Promoter methylation and loss of coding exons of the fragile histidine triad (FHIT) gene in intrahepatic cholangiocarcinomas.

AIMS: About 10-30% of primary liver cancers represent intrahepatic cholangiocarcinomas (IHCC). Since chromosomal losses of 3p are detectable in about 40% of cholangiocarcinomas our study aimed at the identification of mechanisms leading to functional deletion of tumor suppressor genes in this region. Our efforts focussed on genomic losses and epigenetic inactivation of two tumor suppressor genes, the fragile histidine triad (FHIT) and the ras association domain family 1 (RASSF1A) genes, both located on the short arm of chromosome 3. METHODS: Methylation-specific PCR (MSP) and combined bisulfite-dependent restriction analysis (COBRA) were applied to detect epigenetic silencing of gene promoters. Genomic duplex PCR was used to identify exon losses of the FHIT gene. Nineteen paraffin-embedded samples of intrahepatic cholangiocarcinomas were studied. RESULTS: Here we report for the first time that in addition to frequent losses of the exons 5 and 6, hypermethylation of the FHIT promoter occured in a significant portion of IHCC. Methylation specific PCR (MSP) detected epigenetic inactivation of the FHIT/FRA3B locus in 8 of 19 (42%) cases. Combined bisulfite restriction analysis (COBRA) revealed that high levels of methylated FHIT promoter sequences were present in 6 of the 8 methylation positive samples. In agreement with previous reports MSP identified hypermethylation of the RASSF1A gene in 13 of 19 (68%) IHCC specimens examined. CONCLUSIONS: Epigenetic silencing of the FHIT tumor suppressor gene is a novel inactivation mechanism to be considered in the development of intrahepatic cholangiocarcinomas. However, a statistically significant inverse correlation between K-Ras activation and RASSF1A inactivation was not found.     

Decrease in cytosine methylation at CpG island shores and increase in DNA fragmentation during zebrafish aging

Age-related changes in DNA methylation have been demonstrated in mammals, but it remains unclear as to the generality of this phenomenon in vertebrates, which is a criterion for the fundamental cause of senescence. Here we showed that the zebrafish genome gradually and clearly lost methylcytosine in somatic cells, but not in male germ cells during aging, and that age-dependent hypomethylation preferentially occurred at a particular domain called the CpG island shore, which is associated with vertebrates’ genes and has been shown to be hypomethylated in humans with age. We also found that two CpG island shores hypomethylated in zebrafish oocytes were de novo methylated in fertilized eggs, which suggests that the zebrafish epigenome is reset upon fertilization, enabling new generations to restart with a heavily methylated genome. Furthermore, we observed an increase in cleavage of the zebrafish genome to an oligonucleosome length in somatic cells from the age of 12 months, which is suggestive of an elevated rate of apoptosis in the senescent stage.     

Connecting the epigenome,metabolome and proteome for a deeper understanding of disease

Epigenome-wide association studies (EWAS) identify genes that are dysregulated by the studied clinical endpoints, thereby indicating potential new diagnostic biomarkers, drug targets and therapy options. Combining EWAS with deep molecular phenotyping, such as approaches enabled by metabolomics and proteomics, allows further probing of the underlying disease-associated pathways. For instance, methylation of the TXNIP gene is associated robustly with prevalent type 2 diabetes and further with metabolites that are short-term markers of glycaemic control. These associations reflect TXNIP’s function as a glucose uptake regulator by interaction with the major glucose transporter GLUT1 and suggest that TXNIP methylation can be used as a read-out for the organism’s exposure to glucose stress. Another case is the association between DNA methylation of the AHRR and F2RL3 genes with smoking and a protein that is involved in the reprogramming of the bronchial epithelium. These examples show that associations between DNA methylation and intermediate molecular traits can open new windows into how the body copes with physiological challenges. This knowledge, if carefully interpreted, may indicate novel therapy options and, together with monitoring of the methylation state of specific methylation sites, may in the future allow the early diagnosis of impending disease. It is essential for medical practitioners to recognize the potential that this field holds in translating basic research findings to clinical practice. In this review, we present recent advances in the field of EWAS with metabolomics and proteomics and discuss both the potential and the challenges of translating epigenetic associations, with deep molecular phenotypes, to biomedical applications.     

