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1.  DNMT3L Modulates Significant and Distinct Flanking Sequence Preference for DNA Methylation by DNMT3A and DNMT3B In Vivo 
PLoS Genetics  2010;6(9):e1001106.
The DNTM3A and DNMT3B de novo DNA methyltransferases (DNMTs) are responsible for setting genomic DNA methylation patterns, a key layer of epigenetic information. Here, using an in vivo episomal methylation assay and extensive bisulfite methylation sequencing, we show that human DNMT3A and DNMT3B possess significant and distinct flanking sequence preferences for target CpG sites. Selection for high or low efficiency sites is mediated by the base composition at the −2 and +2 positions flanking the CpG site for DNMT3A, and at the −1 and +1 positions for DNMT3B. This intrinsic preference reproducibly leads to the formation of specific de novo methylation patterns characterized by up to 34-fold variations in the efficiency of DNA methylation at individual sites. Furthermore, analysis of the distribution of signature methylation hotspot and coldspot motifs suggests that DNMT flanking sequence preference has contributed to shaping the composition of CpG islands in the human genome. Our results also show that the DNMT3L stimulatory factor modulates the formation of de novo methylation patterns in two ways. First, DNMT3L selectively focuses the DNA methylation machinery on properly chromatinized DNA templates. Second, DNMT3L attenuates the impact of the intrinsic DNMT flanking sequence preference by providing a much greater boost to the methylation of poorly methylated sites, thus promoting the formation of broader and more uniform methylation patterns. This study offers insights into the manner by which DNA methylation patterns are deposited and reveals a new level of interplay between members of the de novo DNMT family.
Author Summary
The methylation of cytosine bases in DNA represents an extra layer of heritable biological information necessary for regulating gene expression and ensuring genomic stability in mammals. In this paper, we examine the function of the proteins responsible for laying down the initial DNA methylation patterns in the human genome. These proteins, called de novo DNA methyltransferases (DNMTs), comprise two active enzymes, DNMT3A and DNMT3B, and one stimulatory factor, DNMT3L. Our study clearly establishes that DNMT3A and DNMT3B do not methylate DNA at random but rather that they show strong and distinct preferences for their target sites in vivo. These preferences lead to the deposition of unique and reproducible patterns of methylation and may have contributed to shaping segments of the human genome. In contrast, we show that DNMT3L stimulates DNA methylation mostly at sites that are poorly methylated on their own, thus leading to patterns that are more uniform. This modulation is proposed to result from DNMT3L anchoring the DNA methylation machinery onto chromatin, the physiological form under which DNA exists in our cells. This study furthers our understanding of how genomic DNA methylation patterns are established in vivo.
doi:10.1371/journal.pgen.1001106
PMCID: PMC2936528  PMID: 20838592
2.  Dnmt3a Protects Active Chromosome Domains against Cancer-Associated Hypomethylation 
PLoS Genetics  2012;8(12):e1003146.
Changes in genomic DNA methylation patterns are generally assumed to play an important role in the etiology of human cancers. The Dnmt3a enzyme is required for the establishment of normal methylation patterns, and mutations in Dnmt3a have been described in leukemias. Deletion of Dnmt3a in a K-ras–dependent mouse lung cancer model has been shown to promote tumor progression, which suggested that the enzyme might suppress tumor development by stabilizing DNA methylation patterns. We have used whole-genome bisulfite sequencing to comprehensively characterize the methylomes from Dnmt3a wildtype and Dnmt3a-deficient mouse lung tumors. Our results show that profound global methylation changes can occur in K-ras–induced lung cancer. Dnmt3a wild-type tumors were characterized by large hypomethylated domains that correspond to nuclear lamina-associated domains. In contrast, Dnmt3a-deficient tumors showed a uniformly hypomethylated genome. Further data analysis revealed that Dnmt3a is required for efficient maintenance methylation of active chromosome domains and that Dnmt3a-deficient tumors show moderate levels of gene deregulation in these domains. In summary, our results uncover conserved features of cancer methylomes and define the role of Dnmt3a in maintaining DNA methylation patterns in cancer.
Author Summary
Dnmt3a is generally assumed to be a de novo DNA methyltransferase that plays an important role in establishing DNA methylation patterns during embryogenesis. However, mutations in the human DNMT3A gene have been detected in various cancers, suggesting that the enzyme might also be relevant for DNA methylation in adult tissues and in tumors. We have established genome-wide methylation profiles at single base pair resolution to define Dnmt3a-dependent methylation changes in a mouse tumor model. Our results show that mouse tumors with a functional Dnmt3a enzyme are characterized by regional hypomethylation, while Dnmt3a-deficient tumors showed a uniformly hypomethylated genome. Further data analysis revealed that Dnmt3a is required for maintaining normal DNA methylation patterns specifically in gene bodies and in active chromosome domains. Our study thus defines the role of Dnmt3a in maintaining DNA methylation patterns and provides a paradigm for understanding the effects of DNMT3A mutations on human cancer methylomes.
doi:10.1371/journal.pgen.1003146
PMCID: PMC3527206  PMID: 23284304
3.  Regulation of Lineage Specific DNA Hypomethylation in Mouse Trophectoderm 
PLoS ONE  2013;8(6):e68846.
Background
DNA methylation is reprogrammed during early embryogenesis by active and passive mechanisms in advance of the first differentiation event producing the embryonic and extraembryonic lineage cells which contribute to the future embryo proper and to the placenta respectively. Embryonic lineage cells re-acquire a highly methylated genome dependent on the DNA methyltransferases (DNMTs) Dnmt3a and Dnmt3b that are required for de novo methylation. By contrast, extraembryonic lineage cells remain globally hypomethylated but the mechanisms that underlie this hypomethylation remain unknown.
Methodology/Principal Findings
We have employed an inducible system that supports differentiation between these two lineages and recapitulates the DNA methylation asymmetry generated in vivo. We find that in vitro down-regulation of Oct3/4 in ES cells recapitulates the decline in global DNA methylation associated with trophoblast. The de novo DNMTs Dnmt3a2 and Dnmt3b are down-regulated during trophoblast differentiation. Dnmt1, which is responsible for maintenance methylation, is expressed comparably in embryonic and trophoblast lineages, however importantly in trophoblast giant cells Dnmt1fails to be attracted to replication foci, thus allowing loss of DNA methylation while implicating a passive demethylation mechanism. Interestingly, Dnmt1 localization was restored by exogenous Np95/Uhrf1, a Dnmt1 chaperone required for Dnmt1-targeting to replication foci, yet DNA methylation levels remained low. Over-expression of de novo DNMTs also failed to increase DNA methylation in target sequences.
Conclusions/Significance
We propose that induced trophoblast cells may have a mechanism to resist genome-wide increases of DNA methylation, thus reinforcing the genome-wide epigenetic distinctions between the embryonic and extraembryonic lineages in the mouse. This resistance may be based on transcription factors or on global differences in chromatin structure.
doi:10.1371/journal.pone.0068846
PMCID: PMC3692478  PMID: 23825703
4.  5-Aza-Deoxycytidine Induces Selective Degradation of DNA Methyltransferase 1 by a Proteasomal Pathway That Requires the KEN Box, Bromo-Adjacent Homology Domain, and Nuclear Localization Signal 
Molecular and Cellular Biology  2005;25(11):4727-4741.
