Biochemical characterization of ParI, an orphan C5-DNA methyltransferase from Psychrobacter arcticus 273-4.

Cytosine-specific DNA methyltransferases are important enzymes in most living organisms. In prokaryotes, most DNA methyltransferases are members of the type II restriction-modification system where they methylate host DNA, thereby protecting it from digestion by the accompanying restriction endonucleases. DNA methyltransferases can also act as solitary enzymes having important roles in controlling gene expression, DNA replication, cell cycle and DNA post-replicative mismatch repair. They have potential applications in biotechnology, such as in labeling of biopolymers, DNA mapping or epigenetic analysis, as well as for general DNA-protein interaction studies. The parI gene from the psychrophilic bacterium Psychrobacter arcticus 273-4 encodes a cytosine-specific DNA methyltransferase. In this work, recombinant ParI was expressed and purified in fusion to either an N-terminal hexahistidine affinity tag, or a maltose binding protein following the hexahistidine affinity tag, for solubility improvement. After removal of the fusion partners, recombinant ParI was found to be monomeric by size exclusion chromatography, with its molecular mass estimated to be 54 kDa. The apparent melting temperature of the protein was 53 °C with no detectable secondary structures above 65 °C. Both recombinant and native ParI showed methyltransferase activity in vivo. In addition, MBP- and His-tagged ParI also demonstrated in vitro activity. Although the overall structure of ParI exhibits high thermal stability, the loss of in vitro activity upon removal of solubility tags or purification from the cellular milieu indicates that the catalytically active form is more labile. Horizontal gene transfer may explain the acquisition of a protein-encoding gene that does not display common cold-adapted features.

Functional characterization of Nicotiana benthamiana chromomethylase 3 in developmental programs by virus-induced gene silencing.

DNA methylation is essential for normal developmental processes and genome stability. DNA methyltransferases are key enzymes catalyzing DNA methylation. Chromomethylase (CMT) genes are specific to the plant kingdom and encode chromodomain-containing methyltransferases. However, the function of CMT genes in plants remains elusive. In this study, we isolated and characterized a CMT gene from Nicotiana benthamiana, designated NbCMT3. Alignment of the NbCMT3 amino acid sequence with other plant CMT3s showed conservation of bromo-adjacent-homology and methyltransferase catalytic domains. We investigated the expression patterns of NbCMT3 and its function in developmental programs. NbCMT3 was expressed predominately in proliferating tissues such as apical shoots and young leaves. NbCMT3 protein showed a nuclear location, which could be related to its putative cellular functions. Knocking down NbCMT3 expression by virus-induced gene silencing revealed its vital role(s) in leaf morphogenesis. The formation of palisade cells was defective in NbCMT3-silenced plants as compared with controls. NbCMT3 has a role in developmental programs.

ICF syndrome mutations cause a broad spectrum of biochemical defects in DNMT3B-mediated de novo DNA methylation.

The DNMT3B de novo DNA methyltransferase (DNMT) plays a major role in establishing DNA methylation patterns in early mammalian development, but its catalytic mechanism remains poorly characterized. Here, we provide a comprehensive biochemical analysis of human DNMT3B function through the characterization of a series of site-directed DNMT3B variants associated with immunodeficiency, centromere instability, and facial anomalies (ICF) syndrome. Our data reveal several novel and important aspects of DNMT3B function. First, DNMT3B, unlike DNMT3A, requires a DNA cofactor in order to stably bind to S-adenosyl-l-methionine (SAM), suggesting that it proceeds according to an ordered catalytic scheme. Second, ICF mutations cause a broad spectrum of biochemical defects in DNMT3B function, including defects in homo-oligomerization, SAM binding, SAM utilization, and DNA binding. Third, all tested ICF mutations, including the A766P and R840Q variants, result in altered catalytic properties without interfering with DNMT3L-mediated stimulation; this indicates that DNMT3L is not involved in the pathogenesis of ICF syndrome. Finally, our study reveals a novel level of coupling between substrate binding, oligomerization, and catalysis that is likely conserved within the DNMT3 family of enzymes.

Novel and selective DNA methyltransferase inhibitors: Docking-based virtual screening and experimental evaluation.