CpG island methylator phenotype and its relationship with prognosis in adult acute leukemia patients

Objective

To investigated the relationship between CpG island methylator phenotype (CIMP) and prognosis in adults with acute leukemia.

Methods

Bone marrow samples from 53 acute myeloid leukemia and 50 acute lymphoblastic leukemia patients were collected. The methylation status of 18 tumor suppressor genes was determined using methylation-specific polymerase chain reaction.

Results

Greater than 30% of acute leukemia patients had methylated p15, p16, CDH1, CDH13, RUNX3, sFRP1, ID4, and DLC-1 genes; methylation of ≥4 were defined as CIMP positive. Age, type of leukemia, white blood cell count, and CIMP status were significantly associated with recurrence-free survival (RFS) and overall survival (OS) (P < 0.05). CIMP status was an independent prognostic factor for OS (hazard ratio: 2.07, 95% confidence interval: 1.03–4.15, P = 0.040). CIMP-negative patients had significantly improved RFS and OS (P < 0.05). p16 and DLC1 methylation was significantly associated with RFS and OS (P < 0.05).

Conclusions

CIMP may serve as an independent risk factor for evaluating the prognosis of patients with acute leukemia.     

甲基转移酶基因甲基化在结直肠肿瘤中的作用

目的探讨6氧甲基鸟嘌呤DNA甲基转移酶(MGMT)基因启动子CpG岛高甲基化在结直肠肿瘤发生、发展中的作用。方法用甲基特异性PCR检测20例正常大肠黏膜、27例散发性结直肠腺瘤和62例散发性结直肠腺癌组织DNA中MGMT基因启动子的甲基化,同时用免疫组化方法检测MGMT蛋白的表达情况。结果正常大肠黏膜组织均未显示甲基化条带,分别有40.7%(11/27)的腺瘤组织和43.5%(27/62)的腺癌组织存在MGMT基因启动子CpG岛高甲基化。正常大肠黏膜胞核和胞质表达MGMT蛋白,22.2%(6/27)的腺瘤和45.2%(28/62)的腺癌MGMT蛋白表达缺失,其差异有显著性(P=0.041)。6例MGMT表达缺失的腺瘤中5例存在甲基化(P=0.027),28例表达缺失的腺癌中24例存在甲基化(P<0.001)。结论结直肠肿瘤中存在高频率MGMT基因启动子高甲基化和蛋白表达缺失,腺癌中蛋白表达缺失比腺瘤中更常见。MGMT蛋白表达缺失与MGMT基因启动子区域高甲基化有关。MGMT基因表型遗传性失活可能在结直肠肿瘤发生过程中起重要作用。     

A system for the targeted amplification of bacterial gene clusters multiplies antibiotic yield in Streptomyces coelicolor

Gene clusters found in bacterial species classified as Streptomyces encode the majority of known antibiotics as well as many pharmaceutically active compounds. A site-specific recombination system similar to those that mediate plasmid conjugation was engineered to catalyze tandem amplification of one of these gene clusters in a heterologous Streptomyces species. Three genetic elements were known to be required for DNA amplification in S. kanamyceticus: the oriT-like recombination sites RsA and RsB, and ZouA, a site-specific relaxase similar to TraA proteins that catalyze plasmid transfer. We inserted RsA and RsB sequences into the S. coelicolor genome flanking a cluster of 22 genes (act) responsible for biosynthesis of the polyketide antibiotic actinorhodin. Recombination between RsA and RsB generated zouA-dependent DNA amplification resulting in 4-12 tandem copies of the act gene cluster averaging nine repeats per genome. This resulted in a 20-fold increase in actinorhodin production compared with the parental strain. To determine whether the recombination event required taxon-specific genetic effectors or generalized bacterial recombination (recA), it was also analyzed in the heterologous host Escherichia coli. zouA was expressed under the control of an inducible promoter in wild-type and recA mutant strains. A plasmid was constructed with recombination sites RsA and RsB bordering a drug resistance marker. Induction of zouA expression generated hybrid RsB/RsA sites, evidence of site-specific recombination that occurred independently of recA. ZouA-mediated DNA amplification promises to be a valuable tool for increasing the activities of commercially important biosynthetic, degradative, and photosynthetic pathways in a wide variety of organisms.     