5-Azacytidine- and 5-aza-deoxycytidine (5-aza-CdR)-mediated reactivation of tumor suppressor genes silenced by promoter methylation has provided an alternate approach in cancer therapy. Despite the importance of epigenetic therapy, the mechanism of action of DNA-hypomethylating agents in vivo has not been completely elucidated. Here we report that among three functional DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), the maintenance methyltransferase, DNMT1, was rapidly degraded by the proteasomal pathway upon treatment of cells with these drugs. The 5-aza-CdR-induced degradation, which occurs in the nucleus, could be blocked by proteasomal inhibitors and required a functional ubiquitin-activating enzyme. The drug-induced degradation occurred even in the absence of DNA replication. Treatment of cells with other nucleoside analogs modified at C-5, 5-fluorodeoxyuridine and 5-fluorocytidine, did not induce the degradation of DNMT1. Mutation of cysteine at the catalytic site of Dnmt1 (involved in the formation of a covalent intermediate with cytidine in DNA) to serine (CS) did not impede 5-aza-CdR-induced degradation. Neither the wild type nor the catalytic site mutant of Dnmt3a or Dnmt3b was sensitive to 5-aza-CdR-mediated degradation. These results indicate that covalent bond formation between the enzyme and 5-aza-CdR-incorporated DNA is not essential for enzyme degradation. Mutation of the conserved KEN box, a targeting signal for proteasomal degradation, to AAA increased the basal level of Dnmt1 and blocked its degradation by 5-aza-CdR. Deletion of the catalytic domain increased the expression of Dnmt1 but did not confer resistance to 5-aza-CdR-induced degradation. Both the nuclear localization signal and the bromo-adjacent homology domain were essential for nuclear localization and for the 5-aza-CdR-mediated degradation of Dnmt1. Polyubiquitination of Dnmt1 in vivo and its stabilization upon treatment of cells with a proteasomal inhibitor indicate that the level of Dnmt1 is controlled by ubiquitin-dependent proteasomal degradation. Overexpression of the substrate recognition component, Cdh1 but not Cdc20, of APC (anaphase-promoting complex)/cyclosome ubiquitin ligase reduced the level of Dnmt1 in both untreated and 5-aza-CdR-treated cells. In contrast, the depletion of Cdh1 with small interfering RNA increased the basal level of DNMT1 that blocked 5-aza-CdR-induced degradation. Dnmt1 interacted with Cdh1 and colocalized in the nucleus at discrete foci. Both Dnmt1 and Cdh1 were phosphorylated in vivo, but only Cdh1 was significantly dephosphorylated upon 5-aza-CdR treatment, suggesting its involvement in initiating the proteasomal degradation of DNMT1. These results demonstrate a unique mechanism for the selective degradation of DNMT1, the maintenance DNA methyltransferase, by well-known DNA-hypomethylating agents.
doi:10.1128/MCB.25.11.4727-4741.2005
PMCID: PMC1140649  PMID: 15899874
5.  Loss of DNMT1o Disrupts Imprinted X Chromosome Inactivation and Accentuates Placental Defects in Females 
PLoS Genetics  2013;9(11):e1003873.
The maintenance of key germline derived DNA methylation patterns during preimplantation development depends on stores of DNA cytosine methyltransferase-1o (DNMT1o) provided by the oocyte. Dnmt1omat−/− mouse embryos born to Dnmt1Δ1o/Δ1o female mice lack DNMT1o protein and have disrupted genomic imprinting and associated phenotypic abnormalities. Here, we describe additional female-specific morphological abnormalities and DNA hypomethylation defects outside imprinted loci, restricted to extraembryonic tissue. Compared to male offspring, the placentae of female offspring of Dnmt1Δ1o/Δ1o mothers displayed a higher incidence of genic and intergenic hypomethylation and more frequent and extreme placental dysmorphology. The majority of the affected loci were concentrated on the X chromosome and associated with aberrant biallelic expression, indicating that imprinted X-inactivation was perturbed. Hypomethylation of a key regulatory region of Xite within the X-inactivation center was present in female blastocysts shortly after the absence of methylation maintenance by DNMT1o at the 8-cell stage. The female preponderance of placental DNA hypomethylation associated with maternal DNMT1o deficiency provides evidence of additional roles beyond the maintenance of genomic imprints for DNA methylation events in the preimplantation embryo, including a role in imprinted X chromosome inactivation.
Author Summary
During oocyte growth and maturation, vital proteins and enzymes are produced to ensure that, when fertilized, a healthy embryo will arise. When this natural process is interrupted, one or more of these essential elements can fail to be produced thus compromising the health of the future embryo. We are using a mouse model, lacking an enzyme (DNMT1o) produced in the oocyte and only required post-fertilization in the early embryo for the maintenance of inherited DNA methylation marks. Here, we reveal that oocytes lacking DNMT1o, when fertilized, generated conceptuses with a wide variety of placental abnormalities. These placental abnormalities were more frequent and severe in females, and showed specific genomic regions constantly deprived of their normal methylation marks. The affected genomic regions were concentrated on the X chromosome. Interestingly, we also found that a region important for the regulation of the X chromosome inactivation process was hypomethylated in female blastocysts and was associated with sex-specific abnormalities in the placenta, relaxation of imprinted X chromosome inactivation, and disruption of DNA methylation later in development. Our findings provide a novel unanticipated role for DNA methylation events taking place within the first few days of life specifically in female preimplantation embryos.
doi:10.1371/journal.pgen.1003873
PMCID: PMC3836718  PMID: 24278026
6.  Role of DNA Methyltransferases in Regulation of Human Ribosomal RNA Gene Transcription* 
The Journal of biological chemistry  2006;281(31):22062-22072.
We have previously demonstrated that the expression of human ribosomal RNA genes (rDNA) in normal and cancer cells is differentially regulated by methylation of the promoter CpG islands. Furthermore, we showed that the methyl CpG-binding protein MBD2 plays a selective role in the methylation-mediated block in rDNA expression. Here, we analyzed the role of three functional mammalian DNA methyltransferases (DNMTs) in regulating the rDNA promoters activity. Immunofluorescence analysis and biochemical fractionation showed that all three DNMTs (DNMT1, DNMT3A, and DNMT3B) are associated with the inactive rDNA in the nucleolus. Although DNMTs associate with both methylated and unmethylated rDNA promoters, DNMT1 preferentially associates with the methylated genes. The rDNA primary transcript level was significantly elevated in DNMT1−/− or DNMT3B−/− human colon carcinoma (HCT116) cells. Southern blot analysis demonstrated a moderate level of rDNA promoter hypomethylation in DNMT1−/− cells and a dramatic loss of rDNA promoter methylation in double knockout cells. Transient overexpression of DNMT1 or DNMT3B suppressed the luciferase expression from both methylated and unmethylated pHrD-IRES-Luc, a reporter plasmid where the rDNA promoter drives luciferase expression. DNMT1-mediated suppression of the unmethylated promoter involves de novo methylation of the promoter, whereas histone deacetylase 2 cooperates with DNMT1 to inhibit the methylated rDNA promoter. Unlike DNMT1, both the wild type and catalytically inactive DNMT3B mutant can suppress rDNA promoter irrespective of its methylation status. DNMT3B-mediated suppression of the rDNA promoter also involves histone deacetylation. Treatment of HCT116 cells with Decitabine (a DNMT inhibitor) or trichostatin A (a histone deacetylase inhibitor) up-regulated endogenous rDNA expression. These inhibitors synergistically activated methylated pHrD-IRES-Luc, whereas they exhibited additive effects on the unmethylated promoter. These results demonstrate localization of DNMTs with the inactive rDNA in the nucleolus, the specific role of DNMT1 and DNMT3B in rDNA expression and the differential regulation of rDNA expression from the methylated and unmethylated rDNA promoters.
doi:10.1074/jbc.M601155200
PMCID: PMC2243234  PMID: 16735507
7.  DNMT1 genetic polymorphisms affect breast cancer risk in the central European Caucasian population 
Clinical Epigenetics  2013;5(1):7.
