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PubMed2005

Cyclin D1 gene activation in human myeloma cells is independent of DNA hypomethylation or histone hyperacetylation.

OBJECTIVE: Overexpression of cyclin D1 is a common event in solid and hematological cancers. In multiple myeloma (MM), a B-cell hemopathy, one mechanism responsible for cyclin D1 expression is the translocation t(11;14)(q13;q32). But this translocation does not account for cyclin D1 gene activation in all MM. We have hypothesized that epigenetic events could regulate cyclin D1 gene transcription. METHODS: Using 6 MM cell lines representative of different cyclin D1 expression levels and exhibiting various chromosome 11 abnormalities, as well as normal B cells, we studied DNA methylation and histone acetylation of the cyclin D1 promoter. RESULTS: With the bisulfite sequencing technique, we have studied the DNA methylation status of the core minimal cyclin D1 promoter containing Sp1- and CRE-binding sites. Our results show that this region is not methylated in 6 human MM cell lines as well as in normal B lymphocytes, independently of cyclin D1 expression. Treatment with the DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-Aza) had no effect on cyclin D1 gene transcription. Chromatin immunoprecipitation (ChIP) experiments indicated that acetylated histones H4 are located at both the active and inactive cyclin D1 promoter. Treatment with a histone deacetylase inhibitor, trichostatin A (TSA), had no effect on gene transcription, nor had combined TSA plus 5-Aza treatment. CONCLUSION: The cyclin D1 gene is silenced within the B lineage by a mechanism different from DNA methylation or histone deacetylation.

Acetylation↗
PubMed2007

The gamma-globin gene promoter progressively demethylates as the hematopoietic stem progenitor cells differentiate along the erythroid lineage in baboon fetal liver and adult bone marrow.

OBJECTIVE: To determine whether the difference in gamma-globin gene promoter methylation in terminal erythroblasts at the fetal and adult stages of development is a result of fetal stage-specific demethylation or adult stage-specific de novo methylation during erythropoiesis. MATERIALS AND METHODS: Fetal liver- (FL, n = 2) and adult bone marrow- (ABM, n = 3) derived hematopoietic stem/progenitor cells and mature erythroblasts were purified by passage through a Miltenyi Magnetic Column followed by fluorescein-activated cell sorting (FACS) into subpopulations, defined by expression of CD34 and CD36 antigens. CD34(+)CD36(-), CD34(+)CD36(+), and CD34(-)CD36(+) subpopulations were purified by FACS and their degree of differentiation verified using the colony-forming cell assay. The methylation pattern of 5 CpG sites in the gamma-globin promoter region of these purified cell populations was determined using bisulfite sequencing. RESULTS: The gamma-globin promoter was highly methylated in the earliest stage of hematopoietic stem progenitor cells (CD34(+)CD36(-)) and methylation progressively decreased as erythroid differentiation progressed in FL and appears so in ABM as well. CONCLUSIONS: These data support a model in which differences in the methylation pattern of the gamma-globin gene in differentiating erythroblasts at different stages of development is the result of fetal stage-specific demethylation associated with transcriptional activation, rather than de novo methylation in the adults. The difference in the extent of gamma-globin gene demethylation in FL and ABM is correlated with the difference in gamma-globin expression at these developmental stages.

Animals↗
PubMed2026

Long-term (>7-year) parental consumption of genetically modified maize (Cry1Ab/Cry2Aj and EPSPS) induces no adverse sperm DNA methylation alterations across two generations of cynomolgus monkeys.

This study assessed the long-term safety of genetically modified (GM) maize from a male reproductive perspective, using a non-human primate model. We analyzed the sperm DNA methylation profiles in cynomolgus monkeys fed GM maize, non-GM parental maize, or a conventional diet over two generations (F0/F1). Whole-genome bisulfite sequencing (WGBS) revealed no significant differences in global methylation levels among groups. The identified differentially methylated regions (DMRs) were short, enriched in non-regulatory genomic areas, and did not cluster after treatment. Functional enrichment analysis showed that DMR-associated genes were consistently involved in the same core biological pathways (e.g., mTOR and Wnt signaling) across all dietary comparisons. These findings indicate that GM maize consumption did not induce specific adverse epigenetic alterations in sperm, with the observed changes reflecting common physiological adaptations to dietary variations rather than GM-related effects.

