Murine diet/tissue and human brain tumorigenesis alter <Emphasis Type="Italic">Mthfr/MTHFR</Emphasis> 5′-end methylation

Polymorphisms and decreased activity of methylenetetrahydrofolate reductase (MTHFR) are linked to disease, including cancer. However, epigenetic regulation has not been thoroughly studied. Our goal was to generate DNA methylation profiles of murine/human MTHFR gene regions and examine methylation in brain and liver tumors. Pyrosequencing in four murine tissues revealed minimal DNA methylation in the CpG island. Higher methylation was seen in liver or intestine in the CpG island shore 5′ to the upstream translational start site or in another region 3′ to the downstream start site. In the latter region, there was negative correlation between expression and methylation. Three orthologous regions were investigated in human MTHFR, as well as a fourth region between the two translation start sites. We found significantly increased methylation in three regions (not the CpG island) in pediatric astrocytomas compared with control brain, with decreased expression in tumors. Methylation in hepatic carcinomas was also increased in the three regions compared with normal liver, but the difference was significant for only one CpG. This work, the first overview of the Mthfr/MTHFR epigenetic landscape, suggests regulation through methylation in some regions, demonstrates increased methylation/decreased expression in pediatric astrocytomas, and should serve as a resource for future epigenetic studies.     

Novel markers of gene methylation and expression in breast cancer

An optimized methylation-sensitive restriction fingerprinting technique was used to search for differentially methylated CpG islands in the tumor genome and detected seven genes subject to abnormal epigenetic regulation in breast cancer: SEMA6B, BIN1, VCPIP1, LAMC3, KCNH2, CACNG4, and PSMF1. For each gene, the rate of promoter methylation and changes in expression were estimated in tumor and morphologically intact paired specimens of breast tissue (N = 100). Significant methylation rates of 38, 18, and 8% were found for SEMA6B, BIN1, and LAMC3, respectively. The genes were not methylated in morphologically intact breast tissue. The expression of SEMA6B, BIN1, VCPIP1, LAMC3, KCNH2, CACNG4, and PSMF1 was decreased in 44–94% of tumor specimens by the real-time RT-PCR assay. The most profound changes in SEMA6B and LAMC3 suggest that these genes can be included in biomarker panels for breast cancer diagnosis. Fine methylation mapping of the most frequently methylated CpG islands (SEMA6B, BIN1, and LAMC3) provides a fundamental basis for developing efficient methylation tests for these genes.     

High CpG island methylation of <Emphasis Type="Italic">p16</Emphasis> gene and loss of p16 protein expression associate with the development and progression of tetralogy of Fallot

We examined CpG island methylation in p16 gene and its effect on p16 protein expression in tetralogy of Fallot (ToF) patients to explore its potential implications in the development and progression of ToF. The study subjects consisted of 75 healthy controls and 63 ToF patients recruited at Linyi People’s Hospital between January 2012 and June 2014. The 4 mL of peripheral venous blood of each subject was obtained and saved in ethylene diamine tetraacetic acid (EDTA) tubes. Methylation-specific polymerase chain reaction (MSP) was employed to detect CpG island methylation in p16 promoter region and Western blotting was used to detect p16 expression of all subjects. Real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) was performed to test p16 mRNA expression. The results showed that p16-methylation rates in ToF group were significantly higher than the control group (ToF group, 58.73%; control group, 13.33%; P < 0.001). Remarkably, Western blotting and FQ-PCR results derived from RVOT revealed that p16 protein expression was significantly lower in ToF group compared to the control group (0.76 ± 0.21 versus 2.31 ± 0.35; P < 0.001), and p16 gene expression was also markedly decreased in ToF group (1.212 ± 0.152 versus 1.346 ± 0.191, P < 0.001). Additionally, our analysis suggested that CpG island methylation in p16 promoters in ToF patients was negatively correlated with p16 protein and gene expression (both P < 0.05). Our study reports that high CpG island methylation of p16 gene and loss of p16 protein expression associate with the development and progression of ToF, which may have significant therapeutic applications for ToF.     