Introduction
DNA methylation of CpG islands within the promoter region of genes is an epigenetic modification with an important role in the development of cancer and it is typically mediated by DNA methyltransferases (DNMTs). In cancer cells, global hypomethylation of the genome as a whole and regional hypermethylation of CpG islands have been reported. Four groups of DNMTs have been identified: DNMT1, DNMT2 (TRDMT1), DNMT3A and DNMT3B. DNMT2 uses the catalytic mechanism of DNMTs, but does in fact methylate RNA. Little is known about the significance of these genes in human breast cancer. In the study presented herein, we analyzed five distinct DNMT single SNPs with regard to potential associations with breast cancer risk.
Case description
In this study, we genotyped 221 female Caucasian breast cancer patients and 221 female Caucasian healthy controls, and we used five allele-specific real-time polymerase chain reaction (qPCR) assays. We selected one locus within the DNMT1 gene and two loci within the DNMT3A and DNMT3B genes, respectively. Statistics were calculated using the chi-squared and Fisher’s exact tests, and correlated with clinical parameters such as age, diagnosis, histology, TNM stage, hormonal receptor status, human epidermal growth factor receptor 2 (HER2) status, response to treatment and survival. Statistically significant results were obtained for correlations with the DNMT1 gene.
Discussion and Evaluation
Five genomic loci within the DNMT1, DNMT3A and DNMT3B genes were assessed. Statistical significance (P = 0.030) was identified for DNMT1 SNP (A201G, rs2228612): six women within the control group were GG homozygous (variant), while this mutation was absent in the breast cancer group.
Conclusions
We conclude that women with the DNMT1 SNP (A201G, rs2228612) GG homozygous genotype (variant) have a lower risk of developing breast cancer compared to heterozygous or wildtype genotypes. To date, alterations within the DNMT1 gene have not been reported to be associated with cancer in the Caucasian population.
doi:10.1186/1868-7083-5-7
PMCID: PMC3646668  PMID: 23638630
DNMT; SNP; Breast cancer
8.  Mouse Oocyte Methylomes at Base Resolution Reveal Genome-Wide Accumulation of Non-CpG Methylation and Role of DNA Methyltransferases 
PLoS Genetics  2013;9(4):e1003439.
DNA methylation is an epigenetic modification that plays a crucial role in normal mammalian development, retrotransposon silencing, and cellular reprogramming. Although methylation mainly occurs on the cytosine in a CG site, non-CG methylation is prevalent in pluripotent stem cells, brain, and oocytes. We previously identified non-CG methylation in several CG-rich regions in mouse germinal vesicle oocytes (GVOs), but the overall distribution of non-CG methylation and the enzymes responsible for this modification are unknown. Using amplification-free whole-genome bisulfite sequencing, which can be used with minute amounts of DNA, we constructed the base-resolution methylome maps of GVOs, non-growing oocytes (NGOs), and mutant GVOs lacking the DNA methyltransferase Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3L. We found that nearly two-thirds of all methylcytosines occur in a non-CG context in GVOs. The distribution of non-CG methylation closely resembled that of CG methylation throughout the genome and showed clear enrichment in gene bodies. Compared to NGOs, GVOs were over four times more methylated at non-CG sites, indicating that non-CG methylation accumulates during oocyte growth. Lack of Dnmt3a or Dnmt3L resulted in a global reduction in both CG and non-CG methylation, showing that non-CG methylation depends on the Dnmt3a-Dnmt3L complex. Dnmt3b was dispensable. Of note, lack of Dnmt1 resulted in a slight decrease in CG methylation, suggesting that this maintenance enzyme plays a role in non-dividing oocytes. Dnmt1 may act on CG sites that remain hemimethylated in the de novo methylation process. Our results provide a basis for understanding the mechanisms and significance of non-CG methylation in mammalian oocytes.
Author Summary
Methylation of cytosine bases in DNA is an epigenetic modification crucial for normal development, retrotransposon silencing, and cellular reprogramming. In mammals, the vast majority of 5-methylcytosine occurs at CG dinucleotides, and thus most studies to date have focused on this dinucleotide. However, recent studies have shown that 5-methylcytosine is abundant at non-CG (CA, CT, and CC) sites in certain tissues and certain cell types in human and mouse. We previously identified non-CG methylation in CG-rich sequences, including the imprint control regions in mouse germinal vesicle oocytes, but its global distribution and the enzymes responsible are unknown. Using advanced high-throughput sequencing technology applicable to minute amounts of DNA, we obtained high-resolution methylation maps of newborn non-growing oocytes, adult germinal vesicle oocytes, and mutant germinal vesicle oocytes lacking any of the four DNA methyltransferase family proteins. Our results revealed that non-CG methylation accumulates genome-wide in close proximity to highly methylated CG sites during the oocyte growth stage. We also found that the de novo DNA methyltransferase proteins Dnmt3a and Dnmt3L are responsible for non-CG methylation in oocytes. Unexpectedly, we found that the maintenance methyltransferase Dnmt1 has a role in de novo CG methylation. Our study provides a basis for understanding the mechanisms and significance of non-CG methylation in mammalian oocytes.
doi:10.1371/journal.pgen.1003439
PMCID: PMC3630097  PMID: 23637617
9.  Investigating the Potential Role of Genetic and Epigenetic Variation of DNA Methyltransferase Genes in Hyperplastic Polyposis Syndrome 
PLoS ONE  2011;6(2):e16831.
Background
Hyperplastic Polyposis Syndrome (HPS) is a condition associated with multiple serrated polyps, and an increased risk of colorectal cancer (CRC). At least half of CRCs arising in HPS show a CpG island methylator phenotype (CIMP), potentially linked to aberrant DNA methyltransferase (DNMT) activity. CIMP is associated with methylation of tumor suppressor genes including regulators of DNA mismatch repair (such as MLH1, MGMT), and negative regulators of Wnt signaling (such as WIF1). In this study, we investigated the potential for interaction of genetic and epigenetic variation in DNMT genes, in the aetiology of HPS.