燃煤型砷中毒患者p16基因缺失及启动区CpG岛甲基化的观察

目的检测燃煤型砷中毒(地砷病)患者p16基因缺失及启动区甲基化的变异,探讨p16基因异常改变在地砷病发生发展乃至癌变过程中的作用。方法采用多重PCR法对103例砷中毒患者和110例正常对照人群p16基因第1、第2外显子缺失情况进行检测;同时采用甲基化特异性PCR法(MSP)和PCR产物测序技术对95例砷中毒患者和100例正常对照人群p16基因启动区甲基化情况进行分析。结果(1)正常对照组p16基因缺失率为5．45％;病例组为14．56％,以第2外显子缺失为主,其中轻、中、重度中毒组p16基因缺失率分别为9．09％、12．50％和19．51％;非癌变组和癌变组p16基因缺失率分别为8．70％和38．89％。以上差异有统计学意义(P<0．05或P<0．01)。(2)对照组p16基因甲基化阳性率为2．00％;病例组为42．11％,其中轻、中、重度中毒组p16基因甲基化阳性率分别为26．32％、41．67％和50．00％;非癌变组和癌变组阳性率分别为21．74％和55．56％。以上差异均有统计学意义(P<0．05或P<0．01)。结论p16基因缺失及启动区甲基化在地砷病的发生发展乃至癌变过程中起重要作用。     

Identification of methylation profile of HOX genes in extrahepatic cholangiocarcinoma

AIM:To identify methylation profile and novel tumor marker of extrahepatic cholangiocarcinoma(CCA)with high throughout microarray.METHODS:Differential methylation profile was compared between normal bile duct epithelial cell lines and CCA cell lines by methyl-DNA immunoprecipitation (MeDIP)microarray.Bisulfite-polymerase chain reaction (BSP)was performed to identify the methylated allels of target genes.Expression of target genes was investigated before and after the treatment with DNA demethylating agent.E...     

MicroRNA-132 dysregulation in schizophrenia has implications for both neurodevelopment and adult brain function

Schizophrenia is characterized by affective, cognitive, neuromorphological, and molecular abnormalities that may have a neurodevelopmental origin. MicroRNAs (miRNAs) are small noncoding RNA sequences critical to neurodevelopment and adult neuronal processes by coordinating the activity of multiple genes within biological networks. We examined the expression of 854 miRNAs in prefrontal cortical tissue from 100 control, schizophrenic, and bipolar subjects. The cyclic AMP-responsive element binding- and NMDA-regulated microRNA miR-132 was significantly down-regulated in both the schizophrenic discovery cohort and a second, independent set of schizophrenic subjects. Analysis of miR-132 target gene expression in schizophrenia gene-expression microarrays identified 26 genes up-regulated in schizophrenia subjects. Consistent with NMDA-mediated hypofunction observed in schizophrenic subjects, administration of an NMDA antagonist to adult mice results in miR-132 down-regulation in the prefrontal cortex. Furthermore, miR-132 expression in the murine prefrontal cortex exhibits significant developmental regulation and overlaps with critical neurodevelopmental processes during adolescence. Adult prefrontal expression of miR-132 can be down-regulated by pharmacologic inhibition of NMDA receptor signaling during a brief postnatal period. Several key genes, including DNMT3A, GATA2, and DPYSL3, are regulated by miR-132 and exhibited altered expression either during normal neurodevelopment or in tissue from adult schizophrenic subjects. Our data suggest miR-132 dysregulation and subsequent abnormal expression of miR-132 target genes contribute to the neurodevelopmental and neuromorphological pathologies present in schizophrenia.     

Mechanisms of activation of the paternally expressed genes by the Prader-Willi imprinting center in the Prader-Willi/Angelman syndromes domains