Functional Analyses of <Emphasis Type="Italic">PtROS1</Emphasis>-RNAi in Poplars and Evaluation of Its Effect on DNA Methylation

DNA methylation occurs mostly at the C5 position of dinucleotide symmetric CpG sites in genomic DNA. A balance is maintained in the plant genome between DNA methylation mediated by RNA-directed DNA methylation (RdDM) and DNA demethylation mediated by the DEMETER (DME) protein family and REPRESSOR OF SILENCING (ROS1). We used double-stranded RNA (dsRNA) silencing to suppress ROS1 protein expression in ‘Nanlin895’ (Populus deltoides × Populus euramericana ‘Nanlin895’). Leaves of WT and transformant poplars revealed more symmetric methylation on CpG sites than roots and stems. In addition, leaves of transformant poplars revealed more methylated CpG sites in both 5.8S rDNA and histone H3 compared to WT types via 0, 50 and 100 mM NaCl treatments. In asymmetric methylation sites, transformant poplars exhibited more methylated CpHpG and CpHpH contexts than WT poplars. On the other hand, hypermethylation induced by PtROS1-RNAi construct resulted in pleiotropic phenotypic changes in transgenic poplars. The percentage of wavy leaves was increased maximum by ~45% in transgenic poplars. Also, the number of leaves was increased by ~200 number in transformants. Furthermore, shooting (%) and rooting (%) was decreased in transgenic poplars versus WT.     

hTERT promoter methylation status in peripheral blood leukocytes as a molecular marker of head and neck cancer progression

Cancer cells, including head and neck cancer cell carcinoma (HNSCC), are characterized by an increased telomerase activity. This enzymatic complex is active in approximately 80–90% of all malignancies, and is regulated by various factors, including methylation status of hTERT gene promoter. hTERT methylation pattern has been thoroughly studied so far. It was proved that hTERT is aberrantly methylated in tumor tissue versus healthy counterparts. However, such effect has not yet been investigated in PBLs (peripheral blood leukocytes) of cancer patients. The aim of this study was to analyze the hTERT gene promoter methylation status in blood leukocytes. DNA was extracted from PBL of 92 patients with histologically diagnosed HNSCC and 53 healthy controls. Methylation status of whole hTERT promoter fragment with independent analysis of each 19 CpG sites was performed using bisulfide conversion technique followed by sequencing of PCR products. Not significant (p?=?0.0532) differences in the general frequency of hTERT CpG sites methylation were detected between patients and healthy controls. However, it was discovered that some of analyzed positions (CpG islands: 1 [p?=?0.0235], 5 [p?=?0.0462], 8 [p?=?0.0343]) are significantly more often methylated in HNSCC patients than in controls. The opposite finding was observed in case of CpG position 2 (p?=?0.0210). Furthermore, closer analysis of single CpG positions revealed differences in methylation status dependent on anatomical site and TNM classification. To conclude, hTERT promoter methylation status (general or single CpG sites) would be considered as a molecular markers of HNSCC diagnostics.     

Plant DNA methyltransferase genes: Multiplicity,expression, methylation patterns

Expression and methylation patterns of genes encoding DNA methyltransferases and their functionally related proteins were studied in organs of Arabidopsis thaliana plants. Genes coding for the major maintenance-type DNA methyltransferases, MET1 and CMT3, and the major de novo-type DNA methyltransferase, DRM2, are actively expressed in all organs. Similar constitutively active expression was observed for genes encoding their functionally related proteins, a histone H3K9 methyltransferase KYP and a catalytically non-active protein DRM3. Expression of the MET1 and CMT3 genes is significantly lower in developing endosperm compared with embryo. Vice versa, expression of the MET2a, MET2b, MET3, and CMT2 genes in endosperm is much more active compared with embryo. A special maintenance DNA methylation system seems to operate in endosperm. The DNMT2 and N6AMT genes encoding putative methyltransferases are constitutively expressed at low levels. CMT1 and DRM1 genes are expressed rather weakly in all investigated organs. Most of the studied genes have methylation patterns conforming to the “body-methylated gene” prototype. A peculiar feature of the MET family genes is methylation at all three possible site types (CG, CHG, and CHH). The most weakly expressed among genes of their respective families, CMT1 and DRM1, are practically unmethylated. The MET3 and N6AMT genes have unusual methylation patterns, promoter region, and most of the gene body devoid of any methylation, and the 3'-end proximal part of the gene body is highly methylated.     