Animals↗
PubMed2005

Epigenetic and classical activation of Entamoeba histolytica heat shock protein 100 (EHsp100) expression.

The protozoan parasite Entamoeba histolytica expresses a cytosine-5 DNA methyltransferase (Ehmeth) that belongs to the DNMT2 protein family. The biological function of members of this DNMT2 family is unknown. In the present study, the 5' region of E. histolytica heat shock protein 100 (5'EHsp100) was isolated by affinity chromatography with 5-methylcytosine antibodies as ligand. The methylation status of 5'EHsp100 was confirmed by sodium bisulfite sequencing. We showed that the expression of EHsp100 was induced by heat shock, 5-azacytidine (5-AzaC), an inhibitor of DNA methyltransferase and Trichostatin A (TSA), an inhibitor of histone deacetylase. The effect of TSA on EHsp100 expression was rapidly reversed by removing the drug from the culture. In contrast, EHsp100 expression was still detectable one month after removing 5-AzaC from the media. Whereas 5-AzaC and TSA caused demethylation in the promoter region of EHsp100, no demethylation was observed following heat shock. Remarkably, DNA that includes three putative heat shock elements identified in the promoter region of EHsp100 bound to a protein of 37kDa present in the nuclear fraction of heat-shocked trophozoites but absent in the nuclear fraction of 5-AzaC and TSA treated trophozoites. Our data suggest that EHsp100 expression can be regulated by both a classical and an epigenetic mechanism.

Animals↗
PubMed2006

Developmental methylation of the regulatory region of HoxB5 gene in mouse correlates with its tissue-specific expression.

We have studied the dynamics of de novo CpG methylation in the regulatory region of one of the homeobox gene HoxB5 during mouse development by sodium bisulfite sequencing. Methylation pattern was examined at embryonic day 18.5 and adult in kidney and spleen while in the liver the same exercise has been done in 11.5 dpc, 18.5 dpc, 5 dpp and in adult. In the liver at 11.5 dpc, all the 47 contiguous sites (including a CpG island from 2035 to 2330 bp) at 5' regulatory region of HoxB5 were unmethylated. Random methylation commences from 18.5 dpc and continues in 5 dpp and in the adult. In the kidney at 18.5 dpc, 26 CpGs were examined (excluding the CpG island region) and all of them were unmethylated but the fetal spleen had at least a few sites considerably methylated. In the adult there was a low level methylation in the kidney, on the other hand, in the spleen, all the CpGs were methylated except a few sites and certain sites were totally methylated. Thus in the adult, the level of methylation was much higher than in the fetal stage. On the other hand semi-quantitative RT-PCR revealed that the extent of expression of HoxB5 was higher in embryonic stages than in the adult. Thus HoxB5 is a good paradigm to support that the developmental methylation of HoxB5 and its expression pattern show an inverse correlation.

Animals↗
PubMed2005

Epigenetic inactivation of DLC-1 in supratentorial primitive neuroectodermal tumor.

Supratentorial primitive neuroectodermal tumors (SPNETs) and medulloblastomas (MBs) are histologically similar intracranial tumors found in different anatomic locations of the brain. Our group has previously demonstrated that loss of chromosome 8p is a frequent event in MBs. The aim of this study was to evaluate whether DLC-1, a newly identified tumor-suppressor gene on chromosome 8p22, is involved in the tumorigenesis of MBs and the histologically similar SPNETs. We first assessed for alterations of gene expression in microdissected tumors and detected lack of DLC-1 transcript in 1 of 9 MBs (case M44) and 1 of 3 SPNETs (case M1). Neither somatic base substitutions nor homozygous deletion were found in tumors without DLC-1 transcript. We then explored the possibility of hypermethylation of the CpG island in DLC-1 as the mechanism of suppressed expression. Methylation-specific polymerase chain reaction revealed promotor hypermethylation of DLC-1 in M1 but not in M44. Bisulfite sequencing further verified a densely methylated pattern of 35 CpG sites studied in M1 that were not found in normal brain, indicating that inactivation of DLC-1 by hypermethylation is involved in SPNET. Based on this finding, we examined an additional 20 MBs, 8 SPNETs, and 4 MB and 2 SPNET cell lines for hypermethylation of the CpG island of DLC-1, finding that none of these samples exhibited DLC-1 methylation. In conclusion, our results demonstrate that transcriptional silencing of DLC-1 through promoter hypermethylation may contribute to tumorigenesis in a subset of SPNETs, and that loss of DLC-1 expression in MBs may be related to mechanisms other than promoter hypermethylation, genomic deletion, and mutation.

