A Downstream CpG Island Controls Transcript Initiation and Elongation and the Methylation State of the Imprinted Airn Macro ncRNA Promoter
Martha V. KoernerFlorian M. PaulerQuanah J. HudsonFederica SantoroAnna SawickaPhilipp GuenzlStefan H. StrickerYvonne M. SchichlPaulina A. LatosRuth M. KlementKatarzyna E. WarczokJacek WojciechowskiChristian SeiserRóbert KrálovicsDenise P. Barlow
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Abstract:
A CpG island (CGI) lies at the 5′ end of the Airn macro non-protein-coding (nc) RNA that represses the flanking Igf2r promoter in cis on paternally inherited chromosomes. In addition to being modified on maternally inherited chromosomes by a DNA methylation imprint, the Airn CGI shows two unusual organization features: its position immediately downstream of the Airn promoter and transcription start site and a series of tandem direct repeats (TDRs) occupying its second half. The physical separation of the Airn promoter from the CGI provides a model to investigate if the CGI plays distinct transcriptional and epigenetic roles. We used homologous recombination to generate embryonic stem cells carrying deletions at the endogenous locus of the entire CGI or just the TDRs. The deleted Airn alleles were analyzed by using an ES cell imprinting model that recapitulates the onset of Igf2r imprinted expression in embryonic development or by using knock-out mice. The results show that the CGI is required for efficient Airn initiation and to maintain the unmethylated state of the Airn promoter, which are both necessary for Igf2r repression on the paternal chromosome. The TDRs occupying the second half of the CGI play a minor role in Airn transcriptional elongation or processivity, but are essential for methylation on the maternal Airn promoter that is necessary for Igf2r to be expressed from this chromosome. Together the data indicate the existence of a class of regulatory CGIs in the mammalian genome that act downstream of the promoter and transcription start.Keywords:
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Genomic Imprinting
The aetiology of OSCC remains unclear, however, aberrant methylation of CpG island promoters of tumor suppressor genes have been identified as contributory developmental pathways in several cancers. The aim of this study was to determine the presence of RUNX3 gene methylation and how its association with patients? demographic variables such as gender, age, histologic class and tumor location could be of diagnostic value for OSCC. Sixty-seven formalin-fixed paraffin-embedded (FFPE) solid tissue blocks of OSCC, and nine blocks of benign oral lesions of epithelial origin retrieved from the archives of the Department of Oral Pathology, University College Hospital, Ibadan, South-West Nigeria were used for the analyses. Frequency of CpG island methylation in the promoter region of RUNX3 was determined by methylation-specific polymerase chain reaction (MSP). Association between gender, age, tumor location, histologic class and promoter methylation in RUNX3 was assessed with Pearson?s ?2 test. Overall, 45% (30/67) of OSCC demonstrated methylation in the RUNX3 promoter indicating a high frequency of methylation of the CpG island promoter region of RUNX3. There was no association between gender, age, histologic class and promoter methylation in RUNX3 (P > 0.05), however a significant association was observed between tumor location and promoter methylation of RUNX3 (P < 0.05). Aberrant methylation of the CpG island promoter region of RUNX3 together with tumor location could therefore be critical in the development and diagnosis of OSCC.
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Promoters play a central role in gene regulation, yet our power to discriminate them from non-promoter sequences in higher eukaryotes is mainly restricted to those associated with CpG islands. Here, we examined in silico the promoters of 30,954 human and 18,083 mouse transcripts in the DBTSS database, to assess the impact of particular sequence and structural features (propeller twist, bendability and nucleosome positioning preference) on promoter classification and prediction. Our analysis showed that a stricter-than-traditional definition of CpG islands captures low and high CpG count promoter classes more accurately than the traditional one. We observed that both human and mouse promoter sequences are flexible with the exception of the TATA box and TSS, which are rigid regions irrespective of association with a CpG island. Therefore varying levels of structural flexibility in promoters may affect their accessibility to proteins, and hence their specificity. For all features investigated, averaged values across core promoters discriminated CpG island associated promoters from background, whereas the same did not hold for promoters without a CpG island. However, local changes around - 34 to - 23 (expected position of TATA box) and the TSS were informative in discriminating promoters (both classes) from non-promoter sequences. Additionally, we investigated ATG deserts and observed that they occur in all promoter sets except those with a TATA-box and without a CpG island in human. Interestingly, all mouse promoter sets showed ATG codon depletion irrespective of the presence of a TATA-box, possibly reflecting a weaker contribution to TSS specificity in mouse.