Methods
We utilized high resolution melting (HRM) analysis to screen 45 cases with HPS for novel sequence variants in DNMT1, DNMT3A, DNMT3B, and DNMT3L. 21 polyps from 13 patients were screened for BRAF and KRAS mutations, with assessment of promoter methylation in the DNMT1, DNMT3A, DNMT3B, DNMT3L MLH1, MGMT, and WIF1 gene promoters.
Results
No pathologic germline mutations were observed in any DNA-methyltransferase gene. However, the T allele of rs62106244 (intron 10 of DNMT1 gene) was over-represented in cases with HPS (p<0.01) compared with population controls. The DNMT1, DNMT3A and DNMT3B promoters were unmethylated in all instances. Interestingly, the DNMT3L promoter showed low levels of methylation in polyps and normal colonic mucosa relative to matched disease free cells with methylation level negatively correlated to expression level in normal colonic tissue. DNMT3L promoter hypomethylation was more often found in polyps harbouring KRAS mutations (p = 0.0053). BRAF mutations were common (11 out of 21 polyps), whilst KRAS mutations were identified in 4 of 21 polyps.
Conclusions
Genetic or epigenetic alterations in DNMT genes do not appear to be associated with HPS, but further investigation of genetic variation at rs62106244 is justified given the high frequency of the minor allele in this case series.
doi:10.1371/journal.pone.0016831
PMCID: PMC3037390  PMID: 21347319
10.  Tumor Suppressor Gene Inactivation during Cadmium-Induced Malignant Transformation of Human Prostate Cells Correlates with Overexpression of de Novo DNA Methyltransferase 
Environmental Health Perspectives  2007;115(10):1454-1459.
Background
Aberrant DNA methylation is common in carcinogenesis. The typical pattern appears to involve reduced expression of maintenance DNA methyltransferase, DNMT1, inducing genomic hypomethylation, whereas increased expression of de novo DNMT3a or 3b causes gene-specific hypermethylation.
Objectives
During cadmium-induced malignant transformation, an unusual pattern of genomic hypermethylation occurred that we studied to provide insight into the roles of specific DNMTs in oncogenesis.
Methods
Gene expression and DNA methylation were assessed in control and chronic cadmium-transformed prostate epithelial cells (CTPE) using reverse transcription–polymerase chain reaction (RT-PCR), Western blot analysis, methylation-specific PCR, and methyl acceptance assay.
Results
During the 10-weeks of cadmium exposure that induced malignant transformation, progressive increases in generalized DNMT enzymatic activity occurred that were associated with over-expression of DNMT3b without changes in DNMT1 expression. Increased DNMT3b expression preceded increased DNMT enzymatic activity. Procainamide, a specific DNMT1 inhibitor, reversed cadmium-induced genomic DNA hypermethylation. Reduced expression of the tumor suppressor genes, RASSF1A and p16, began about the time DNMT3b overexpression first occurred and progressively decreased thereafter. RASSF1A and p16 promoter regions were heavily methylated in CTPE cells, indicating silencing by hypermethylation, while the DNA demethylating agent, 5-aza-2′-deoxycytidine, reversed this silencing. DNMT1 inhibition only modestly increased RASSF1A and p16 expression in CTPE cells and did not completely reverse silencing.
Conclusions
These data indicate that DNMT3b overexpression can result in generalized DNA hypermethylation and gene silencing but that DNMT1 is required to maintain these effects. The pattern of genomic DNA hypermethylation together with up-regulation of DNMT3b may provide a unique set of biomarkers to specifically identify cadmium-induced human prostate cancers.
doi:10.1289/ehp.10207
PMCID: PMC2022656  PMID: 17938735
cadmium; carcinogenesis; DNA methylation; DNMT3b; p16; prostate; RASSF1A
11.  Clinicopathological Significance and Prognostic Value of DNA Methyltransferase 1, 3a, and 3b Expressions in Sporadic Epithelial Ovarian Cancer 
PLoS ONE  2012;7(6):e40024.
Altered DNA methylation of tumor suppressor gene promoters plays a role in human carcinogenesis and DNA methyltransferases (DNMTs) are responsible for it. This study aimed to determine aberrant expression of DNMT1, DNMT3a, and DNMT3b in benign and malignant ovarian tumor tissues for their association with clinicopathological significance and prognostic value. A total of 142 ovarian cancers and 44 benign ovarian tumors were recruited for immunohistochemical analysis of their expression. The data showed that expression of DNMT1, DNMT3a, and DNMT3b was observed in 76 (53.5%), 92 (64.8%) and 79 (55.6%) of 142 cases of ovarian cancer tissues, respectively. Of the serious tumors, DNMT3a protein expression was significantly higher than that in benign tumor samples (P = 0.001); DNMT3b was marginally significant down regulated in ovarian cancers compared to that of the benign tumors (P = 0.054); DNMT1 expression has no statistical difference between ovarian cancers and benign tumor tissues (P = 0.837). Of the mucious tumors, the expression of DNMT3a, DNMT3b, and DNMT1 was not different between malignant and benign tumors. Moreover, DNMT1 expression was associated with DNMT3b expression (P = 0.020, r = 0.195). DNMT1 expression was associated with age of the patients, menopause status, and tumor localization, while DNMT3a expression was associated with histological types and serum CA125 levels and DNMT3b expression was associated with lymph node metastasis. In addition, patients with DNMT1 or DNMT3b expression had a trend of better survival than those with negative expression. Co-expression of DNMT1 and DNMT3b was significantly associated with better overall survival (P = 0.014). The data from this study provided the first evidence for differential expression of DNMTs proteins in ovarian cancer tissues and their associations with clinicopathological and survival data in sporadic ovarian cancer patients.
doi:10.1371/journal.pone.0040024
PMCID: PMC3386927  PMID: 22768205
12.  Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation* 
The Journal of Biological Chemistry  2014;289(12):8277-8287.
Background: SET7 monomethylates DNMT1, promoting its proteasomal degradation, yet methylated DNMT1 still remains throughout the cell cycle.
Results: The methyllysine reader PHF20L1 stabilizes methylated DNMT1. Disruption of PHF20L1 induces DNMT1 degradation and genome hypomethylation.
Conclusion: PHF20L1, an epigenetic reader, cooperates with writer and eraser to regulate epigenetic inheritance.
Significance: PHF20L1 can be targeted as a means of regulating DNMT1 activity and DNA methylation in cells.
Inheritance of DNA cytosine methylation pattern during successive cell division is mediated by maintenance DNA (cytosine-5) methyltransferase 1 (DNMT1). Lysine 142 of DNMT1 is methylated by the SET domain containing lysine methyltransferase 7 (SET7), leading to its degradation by proteasome. Here we show that PHD finger protein 20-like 1 (PHF20L1) regulates DNMT1 turnover in mammalian cells. Malignant brain tumor (MBT) domain of PHF20L1 binds to monomethylated lysine 142 on DNMT1 (DNMT1K142me1) and colocalizes at the perinucleolar space in a SET7-dependent manner. PHF20L1 knockdown by siRNA resulted in decreased amounts of DNMT1 on chromatin. Ubiquitination of DNMT1K142me1 was abolished by overexpression of PHF20L1, suggesting that its binding may block proteasomal degradation of DNMT1K142me1. Conversely, siRNA-mediated knockdown of PHF20L1 or incubation of a small molecule MBT domain binding inhibitor in cultured cells accelerated the proteasomal degradation of DNMT1. These results demonstrate that the MBT domain of PHF20L1 reads and controls enzyme levels of methylated DNMT1 in cells, thus representing a novel antagonist of DNMT1 degradation.
doi:10.1074/jbc.M113.525279
PMCID: PMC3961655  PMID: 24492612
DNA-binding Protein; DNA Methylation; DNA Methyltransferase; Protein Degradation; Protein Methylation; MBT Domain; PHF20L1; SET7; UNC1215
13.  Nucleosomes Containing Methylated DNA Stabilize DNA Methyltransferases 3A/3B and Ensure Faithful Epigenetic Inheritance 
PLoS Genetics  2011;7(2):e1001286.
