A DNA Methylation Site in the Male-Specific P450 (<i>Cyp</i> 2d-9) Promoter and Binding of the Heteromeric Transcription Factor GABP

Yokomori, Norihiko; Kobayashi, Ryûji; Moore, Rick; Sueyoshi, Tatsuya; Negishi, Masahiko

doi:10.1128/mcb.15.10.5355

Cited by 81 publications

(61 citation statements)

References 39 publications

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“…DNA methylation was recently shown to contribute to the cell-specific expression of the gene encoding l-histidine decarboxylase and cyclin D1 [16,20]. We have also found that demethylation of specific CpG sites, as well as a methylation-sensitive transcription factor, contribute to the sex-specific expression of the Cyp2d-9 gene and to the cell-specific expression of the thyrotropin receptor gene [21,22]. The differentiation of 3T3-L1 preadipocytes to adipocytes is accompanied by the activation of numerous adipocyte-specific genes [23].…”

Section: Discussionmentioning

confidence: 59%

DNA demethylation modulates mouse leptin promoter activity during the differentiation of 3T3-L1 cells

2002

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During the differentiation of 3T3-L1 preadipocytes to adipocytes, transcriptional activators and repressors regulate the expression of many adipocyte-specific genes [1±4]. In addition, DNA methylation could also play an important role during the process of adipocyte differentiation. We recently showed that methylation of specific CpG sites of the GLUT4 gene and a methylation sensitive transcription factor, contribute to GLUT4 gene regulation during 3T3-L1 differentiation [5]. Similar mechanisms could also regulate the expression of other genes induced during adipocyte differentiation.Leptin, a major hormonal regulator of appetite and fat cell mass, is one of these adipocyte-specific genes [6,7]. It is secreted by adipose tissue in response to a high content of body fat. Thus, mice lacking a functional leptin gene develop hyperphagia, hy- Abstract Aims/hypothesis. The mouse leptin gene, a major hormonal regulator of appetite and fat cell mass, expresses during the differentiation of 3T3-L1 preadipocytes to adipocytes. To determine if DNA methylation is involved in regulating the expression of the leptin gene, we examined the methylation status and methylation-sensitive transcription factors during 3T3-L1 differentiation. Methods. DNase I footprinting, electrophoretic mobility-shift assays, and a Southwestern analysis were carried out using nuclear extracts from preadipocytes and adipocytes. Promoter activity was measured by luciferase assays. The CpG methylation pattern was determined.Results. Transient transfection of reporter constructs with the leptin promoter showed that preadipocytes that do not transcribe the leptin gene show enough transactivation, suggesting the presence of an additional regulatory mechanism. We identified eight CpG sites in the promoter up to nt ±161, all of which were highly methylated ( > 92 %) in preadipocytes. Seven of these sites showed a varying degree of demethylation during differentiation, while the site at nt ±54 remained methylated. In electrophoretic mobilityshift assays, DNA fragments from nt ±115 to nt ±70 generated a methylation-sensitive band with nuclear extracts from preadipocytes when the CpG sites were methylated. Southwestern analysis identified a 52 kM r protein that binds strongly to the methylated probes. Promoter activity was reduced by methylation of the CpG sites up to nt ±115, but not up to nt ±70. Conclusion/interpretation. These results suggest that methylation of specific CpG sites between nt ±115 and nt ±70 and a methylation-sensitive protein could contribute to leptin gene expression during adipocyte differentiation in 3T3-L1 cells. [Diabetologia (2002) 45: 140±148]

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Section: Discussionmentioning

confidence: 59%

DNA demethylation modulates mouse leptin promoter activity during the differentiation of 3T3-L1 cells

2002

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“…Of additional interest, both families of transcription factors already known to modulate HPSE expression also relate to DNA methylation. GABP, ETS-1 and other ETS factors bind to target sequences in humans and rodents, depending on the methylation status of nearby cytosines (Yokomori et al, 1995(Yokomori et al, , 1998De Smet et al, 1996;Umezawa et al, 1997;Guo et al, 2002), whereas Sp1 sites were shown to protect at least some CpG islands from spread of methylation present in adjacent repetitive sequences (Brandeis et al, 1994;Macleod et al, 1994;Siegfried et al, 1999). Thus, it is reasonable to propose that the promoter activity of mammalian heparanase is determined by interplay of genetic and epigenetic factors.…”