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Objective To investigate the methylation status of 5'CpG island of the promoter region of HOXA10 gene,and to explore the pathogenic role of the methylation status on endometriosis.Methods Thirty patients with endometriosis,who were admitted to Zhujiang Hospital of Southern Medical University from Sep.2007 to May 2008,and 25 healthy women were involved in present study.Of the 30 patients with endometriosis,10 were in stages Ⅰ-Ⅱ,and 20 in stages Ⅲ-Ⅳ.The specimens of endometrial tissue were collected from 30 patients with endometriosis and 25 healthy women,and the methylation status in 5'CpG island of HOXA10 gene promoter region was detected by using methylation-specific PCR technique(MSP).Results Different methylation levels in 5'CpG island in promoter region of HOXA10 gene were detected from the patients in different clinical stage of endometriosis.Methylation in 5'CpG island of the promoter region of HOXA10 gene was observed in 11 of 20(55%) patients in endometriosis stages Ⅲ-Ⅳ,and 1 of 10(10%) patients in endometriosis stages Ⅰ-Ⅱ.The methylation level in 5'CpG island of the promoter region of HOXA10 gene of the patients in endometriosis stages Ⅲ-Ⅳ was higher than that of the patients in endometriosis stages Ⅰ-Ⅱ(P0.05).However,no methylation was found in the endometrial tissue of 25 healthy women.Conclusion The methylation in 5'CpG island of the promoter region of HOXA10 gene may play an important role in the pathogenesis and development of endometriosis.
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The 5′ region for the endothelin receptor B (EDNRB) gene is a complex CpG island giving rise to four individual transcripts initiating within the island. Here, for the first time, we analyze the relationship between methylation and gene expression in a CpG island located in the 5′ region of a gene with multiple transcription start sites. The CpG island was unmethylated in normal prostate and bladder tissue, whereas it became methylated in apparently normal colonic epithelium. Tumors derived from these tissues were frequently hypermethylated relative to the respective normal tissues. Analysis of 11 individual CpG sites located throughout the CpG island showed that specific sites with high methylation levels in several tumors were also methylated in normal tissues, suggesting that they might serve as foci for further de novo methylation. This region also had high levels of methylation in several cancer cell lines, and we found that a low methylation level in a small region within the 5′ region correlated with expression of the 5′-most transcript. Interestingly, almost complete methylation 200–1000 bp downstream of the transcriptional start site did not block expression of this transcript. Finally, we show that treatment with 5-aza-2′-deoxycytidine can induce transcriptional activation of the four EDNRB transcripts. Our results show the existence of differential, tissue-dependent methylation at the EDNRB 5′ region, suggest the existence of a spreading mechanism for de novo methylation, starting from methylation hotspots, and show that hypermethylation immediately 3′ to a transcriptional start site does not prevent initiation.