The Prader-Willi syndrome/Angelman syndrome (PWS/AS) imprinted domain is regulated by a bipartite imprinting control center (IC) composed of a sequence around the SNRPN promoter (PWS-IC) and a 880-bp sequence located 35 kb upstream (AS-IC). The AS-IC imprint is established during gametogenesis and confers repression upon PWS-IC on the maternal allele. Mutation at PWS-IC on the paternal allele leads to gene silencing across the entire PWS/AS domain. This silencing implies that PWS-IC functions on the paternal allele as a bidirectional activator. Here we examine the mechanism by which PWS-IC activates the paternally expressed genes (PEGs) using transgenes that include the PWS-IC sequence in the presence or absence of AS-IC and NDN, an upstream PEG, as an experimental model. We demonstrate that PWS-IC is in fact an activator of NDN. This activation requires an unmethylated PWS-IC in the gametes and during early embryogenesis. PWS-IC is dispensable later in development. Interestingly, a similar activation of a nonimprinted gene (APOA1) was observed, implying that PWS-IC is a universal activator. To decipher the mechanism by which PWS-IC confers activation of remote genes, we performed methylated DNA immunoprecipitation (MeDIP) array analysis on lymphoblast cell lines that revealed dispersed, rather than continued differential methylation. However, chromatin conformation capture (3c) experiments revealed a physical interaction between PWS-IC and the PEGs, suggesting that activation of PEGs may require their proximity to PWS-IC.     

Hypermethylation of CpG island in O6-methylguanine-DNA methyltransferase gene was associated with K-ras G to A mutation in colorectal tumor

AIM:To investigate the functions of promoter hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT) gene in colorectal tumorigenesis and progression. METHODS: The promoter hypermethylation of MGMT gene was detected in 27 sporadic colorectal adenomas, 62 sporadic colorectal carcinomas and 20 normal colorectal mucosa tissues by methylation-specific PCR. At the same time, the expression of MGMT protein was carried out in the same samples using immunohistochemistry. Mutant-allele-specific amplification was used to detect K-rasG to A point mutation in codon 12. RESULTS: None of the normal colorectal mucosa tissues showed methylated bands. Promoter hypermethylation was detected in 40.7% (11 of 27) of adenomas and 43.5% (27 of 62) of carcinomas. MGMT proteins were expressed in nucleus and cytoplasm of normal colorectal mucosa tissues. Loss of MGMT expression was found in 22.2% (6 of 27) of adenomas and 45.2% (28 of 62) of carcinomas. The difference between them was significant (P= 0.041). In the 6 adenomas and 28 carcinomas losing MGMT expression, 5 and 24 cases presented methylation, respectively (P = 0.027, P<0.001). Thirteen of the 19 colorectal tumors with K-rasG to A point mutation in codon 12 had methylated MGMT(P= 0.011). The frequencies of K-rasG to A point mutation were 35.3% (12 of 34) and 12.7% (7 of 55) in tumors losing MGMT expression and with normal expression, respectively. CONCLUSION: Promoter hypermethylation and loss of expression of MGMT gene were common events in colorectal tumorigenesis, and loss of expression of MGMT occurs more frequently in carcinomas than in adenomas in sporadic patients. Hypermethylation of the CpG island of MGMT gene was associated with loss of MGMT expression and K-rasG to A point mutation in colorectal tumor. The frequency of K-rasG to A point mutation was increased in tumors losing MGMT expression. It suggests that epigenetic inactivation of MGMT plays an important role in colorectal neoplasia.     

Convergent gene loss following gene and genome duplications creates single-copy families in flowering plants

The importance of gene gain through duplication has long been appreciated. In contrast, the importance of gene loss has only recently attracted attention. Indeed, studies in organisms ranging from plants to worms and humans suggest that duplication of some genes might be better tolerated than that of others. Here we have undertaken a large-scale study to investigate the existence of duplication-resistant genes in the sequenced genomes of 20 flowering plants. We demonstrate that there is a large set of genes that is convergently restored to single-copy status following multiple genome-wide and smaller scale duplication events. We rule out the possibility that such a pattern could be explained by random gene loss only and therefore propose that there is selection pressure to preserve such genes as singletons. This is further substantiated by the observation that angiosperm single-copy genes do not comprise a random fraction of the genome, but instead are often involved in essential housekeeping functions that are highly conserved across all eukaryotes. Furthermore, single-copy genes are generally expressed more highly and in more tissues than non–single-copy genes, and they exhibit higher sequence conservation. Finally, we propose different hypotheses to explain their resistance against duplication.     

