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Unlabelled: c-Myc is a well-known oncogene frequently up-regulated in different malignancies, whereas liver-specific microRNA (miR)-122, a bona fide tumor suppressor, is down-regulated in hepatocellular cancer (HCC). Here we explored the underlying mechanism of reciprocal regulation of these two genes. Real-time reverse-transcription polymerase chain reaction (RT-PCR) and northern blot analysis demonstrated reduced expression of the primary, precursor, and mature miR-122 in c-MYC-induced HCCs compared to the benign livers, indicating transcriptional suppression of miR-122 upon MYC overexpression. Indeed, chromatin immunoprecipitation (ChIP) assay showed significantly reduced association of RNA polymerase II and histone H3K9Ac, markers of active chromatin, with the miR-122 promoter in tumors relative to the c-MYC-uninduced livers, indicating transcriptional repression of miR-122 in c-MYC-overexpressing tumors. The ChIP assay also demonstrated a significant increase in c-Myc association with the miR-122 promoter region that harbors a conserved noncanonical c-Myc binding site in tumors compared to the livers. Ectopic expression and knockdown studies showed that c-Myc indeed suppresses expression of primary and mature miR-122 in hepatic cells. Additionally, Hnf-3β, a liver enriched transcription factor that activates miR-122 gene, was suppressed in c-MYC-induced tumors. Notably, miR-122 also repressed c-Myc transcription by targeting transcriptional activator E2f1 and coactivator Tfdp2, as evident from ectopic expression and knockdown studies and luciferase reporter assays in mouse and human hepatic cells., Conclusion: c-Myc represses miR-122 gene expression by associating with its promoter and by down-regulating Hnf-3β expression, whereas miR-122 indirectly inhibits c-Myc transcription by targeting Tfdp2 and E2f1. In essence, these results suggest a double-negative feedback loop between a tumor suppressor (miR-122) and an oncogene (c-Myc)., (© 2013 by the American Association for the Study of Liver Diseases.)

Unlabelled: Considerable effort has been made in elucidating the mechanism and functional significance of high levels of aerobic glycolysis in cancer cells, commonly referred to as the Warburg effect. Here we investigated whether the gluconeogenic pathway is significantly modulated in hepatocarcinogenesis, resulting in altered levels of glucose homeostasis. To test this possibility, we used a mouse model (mice fed a choline-deficient diet) that develops nonalcoholic steatohepatitis (NASH), preneoplastic nodules, and hepatocellular carcinoma (HCC), along with human primary HCCs and HCC cells. This study demonstrated marked reduction in the expressions of G6pc, Pepck, and Fbp1 encoding the key gluconeogenic enzymes glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, fructose-1,6-phosphatase, respectively, and the transcription factor Pgc-1α in HCCs developed in the mouse model that correlated with reduction in serum glucose in tumor-bearing mice. The messenger RNA (mRNA) levels of these genes were also reduced by ≈80% in the majority of primary human HCCs compared with matching peritumoral livers. The expression of microRNA (miR)-23a, a candidate miR targeting PGC-1α and G6PC, was up-regulated in the mouse liver tumors as well as in primary human HCC. We confirmed PGC-1α and G6PC as direct targets of miR-23a and their expressions negatively correlated with miR-23a expression in human HCCs. G6PC expression also correlated with tumor grade in human primary HCCs. Finally, this study showed that the activation of interleukin (IL)-6-Stat3 signaling caused the up-regulation of miR-23a expression in HCC., Conclusion: Based on these data, we conclude that gluconeogenesis is severely compromised in HCC by IL6-Stat3-mediated activation of miR-23a, which directly targets PGC-1α and G6PC, leading to decreased glucose production., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Aberrations in methylation profile of the genome occur in human cancers induced by folate deficiency. To elucidate the underlying mechanism, male F344 rats were fed a diet deficient in l-methionine and devoid of folic acid and choline (FMD diet), which is known to induce hepatocellular carcinomas. We investigated changes in the DNA methylation machinery, namely, de novo DNA methyltransferases (Dnmt3a and 3b), maintenance DNA methyltransferase (Dnmt1), and methyl CpG binding proteins (MBDs), in rat livers during early stages of tumorigenesis. RT-PCR and Western blot analyses revealed differential expression of these proteins in the livers of rats fed the FMD diet. Although the hepatic Dnmt1 mRNA level declined with age (P < 0.001), it was elevated (P < 0.001) in deficient rats compared with controls. The changes in hepatic Dnmt1 protein level with the diet correlated with its mRNA levels (r = 0.60, P = 0.002). Similarly, the Dnmt3a mRNA level was elevated in rats fed the FMD diet (P < 0.001), whereas the Dnmt3b level (mRNA and protein) was not affected by diet or age. Compared with controls, hepatic MBD1-3 RNA levels increased (P < 0.001) and the protein levels of MBD1, 2, and 4 were elevated (P < 0.001) in the deficient rats. In both diet groups, hepatic MBD2 protein decreased (P < 0.001), whereas MeCP2 protein increased (P < 0.001) with age. These results demonstrate that a combined folate and methyl deficiency alters components of the DNA methylation machinery by both transcriptional and posttranscriptional mechanisms during early stages of hepatocarcinogenesis.