Adolescent↗
PubMed2026

Epigenetic activation of NK-cell effector programs and caspase-8-dependent apoptosis mediates the antitumor activity of LGP in NSCLC.

BACKGROUND: Effective activation of natural killer (NK) cell cytotoxicity and caspase-8-dependent extrinsic apoptosis remains a major challenge in non-small cell lung cancer (NSCLC). Epigenetic mechanisms regulating NK cell function within the tumor microenvironment are poorly understood and rarely targeted therapeutically. METHODS: The antitumor activity of Li-Ginseng Powder (LGP), a specifically processed Panax ginseng formulation enriched in rare ginsenosides (Rh4, Rg3, Rg5, Rk1, and Rk3), was evaluated in human lung cancer A549 cells and A549 xenograft mouse models. NK cell infiltration and activation were assessed by flow cytometry, immunoblotting, and immunohistochemistry. Whole-genome bisulfite sequencing (WGBS) was performed to analyze DNA methylation changes. The effects of LGP ginsenosides (LGG) on tumor cell apoptosis and death receptor signaling were examined in vitro. RESULTS: LGP significantly suppressed tumor growth and enhanced systemic and intratumoral NK cell activation. Promoter demethylation of NK cell effector genes, including Ncr1, Gzmb, Nktr, and Itgal, was associated with increased NK cell infiltration and activation, elevated granule-mediated cytotoxicity, and enhanced IFN-γ signaling. In parallel, LGP treatment induced caspase-8-dependent apoptosis associated with increased expression of membrane death receptors, their ligands, FADD, and procaspase-8 in tumor tissues. In vitro, LGG upregulated these apoptosis-initiating proteins and triggered caspase-8 activation in A549 cells independent of promoter methylation changes. Collectively, these immune-associated and tumor-intrinsic responses contributed to robust tumor suppression with a favorable systemic safety profile. CONCLUSIONS: LGP exerts dual antitumor effects characterized by enhanced NK-cell activation and increased sensitivity of tumor cells to caspase-8-dependent extrinsic apoptosis. These coordinated immune-associated and apoptosis-sensitizing effects underscore the therapeutic potential of LGP for the treatment of NSCLC.

DNA methylation↗
PubMed2004

Hypermethylation of NAD(P)H: quinone oxidoreductase 1 (NQO1) gene in human hepatocellular carcinoma.

BACKGROUND/AIMS: NAD(P)H: quinone oxydoreductase 1 (NQO1) and glutathione S-transferase P1 (GSTP1) belong to phase II xenobiotic-metabolizing enzymes. GSTP1 inactivation via CpG island hypermethylation in hepatocellular carcinoma (HCC) was previously reported, but the involvement of NQO1 in HCC is not well known. In this study, we assessed the transcription and status of methylation of NQO1 gene in human hepatoma cells and primary human HCC tissues. METHODS: NQO1 transcription and DNA hypermethylation in hepatoma cells with or without 5-aza-deoxycytidine (5-Aza-CdR) treatment were investigated by reverse-transcription PCR (RT-PCR), sodium bisulfite sequencing and methylation-specific PCR (MSP). The methylation status of NQO1 and GSTP1, and NQO1 mRNA in 44 HCC cases was also analyzed by MSP and real-time PCR, respectively. RESULTS: NQO1 transcription was down-regulated and the CpG island DNA was hypermethylated in Hep3B and HuH6 cells. After treatment with 5-Aza-CdR, NQO1 transcription was restored and CpG island DNA was demethylated in these cells. MSP analysis revealed that NQO1 hypermethylation occurred in 50.0% of HCC. All of the tumors that exhibited lesser amounts of NQO1 mRNA than corresponding non-tumorous tissues showed NQO1 hypermethylation. CONCLUSIONS: NQO1 transcription might be inappropriately suppressed by promoter hypermethylation in a subset of HCC, as well as GSTP1 gene.