Prognostic potential of <Emphasis Type="Italic">KLOTHO</Emphasis> and <Emphasis Type="Italic">SFRP1</Emphasis> promoter methylation in head and neck squamous cell carcinoma

Hypermethylation in the CpG island promoter regions of tumor suppressors is known to play a significant role in the development of HNSCC and the detection of which can aid the classification and prognosis of HNSCC. This study aims to profile the methylation patterns in a panel of key genes including CDKN2A, CDKN2B, KLOTHO (KL), RASSF1A, RARB, SLIT2, and SFRP1, in a group of HNSCC samples from Saudi Arabia. The extent of methylation in these genes is determined using the MethyLight assay and correlated with known clinicopathological parameters in our samples of 156 formalin-fixed and paraffin-embedded HNSCC tissues. SLIT2 methylation had the highest frequency (64.6%), followed by RASSF1A (41.3%), RARB (40.7%), SFRP1 (34.9), KL (30.7%), CKDN2B (29.6%), and CKDN2A (29.1%). KL and SFRP1 methylation were more predominant in nasopharyngeal tumors (P = 0.001 and P = 0.031 respectively). Kaplan Meier analysis showed that patients with moderately differentiated tumors who display SFRP1 methylation have significantly worse overall survival in comparison with other samples. In contrast, better clinical outcomes were seen in patients with KL methylation. In conclusion, our findings suggest that the detection of frequent methylation in SFRP1 and KL genes’ promoters could serve as prognostic biomarkers for HNSCC.     

Methylation of the <Emphasis Type="Italic">RASSF1A</Emphasis> promoter region and the allelic imbalance frequencies in chromosome 3 critical regions correlate with progression of clear cell renal carcinoma

The short arm of chromosome 3 (3p) contains several critical regions that have increased frequencies of allelic deletions and harbor a set of tumor suppressor genes. In particular, the range of functions performed by RASSF1A (LUCA region, 3p21.31) includes those potentially associated with carcinogenesis. Among 3p genes, RASSF1A has the highest methylation frequency in epithelial tumors of various locations. For the first time, two different methods (methylation-specific PCR and methylation-sensitive restriction analysis) independently showed that the methylation level of the CpG island in the RASSF1A promoter region significantly correlated with grade and clinical stage of clear cell renal cell carcinoma (RCC). An analysis of 23 3p polymorphic markers in a representative set of 80 RCC cases characterized clinically and histologically revealed that RCC progression significantly correlated with the frequency of allelic imbalances in some critical regions of 3p (LUCA and AP20), but not in 3p as a whole. These data suggest that RCC progression is associated with the methylation of the RASSF1A promoter and, possibly, with structural and functional alterations in other 3p genes. In addition, significant correlation between RASSF1A methylation and allelic losses at the nearby polymorphic marker locus suggests the “two hit” model for the inactivation of this tumor suppressor gene in RCC.     

Modification effect of fenofibrate therapy,a longitudinal epigenomic-wide methylation study of triglycerides levels in the GOLDN study

Background

Identification of interactions between epigenetic factors and treatments might lead to personalized intervention of diseases. This paper aims to examine the modification effect of fenofibrate therapy on the association of methylation levels and fasting blood triglycerides (TG), and the related biological pathways among methylation sites.