Dnmt3a and Dnmt3b are de novo DNA methyltransferases that also act as transcriptional repressors independent of methyltransferase activity. To elucidate the underlying mechanism of transcriptional repression, Dnmt3a was purified from mouse lymphosarcoma cells (P1798) by extensive fractionation on five different chromatographic matrices followed by glycerol density gradient centrifugation. Liquid chromatography electrospray tandem mass spectrometry analysis of Dnmt3a-associated polypeptides identified the methyl CpG binding protein Mbd3, histone deacetylase 1(Hdac1), and components of Brg1 complex (Brg1, Baf155, and Baf57) in the purified preparation. Association of Dnmt3a with Mbd3 and Brg1 was confirmed by coimmunoprecipitation and coimmunolocalization studies. Glutathione S-transferase pulldown assay showed that the NH2-terminal ATRX homology domain of Dnmt3a interacts with the methyl CpG binding domain of Mbd3 and with both bromo and ATPase domains of Brg1. Chromatin immunoprecipitation assay revealed that all three proteins are associated with transcriptionally silent methylated metallothionein (MT-I) promoter in the mouse lymphosarcoma cells. To understand the functional significance of their association with the promoter, their role on the MT-I promoter activity was analyzed by transient transfection assay. The results showed that Mbd3 and Dnmt3a specifically inhibited the methylated promoter, and the catalytic activity of Dnmt3a was dispensable for the suppression. In contrast, the wild-type but not the ATPase-inactive mutant of Brg1 suppressed MT-I promoter irrespective of its methylation status, implicating involvement of ATP-dependent chromatin remodeling in the process. Coexpression of two of the three interacting proteins at a time augmented their repressor function. This study shows physical and functional interaction of Dnmt3a with components of nucleosome remodeling machinery.

Spinal muscular atrophy, an autosomal recessive disorder, is caused by loss of the SMN1 (survival motor neuron) gene while retaining the SMN2 gene. SMN1 produces a majority of full-length SMN transcript, whereas SMN2 generates mostly an isoform lacking exon 7. Here, we demonstrate a novel cAMP-response element, CRE-II, in the SMN promoter that interacts with the cAMP-response element-binding (CREB) family of proteins. In vitro DNase I protection analysis and in vivo genomic footprinting of the SMN promoter using the brain and liver nuclei from SMN2 transgenic mice revealed footprinting at the CRE-II site. Site-directed mutation of the CRE-II element caused a marked reduction in the SMN promoter activity revealed by transient transfection assay. Activation of the cAMP pathway by dibutyryl cAMP (0.5 mm) alone or in combination with forskolin (20 microm) caused a 2-5-fold increase in the SMN promoter activity but had no effect on the CRE-II mutated promoter. Electrophoretic mobility shift assay and a UV-induced DNA-protein cross-linking experiment confirmed that CREB1 binds specifically to the CRE-II site. Transient overexpression of CREB1 protein resulted in a 4-fold increase of the SMN promoter activity. Intraperitoneal injection of epinephrine in mice expressing two copies of the human SMN2 gene resulted in a 2-fold increase in full-length SMN transcript in the liver. Combined treatment with dibutyryl cAMP and forskolin significantly increased the level of both the full-length and exon 7-deleted SMN (exonDelta7SMN) transcript in primary hepatocytes from mice expressing two copies of human SMN2 gene. Similar treatments of type I spinal muscular atrophy mouse and human fibroblasts as well as HeLa cells resulted in an augmented level of SMN transcript. These findings suggest that the CRE-II site in SMN promoter positively regulates the expression of the SMN gene, and treatment with cAMP-elevating agents increases expression of both the full-length and exonDelta7SMN transcript.