瘦素启动子甲基化在骨关节炎发病中的作用

目的 探讨瘦素启动子甲基化在骨关节炎(OA)发病中的作用.方法 取OA组和对照组(创伤行截肢手术,除外OA等关节炎性疾病)患者膝骨关节标本,培养膝关节软骨细胞,采用不同浓度(5.0 μmol/L、10.0 μmol/L、20.0 μmol/L)不同时间(12 h、24 h、48 h、72 h、96h、120 h、168 h)5-氮杂-2'-脱氧胞苷(5-Aza-CdR)刺激软骨细胞,实时定量PCR检测膝关节软骨细胞瘦素mRNA表达,同时使用Epityper DNA甲基化分析技术枪测该基因启动子部分区域(-280～+79)的甲基化状态.结果 (1)10μmol/L 5-Aza-CdR刺激OA组软骨细胞,其瘦素mRNA表达水平增高,72 h最为显著.(2)OA组5-Aza-CdR刺激72 h后软骨细胞瘦素mRNA表达最高,对照组无5-Aza-CdR刺激软骨细胞瘦素mRNA表达最低.(3)使用非先导分层聚类分析法对瘦素启动子区域进行分析,2组甲基化模式存在差异,且 5-Aza-CdR刺激前后甲基化模式也不相同.结论 瘦素启动子部分位点的去甲基化可能是引起该基因异常表达导致OA发生的始动因素之一.

Abstract：

Objective To investigate the effects of 5-Aza-CdR( methylation transferase inhibitor) on the expression levels of leptin gene in chondrocytes and methylation states of leptin promoter region between osteoarthritis (OA) group and control. Methods The chondrocytes in osteoarthritis group were treated with 5-Aza-CdR with different doses and time-points, and the expression level of leptin was detected by real-time polymerase chain reaction for picking up the optimum dose and time-point. Next, the chondrocytes in 5 osteoarthritis patients and 5 control patients (amputation due to severe trauma) were treated with 5-Aza-CdR. Lastly, leptin mRNA expression levels in the four groups osteoarthritis and control chondrocytes treated with/without 5-Aza-CdR were measured by real-time PCR and the methylation state of promoter region ( - 280- + 79) was detected by epityper quantitative DNA methylation analysis. Results ( 1 ) After treating the chondrocytes in OA groups with 10 μmol/L 5-Aza-CdR for 72 h, the mRNA expression levels of leptin were increased significantly. ( 2 ) The mRNA expression levels of leptin were significantly different among the four groups ( P ＜ 0. 05 ), and the chondrocytes in osteoarthritis groups treated with 5-Aza-CdR showed a marked induction of leptin mRNA expression. (3) Analysis of quantitative methylation data using an unsupervised hierarchical clustering algorithm, showed that methylation patterns of leptin promoter was different between control and osteoarthritis chondrocyte treated with/without 5-Aza-CdR. Conclusion Demethylation of leptin promoter might up-regulate leptin gene expression level and it might contribute to osteoarthritis.     

H3K4 methyltransferase Set1 is involved in maintenance of ergosterol homeostasis and resistance to Brefeldin A

Set1 is a conserved histone H3 lysine 4 (H3K4) methyltransferase that exists as a multisubunit complex. Although H3K4 methylation is located on many actively transcribed genes, few studies have established a direct connection showing that loss of Set1 and H3K4 methylation results in a phenotype caused by disruption of gene expression. In this study, we determined that cells lacking Set1 or Set1 complex members that disrupt H3K4 methylation have a growth defect when grown in the presence of the antifungal drug Brefeldin A (BFA), indicating that H3K4 methylation is needed for BFA resistance. To determine the role of Set1 in BFA resistance, we discovered that Set1 is important for the expression of genes in the ergosterol biosynthetic pathway, including the rate-limiting enzyme HMG-CoA reductase. Consequently, deletion of SET1 leads to a reduction in HMG-CoA reductase protein and total cellular ergosterol. In addition, the lack of Set1 results in an increase in the expression of DAN1 and PDR11, two genes involved in ergosterol uptake. The increase in expression of uptake genes in set1Δ cells allows sterols such as cholesterol and ergosterol to be actively taken up under aerobic conditions. Interestingly, when grown in the presence of ergosterol set1Δ cells become resistant to BFA, indicating that proper ergosterol levels are needed for antifungal drug resistance. These data show that H3K4 methylation impacts gene expression and output of a biologically and medically relevant pathway and determines why cells lacking H3K4 methylation have antifungal drug sensitivity.