The DNA methyltransferase (DNMT) enzyme family consists of four members with diverse functions and represents one of the most promising targets for the development of novel anticancer drugs. However, the standard drugs for DNMT inhibition are non-selective cytosine analogues with considerable cytotoxic side-effects that have been developed several decades ago. In this work, we conducted a virtual screening of more than 65,000 lead-like compounds selected from the National Cancer Institute collection using a multistep docking approach with a previously validated homology model of the catalytic domain of human DNMT1. Experimental evaluation of top-ranked molecules led to the discovery of novel small molecule DNMT1 inhibitors. Virtual screening hits were further evaluated for DNMT3B inhibition revealing several compounds with selectivity towards DNMT1. These are the first small molecules reported with biochemical selectivity towards an individual DNMT enzyme capable of binding in the same pocket as the native substrate cytosine, and are promising candidates for further rational optimization and development as anticancer drugs. The availability of enzyme-selective inhibitors will also be of great significance for understanding the role of individual DNMT enzymes in epigenetic regulation.

A novel DNMT3B splice variant expressed in tumor and pluripotent cells modulates genomic DNA methylation patterns and displays altered DNA binding.

DNA methylation is an epigenetic mark essential for mammalian development, genomic stability, and imprinting. DNA methylation patterns are established and maintained by three DNA methyltransferases: DNMT1, DNMT3A, and DNMT3B. Interestingly, all three DNMTs make use of alternative splicing. DNMT3B has nearly 40 known splice variants expressed in a tissue- and disease-specific manner, but very little is known about the role of these splice variants in modulating DNMT3B function. We describe here the identification and characterization of a novel alternatively spliced form of DNMT3B lacking exon 5 within the NH(2)-terminal regulatory domain. This variant, which we term DNMT3B3Delta5 because it is closely related in structure to the ubiquitously expressed DNMT3B3 isoform, is highly expressed in pluripotent cells and brain tissue, is downregulated during differentiation, and is conserved in the mouse. Creation of pluripotent iPS cells from fibroblasts results in marked induction of DNMT3B3Delta5. DNMT3B3Delta5 expression is also altered in human disease, with tumor cell lines displaying elevated or reduced expression depending on their tissue of origin. We then compared the DNA binding and subcellular localization of DNMT3B3Delta5 versus DNMT3B3, revealing that DNMT3B3Delta5 possessed significantly enhanced DNA binding affinity and displayed an altered nuclear distribution. Finally, ectopic overexpression of DNMT3B3Delta5 resulted in repetitive element hypomethylation and enhanced cell growth in a colony formation assay. Taken together, these results show that DNMT3B3Delta5 may play an important role in stem cell maintenance or differentiation and suggest that sequences encoded by exon 5 influence the functional properties of DNMT3B.

A new class of quinoline-based DNA hypomethylating agents reactivates tumor suppressor genes by blocking DNA methyltransferase 1 activity and inducing its degradation.

Reactivation of silenced tumor suppressor genes by 5-azacytidine (Vidaza) and its congener 5-aza-2'-deoxycytidine (decitabine) has provided an alternate approach to cancer therapy. We have shown previously that these drugs selectively and rapidly induce degradation of the maintenance DNA methyltransferase (DNMT) 1 by a proteasomal pathway. Because the toxicity of these compounds is largely due to their incorporation into DNA, it is critical to explore novel, nonnucleoside compounds that can effectively reactivate the silenced genes. Here, we report that a quinoline-based compound, designated SGI-1027, inhibits the activity of DNMT1, DNMT3A, and DNMT3B as well M. SssI with comparable IC(50) (6-13 micromol/L) by competing with S-adenosylmethionine in the methylation reaction. Treatment of different cancer cell lines with SGI-1027 resulted in selective degradation of DNMT1 with minimal or no effects on DNMT3A and DNMT3B. At a concentration of 2.5 to 5 micromol/L (similar to that of decitabine), complete degradation of DNMT1 protein was achieved within 24 h without significantly affecting its mRNA level. MG132 blocked SGI-1027-induced depletion of DNMT1, indicating the involvement of proteasomal pathway. Prolonged treatment of RKO cells with SGI-1027 led to demethylation and reexpression of the silenced tumor suppressor genes P16, MLH1, and TIMP3. Further, this compound did not exhibit significant toxicity in a rat hepatoma (H4IIE) cell line. This study provides a novel class of DNA hypomethylating agents that have the potential for use in epigenetic cancer therapy.

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.

Molecular characterization of the DNA methyltransferase M1.NcuI from Neisseria cuniculi ATCC 14688.