How epigenetic information is propagated during somatic cell divisions is still unclear but is absolutely critical for preserving gene expression patterns and cellular identity. Here we show an unanticipated mechanism for inheritance of DNA methylation patterns where the epigenetic mark not only recruits the catalyzing enzyme but also regulates the protein level, i.e. the enzymatic product (5-methylcytosine) determines the level of the methylase, thus forming a novel homeostatic inheritance system. Nucleosomes containing methylated DNA stabilize de novo DNA methyltransferases, DNMT3A/3B, allowing little free DNMT3A/3B enzymes to exist in the nucleus. Stabilization of DNMT3A/3B on nucleosomes in methylated regions further promotes propagation of DNA methylation. However, reduction of cellular DNA methylation levels creating more potential CpG substrates counter-intuitively results in a dramatic decrease of DNMT3A/3B proteins due to diminished nucleosome binding and subsequent degradation of the unstable free proteins. These data show an unexpected self-regulatory inheritance mechanism that not only ensures somatic propagation of methylated states by DNMT1 and DNMT3A/3B enzymes but also prevents aberrant de novo methylation by causing degradation of free DNMT3A/3B enzymes.
Author Summary
Proper inheritance of DNA methylation patterns is essential for preserving cellular identity and preventing malignant cellular transformation. In mammals, DNMT3A/3B, the de novo methyltransferases, establish the DNA methylation patterns during development and then maintain them in co-operation with the maintenance methyltransferase, DNMT1, through cell divisions. However, the mechanisms by which DNMT3A/3B assist DNMT1 in faithful inheritance of methylation patterns in somatic cells while guarding against aberrant de novo DNA methylation are still unclear. In this study, we present a novel principle of enzyme regulation where the levels of the catalyzing enzymes, DNMT3A/3B, are determined by the level of their own enzymatic product, i.e. 5-methylcytosine itself. Through biochemical analyses, we have shown that binding of DNMT3A/3B to nucleosomes with methylated DNA stabilizes these proteins, enabling faithful propagation of methylation patterns through cell divisions. However, reduction in DNA methylation results in diminished nucleosome binding of DNMT3A/3B and subsequent degradation of the free DNMT3A/3B proteins. This novel self-regulatory inheritance mechanism not only ensures faithful somatic propagation of methylated states but also prevents aberrant de novo methylation by causing degradation of free DNMT3A/3B enzymes.
doi:10.1371/journal.pgen.1001286
PMCID: PMC3033376  PMID: 21304883
14.  A novel DNMT3B splice variant expressed in tumor and pluripotent cells modulates genomic DNA methylation patterns and displays altered DNA binding 
Molecular cancer research : MCR  2009;7(10):1622-1634.
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 N-terminal regulatory domain. This variant, which we term DNMT3B3Δ5 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 DNMT3B3Δ5. DNMT3B3Δ5 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 DNMT3B3Δ5 versus DNMT3B3, revealing that DNMT3B3Δ5 possessed significantly enhanced DNA binding affinity and displayed an altered nuclear distribution. Finally, ectopic overexpression of DNMT3B3Δ5 resulted in repetitive element hypomethylation and enhanced cell growth in a colony formation assay. Taken together, these results demonstrate that DNMT3B3Δ5 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.
doi:10.1158/1541-7786.MCR-09-0018
PMCID: PMC2783805  PMID: 19825994
15.  Essential Role for Dnmt1 in the Prevention and Maintenance of MYC-Induced T-Cell Lymphomas 
Molecular and Cellular Biology  2013;33(21):4321-4333.
DNA cytosine methylation is an epigenetic modification involved in the transcriptional repression of genes controlling a variety of physiological processes, including hematopoiesis. DNA methyltransferase 1 (Dnmt1) is a key enzyme involved in the somatic inheritance of DNA methylation and thus plays a critical role in epigenomic stability. Aberrant methylation contributes to the pathogenesis of human cancer and of hematologic malignancies in particular. To gain deeper insight into the function of Dnmt1 in lymphoid malignancies, we genetically inactivated Dnmt1 in a mouse model of MYC-induced T-cell lymphomagenesis. We show that loss of Dnmt1 delays lymphomagenesis by suppressing normal hematopoiesis and impairing tumor cell proliferation. Acute inactivation of Dnmt1 in primary lymphoma cells rapidly induced apoptosis, indicating that Dnmt1 is required to sustain T-cell lymphomas. Using high-resolution genome-wide profiling, we identified differentially methylated regions between control and Dnmt1-deficient lymphomas, demonstrating a locus-specific function for Dnmt1 in both maintenance and de novo promoter methylation. Dnmt1 activity is independent of the presence of Dnmt3a or Dnmt3b in de novo promoter methylation of the H2-Ab1 gene. Collectively, these data show for the first time that Dnmt1 is critical for the prevention and maintenance of T-cell lymphomas and contributes to aberrant methylation by both de novo and maintenance methylation.
doi:10.1128/MCB.00776-13
PMCID: PMC3811897  PMID: 24001767
16.  FOXM1 Induces a Global Methylation Signature That Mimics the Cancer Epigenome in Head and Neck Squamous Cell Carcinoma 
PLoS ONE  2012;7(3):e34329.
The oncogene FOXM1 has been implicated in all major types of human cancer. We recently showed that aberrant FOXM1 expression causes stem cell compartment expansion resulting in the initiation of hyperplasia. We have previously shown that FOXM1 regulates HELLS, a SNF2/helicase involved in DNA methylation, implicating FOXM1 in epigenetic regulation. Here, we have demonstrated using primary normal human oral keratinocytes (NOK) that upregulation of FOXM1 suppressed the tumour suppressor gene p16INK4A (CDKN2A) through promoter hypermethylation. Knockdown of HELLS using siRNA re-activated the mRNA expression of p16INK4A and concomitant downregulation of two DNA methyltransferases DNMT1 and DNMT3B. The dose-dependent upregulation of endogenous FOXM1 (isoform B) expression during tumour progression across a panel of normal primary NOK strains (n = 8), dysplasias (n = 5) and head and neck squamous cell carcinoma (HNSCC) cell lines (n = 11) correlated positively with endogenous expressions of HELLS, BMI1, DNMT1 and DNMT3B and negatively with p16INK4A and involucrin. Bisulfite modification and methylation-specific promoter analysis using absolute quantitative PCR (MS-qPCR) showed that upregulation of FOXM1 significantly induced p16INK4A promoter hypermethylation (10-fold, P<0.05) in primary NOK cells. Using a non-bias genome-wide promoter methylation microarray profiling method, we revealed that aberrant FOXM1 expression in primary NOK induced a global hypomethylation pattern similar to that found in an HNSCC (SCC15) cell line. Following validation experiments using absolute qPCR, we have identified a set of differentially methylated genes, found to be inversely correlated with in vivo mRNA expression levels of clinical HNSCC tumour biopsy samples. This study provided the first evidence, using primary normal human cells and tumour tissues, that aberrant upregulation of FOXM1 orchestrated a DNA methylation signature that mimics the cancer methylome landscape, from which we have identified a unique FOXM1-induced epigenetic signature which may have clinical translational potentials as biomarkers for early cancer screening, diagnostic and/or therapeutic interventions.
