microRNA-199a-3p,DNMT3A,and aberrant DNA methylation in testicular cancer

It was previously demonstrated that miR-199a was downregulated in testicular germ cell tumor (TGCT), probably due to hypermethylation of its promoter. Further study found that re-expression of miR-199a in testicular cancer cells (NT2) led to suppression of cell growth, cancer migration, invasion and metastasis. More detailed analyses showed that these properties of miR-199a could be assigned to miR-199a-5p, one of its two derivatives. The biological role of the other derivative, miR-199a-3p in TGCT, remains largely uncharacterized. In this report, we identified DNA (cytosine-5)-methyltransferase 3A (DNMT3A), the de novo methyltransferase, as a direct target of miR-199a-3p using a 3′-UTR reporter assay. Transient expression of miR-199a-3p in NT2 cells led to decrease, while knocking down of miR-199a-3p in a normal human testicular cell line (HT) led to elevation, of DNMT3A2 (DNMT3A gene isoform 2) mRNA and protein levels. In clinical samples, DNMT3A2 was significantly overexpressed in malignant testicular tumor, and the expression of DNMT3A2 was inversely correlated with the expression of miR-199a-3p. However, DNMT3A did not affect miR-199a expression in NT2 cells. Further characterization of miR-199a-3p revealed that it negatively regulated DNA methylation, partly through targeting DNMT3A. Overexpression of miR-199a-3p restored the expression of APC and MGMT tumor-suppressor genes in NT2 cells by affecting DNA methylation of their promoter regions. Our studies demonstrated the deregulation of miR-199a-3p expression in TGCT may provide novel mechanistic insights into TGCT carcinogenesis and suggested a potentially therapeutic use of synthetic miR-199a-3p oligonucleotides as effective hypomethylating compounds in the treatment of TGCT.     

Oligomerization of DNMT3A controls the mechanism of de novo DNA methylation

DNMT3A is one of two human de novo DNA methyltransferases essential for regulating gene expression through cellular development and differentiation. Here we describe the consequences of single amino acid mutations, including those implicated in the development of acute myeloid leukemia (AML) and myelodysplastic syndromes, at the DNMT3A·DNMT3A homotetramer and DNMT3A·DNMT3L heterotetramer interfaces. A model for the DNMT3A homotetramer was developed via computational interface scanning and tested using light scattering and electrophoretic mobility shift assays. Distinct oligomeric states were functionally characterized using fluorescence anisotropy and steady-state kinetics. Replacement of residues that result in DNMT3A dimers, including those identified in AML patients, show minor changes in methylation activity but lose the capacity for processive catalysis on multisite DNA substrates, unlike the highly processive wild-type enzyme. Our results are consistent with the bimodal distribution of DNA methylation in vivo and the loss of clustered methylation in AML patients. Tetramerization with the known interacting partner DNMT3L rescues processive catalysis, demonstrating that protein binding at the DNMT3A tetramer interface can modulate methylation patterning. Our results provide a structural mechanism for the regulation of DNMT3A activity and epigenetic imprinting.     

The de novo DNA methyltransferase DNMT3A in development and cancer

DNA methylation, one of the best-characterized epigenetic modifications, plays essential roles in development, aging and diseases. The de novo DNA methyltransferase DNMT3A is responsible for the establishment of de novo genomic DNA methylation patterns and, as such, involved in normal development as well as in many diseases including cancer. In recent years, our understanding of this important protein has made significant progress, which was facilitated by stunning development in the analysis of the DNA methylome of multiple organs and cell types. In this review, recent developments in the characterization of DNMT3A were discussed with special emphasis on the roles of DNMT3A in development and cancer.     

The de novo DNA methyltransferase DNMT3A in development and cancer

DNA methylation, one of the best-characterized epigenetic modifications, plays essential roles in development, aging and diseases. The de novo DNA methyltransferase DNMT3A is responsible for the establishment of de novo genomic DNA methylation patterns and, as such, involved in normal development as well as in many diseases including cancer. In recent years, our understanding of this important protein has made significant progress, which was facilitated by stunning development in the analysis of the DNA methylome of multiple organs and cell types. In this review, recent developments in the characterization of DNMT3A were discussed with special emphasis on the roles of DNMT3A in development and cancer.     