Acetylation↗
PubMed2005

Patterned CpG methylation of silenced B cell gene promoters in classical Hodgkin lymphoma-derived and primary effusion lymphoma cell lines.

Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) and primary effusion lymphoma (PEL) are derived from germinal center (GC) and post-GC B cells, respectively. Neither express many of the B cell genes or surface markers typically expressed by other GC-derived B cell lymphomas or normal B cells. This loss of B cell gene expression is not due to a lack of essential transcription factors, as studies have shown that the ectopic expression of missing transcription factors failed to reactivate endogenous target genes. These results implicate epigenetic mechanisms extinguishing B cell gene expression. Silenced endogenous B cell genes representing a surface receptor, B29 (Igbeta, CD79b), a signaling molecule, TCL1, and a transcription factor, Bob1 (OCA-B, OBF-1), were reactivated by 5-aza-2'-deoxycytidine, indicating that gene silencing in HRS and PEL cells is due to DNA methylation. Genomic bisulfite sequencing corroborated this prediction and revealed three distinct patterns of methylation for the silenced B29 and TCL1 promoters. These distinct patterns consisted of 5' promoter CpG methylation alone, 5' and 3' promoter CpG methylation sparing sites in the central cores, and complete CpG methylation throughout the promoter regions. The silenced Bob1 promoter showed one pattern of dense CpG methylation at essentially all sites. These consistent patterns predict that, although gene silencing in many HRS and PEL cells mimics appropriate gene silencing, in some cases of complete CpG methylation throughout entire promoters both the activation and targeting of methylation is abnormal.

B-Lymphocytes↗
PubMed2026

Transmitochondrial pigs reveal causal effects of mitochondrial DNA on backfat thickness via nuclear epigenetic reprogramming.

Mitochondrial DNA (mtDNA) polymorphisms have been associated with production traits in farm animals, including backfat thickness in pigs, yet direct in vivo evidence establishing a causal link between specific mtDNA haplotypes and fat deposition remains limited. In this study, we generated transmitochondrial pigs (mitopigs) by combining the Dapulian nuclear genome with Wuzhishan mtDNA via somatic cell nuclear transfer, introducing 23 mtDNA mutations relative to controls. Mitopigs exhibited significantly increased backfat thickness at 5 months, a difference that persisted in their offspring, without significant differences in body weight, body size, or litter size. Fibroblasts derived from mitopigs exhibited reduced mtDNA copy numbers, decreased expression of mitochondrial biogenesis genes (PPARA, PPARGC1A, RRM2B, and LRPPRC), impaired mitochondrial respiration, elevated reactive oxygen species (ROS), and upregulated adipogenic transcription factors (CEBPA, CEBPB, and PPARG). Consistent with these fibroblast findings, backfat tissue of mitopigs showed corresponding upregulation of adipogenic transcription factors and downregulation of mitochondrial biogenesis genes. Integrated transcriptomic and whole-genome bisulfite sequencing (WGBS) analyses revealed nuclear transcriptional reprogramming that was closely associated with differential DNA methylation, predominantly affecting mitochondrial function and lipid metabolism pathways. Mitopig fibroblasts also showed a pro-inflammatory response to lipopolysaccharide stimulation, with elevated expression of IL-12, NOS2, RELA, and TNF-α. Our findings provide direct in vivo evidence that mtDNA variants regulate adiposity in pigs through mitochondrial dysfunction, oxidative stress, and nuclear epigenetic modulation, highlighting the potential for incorporating mtDNA haplotype information into pig breeding programs as a complementary strategy to nuclear genomic selection.

Adipogenesis↗
PubMed2005

ADAM23 methylation and expression analysis in brain tumors.