doi:10.1371/journal.pone.0034329
PMCID: PMC3312909  PMID: 22461910
17.  Haploinsufficiency of the paternal-effect gene Dnmt3L results in transient DNA hypomethylation in progenitor cells of the male germline 
STUDY QUESTION
How does haploinsufficiency of the paternal-effect gene Dnmt3L affect DNA methylation establishment and stability in the male germline?
SUMMARY ANSWER
Reduced expression of DNMT3L in male germ cells, associated with haploinsufficiency of the paternal-effect gene Dnmt3L, results in abnormal hypomethylation of prenatal germline progenitor cells.
WHAT IS KNOWN ALREADY
The DNA methyltransferase regulator Dnmt3-Like (Dnmt3L) is a paternal-effect gene required for DNA methylation acquisition in male germline stem cells and their precursors. In males, DNMT3L deficiency causes meiotic abnormalities and infertility. While Dnmt3L heterozygous males are fertile, they have abnormalities in X chromosome compaction and postmeiotic gene expression and sire offspring with sex chromosome aneuploidy. It has been proposed that the paternal effects of Dnmt3L haploinsufficiency are due to epigenetic defects in early male germ cells. DNA methylation is an essential epigenetic modification essential for normal germ cell development. Since patterns of DNA methylation across the genome are initially acquired in prenatal male germ cells, perturbations in methylation could contribute to the epigenetic basis of the paternal effects in Dnmt3L+/− males.
STUDY DESIGN, SIZE, DURATION
This is a cross-sectional study of DNA methylation in Dnmt3L+/+ versus Dnmt3L+/− male germ cells collected from mice at 16.5 days post-coitum (dpc), Day 6 and Day 70 (n = 3 per genotype, each n represents a pool of 2–20 animals). Additionally, DNA methylation was compared in enriched populations of spermatogonial stem cells (SSC)/progenitor cells from Dnmt3L+/+ and Dnmt3L+/− males following ∼2 months in culture.
MATERIALS, SETTING, METHODS
DNA methylation at intergenic loci along chromosomes 9 and X was examined by quantitative analysis of DNA methylation by real-time polymerase chain reaction at the time of initial acquisition of epigenetic patterns in the prenatal male germline (16.5 dpc) and compared with patterns in early post-natal spermatogonia (Day 6) and in spermatozoa in mice. DNA methylation status at CpG-rich sites across the genome was assessed in spermatogonial precursors from Day 4 male mice using restriction landmark genomic scanning.
MAIN RESULTS AND THE ROLE OF CHANCE
At 16.5 dpc, 42% of intergenic loci examined along chromosome 9 and 10% of those along chromosome X were hypomethylated in Dnmt3L heterozygotes. By Day 6 and in spermatozoa, germ cell DNA methylation was similar in heterozygous and wild-type mice. DNA methylation stability of acquired patterns in wild-type and Dnmt3L+/− SSC/progenitor cell culture was analyzed at numerous loci across the genome in cells cultured in vitro and collected at passages 6–28. While the methylation of most loci was stable in culture over time, differences at ∼1% of sites were found between Dnmt3L+/− and Dnmt3L+/+ cultures.
LIMITATIONS, REASONS FOR CAUTION
Evaluation of DNA methylation in SSCs can only be performed after a period of culture limiting the investigation to changes observed during culture when compared with DNA methylation differences between genotypes that could be present at the beginning of culture establishment.
WIDER IMPLICATIONS OF THE FINDINGS
The DNA methylation defects described here in prenatal male germline progenitor cells and SSC culture are the earliest epigenetic perturbations yet identified for a mammalian paternal-effect gene and may influence downstream epigenetic events in germ cells at later stages of development. Together, the results provide evidence of a ‘window’ of susceptibility in prenatal male germ cell precursors for the induction of epimutations due to genetic perturbations and, potentially, in utero environmental exposures.
STUDY FUNDING/COMPETING INTEREST(S)
Canadian Institutes of Health Research (CIHR) provided funding for J.M.T. (MOP229913) and M.C.N. (MOP86532). The authors have no conflicts of interest to declare.
doi:10.1093/humrep/des395
PMCID: PMC3695691  PMID: 23159436
epigenetics; DNA methylation; Dnmt3L; spermatogonial stem cells; paternal effect
18.  The De Novo Methyltransferases DNMT3a and DNMT3b Target the Murine Gammaherpesvirus Immediate-Early Gene 50 Promoter during Establishment of Latency▿  
Journal of Virology  2010;84(10):4946-4959.
The role of epigenetic modifications in the regulation of gammaherpesvirus latency has been a subject of active study for more than 20 years. DNA methylation, associated with transcriptional silencing in mammalian genomes, has been shown to be an important mechanism in the transcriptional control of several key gammaherpesvirus genes. In particular, DNA methylation of the functionally conserved immediate-early replication and transcription activator (RTA) has been shown to regulate Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus Rta expression. Here we demonstrate that the murine gammaherpesvirus (MHV68) homolog, encoded by gene 50, is also subject to direct repression by DNA methylation, both in vitro and in vivo. We observed that the treatment of latently MHV68-infected B-cell lines with a methyltransferase inhibitor induced virus reactivation. In addition, we show that the methylation of the recently characterized distal gene 50 promoter represses activity in a murine macrophage cell line. To evaluate the role of de novo methyltransferases (DNMTs) in the establishment of these methylation marks, we infected mice in which conditional DNMT3a and DNMT3b alleles were selectively deleted in B lymphocytes. DNMT3a/DNMT3b-deficient B cells were phenotypically normal, displaying no obvious compromise in cell surface marker expression or antibody production either in naïve mice or in the context of nonviral and viral immunogens. However, mice lacking functional DNMT3a and DNMT3b in B cells exhibited hallmarks of deregulated MHV68 lytic replication, including increased splenomegaly and the presence of infectious virus in the spleen at day 18 following infection. In addition, total gene 50 transcript levels were elevated in the spleens of these mice at day 18, which correlated with the hypomethylation of the distal gene 50 promoter. However, by day 42 postinfection, aberrant virus replication was resolved, and we observed wild-type frequencies of viral genome-positive splenocytes in mice lacking functional DNMT3a and DNMT3b in B lymphocytes. The latter correlated with increased CpG methylation in the distal gene 50 promoter, which was restored to levels similar to those of littermate controls harboring functional DNMT3a and DNMT3b alleles in B lymphocytes, suggesting the existence of an alternative mechanism for the de novo methylation of the MHV68 genome. Importantly, this DNMT3a/DNMT3b-independent methylation appeared to be targeted specifically to the gene 50 promoter, as we observed that the promoters for MHV68 gene 72 (v-cyclin) and M11 (v-bcl2) remained hypomethylated at day 42 postinfection. Taken together, these data provide the first evidence of the importance of DNA methylation in regulating gammaherpesvirus RTA/gene 50 transcription during virus infection in vivo and provide insight into the hierarchy of host machinery required to establish this modification.
doi:10.1128/JVI.00060-10
PMCID: PMC2863815  PMID: 20200245
19.  Low-Level Environmental Cadmium Exposure Is Associated with DNA Hypomethylation in Argentinean Women 
Environmental Health Perspectives  2012;120(6):879-884.