H3K36me2/3 Binding and DNA Binding of the DNA Methyltransferase DNMT3A PWWP Domain Both Contribute to its Chromatin Interaction

The PWWP domain of DNMT3 DNA methyltransferases binds to histone H3 tails containing methylated K36, and this activity is important for heterochromatic targeting. Here, we show that the PWWP domain of mouse DNMT3A binds to H3K36me2 and H3K36me3 with a slight preference for H3K36me2. PWWP domains have also been reported to bind to DNA, and the close proximity of H3K36 and nucleosomal DNA suggests a combined binding to H3K36me2/3 and DNA. We show here that the DNMT3A PWWP domain binds to DNA with a weak preference for AT-rich sequences and that the designed charge reversal R362E mutation disrupts DNA binding. The K295E mutation, as well as K295I recently identified in paraganglioma, a rare neuroendocrine neoplasm, disrupts both DNA and H3K36me2/3 binding, which is in agreement with the proximity of K295 to residues involved in K36me2/3 methyllysine binding. Nucleosome pulldown experiments show that DNA binding and H3K36me2/3 binding are important for the interaction of the DNMT3A PWWP domain with nucleosomes. Localization studies of transiently transfected fluorescently-tagged wild-type and PWWP-mutated full-length DNMT3A indicate that both interactions contribute to the subnuclear localization of DNMT3A in mouse cells. In summary, our data demonstrate that the combined binding of the DNMT3A PWWP domain to the H3 tail containing K36me2/3 and to the nucleosomal or linker DNA is important for its chromatin interaction and subnuclear targeting of DNMT3A in living cells.     

Genetic variation in DNMT3B and increased global DNA methylation is associated with suicide attempts in psychiatric patients

Recently, a significant epigenetic component in the pathology of suicide has been realized. Here we investigate candidate functional SNPs in epigenetic‐regulatory genes, DNMT1 and DNMT3B, for association with suicide attempt (SA) among patients with co‐existing psychiatric illness. In addition, global DNA methylation levels [5‐methyl cytosine (5‐mC%)] between SA and psychiatric controls were quantified using the Methylflash Methylated DNA Quantification Kit. DNA was obtained from blood of 79 suicide attempters and 80 non‐attempters, assessed for DSM‐IV Axis I disorders. Functional SNPs were selected for each gene (DNMT1; n = 7, DNMT3B; n = 10), and genotyped. A SNP (rs2424932) residing in the 3′ UTR of the DNMT3B gene was associated with SA compared with a non‐attempter control group (P = 0.001; Chi‐squared test, Bonferroni adjusted P value = 0.02). Moreover, haplotype analysis identified a DNMT3B haplotype which differed between cases and controls, however this association did not hold after Bonferroni correction (P = 0.01, Bonferroni adjusted P value = 0.56). Global methylation analysis showed that psychiatric patients with a history of SA had significantly higher levels of global DNA methylation compared with controls (P = 0.018, Student's t‐test). In conclusion, this is the first report investigating polymorphisms in DNMT genes and global DNA methylation quantification in SA risk. Preliminary findings suggest that allelic variability in DNMT3B may be relevant to the underlying diathesis for suicidal acts and our findings support the hypothesis that aberrant DNA methylation profiles may contribute to the biology of suicidal acts. Thus, analysis of global DNA hypermethylation in blood may represent a biomarker for increased SA risk in psychiatric patients.     

Multi-target siRNA based on DNMT3A/B homologous conserved region influences cell cycle and apoptosis of human prostate cancer cell line TSU-PR1

Abnormal genome hypermethylation participates in the tumorigenesis and development of prostate cancer. Prostate cancer cells highly express DNA methyltransferase 3 (DMNT3) family genes, essential for maintaining genome methylation. In the present study, multi-target siRNA, based on the homologous region of the DNMT3 family, was designed for the in vitro investigation of its effects on the proliferation, migration, and invasion of TSU-PR1 prostate cancer cells. The consequential cell-cycle derangement, through DNMT3A/B or only DNMT3B silencing, was partially efficient, without affecting apoptosis. DNMT3A silencing had absolutely no effect on changing TSU-PR1 cell biological behavior. Hence, DNMT3B alone apparently plays a key role in maintaining the unfavorable behavior of prostate-cancer cells, thereby implying its potential significance as a promising therapeutic target, with DNMT3A simply in the role of helper.     