Results

Mixed-effects models were employed to assess pre- and posttreatment associations and drug modification effects simultaneously. Five cytosine-phosphate-guanine (CpG) sites were found to be associated with TG levels before and after the fenofibrate therapy: cg00574958, cg17058475, and cg01082498 on CPT1A gene, chromosome 11; cg03725309 on SARS, chromosome 1; and cg06500161 on ABCG1, chromosome 21. In addition, fenofibrate therapy modified the methylation levels on the following 4 CpG sites: cg20015535 (gene EGLN1, chromosome 1); cg24870738 (gene RNF220, chromosome 1); cg06891775 (gene LOC283050, chromosome 10); and cg00607630 (gene USP7, chromosome 16). Further, gene set enrichment analysis (GSEA) identified cancer- and metabolism-related pathways that were associated with TG-related CpG sites.

Conclusions

We identified modification effects of fenofibrate on the associations between blood TG levels and several CpG sites. Pathway enrichment analysis indicated the alternations in some metabolism and cancer-related pathways. Our findings have important implications for future research in pharmacoepigenetics and personalized medicine.

     

Dynamics of LINE-1 retrotransposon methylation levels in circulating DNA from lung cancer patients undergoing antitumor therapy

Malignant cell transformation is accompanied with abnormal DNA methylation, such as the hypermethylation of certain gene promoters and hypomethylation of retrotransposons. In particular, the hypomethylation of the human-specific family of LINE-1 retrotransposons was observed in lung cancer tissues. It is also known that the circulating DNA (cirDNA) of blood plasma and cell-surface-bound circulating DNA (csb-cirDNA) of cancer patients accumulate tumor-specific aberrantly methylated DNA fragments, which are currently considered to be valuable cancer markers. This work compares LINE-1 retrotransposon methylation patterns in cirDNA of 16 lung cancer patients before and after treatment. CirDNA was isolated from blood plasma, and csb-cirDNA fractions were obtained by successive elution with EDTA-containing phosphate buffered saline and trypsin. Concentrations of methylated LINE-1 region 1 copies (LINE-1-met) were assayed by real-time methylation-specific PCR. LINE-1 methylation levels were normalized to the concentration of LINE-1 region 2, which was independent of the methylation status (LINE-1-Ind). The concentrations of LINE-1-met and LINE-1-Ind in csb-cirDNA of lung cancer patients exhibited correlations before treatment (r = 0.54), after chemotherapy (r = 0.72), and after surgery (r = 0.83) (P < 0.05, Spearman rank test). In the total group of patients, the level of LINE-1 methylation (determined as the LINE-1-met/LINE-1-Ind ratio) was shown to increase significantly during the follow-up after chemotherapy (P < 0.05, paired t test) and after surgery compared to the level of methylation before treatment (P < 0.05, paired t test). The revealed association between the level of LINE-1 methylation and the effect of antitumor therapy was more pronounced in squamous cell lung cancer than in adenocarcinoma (P < 0.05 and P > 0.05, respectively). These results suggest a need for the further investigation of dynamic changes in levels of LINE-1 methylation depending on the antitumor therapy.     

Methylation of the genes for the microRNAs miR-129-2 and miR-9-1, changes in their expression,and activation of their potential target genes in clear cell renal cell carcinoma

Methylation of promoter CpG islands and microRNA (miRNA) interactions with mRNAs of target genes are epigenetic mechanisms that play a crucial role in deregulation of gene expression and signaling pathways in tumors. Altered expression of six chromosome 3p genes (RARB(2), SEMA3B, RHOA, GPX1, NKIRAS1, and CHL1) and two miRNA genes (MIR-129-2 and MIR-9-1) was observed in primary clear cell renal cell carcinomas (ccRCCs, 31–48 samples) by RT-PCR and qPCR. Significant downregulation (p < 0.05, Fisher’s exact test) was observed for SEMA3B, NKIRAS1, and CHL1; and differential expression, for the other chromosome 3p and miRNA genes. Methylation-specific PCR with primers to RARB(2), SEMA3B, MIR-129-2, and MIR-9-1 showed that their methylation frequency was significantly (p < 0.05, Fisher’s exact test) elevated in the ccRCC samples. Significant correlations between promoter methylation and expression were confirmed for SEMA3B and observed for the first time for RARB(2), GPX1, and MIR-129-2 in ccRCC (Spearman’s correlation coefficient r _s ranging 0.31–0.60, p < 0.05). The MIR-129-2 and RARB(2) methylation frequencies significantly correlated with ccRCC progression. MIR-129-2 methylation correlated with upregulation of RARB(2), RHOA, NKIRAS1, and CHL1 (r _s ranging 0.35–0.53, p < 0.05). The findings implicate methylation in regulating RARB(2), SEMA3B, GPX1, and MIR-129-2 and indicate that miR-129-2 and methylation of its gene affect RARB(2), RHOA, NKIRAS1, and CHL1 expression.     