The ADAMs comprises a family of cell surface proteins with putative roles in cell-cell and/or cell-matrix interactions and in protease activities. In this work, we have examined the expression level and the methylation status of the 5' upstream region of the adhesion molecule ADAM23 in two brain tumor cell lines (A172 and T98G) as well as in three primary brain tumors (one grade II astrocytoma and two meningiomas) and 15 glioblastoma xenografts. Using bisulfite sequencing we verified that the percentage of methylated dinucleotides is higher in T98G when compared to A172 and that methylation significantly correlates with ADAM23 mRNA and protein expression. However, we were unable to detect methylation and down-regulation of the ADAM23 gene in brain tumors. Together, these results indicate that ADAM23 down-regulation by methylation in brain tumors is a rare event and it may help explain why brain tumor metastases are rarely found elsewhere in the body.

ADAM Proteins↗
PubMed2006

DNA methylation paradigm shift: 15-lipoxygenase-1 upregulation in prostatic intraepithelial neoplasia and prostate cancer by atypical promoter hypermethylation.

Fifteen (15)-lipoxygenase type 1 (15-LO-1, ALOX15), a highly regulated, tissue- and cell-type-specific lipid-peroxidating enzyme has several functions ranging from physiological membrane remodeling, pathogenesis of atherosclerosis, inflammation and carcinogenesis. Several of our findings support a possible role for 15-LO-1 in prostate cancer (PCa) tumorigenesis. In the present study, we identified a CpG island in the 15-LO-1 promoter and demonstrate that the methylation status of a specific CpG within this island region is associated with transcriptional activation or repression of the 15-LO-1 gene. High levels of 15-LO-1 expression was exclusively correlated with one of the CpG dinucleotides within the 15-LO-1 promoter in all examined PCa cell-lines expressing 15-LO-1 mRNA. We examined the methylation status of this specific CpG in microdissected high grade prostatic intraepithelial neoplasia (HGPIN), PCa, metastatic human prostate tissues, normal prostate cell lines and human donor (normal) prostates. Methylation of this CpG correlated with HGPIN, PCa and metastatic human prostate tissues, while this CpG was unmethylated in all of the normal prostate cell lines and human donor (normal) prostates that either did not display or had minimal basal 15-LO-1 expression. Immunohistochemistry for 15-LO-1 was performed in prostates from PCa patients with Gleason scores 6, 7 [(4+3) and (3+4)], >7 with metastasis, (8-10) and 5 normal (donor) individual males. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect 15-LO-1 in PrEC, RWPE-1, BPH-1, DU-145, LAPC-4, LNCaP, MDAPCa2b and PC-3 cell lines. The specific methylated CpG dinucleotide within the CpG island of the 15-LO-1 promoter was identified by bisulfite sequencing from these cell lines. The methylation status was determined by COBRA analyses of one specific CpG dinucleotide within the 15-LO-1 promoter in these cell lines and in prostates from patients and normal individuals. Fifteen-LO-1, GSTPi and beta-actin mRNA expression in BPH-1, LNCaP and MDAPCa2b cell lines with or without 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin-A (TSA) treatment were investigated by qRT-PCR. Complete or partial methylation of 15-LO-1 promoter was observed in all PCa patients but the normal donor prostates showed significantly less or no methylation. Exposure of LNCAP and MDAPCa2b cell lines to 5-aza-dC and TSA resulted in the downregulation of 15-LO-1 gene expression. Our results demonstrate that 15-LO-1 promoter methylation is frequently present in PCa patients and identify a new role for epigenetic phenomenon in PCa wherein hypermethylation of the 15-LO-1 promoter leads to the upregulation of 15-LO-1 expression and enzyme activity contributes to PCa initiation and progression.

Adult↗
PubMed2026

Integrative WGBS and ATAC-seq profiling reveals epigenetic and chromatin accessibility signatures associated with clutch length in goose ovaries.