Background: Cadmium, a common food pollutant, alters DNA methylation in vitro. Epigenetic effects might therefore partly explain cadmium’s toxicity, including its carcinogenicity; however, human data on epigenetic effects are lacking.
Objective: We evaluated the effects of dietary cadmium exposure on DNA methylation, considering other environmental exposures, genetic predisposition, and gene expression.
Methods: Concentrations of cadmium, arsenic, selenium, and zinc in blood and urine of nonsmoking women (n = 202) from the northern Argentinean Andes were measured by inductively coupled mass spectrometry. Methylation in CpG islands of LINE-1 (long interspersed nuclear element-1; a proxy for global DNA methylation) and promoter regions of p16 [cyclin-dependent kinase inhibitor 2A (CDKN2A)] and MLH1 (mutL homolog 1) in peripheral blood were measured by bisulfite polymerase chain reaction pyrosequencing. Genotyping (n = 172) for the DNA (cytosine-5-)-methyltransferase 1 gene (DNMT1 rs10854076 and rs2228611) and DNA (cytosine-5-)-methyltransferase 3 beta gene (DNMT3B rs2424913 and rs2424932) was performed with Sequenom iPLEX GOLD SNP genotyping; and gene expression (n = 90), with DirectHyb HumanHT-12 (version 3.0).
Results: Cadmium exposure was low: median concentrations in blood and urine were 0.36 and 0.23 µg/L, respectively. Urinary cadmium (natural log transformed) was inversely associated with LINE-1 methylation (β = –0.50, p = 0.0070; β = –0.44, p = 0.026, adjusted for age and coca chewing) but not with p16 or MLH1 methylation. Both DNMT1 rs10854076 and DNMT1 rs2228611 polymorphisms modified associations between urinary cadmium and LINE-1 (p-values for interaction in adjusted models were 0.045 and 0.064, respectively). The rare genotypes demonstrated stronger hypomethylation with increasing urinary cadmium concentrations. Cadmium was inversely associated with DNMT3B (rS = –0.28, p = 0.0086) but not with DNMT1 expression (rS = –0.075, p = 0.48).
Conclusion: Environmental cadmium exposure was associated with DNA hypomethylation in peripheral blood, and DNMT1 genotypes modified this association. The role of epigenetic modifications in cadmium-associated diseases needs clarification.
doi:10.1289/ehp.1104600
PMCID: PMC3385444  PMID: 22382075
cadmium; DNMT1; DNMT3B; epigenetic; genotype; LINE-1; MLH1; p16; pyrosequencing
20.  Transition of LINE-1 DNA Methylation Status and Altered Expression in First and Third Trimester Placentas 
PLoS ONE  2014;9(5):e96994.
DNA methylation plays a critical role in the regulation of gene expression, genomic DNA stability, cell proliferation, and malignant transformation. Common cellular features including fast tissue expansion, invasive growth, and active angiogenesis, have been noticed between placental development and tumorigenesis by many investigators. While the DNA hypomethylation and transcriptional activation of LINE-1 has been found to be a feature of tumorigenesis, it is not clear if similar changes could be involved in placental development. In this study, we assessed LINE-1 methylation in human placentas from different gestational ages and observed a significant decrease of LINE-1 methylation levels in third trimester placentas compared to first trimester placentas. Accompanying with this change is the significantly increased LINE-1 mRNA levels in third trimester placentas. Since no global DNA methylation change was detected between first and third trimesters, LINE-1 methylation changes appeared to be a specific epigenetic entity contributing to placental development. Indeed, further analyses showed that LINE-1 upregulation was correlated with higher levels of PCNA, suggesting a link between LINE-1 activation and fast proliferation of certain cellular components in third trimester placentas. Measurement of the DNMT1, DNMT3A, and DNMT3B expression found a significant reduction of DNMT3B between third and first trimesters, pointing to the possible involvement of this enzyme in the regulation of LINE-1 methylation. Taken together these results provided evidence for a dynamic temporal regulation of LINE-1 methylation and activation during placental development. These studies have laid a foundation for future investigation on the function of LINE-1 expression in human placenta under different patho-physiological conditions.
doi:10.1371/journal.pone.0096994
PMCID: PMC4018393  PMID: 24821186
21.  Mouse ES cells overexpressing DNMT1 produce abnormal neurons with upregulated NR1 
High levels of DNA methyltransferase 1 (DNMT1), hypermethylation, and downregulation of GAD67 and reelin have been described in GABAergic interneurons of patients with schizophrenia (SZ) and bipolar (BP) disorders. However, overexpression of DNMT1 is lethal, making it difficult to assess the direct effect of high levels of DNMT1 on neuronal development in vivo. We therefore used Dnmt1tet/tet mouse ES cells that overexpress DNMT1 as an in vitro model to investigate the impact of high levels of DNMT1 on neuronal differentiation. Although there is down-regulation of DNMT1 during early stages of differentiation in wild type and Dnmt1tet/tet ES cell lines, neurons derived from Dnmt1tet/tet cells showed abnormal dendritic arborization and branching. The Dnmt1tet/tet neuronal cells also showed elevated levels of functional N-methyl D-aspartate receptor (NMDAR), a feature also reported in some neurological and neurodegenerative disorders. Considering the roles of reelin and GAD67 in neuronal networking and excitatory/inhibitory balance respectively, we studied methylation of these genes' promoters in Dnmt1tet/tet ES cells and neurons. Both reelin and GAD67 promoters were not hypermethylated in the Dnmt1tet/tet ES cells and neurons, suggesting that overexpression of DNMT1 may not directly result in methylation-mediated repression of these two genes. Taken together, our results suggest that overexpression of DNMT1 in ES cells results in an epigenetic change prior to the onset of differentiation. This epigenetic change in turn results in abnormal neuronal differentiation and upregulation of functional NMDA receptor.
doi:10.1016/j.diff.2011.03.003
PMCID: PMC3115397  PMID: 21492995
DNA methylation; Dnmt1; neuronal differentiation; NMDA receptor; epigenetic
22.  Expression of DNMT1 and DNMT3a Are Regulated by GLI1 in Human Pancreatic Cancer 
PLoS ONE  2011;6(11):e27684.