The methyltransferase M1.NcuI is a member of the restriction-modification system in Neisseria cuniculi ATCC14688 and recognizes the asymmetric pentanucleotide sequence 5'-GAAGA-3'/3'-CTTCT-5'. We purified M1.NcuI to electrophoretic homogeneity using a four-step chromatographic procedure. M1.NcuI is a protein with M(r)=32,000+/-1000 under denaturing conditions. It modifies the recognition sequence by transferring the methyl group from S-adenosyl-l-methionine to the 3' adenine of the pentanucleotide sequence 5'-GAAGA-3'. M1.NcuI, like many other methyltransferases, occurs as a monomer in solution, as determined by gel filtration. Divalent cations inhibit the methylation activity of M1.NcuI. Optimal enzyme activity was observed at a pH of 8.0. M1.NcuI cross-reacted with anti-M1.MboII serum which reflects the similarity of M1.NcuI with M1.MboII at the amino acid level. The gene coding for the enzyme, designated ncuIM1, was cloned, sequenced and overexpressed in Escherichia coli. The structural gene is 780 nucleotides in length coding for a protein of 259 amino acids (M(r) 30,098). The presence and distribution of nine highly conserved amino acid sequence motifs and a putative target recognition domain in the enzyme structure suggest that M1.NcuI, similar to M1.MboII and M1.HpyAII, belongs to N(6)-adenine beta-class DNA methyltransferases.

VIM1, a methylcytosine-binding protein required for centromeric heterochromatinization.

Epigenetic regulation in eukaryotes is executed by a complex set of signaling interactions among small RNA species and chromatin marks, including histone modification and DNA methylation. We identified vim1 (VARIANT IN METHYLATION 1), an Arabidopsis mutation causing cytosine hypomethylation and decondensation of centromeres in interphase. VIM1 is a member of a small gene family, encoding proteins containing PHD, RING, and SRA (SET- and RING-associated) domains, which are found together in mammalian proteins implicated in regulation of chromatin modification, transcription, and the cell cycle. VIM1 is an unconventional methylcytosine-binding protein that interacts in vitro with 5mCpG- and 5mCpHpG-modified DNA (via its SRA domain), as well as recombinant histones (H2B, H3, H4, and HTR12) in plant extracts. VIM1 associates with methylated genomic loci in vivo and is enriched in chromocenters. Our findings suggest that VIM1 acts at the DNA methylation-histone interface to maintain centromeric heterochromatin.

DNMT1 and HDAC1 gene expression in impaired spermatogenesis and testicular cancer.

DNA methylation catalyzed by DNA methyltransferases (DNMTs) and histone deacetylation catalyzed by histone deacetylases (HDACs) play an important role for the regulation of gene expression during carcinogenesis and spermatogenesis. We therefore studied the cell-specific expression of DNMT1 and HDAC1 for the first time in human testicular cancer and impaired human spermatogenesis. During normal spermatogenesis, DNMT1 and HDAC1 were colocalized in nuclei of spermatogonia. While HDAC1 was additionally present in nuclei of Sertoli cells, DNMT1 was restricted to germ cells exhibiting a different expression pattern of mRNA (in pachytene spermatocytes and round spermatids) and protein (in round spermatids). Interestingly, in infertile patients revealing round spermatid maturation arrest, round spermatids lack DNMT1 protein, while pachytene spermatocytes became immunopositive for DNMT1. In contrast, no changes in the expression pattern could be observed for HDAC1. This holds true also in testicular tumors, where HDAC1 has been demonstrated in embryonal carcinoma, seminoma and teratoma. Interestingly, DNMT1 was not expressed in seminoma, but upregulated in embryonal carcinoma.

Reversible inactivation of the CG specific SssI DNA (cytosine-C5)-methyltransferase with a photocleavable protecting group.

Caging of proteins by conjugation with a photocleavable group is a powerful approach for reversibly blocking enzymatic activity. Here we describe the covalent modification of the bacterial SssI DNA methyltransferase (M.SssI) with the cysteine-specific reagent 4,5-dimethoxy-2-nitrobenzylbromide (DMNBB). M.SssI contains two cysteine residues; replacement of the active-site Cys141 with Ser resulted in an approximately 100-fold loss of enzymatic activity; this indicates an important role for this residue in catalysis. However, replacement of Cys368 with Ala did not affect methyltransferase activity. Treatment of the Cys368Ala mutant enzyme with DMNBB led to an almost complete loss of activity. Irradiation of the inactivated enzyme with near-ultraviolet light (320-400 nm) restored 60 % of the catalytic activity. This indicates that caging by DMNBB can be used for the reversible inactivation of M.SssI.

Purification and identification of proteins that bind to the hereditary persistence of fetal hemoglobin -198 mutation in the gamma-globin gene promoter.