Comparative WGBS identifies genes that influence non-ripe phenotype in tomato epimutant <Emphasis Type="Italic">Colourless non-ripening</Emphasis>

Whole-genome bisulfite sequencing (WGBS) allows single-base resolution and genome-wide profiling of DNA methylation in plants and animals. This technology provides a powerful tool to identify genes that are potentially controlled by dynamic changes of DNA methylation and demethylation. However, naturally occurring epimutants are rare and genes under epigenetic regulation as well as their biological relevances are often difficult to define. In tomato, fruit development and ripening are a complex process that involves epigenetic control. We have taken the advantage of the tomato epimutant Colourless non-ripening (Cnr) and performed comparative mining of the WGBS datasets for the Cnr and SlCMT3-silenced Cnr fruits. We compared DNA methylation profiles for the promoter sequences of approximately 5,000 bp immediately upstream of the coding region of a list of 20 genes. Differentially methylated regions were found for some of these genes. Virus-induced gene silencing (VIGS) of differentially methylated gene SlDET1 or SlPDS resulted in unusual brown pigmentation in Cnr fruits. These results suggest that comparative WGBS coupled with VIGS can be used to identify genes that may contribute to the colourless unripe phenotype of fruit in the Cnr epimutant.     

Polymorphic Imprinting of <Emphasis Type="Italic">SLC38A4</Emphasis> Gene in Bovine Placenta

Imprinted genes are characterized by monoallelic expression that is dependent on parental origin. Comparative analysis of imprinted genes between species is a powerful tool for understanding the biological significance of genomic imprinting. The slc38a4 gene encodes a neutral amino acid transporter and is identified as imprinted in mice. In this study, the imprinting status of SLC38A4 was assessed in bovine adult tissues and placenta using a polymorphism-based approach. Results indicate that SLC38A4 is not imprinted in eight adult bovine tissues including heart, liver, spleen, lung, kidney, muscle, fat, and brain. It was interesting to note that SLC38A4 showed polymorphic status in five heterogeneous placentas, with three exhibiting paternal monoallelic expression and two exhibiting biallelic expression. Monoallelic expression of imprinted genes is generally associated with allele-specific differentially methylation regions (DMRs) of CpG islands (CGIs)-encompassed promoter; therefore, the DNA methylation statuses of three CGIs in the SLC38A4 promoter and exon 1 region were tested in three placentas (two exhibiting paternal monoallelic and one showing biallelic expression of SLC38A4) and their corresponding paternal sperms. Unexpectedly, extreme hypomethylation (<?3%) of the DNA was observed in all the three detected placentas and their corresponding paternal sperms. The absence of DMR in bovine SLC38A4 promoter region implied that DNA methylation of these three CGIs does not directly or indirectly affect the polymorphic imprinting of SLC38A4 in bovine placenta. This suggested other epigenetic features other than DNA methylation are needed in regulating the imprinting of bovine SLC38A4, which is different from that of mouse with respect to a DMR existence at the mouse’s slc38a4 promoter region. Although further work is needed, this first characterization of polymorphic imprinting status of SLC38A4 in cattle placenta provides valuable information on investigating the genomic imprinting phenomenon itself.     