Background and Aims
GLI1, as an indispensable transcriptional factor of Hedgehog signaling pathway, plays an important role in the development of pancreatic cancer (PC). DNA methyltransferases (DNMTs) mediate the methylation of quantity of tumor-related genes. Our study aimed to explore the relationship between GLI1 and DNMTs.
Methods
Expressions of GLI1 and DNMTs were detected in tumor and adjacent normal tissues of PC patients by immunohistochemistry (IHC). PANC-1 cells were treated by cyclopamine and GLI1-siRNA, while BxPC-3 cells were transfected with overexpression-GLI1 lentiviral vector. Then GLI1 and DNMTs expression were analyzed by qRT-PCR and western blot (WB). Then we took chromatin immunoprecipitation (ChIP) to demonstrate GLI1 bind to DNMT1. Finally, nested MSP was taken to valuate the methylation levels of APC and hMLH1, when GLI1 expression altered.
Results
IHC result suggested the expressions of GLI1, DNMT1 and DNMT3a in PC tissues were all higher than those in adjacent normal tissues (p<0.05). After GLI1 expression repressed by cyclopamine in mRNA and protein level (down-regulation 88.1±2.2%, 86.4±2.2%, respectively), DNMT1 and DNMT3a mRNA and protein level decreased by 91.6%±2.2% and 83.8±4.8%, 87.4±2.7% and 84.4±1.3%, respectively. When further knocked down the expression of GLI1 by siRNA (mRNA decreased by 88.6±2.1%, protein decreased by 63.5±4.5%), DNMT1 and DNMT3a mRNA decreased by 80.9±2.3% and 78.6±3.8% and protein decreased by 64.8±2.8% and 67.5±5.6%, respectively. Over-expression of GLI1 by GLI1 gene transfection (mRNA increased by 655.5±85.9%, and protein increased by 272.3±14.4%.), DNMT1 and DNMT3a mRNA and protein increased by 293.0±14.8% and 578.3±58.5%, 143.5±17.4% and 214.0±18.9%, respectively. ChIP assays showed GLI1 protein bound to DNMT1 but not to DNMT3a. Results of nested MSP demonstrated GLI1 expression affected the DNA methylation level of APC but not hMLH1 in PC.
Conclusion
DNMT1 and DNMT3a are regulated by GLI1 in PC, and DNMT1 is its direct target gene.
doi:10.1371/journal.pone.0027684
PMCID: PMC3215738  PMID: 22110720
23.  DNMT1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells 
The Journal of Cell Biology  2007;176(5):565-571.
DNA methylation plays a central role in the epigenetic regulation of gene expression in vertebrates. Genetic and biochemical data indicated that DNA methyltransferase 1 (Dnmt1) is indispensable for the maintenance of DNA methylation patterns in mice, but targeting of the DNMT1 locus in human HCT116 tumor cells had only minor effects on genomic methylation and cell viability. In this study, we identified an alternative splicing in these cells that bypasses the disrupting selective marker and results in a catalytically active DNMT1 protein lacking the proliferating cell nuclear antigen–binding domain required for association with the replication machinery. Using a mechanism-based trapping assay, we show that this truncated DNMT1 protein displays only twofold reduced postreplicative DNA methylation maintenance activity in vivo. RNA interference–mediated knockdown of this truncated DNMT1 results in global genomic hypomethylation and cell death. These results indicate that DNMT1 is essential in mouse and human cells, but direct coupling of the replication of genetic and epigenetic information is not strictly required.
doi:10.1083/jcb.200610062
PMCID: PMC2064015  PMID: 17312023
24.  Loss of Dnmt3b function upregulates the tumor modifier Ment and accelerates mouse lymphomagenesis 
DNA methyltransferase 3B (Dnmt3b) belongs to a family of enzymes responsible for methylation of cytosine residues in mammals. DNA methylation contributes to the epigenetic control of gene transcription and is deregulated in virtually all human tumors. To better understand the generation of cancer-specific methylation patterns, we genetically inactivated Dnmt3b in a mouse model of MYC-induced lymphomagenesis. Ablation of Dnmt3b function using a conditional knockout in T cells accelerated lymphomagenesis by increasing cellular proliferation, which suggests that Dnmt3b functions as a tumor suppressor. Global methylation profiling revealed numerous gene promoters as potential targets of Dnmt3b activity, the majority of which were demethylated in Dnmt3b–/– lymphomas, but not in Dnmt3b–/– pretumor thymocytes, implicating Dnmt3b in maintenance of cytosine methylation in cancer. Functional analysis identified the gene Gm128 (which we termed herein methylated in normal thymocytes [Ment]) as a target of Dnmt3b activity. We found that Ment was gradually demethylated and overexpressed during tumor progression in Dnmt3b–/– lymphomas. Similarly, MENT was overexpressed in 67% of human lymphomas, and its transcription inversely correlated with methylation and levels of DNMT3B. Importantly, knockdown of Ment inhibited growth of mouse and human cells, whereas overexpression of Ment provided Dnmt3b+/+ cells with a proliferative advantage. Our findings identify Ment as an enhancer of lymphomagenesis that contributes to the tumor suppressor function of Dnmt3b and suggest it could be a potential target for anticancer therapies.
doi:10.1172/JCI57292
PMCID: PMC3248285  PMID: 22133874
25.  Epigenetic Regulation of Dpp6 Expression by Dnmt3b and Its Novel Role in the Inhibition of RA Induced Neuronal Differentiation of P19 Cells 
PLoS ONE  2013;8(2):e55826.
DNA methylation is an important mechanism of gene silencing in mammals catalyzed by a group of DNA methyltransferases including Dnmt1, Dnmt3a, and Dnmt3b which are required for the establishment of genomic methylation patterns during development and differentiation. In this report, we studied the role of DNA methyltransferases during retinoic acid induced neuronal differentiation of P19 cells. We observed an increase in the mRNA and protein level of Dnmt3b, whereas the expression of Dnmt1 and Dnmt3a was decreased after RA treatment of P19 cells which indicated that Dnmt3b is more important during neuronal differentiation of P19 cells. Dnmt3b enriched chromatin library from RA treated P19 cells identified dipeptidyl peptidase 6 (Dpp6) gene as a novel target of Dnmt3b. Further, quantitative ChIP analysis showed that the amount of Dnmt3b recruited on Dpp6 promoter was equal in both RA treated as well as untreated p19 cells. Bisulfite genomic sequencing, COBRA, and methylation specific PCR analysis revealed that Dpp6 promoter was heavily methylated in both RA treated and untreated P19 cells. Dnmt3b was responsible for transcriptional silencing of Dpp6 gene as depletion of Dnmt3b resulted in increased mRNA and protein expression of Dpp6. Consequently, the average methylation of Dpp6 gene promoter was reduced to half in Dnmt3b knockdown cells. In the absence of Dnmt3b, Dnmt3a was associated with Dpp6 gene promoter and regulated its expression and methylation in P19 cells. RA induced neuronal differentiation was inhibited upon ectopic expression of Dpp6 in P19 cells. Taken together, the present study described epigenetic silencing of Dpp6 expression by DNA methylation and established that its ectopic expression can act as negative signal during RA induced neuronal differentiation of P19 cells.
doi:10.1371/journal.pone.0055826
PMCID: PMC3567024  PMID: 23409053

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