Clutch length is an important reproductive trait in geese, but its epigenetic basis remains poorly characterized. Daily egg production was recorded for 280 individually housed Zi geese, and clutch-related indices were calculated as described in our previous study. Based on these records, six geese with contrasting clutch-length phenotypes were selected and assigned to the long-clutch (LC) and short-clutch (SC) groups. Ovarian tissues from three geese per group were subjected to whole-genome bisulfite sequencing (WGBS) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) to identify candidate epigenetic signatures associated with clutch length. WGBS identified 630,909 differentially methylated regions (DMRs), whereas ATAC-seq identified 902 differentially accessible regions (DARs). Integrated analysis revealed distinct patterns of ovarian DNA methylation and chromatin accessibility between the two groups, suggesting that clutch length variation may be accompanied by epigenomic differences in ovarian tissue. Genes associated with DMRs and/or DARs were enriched in biological processes related to granulosa cell differentiation and endocrine competence, follicular fate regulation, and periovulatory cytoskeletal and signaling remodeling. RERE was prioritized as a candidate locus because it was supported by changes in both DNA methylation and chromatin accessibility, whereas FOXL2, STAR, BAK1, FGF17, PRSS35, ACTR3, and AXIN1 were supported mainly by evidence from a single omics layer. RT-qPCR analysis of selected genes showed expression trends broadly consistent with the corresponding epigenomic differences, providing additional supportive evidence for these candidate associations. Collectively, this study provides an exploratory ovarian epigenomic resource and identifies candidate epigenetic signatures, genes, and biological processes associated with clutch length variation in geese.

DNA methylation↗
PubMed2007

The DNA methylation profile within the 5'-regulatory region of DRD2 in discordant sib pairs with schizophrenia.

Studies of discordance in monozygotic twins have demonstrated that environmental effects play an important role in the pathogenesis of schizophrenia. DNA microarray analysis has revealed upregulation of the DRD2 gene in peripheral blood lymphocytes of schizophrenic patients. We hypothesized that this expression alteration could involve the DNA (CpG) methylation status that is implicated to the transcription status of the gene and in this study we used bisulfited sequence analysis to determine the DNA methylation status of a typical CpGs island within the 5'-regulatory region of DRD2 in peripheral blood lymphocytes from 48 discordant sib pairs suffering from schizophrenia. We found that the methylated cytosines occurred mainly in three clusters. No statistically significant difference in frequency of site-specific cytosine methylation modification of DRD2 between patients and normal controls was found nor did we find any significant association between sex, age on admission or age at onset of schizophrenia and methylated cytosines of DRD2. Our study did not support the hypothesis that site-specific cytosine methylation of DRD2 plays a role in the psychopathology of schizophrenia.

5' Flanking Region↗
PubMed2004

The human papillomavirus-18 genome is efficiently targeted by cellular DNA methylation.

Human papillomaviruses (HPVs) infect epithelia, including the simple and the squamous epithelia of the cervix, where they can cause cancer and precursor lesions. The molecular events leading from asymptomatic HPV infections to neoplasia are poorly understood. There is evidence that progression is modulated by transcriptional mechanisms that control HPV gene expression. Here, we report the frequent methylation of HPV-18 genomes in cell culture and in situ. DNA methylation is generally known to lead to transcriptional repression due to chromatin changes. We investigated two cell lines derived from cervical cancers, namely, C4-1, which contains one HPV-18 genome, and different clones of HeLa, with 50 HPV-18 genomes. By restriction cleavage, we detected strong methylation of the L1 gene and absence of methylation of parts of the long control region (LCR). A 3-kb segment of the HPV-18 genomes downstream of the oncogenes was deleted in both cell lines. Bisulfite sequencing showed that in C4-1 cells and two HeLa clones, 18 of the 19 CpG residues in the 1.2-kb terminal part of the L1 gene were methylated, whereas a third HeLa clone had only eight methylated CpG groups, indicating changes of the methylation pattern after the establishment of the HeLa cell line. In the same four clones, none of the 12 CpG residues that overlapped with the enhancer and promoter was methylated. In six HPV-18 containing cancers and five smears from asymptomatic patients, most of the CpG residues in the L1 gene were methylated. There was complete or partial methylation, respectively, of the HPV enhancer in three of the cancers, and lack of methylation in the remaining eight samples. The promoter sequences were methylated in three of the six cancers and four of the six smears, and unmethylated elsewhere. Our data show that epithelial cells efficiently target HPV-18 genomes for DNA methylation, which may affect late and early gene transcription.

Cell Line, Tumor↗
PubMed2006

Detailed analysis of the methylation patterns of the KvDMR1 imprinting control region of human chromosome 11.