A reinvestigation of somatic hypermethylation at the PTEN CpG island in cancer cell lines

Background

PTEN is an important tumour suppressor gene that is mutated in Cowden syndrome as well as various sporadic cancers. CpG island hypermethylation is another route to tumour suppressor gene inactivation, however, the literature regarding PTEN hypermethylation in cancer is controversial. Furthermore, investigation of the methylation status of the PTEN CpG island is challenging due to sequence homology with the PTEN pseudogene, PTENP1. PTEN shares a CpG island promoter with another gene known as KLLN. Here we present a thorough reinvestigation of the methylation status of the PTEN CpG island in DNA from colorectal, breast, ovarian, glioma, lung and haematological cancer cell lines.

Results

Using a range of bisulphite-based PCR assays we investigated 6 regions across the PTEN CpG island. We found that regions 1-4 were not methylated in cancer cell lines (0/36). By allelic bisulphite sequencing and pyrosequencing methylation was detected in regions 5 and 6 in colorectal, breast and haematological cancer cell lines. However, methylation detected in this region was associated with the PTENP1 promoter and not the PTEN CpG island.

Conclusions

We show that methylation of the PTEN CpG island is a rare event in cancer cell lines and that apparent methylation most likely originates from homologous regions of the PTENP1 pseudogene promoter. Future studies should utilize assays that reliably discriminate between PTEN and PTENP1 to avoid data misinterpretation.     

Characterization and tissue distribution of Lhx9 and Lhx9 <Emphasis Type="Italic">α</Emphasis> in Chinese giant salamander <Emphasis Type="Italic">Andrias davidianus</Emphasis>

Lhx9 is an LIM (named for the first three proteins in which the domain was found, Lin-11, Isl1 and Mec-3) homeodomain protein involved in development and differentiation of the gonad. In this study, we isolated the full-length Lhx9 and Lhx9 α from Andrias davidianus, detected the tissue distribution and analysed the methylation of the promoters. We identified Lhx9 of 1411 bp and Lhx9 α of 1153-bp length, differing in the 3\(^{\prime }\)-flanking region, encoding 399 and 330 amino acids, respectively. The Lhx9 gene was detected primarily in liver, ovary and heart with moderate expression in brain, pituitary, intestine and spleen, and low expression in the remaining examined tissues, while Lhx9 α expression was high in heart, pituitary and liver, and low in spleen and stomach. Significantly higher Lhx9 expression was observed in ovary than in testis, with no differences in Lhx9 α expression between testis and ovary observed. Bisulphite sequencing revealed significantly higher methylation in testis compared to ovary. The methylation level of CpG sites –733, –673, –615 and –594 exhibited significantly higher levels in testis than in ovary, which was negatively correlated with Lhx9 expression. The methylation and expression patterns suggested that promoter methylation suppressed expression of Lhx9 in A. davidianus.     

Opioid Exposure is Associated with Aberrant DNA Methylation of <Emphasis Type="Italic">OPRM1</Emphasis> Promoter Region in a Chinese Han Population

The μ-opioid receptor (OPRM1) plays an important role in opiate addiction. The OPRM1 gene promoter showed hypermethylation in lymphocytes of opiate addicts as well as opioid medications users, while the methylation status displayed ethnic diversity. The purpose of the study was to investigate the methylation pattern of OPRM1 promoter in the Han Chinese population. We analyzed 22 CpG sites located in OPRM1 promoter in 186 former opiate addicts (94 males and 92 females) and 184 healthy controls (102 males and 82 females). The +?126 CpG site was significantly hypermethylated in the former heroin addicts compared with controls (13.67% versus 8.39%, \(P = 3.78 \times 10^{ - 9}\), corrected for 36 tests). Six CpG sites were significantly associated with opioid exposure, including the most significant +126 CpG site (opiate addicts 13.57%, control 8.39%, \(P = 9.19 \times 10^{ - 12}\), corrected for 36 tests), while the +23 GpG site was the only hypomethylated one in former opiate addicts compared with controls (P?=?0.0023 after Bonferroni correction). Our results supported that opioid exposure was associated with methylation status of OPRM1 promoter and showed ethnic dependence.