The robust induction of metallothionein-I and II (MT-I and MT-II) genes by several heavy metals such as zinc and cadmium requires the specific transcription factor metal-responsive transcription factor 1 (MTF1). Chromium (VI), a major environmental carcinogen, not only failed to activate these genes but also inhibited their induction by Zn2+ or Cd2+. The heavy metal-induced expression of another MTF1 target gene, zinc transporter 1 (ZnT-1), was also down-regulated by Cr6+. By contrast, the expression of two MTF1-independent Cd2+-inducible genes, heme oxygenase 1 (HO-1) and HSP-70, was not sensitive to Cr6+. Cr6+ did not also affect the expression of housekeeping genes such as GAPDH or beta-actin. Stable cell lines overexpressing variable levels of MTF1, the key transactivator of the MT genes, demonstrated differential resistance toward the inhibitory effect of Cr6+, indicating MTF1 as a target of chromium toxicity. The basal and inducible binding of MTF1 to metal response elements was not affected by treatment of cells with Cr6+. Transient transfection studies showed that the ability of MTF1 to transactivate the MT-I promoter was significantly compromised by Cr6+. The fusion protein consisting of a Gal-4 DNA binding domain and one or more of the three transactivation domains of MTF1, namely the acidic domain, proline-rich domain, and serine-threonine rich domain, activated the GAL-4-driven luciferase gene to different degrees, but all were sensitive to Cr6+. MTF1 null cells were prone to apoptosis after exposure to Zn2+ or Cd2+ that was augmented in presence Cr6+, whereas the onset of apoptosis was significantly delayed in cells overexpressing MTF1.

Inhibitors of DNA methyltransferase (Dnmt) and histone deacetylases (HDAC) synergistically activate the methylated metallothionein I gene (MT-I) promoter in mouse lymphosarcoma cells. The cooperative effect of these two classes of inhibitors on MT-I promoter activity was robust following demethylation of only a few CpG dinucleotides by brief exposure to 5-azacytidine (5-AzaC) but persisted even after prolonged treatment with the nucleoside analog. HDAC inhibitors (trichostatin A [TSA] and depsipeptide) either alone or in combination with 5-AzaC did not facilitate demethylation of the MT-I promoter. Treatment of cells with HDAC inhibitors increased accumulation of multiply acetylated forms of H3 and H4 histones that remained unaffected after treatment with 5-AzaC. Chromatin immunoprecipitation (ChIP) assay showed increased association of acetylated histone H4 and lysine 9 (K9)-acetyl H3 with the MT-I promoter after treatment with TSA, which was not affected following treatment with 5-AzaC. In contrast, the association of K9-methyl histone H3 with the MT-I promoter decreased significantly after treatment with 5-AzaC and TSA. ChIP assay with antibodies specific for methyl-CpG binding proteins (MBDs) demonstrated that only methyl-CpG binding protein 2 (MeCP2) was associated with the MT-I promoter, which was significantly enhanced after TSA treatment. Association of histone deacetylase 1 (HDAC1) with the promoter decreased after treatment with TSA or 5-AzaC and was abolished after treatment with both inhibitors. Among the DNA methyltransferases, both Dnmt1 and Dnmt3a were associated with the MT-I promoter in the lymphosarcoma cells, and association of Dnmt1 decreased with time after treatment with 5-AzaC. Treatment of these cells with HDAC inhibitors also increased expression of the MTF-1 (metal transcription factor-1) gene as well as its DNA binding activity. In vivo genomic footprinting studies demonstrated increased occupancy of MTF-1 to metal response elements of the MT-I promoter after treatment with both inhibitors. Analysis of the promoter by mapping with restriction enzymes in vivo showed that the MT-I promoter attained a more open chromatin structure after combined treatment with 5-AzaC and TSA as opposed to treatment with either agent alone. These results implicate involvement of multifarious factors including modified histones, MBDs, and Dnmts in silencing the methylated MT-I promoter in lymphosarcoma cells. The synergistic activation of this promoter by these two types of inhibitors is due to demethylation of the promoter and altered association of different factors that leads to reorganization of the chromatin and the resultant increase in accessibility of the promoter to the activated transcription factor MTF-1.