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To examine methylation of the peroxisome proliferator-activated receptor γ (PPARγ) gene and its relationship with child weight status, at birth and 9 years.We measured PPARγ methylation across 23 CpG sites using the Infinium Illumina 450 k array for children from the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) cohort at birth (N = 373) and 9 years (N = 245).Methylation level correlation patterns across the 23 PPARγ CpG sites were conserved between birth and 9-year ages. We found high inter-CpG correlations between sites 1-3 (methylation block 1) and also between sites 18-23 (methylation block 2) for both time points, although these patterns were less pronounced at 9 years. Additionally, sites 1-3 (north shore) had the highest intra-CpG correlations over time (r = 0.24, 0.42, and 0.3; P = 0.002, P < 0.001, P < 0.001, respectively). PPARγ methylation levels tended to increase with age, and the largest differences were observed for north shore sites (7.4%). Adjusting for sex, both site 1 and site 20 (gene body) methylation at birth was significantly and inversely associated with birth weight (β = -0.13, P = 0.033; β = -0.09, P = 0.025, respectively). Similarly, we found that site 1 and site 20 methylation at 9 years was significantly and inversely associated with 9-year BMI z-score (β = -0.41, P = 0.015; β = -0.23, P = 0.045, respectively).Our results indicate that PPARγ methylation is highly organized and conserved over time, and highlight the potential functional importance of north shore sites, adding to a better understanding of regional human methylome patterns. Overall, our results suggest that PPARγ methylation may be associated with child body size.
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Abstract Background The ESR 1 gene suffers methylation changes in many types of cancers, including breast cancer (BC), the most frequently diagnosed cancer in women that is also present in men. Methylation at promoter A of ESR 1 is the worse prognosis in terms of overall survival; thus, the early detection, prognostic, and prediction of therapy involve some methylation biomarkers. Methods Therefore, our study aimed to examine the methylation levels at the ESR 1 gene in samples from Mexican BC patients and its possible association with menopausal status. Results We identified a novel 151-bp CpG island in the promoter A of the ESR 1 gene. Interestingly, methylation levels at this CpG island in positive ERα tumors were approximately 50% less than negative ERα or control samples. Furthermore, methylation levels at ESR 1 were associated with menopausal status. In postmenopausal patients, the methylation levels were 1.5-fold higher than in premenopausal patients. Finally, according to tumor malignancy, triple-negative cancer subtypes had higher ESR 1 methylation levels than luminal/HER2+ or luminal A subtypes. Conclusions Our findings suggest that methylation at this novel CpG island might be a promising prognosis marker
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Objective The aim of this study is to investigate the CpG island methylation status of TIMP- 3 in HCC cell lines, to treate the HCC cells that exhibit positive methylation with DNA methyltransferase in- hibitor(5-Aza-CdR), and to observe the influence of 5-Aza-CdR on the expression of TIMP-3 mRNA and its CpG island methylation degree. Methods TIMP-3 CpG methylation status of HCC cells were examined using MSP assay. Methylation-positive HCC cells were treated with DNA methyltransferase inhibitor(5-Aza- CdR)(intervention group), and the difference of TIMP-3 mRNA expression was observed between interven- tion group and control group. Pyrosequencing method was applied to quantitatively examine the CpG island methylation percentage. Results The methylation of TIMP-3 CpG islands in C3A, HepG-2, and Hep-3B was positive. The results showed that TIMP-3 mRNA expression was increased(P 0.05) and the methylation level was decreased(P 0.05) in the intervention group compared to control group. Conclusion 5-Aza-CdR could remarkably reduce the level of the CpG island methylation and increase the expression of TIMP-3 mR- NA in HCC cells.
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Bisulfite sequencing
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Objective To investigate the role of CpG island methylation of INSL3 gene in cryp-torchid mice induced by DEHP. Methods CpG islands in INSL3 gene were predicted with MethPrim-er software. The status of methylation of CpG islands in INSL3 gene was analyzed using methylation-specific PCR(MS-PCR). The levels of INSL3 mRNA in rats of the experimental and control group were detected and compared by RT-PCR methods. Results The methylated CpG islands in the first exon of INSL3 were merely noted in cryptorchid mice, while not in normal mice. The levels of INSL3 mRNA reduced significantly in cryptorchid mice. Conclusions The hypermethylation of CpO islands in the first exon of INSL3 may probably cause low INSL3 expression in cryptorchid mice, which can be closely related with the pathogenesis of cryptorchid.
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Insulin-like factor 3; Cryptorchidsm; DNA methylation
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Pathogenesis
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