Expression of the gamma-globin gene is silenced in adult humans. However, certain point mutations in the gamma-globin gene promoter are capable of maintaining expression of this gene during adult erythropoiesis, a condition called non-deletion hereditary persistence of fetal hemoglobin (HPFH). Among these, the British form of HPFH carrying a T-->C point mutation at position -198 of the Agamma-globin gene promoter results in 4-10% fetal hemoglobin in heterozygotes. In this study, we used nuclear extracts from murine erythroleukemia cells to purify a protein complex that binds the HPFH -198 gamma-globin gene promoter. Members of this protein complex were identified by mass spectrometry and include DNMT1, the transcriptional coactivator p52, the protein SNEV, and RAP74 (the largest subunit of the general transcription factor IIF). Sp1, which was previously considered responsible for HPFH -198 gamma-globin gene activation, was not identified. The potential role of these proteins in the reactivation and/or maintenance of gamma-globin gene expression in the adult transcriptional environment is discussed.

Characterization of DNA methyltransferase genes in Brassica rapa.

DNA methylation is essential for normal development and plays important roles in regulating gene expression in plants. Analysis of the key enzymes catalyzing DNA methylation is important to understand epigenetic phenomena. In this study, three putative methyltransferase genes, BrMET1a, BrMET1b, and BrCMT, were isolated from a genome library of Brassica rapa. Structural conservation of the amino acid sequence between BrMET1a/BrMET1b and AtMET1 and that between BrCMT and AtCMT3 suggests that they may function as DNA methyltransferase. BrMET1a was expressed in vegetative and reproductive organs, while BrMET1b was expressed only in pistils, indicating that these two genes have different functions. BrCMT was expressed especially in stamens at the stage of 2-4 days before anthesis. We isolated three DNA methyltransferase genes in Brassica rapa and indicated differences of expression patterns of these DNA methyltransferase genes and expression levels in different tissues and developmental stages, suggesting that these genes might play important roles in epigenetic gene regulation in B. rapa.

Reconstitution and mechanism of the stimulation of de novo methylation by human DNMT3L.

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.

An orphan DNA (cytosine-5-)-methyltransferase in Vibrio cholerae.

5-Methyl cytosine (m5C) was detected in genomic DNA of the enteric pathogen Vibrio cholerae by HPLC analysis and immunoblotting with m5C-specific antibody. Although cleavage with the restriction endonuclease EcoRII revealed the absence of a Dcm homologue in V. cholerae, analysis of the genome sequence indicated the presence of a gene, designated in this study as vchM, which encodes a DNA (cytosine-5-)-methyltransferase (m5C-MTase) designated M.Vch. M.Vch is not associated with a restriction endonuclease or a mismatch very short patch repair (Vsr)-like endonuclease and is hence an 'orphan' or solitary MTase, although analysis of a phylogenetic tree indicated that related cytosine MTases are all components of restriction-modification systems. M.Vch recognizes and methylates the first 5' C in the degenerate sequence 5'-RCCGGY-3'. RT-PCR analysis suggested that vchM gene expression is increased during the stationary phase of growth. During stationary phase, the spontaneous mutation frequency in the V. cholerae wild-type strain was significantly higher than in the corresponding vchM mutant strain, suggesting that the presence of M.Vch and the absence of a very short patch (VSP) repair-like system imposes upon V. cholerae a mutator phenotype.

Mutational analysis of the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase.

On the basis of amino acid sequence alignments and structural data of related enzymes, we have performed a mutational analysis of 14 amino acid residues in the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase. The target residues are located within the ten conserved amino acid sequence motifs characteristic for cytosine-C5 methyltransferases and in the putative DNA recognition domain of the enzyme (TRD). Mutant proteins were purified and tested for their catalytic properties and their abilities to bind DNA and AdoMet. We prepared a structural model of Dnmt3a to interpret our results. We demonstrate that Phe50 (motif I) and Glu74 (motif II) are important for AdoMet binding and catalysis. D96A (motif III) showed reduced AdoMet binding but increased activity under conditions of saturation with S-adenosyl-L-methionine (AdoMet), indicating that the contact of Asp96 to AdoMet is not required for catalysis. R130A (following motif IV), R241A and R246A (in the TRD), R292A, and R297A (both located in front of motif X) showed reduced DNA binding. R130A displayed a strong reduction in catalytic activity and a complete change in flanking sequence preferences, indicating that Arg130 has an important role in the DNA interaction of Dnmt3a. R292A also displayed reduced activity and changes in the flanking sequence preferences, indicating a potential role in DNA contacts farther away from the CG target site. N167A (motif VI) and R202A (motif VIII) have normal AdoMet and DNA binding but reduced catalytic activity. While Asn167 might contribute to the positioning of residues from motif VI, according to structural data Arg202 has a role in catalysis of cytosine-C5 methyltransferases. The R295A variant was catalytically inactive most likely because of destabilization of the hinge sub-domain of the protein.