Mitochondrial transcription factor A is a key regulator involved in mitochondrial DNA transcription and replication. In a poorly differentiated rat hepatoma, Morris hepatoma 3924A, the mRNA and protein levels of this factor were elevated about 10- and 11-fold, respectively, relative to the host liver. The mRNA levels for the hepatoma cytochrome c oxidase I, II, and NADH dehydrogenase 5, 6, the downstream targets of Tfam, were augmented 10-, 8-, 5-, and 3-fold, respectively. Interestingly, Tfam was also found in the hepatoma nucleus. The mRNA levels for nuclear respiratory factor 1 and 2 (NRF-1 and -2), the proteins that are known to interact with specific regulatory elements on human TFAM promoter, were 5- and 3-fold higher, respectively, in the hepatoma relative to the host liver. Unlike the human promoter, the rat Tfam promoter did not form a specific complex with the NRF-1 in the liver or hepatoma nuclear extracts, which is consistent with the absence of an NRF-1 consensus sequence in the proximal rat promoter. A single specific complex formed between the rat promoter and the NRF-2 protein was comparable in the two extracts. The DNA binding activity of Sp1 in the hepatoma nuclear extract was 4-fold greater than that in the liver extract. In vivo genomic footprinting showed occupancy of NRF-2 and Sp1 consensus sites on the promoter of rat Tfam gene. Tfam was also up-regulated in other hepatoma cells. Together, these results show up-regulation of Tfam in some tumors, particularly the liver tumors. Further, the relatively high level of Sp1 binding to the promoter in the hepatoma could play a major role in the up-regulation of Tfam in these tumor cells.

Metallothionein (MT) promoter was methylated in rat hepatoma and in mouse lymphosarcoma cells by methylation of cytosine within the CpG dinucleotide region. After demethylation of MT-I promoter in mouse lymphosarcoma cells or in the transplanted rat hepatoma with 5-azacytidine, a potent inhibitor of DNA methyltransferase, the promoter was activated in response to heavy metal treatment. MT-I promoter was also suppressed in human prostate cancer lines PC3 and DU145, probably by promoter methylation, whereas cadmium induced MT-I in the human prostate cancer line LNCaP. In the prostate cancer lines where MT-I was suppressed, glutathione-S-transferase-pi (GST-pi) was expressed. On the contrary, GST-pi gene was repressed in the cell line where MT-I was induced, which suggests an inverse relationship between MT-I induction and GST-pi expression in some prostate cancer lines. The expressions of GST-pi and gamma-glutamyl cysteine synthase were also significantly higher (5- to 12-fold) in the lymphosarcoma cells and the hepatoma relative to the parental tissues. The higher expressions of these two genes suggest a compensatory mechanism in the cells where the gene for the antioxidant MT-I/II is not induced. MT-I/II may function as a growth suppressor either alone or in concert with other factor(s), and consequently their lack of expression could facilitate the tumor growth. In addition to suppression of MT-I/II expression by promoter methylation, the lack of MT induction could also be brought about by nuclear factor I (NFI), probably by interaction with the metal transcription factor MTF-1. An inverse relationship was observed between the level of NFI and MT-I expression in some cells, which suggests a role for NFI in the relatively low constitutive levels of MT-I expression in these cells.

Background: A hallmark of cancer cells is hypermethylation of CpG islands (CGIs), which probably arises from upregulation of one or more DNA methyltransferases. The purpose of this study was to identify the targets of DNMT3B, an essential DNA methyltransferase in mammals, in colon cancer. Methodology/Principal Findings: Chromatin immunoprecipitation with DNMT3B specific antibody followed by CGI microarray identified genes with or without CGIs, repeat elements and genomic contigs in RKO cells. ChIP-Chop analysis showed that the majority of the target genes including P16, DCC, DISC1, SLIT1, CAVEOLIN1, GNA11, TBX5, TBX18, HOXB13 and some histone variants, that harbor CGI in their promoters, were methylated in multiple colon cancer cell lines but not in normal colon epithelial cells. Further, these genes were reactivated in RKO cells after treatment with 5-aza-29-deoxycytidine, a DNA hypomethylating agent. COBRA showed that the CGIs encompassing the promoter and/or coding region of DCC, TBX5, TBX18, SLIT1 were methylated in primary colorectal tumors but not in matching normal colon tissues whereas GNA11 was methylated in both. MassARRAY analysis demonstrated that the CGI located ~4.5 kb upstream of HOXB13 +1 site was tumor-specifically hypermethylated in primary colorectal cancers and cancer cell lines. HOXB13 upstream CGI was partially hypomethylated in DNMT1-/- HCT cells but was almost methylation free in cells lacking both DNMT1 and DNMT3B. Analysis of tumor suppressor properties of two aberrantly methylated transcription factors, HOXB13 and TBX18, revealed that both inhibited growth and clonogenic survival of colon cancer cells in vitro, but only HOXB13 abolished tumor growth in nude mice. Conclusions/Significance: This is the first report that identifies several important tumor suppressors and transcription factors as direct DNMT3B targets in colon cancer and as potential biomarkers for this cancer. Further, this study shows that methylation at an upstream CGI of HOXB13 is unique to colon cancer. [ABSTRACT FROM AUTHOR]