Section: Discussionmentioning

confidence: 99%

Role of promoter methylation in regulation of the mammalian heparanase gene

et al. 2003

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Mammalian heparanase (endo-b-glucuronidase) degrades heparan sulfate proteoglycans and is an important modulator of the extracellular matrix and associated factors. The enzyme is preferentially expressed in neoplastic tissues and contributes to tumour metastasis and angiogenesis. To investigate the epigenetic regulation of the heparanase locus, methylation-specific and bisulfite PCR were performed on a panel of 22 human cancer cell lines. Cytosine methylation of the heparanase promoter was associated with inactivation of the affected allele. Despite lack of sequence homology, extensively methylated CpG islands were found both in human choriocarcinoma (JAR) and rat glioma (C-6) cells which lack heparanase activity. Treatment of these cells with demethylating agents (5-azacytidine, 5-aza-2 0 -deoxycytidine) resulted in stable dose-and time-dependant promoter hypomethylation accompanied by reappearance of heparanase mRNA, protein and enzymatic activity. An inhibitor of histone deacetylase, Trichostatin A, failed to induce either of these effects. Upregulation of heparanase expression and activity by demethylating drugs was associated with a marked increase in lung colonization by pretreated C-6 rat glioma cells. The increased metastatic potential in vivo was inhibited in mice treated with laminaran sulfate, a potent inhibitor of heparanase activity. We propose a model wherein expression of mammalian heparanase gene is modulated by the interplay between trans-activating genetic and cis-inhibitory epigenetic elements in its promoter.

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“…Previous work has shown that the interaction of GABP␣ with its recognition sequence is blocked by CpG methylation (42)(43)(44)(45). Yokomori et al (44) suggested that the sensitivity of GABP␣ to DNA methylation may be a mechanism for regulating sex-specific expression at the mouse Cyp 2d-9 locus, which is differentially methylated in males and females.…”

Section: Discussionmentioning

confidence: 99%

Methylation-sensitive Regulation of TMS1/ASC by the Ets Factor, GA-binding Protein-α

Lucas

Crider

Powell

et al. 2009

Journal of Biological Chemistry

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Epigenetic silencing involving the aberrant DNA methylation of promoter-associated CpG islands is one mechanism leading to the inactivation of tumor suppressor genes in human cancers. However, the molecular mechanisms underlying this event remains poorly understood. TMS1/ASC is a novel proapoptotic signaling factor that is subject to epigenetic silencing in human breast and other cancers. The TMS1 promoter is embedded within a CpG island that is unmethylated in normal cells and is spanned by three DNase I-hypersensitive sites (HS). Silencing of TMS1 in cancer cells is accompanied by local alterations in histone modification, remodeling of the HS, and hypermethylation of DNA. In this study, we probed the functional significance of the CpG island-specific HS. We identified a methylation-sensitive complex that bound a 55-bp intronic element corresponding to HS2. Affinity chromatography and mass spectrometry identified a component of this complex to be the GA-binding protein (GABP) ␣. Supershift analysis indicated that the GABP␣ binding partner, GABP␤1, was also present in the complex. The HS2 element conferred a 3-fold enhancement in TMS1 promoter activity, which was dependent on both intact tandem ets binding sites and the presence of GABP␣/␤1 in trans. GABP␣ was selectively enriched at HS2 in human cells, and its occupancy was inversely correlated with CpG island methylation. Down-regulation of GABP␣ led to a concomitant decrease in TMS1 expression. These data indicate that the intronic HS2 element acts in cis to maintain transcriptional competency at the TMS1 locus and that this activity is mediated by the ets transcription factor, GABP␣.Methylation of DNA in the human genome is tightly controlled during development by the action of DNA methyltransferases. DNA methyltransferases catalyze the transfer of a methyl group from S-adenosylmethionine to the carbon-5 position of cytosine in the dinucleotide 5Ј-CpG-3Ј. This epigenetic mark is copied after DNA synthesis, providing a heritable memory of transcriptional status. Overall, CpG dinucleotides are depleted in the human genome except in CpG islands (1). CpG islands are ϳ200 bp to several kb in length and are found mainly in the 5Ј-regions of 70% of human genes (2). Most CpG dinucleotides in the genome are heavily methylated whereas, in contrast, the CpG sites in the CpG islands, especially those associated with gene promoters, are usually unmethylated. Aberrant methylation of such CpG islands is associated with inappropriate gene silencing and has been implicated in the inactivation of tumor suppressor genes in human cancers.At present, the mechanisms underlying cancer-associated CpG island methylation are unknown. There are at least two mechanisms in which aberrant DNA methylation is thought to contribute to stable gene repression. DNA methylation can affect gene expression through its affects on local chromatin structure. Methylated DNA is recognized by methyl-CpGbinding domain proteins, which are components of repressor complexes that contain histone deacetylas...

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A DNA Methylation Site in the Male-Specific P450 (Cyp 2d-9) Promoter and Binding of the Heteromeric Transcription Factor GABP

Cited by 81 publications

References 39 publications

DNA demethylation modulates mouse leptin promoter activity during the differentiation of 3T3-L1 cells

DNA demethylation modulates mouse leptin promoter activity during the differentiation of 3T3-L1 cells

Role of promoter methylation in regulation of the mammalian heparanase gene

Methylation-sensitive Regulation of TMS1/ASC by the Ets Factor, GA-binding Protein-α

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