DNA methylation at differentially methylated regions of imprinted genes is resistant to developmental programming by maternal nutrition

The nutritional environment in which the mammalian fetus or infant develop is recognized as influencing the risk of chronic diseases, such as type 2 diabetes and hypertension, in a phenomenon that has become known as developmental programming. The late onset of such diseases in response to earlier transient experiences has led to the suggestion that developmental programming may have an epigenetic component, because epigenetic marks such as DNA methylation or histone tail modifications could provide a persistent memory of earlier nutritional states. One class of genes that has been considered a potential target or mediator of programming events is imprinted genes, because these genes critically depend upon epigenetic modifications for correct expression and because many imprinted genes have roles in controlling fetal growth as well as neonatal and adult metabolism. In this study, we have used an established model of developmental programming—isocaloric protein restriction to female mice during gestation or lactation—to examine whether there are effects on expression and DNA methylation of imprinted genes in the offspring. We find that although expression of some imprinted genes in liver of offspring is robustly and sustainably changed, methylation of the differentially methylated regions (DMRs) that control their monoallelic expression remains largely unaltered. We conclude that deregulation of imprinting through a general effect on DMR methylation is unlikely to be a common factor in developmental programming.     

Influence of TGFBR2, TGFB3, DNMT1, and DNMT3A Knockdowns on CTGF,TGFBR2, and DNMT3A in Neonatal and Adult Human Dermal Fibroblasts Cell Lines

Dermal fibroblasts are responsible for the production of the extracellular matrix that undergoes significant changes during the skin aging process. These changes are partially controlled by the TGF-β signaling, which regulates tissue homeostasis dependently on several genes, including CTGF and DNA methyltransferases. To investigate the potential differences in the regulation of the TGF-β signaling and related molecular pathways at distinct developmental stages, we silenced the expression of TGFB1, TGFB3, TGFBR2, CTGF, DNMT1, and DNMT3A in the neonatal (HDF-N) and adult (HDF-A) human dermal fibroblasts using the RNAi method. Through Western blot, we analyzed the effects of the knockdowns of these genes on the level of the CTGF, TGFBR2, and DNMT3A proteins in both cell lines. In the in vitro assays, we observed that CTGF level was decreased after knockdown of DNMT1 in HDF-N but not in HDF-A. Similarly, the level of DNMT3A was decreased only in HDF-N after silencing of TGFBR2, TGFB3, or DNMT1. TGFBR2 level was lower in HDF-N after knockdown of TGFB3, DNMT1, or DNMT3A, but it was higher in HDF-A after TGFB1 silencing. The reduction of TGFBR2 after silencing of DNMT3A and vice versa in neonatal cells only suggests the developmental stage-specific interactions between these two genes. However, additional studies are needed to explain the dependencies between analyzed proteins.     

Expression and methylation status of imprinted genes in placentas of deceased and live cloned transgenic calves

Placental deficiencies are linked with developmental abnormalities in cattle produced by somatic cell nuclear transfer (SCNT). To investigate whether the aberrant expression of imprinted genes in placenta was responsible for fetal overgrowth and placental hypertrophy, quantitative expression analysis of six imprinted genes (H19, XIST, IGF2R, SNRPN, PEG3, and IGF2) was conducted in placentas of: 1) deceased (died during perinatal period) transgenic calves (D group, n = 4); 2) live transgenic calves (L group, n = 15); and 3) conventionally produced (control) female calves (N group, n = 4). In this study, XIST, PEG3 and IGF2 were significantly over-expressed in the D group, whereas expression of H19 and IGF2R was significantly reduced in the D group compared to controls. The DNA methylation patterns in the differentially methylated region (DMR) from H19, XIST, and IGF2R were compared using Bisulfite Sequencing PCR (BSP) and Combined Bisulfite Restriction Analysis (COBRA). In the D group, H19 DMR was significantly hypermethylated, but XIST DMR and IGF2R ICR were significantly hypomethylated compared to controls. In contrast, there were no noticeable differences in the expression and DNA methylation status of imprinted genes (except DNA methylation level of XIST DMR) in the L group compared to controls. In conclusion, altered DNA methylation levels in the DMRs of imprinted genes in placentas of deceased transgenic calves, presumably due to aberrant epigenetic nuclear reprogramming during SCNT, may have been associated with abnormal expression of these genes; perhaps this caused developmental insufficiencies and ultimately death in cloned transgenic calves.     

Single substitution in H3.3G34 alters DNMT3A recruitment to cause progressive neurodegeneration

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