Isolation and characterization of biochemical properties of DNA methyltransferase FauIA modifying the second cytosine in the nonpalindromic sequence 5'-CCCGC-3'.

A gene encoding DNA methyltransferase (methylase) FauIA of the restriction-modification system FauI from Flavobacterium aquatile (recognizing sequence 5'-CCCGC-3') was cloned in pJW vector. The latter was used for transformation of E. coli RRI cells followed by subsequent thermoinduction and biomass elaboration. Highly purified DNA methyltransferase FauIA preparation was obtained using chromatography on different sorbents. The molecular mass of the isolated enzyme of about 39 kD corresponds to its theoretical value. The enzyme was characterized by temperature and pH optima of 33 degrees C and pH 7.5, respectively. Methylation of a synthetic oligonucleotide by FauIA methylase followed by its cleavage with various restrictases and analysis of the resultant restriction fragments revealed that FauIA methylase modified the second cytosine residue in the sequence 5'-CCCGC-3'. Kinetic analysis revealed Km and catalytic constant values of 0.16 microM and 0.05 min(-1), respectively.

Restriction and modification of SP10 phage by BsuM of Bacillus subtilis Marburg.

Bacillus subtilis Marburg has only one intrinsic restriction and modification system BsuM that recognizes the CTCGAG (XhoI site) sequence. It consists of two operons, BsuMM operon for two cytosine DNA methyltransferases, and BsuMR operon for a restriction nuclease and two associated proteins of unknown function. In this communication, we analyzed the BsuM system by utilizing phage SP10 that possesses more than twenty BsuM target sequences on the phage genome. SP10 phages grown in the restriction and modification-deficient strain could not make plaques on the restriction-proficient BsuMR(+) indicator strain. An enforced expression of the wild type BsuMM operon in the BsuMR(+) indicator strain, however, allowed more than thousand times more plaques. DNA extracted from SP10 phages, thus, propagated became more but not completely refractory to XhoI digestion in vitro. Thus, the SP10 phage genome DNA is able to be nearly full-methylated but some BsuM sites are considered to be unmethylated.

Significance of codon usage and irregularities of rare codon distribution in genes for expression of BspLU11III methyltransferases.

Genes of adenine-specific DNA-methyltransferase M.BspLU11IIIa and cytosine-specific DNA-methyltransferase M.BspLU11IIIb of the type IIG BspLU11III restriction-modification system from the thermophilic strain Bacillus sp. LU11 were expressed in E. coli. They contain a large number of codons that are rare in E. coli and are characterized by equal values of codon adaptation index (CAI) and expression level measure (E(g)). Rare codons are either diffused (M.BspLU11IIIa) or located in clusters (M.BspLU11IIIb). The expression level of the cytosine-specific DNA-methyltransferase was increased by a factor of 7.3 and that of adenine-specific DNA only by a factor of 1.25 after introduction of the plasmid pRARE supplying tRNA genes for six rare codons in E. coli. It can be assumed that the plasmid supplying minor tRNAs can strongly increase the expression level of only genes with cluster distribution of rare codons. Using heparin-Sepharose and phosphocellulose chromatography and gel filtration on Sephadex G-75 both DNA-methyltransferases were isolated as electrophoretically homogeneous proteins (according to the results of SDS-PAGE).

Isolation and characterization of a novel DNA methyltransferase complex linking DNMT3B with components of the mitotic chromosome condensation machinery.

Proper patterns of genome-wide DNA methylation, mediated by DNA methyltransferases DNMT1, -3A and -3B, are essential for embryonic development and genomic stability in mammalian cells. The de novo DNA methyltransferase DNMT3B is of particular interest because it is frequently overexpressed in tumor cells and is mutated in immunodeficiency, centromere instability and facial anomalies (ICF) syndrome. In order to gain a better understanding of DNMT3B, in terms of the targeting of its methylation activity and its role in genome stability, we biochemically purified endogenous DNMT3B from HeLa cells. DNMT3B co-purifies and interacts, both in vivo and in vitro, with several components of the condensin complex (hCAP-C, hCAP-E and hCAP-G) and KIF4A. Condensin mediates genome-wide chromosome condensation at the onset of mitosis and is critical for proper segregation of sister chromatids. KIF4A is proposed to be a motor protein carrying DNA as cargo. DNMT3B also interacts with histone deacetylase 1 (HDAC1), the co-repressor SIN3A and the ATP-dependent chromatin remodeling enzyme hSNF2H. Further more, DNMT3B co-localizes with condensin and KIF4A on condensed chromosomes throughout mitosis. These studies therefore reveal the first direct link between the machineries regulating DNA methylation and mitotic chromosome condensation in mammalian cells.