipped 180°out of the DNA backbone into an active site pocket of the enzyme (10). After completion of the methyl transfer reaction, the products, methylated DNA and S-adenosyl-L-homocysteine (AdoHcy), are released. Previous studies on the mechanism of methylation were mainly limited to prokaryotic m 5 C MTases although some limited kinetic studies have also been reported with mouse and human DNMT1 (11,12...

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Baculovirus-Mediated High-Level Expression of a Mammalian DNA Methyltransferase

Cited by 12 publications

References 0 publications

Complementation of methylation deficiency in embryonic stem cells by a DNA methyltransferase minigene

Complementation of methylation deficiency in embryonic stem cells by a DNA methyltransferase minigene

Peptide Mapping of the Murine DNA Methyltransferase Reveals a Major Phosphorylation Site and the Start of Translation

Recombinant Human DNA (Cytosine-5) Methyltransferase

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