The paternal repression of several genes in human chromosome 11p15.5 (mouse chromosome 7) is associated with paternal expression of a transcript called KCNQ1OT1 (also known as LIT1). This long transcript originates from a promoter that resides in a CpG island in intron 10 of the KCNQ1 gene and runs in an antisense orientation to the direction of the coding KCNQ1 transcript. The CpG island is maternally methylated but paternally nonmethylated. The CpG island loses its maternal methylation in over 50% of cases of Beckwith-Wiedemann syndrome who lack uniparental disomy. This loss is usually accompanied by biallelic expression of the KCNQ1OT1 transcript. We have examined the methylation status of this CpG island in somatic cell hybrids and diploid lymphoblasts using Southern hybridization and bisulfite sequencing techniques. We find that the maternal copy of the CpG island is methylated at all CpGs examined within the CpG island and uniformly paternally unmethylated. In addition, in BWS patients who have lost methylation of the CpG island, this loss occurs throughout the CpG island. Finally, we find that there is a switch in methylation patterns outside the CpG island from maternal methylation within the island to predominantly paternal methylation at sites flanking the CpG island.

Animals↗
PubMed2026

Integrated GWAS and methylation analysis identify DNMT3A as an important regulator of growth in rabbits.

The parameters of individual growth curve can serve as pseudo-phenotype for genetic evaluation in livestock. In this study, we compared five nonlinear growth models using post-weaning body weights of 706 New Zealand White rabbits. Under the best-fitting model, two parameters of mature weight and maturity rate were subjected to GWAS through single-step genomic BLUP framework that integrated phenotypic records from non-genotyped animals with 41,359 SNPs genotyped in 198 individuals. Association analysis identified 147 relevant genomic regions, and also highlighted DNMT3A as a promising candidate gene for further functional investigation. siRNA-mediated knockdown of DNMT3A significantly impaired myoblast proliferation. Whole-genome bisulfite sequencing of DNMT3A-knockdown myoblasts identified 69,480 differentially methylated regions (DMRs). Integrative analyses revealed substantial overlap between DMR-associated genes and GWAS candidate genes, with significant enrichment in vitamin B6 and tyrosine metabolism pathways. These findings suggest that DNMT3A may regulate rabbit growth via mediating DNA methylation of downstream genes.

Animals↗
PubMed2005

Aberrant expression and mutations of TGF-beta receptor type II gene in endometrial cancer.

OBJECTIVE: Transforming growth factor beta (TGF-beta) is a multifunctional cytokine that strongly inhibits epithelial cell growth. Disabling of TGF-beta signaling is thought to be involved in development of a variety of tumors in which abnormal expression or function of TGF-beta receptor plays critical roles. In the present study, we examined aberrant expression and mutation of the gene TGF-beta receptor type II (TbetaRII) in endometrial cancers of endometrioid subtype. METHODS AND RESULTS: Real-time PCR analysis using surgical tissue specimens of 27 endometrial cancers and 24 normal endometria revealed that endometrial cancers had significantly decreased levels of TbetaRII mRNA expression (mean level 2.44 +/- 2.65), compared to normal endometria (mean level 7.23 +/- 6.07) (P < 0.001). Methylation status of TbetaRII promoter containing 30 CpGs was examined by bisulfite sequencing analysis, and 98% (51/52) of the patients were found to have unmethylated TbetaRII promoter, indicating that promoter hypermethylation is not the major cause of decreased expression of TbetaRII in endometrial cancers. Mutational analysis revealed that 15.1% (8/53) of endometrial cancers had frameshift mutations at polyadenine repeats in exon 3 of the TbetaRII gene. Notably, these mutations were preferentially accumulated in patients with MSI-H phenotype (7/19:37%) (P < 0.001) or with those with methylated MLH1 promoters (6/16:38%) (P < 0.01). Thus, it appears that the TbetaRII gene is a target of mismatch repair deficiency. CONCLUSION: Taken together, we found that the decreased expression of TbetaRII as well as frameshift mutation of TbetaRII via mismatch repair deficiency frequently occurs in this tumor type, possibly causing loss of receptor function and unresponsiveness of TGF-beta signaling that may lead to endometrial carcinogenesis.

Acetylation↗