TY - JOUR
AU - Kakeya, Hideaki
AB - Abstract Human chromosome 7 open reading frame 24 (C7orf24)/γ-glutamyl cyclotransferase has been suggested to be a potential diagnostic marker for several cancers, including carcinomas in the bladder urothelium, breast and endometrial epithelium. We here investigated the epigenetic regulation of the human C7orf24 promoter in normal diploid ARPE-19 and IMR-90 cells and in the MCF-7 and HeLa cancer cell lines to understand the transcriptional basis for the malignant-associated high expression of C7orf24. Chromatin immunoprecipitation analysis revealed that histone modifications associated with active chromatin were enriched in the proximal region but not in the distal region of the C7orf24 promoter in HeLa and MCF-7 cells. In contrast, elevated levels of histone modifications leading to transcriptional repression and accumulation of heterochromatin proteins in the C7orf24 promoter were observed in the ARPE-19 and IMR-90 cells, compared to the levels in HeLa and MCF-7 cancer cells. In parallel, the CpG island of the C7orf24 promoter was methylated to a greater extent in the normal cells than in the cancer cells. These results suggest that the transcriptional silencing of the C7orf24 gene in the non-malignant cells is elicited through heterochromatin formation in its promoter region; aberrant expression of C7orf24 associated with malignant alterations results from changes in chromatin dynamics. C7orf24, DNA methylation, γ-glutamyl cyclotransferase, histone modification, tumour biomarker C7orf24—named after a hypothetical gene product of human chromosome 7 open reading frame 24—was originally identified as a cytoplasmic protein specifically expressed in the urothelial carcinoma (1, 2). Aberrant expression of C7orf24 has also been reported in a range of cancers, including prostate and breast cancers (3–7). In marked contrast, the expression levels of the C7orf24 gene are considerably lower in most normal tissues. Kageyama et al. (2) have also proposed an association of C7orf24 expression with muscle-invasive bladder tumours. In addition, the ectopic expression of C7orf24 has been shown to promote the proliferation of murine normal fibroblasts, and silencing of the gene by siRNA showed an anti-proliferative effect on tumour cell lines (2). These data suggest a potential role for this protein in cell proliferation. Therefore, we propose that C7orf24 could be a potent diagnostic marker and a therapeutic target for tumours in the bladder, prostate, breast and lung. C7orf24 has also been proposed to have a possible role in glutathione homeostasis (8). Oakley et al. expressed recombinant C7orf24 protein in bacteria and showed that it acted as a γ-glutamyl cyclotransferase and catalysed the conversion of γ-glutamyl-l-amino acid into 5-oxoproline and l-amino acid. A series of their X-ray crystal structural studies revealed that C7orf24 shares a remarkable similarity in its tertiary structure with the γ-glutamyl cyclotransferase-like folding family, thus supporting their enzymatic data (8, 9). In a previous study, we found a high expression of C7orf24 in cancer cell lines but not in normal diploid cells—this is in agreement with the C7orf24 expression profiles in clinical specimens (10). As the cellular C7orf24 protein levels correlate well to the mRNA levels, the C7orf24 expression is likely regulated at the transcriptional level. Subsequently, we characterized the C7orf24 promoter to demonstrate the molecular basis underlying transcriptional regulation of this gene. We found transcriptional significance of the three CCAAT boxes located in the proximal promoter of the C7orf24 gene both in non-malignant and in malignant cells. NF-Y was shown to bind to all proximal CCAAT boxes and facilitate recruitment of the general transcription machinery to the initiator, which promoted C7orf24 transcription (10). On the other hand, we did not find any differences in the significance of the CCAAT boxes and NF-Y in C7orf24 transcription between non-malignant and malignant cells. In addition, the transcription of a luciferase gene driven from the C7orf24 promoter was activated in malignant cells but not in non-malignant cells. These results suggested the involvement of some extracellular signals and/or epigenetic regulation in the C7orf24 gene activation in malignant cells, rather than genetic alternation within the transcriptional regulatory region. Therefore, in this article, we investigated whether epigenetic alterations in the human C7orf24 promoter were correlated to the high expression of C7orf24 in malignant cells. Our data revealed a striking contrast in the DNA methylation and histone H3 modification status of the C7orf24 promoter between non-malignant and malignant cells, suggesting that the aberrant expression of C7orf24 in malignant cells results from transcriptional up-regulation caused by chromatin structural alterations in the C7orf24 promoter. Materials and Methods Antibodies Mouse monoclonal antibodies against human heterochromatin protein 1α (HP1α, 39977) and enhancer of zeste homolog 2 (EZH2, 39875), as well as rabbit polyclonal antibodies against modified histone H3 tails, i.e. trimethylated Lys4 (H3K4me3, 39115), trimethylated Lys9 (H3K9me3, 39765), trimethylated Lys27 (H3K27me3, 39155) and acetylated Lys9 (H3K9ac, 39117) were purchased from Active Motif (Carlsbad, CA, USA). Rabbit IgG and mouse IgG2a-κ isotype control antibodies were obtained from Vector Laboratories (I-1000, Burlingame, CA) and BioLegend (400201, San Diego, CA, USA) respectively. Cell culture Human cell lines, normal retinal pigment epithelial cells, ARPE-19, normal lung fibroblast IMR-90 cells, cervical carcinoma HeLa cells and breast adenocarcinoma MCF-7 cells were purchased from the American Tissue Culture Collection (Manassas, VA, USA). All cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum and cultured in a humidified 5% CO2/95% air incubator at 37°C. Quantitative RT-PCR analysis Total RNA was extracted with an RNAspin Mini RNA isolation kit (GE Healthcare, Chalfont St. Giles, UK) according to the manufacturer’s instructions. The RNA was reverse transcribed to cDNA using a random hexanucleotide primer by PrimeScript RT Reagent Kit (Takara Bio, Otsu, Japan) and then PCR-amplified with the following primer pairs: for detection of human C7orf24 mRNA, 5′-GAGAGTTTTCTGTACTTTGCCTACG-3′ (sense) and 5′-TGGGAATTGCCAAAGTCAA-3′ (antisense); for detection of heterogeneous nuclear C7orf24 RNA, 5′-TGGAGAGCTCACAGAATGACC-3′ (sense) and 5′-CCCTTTATGCTTGTCCGTTT-3′ (antisense); and for detection of human G3pdh mRNA, 5′-GGGCCCACTTGAAGGGTGGAGCCAAAAG-3′ (sense) and 5′-CCGTATTCATTGTCATACCAGGAAATG-3′ (antisense). Quantitative RT-PCR analysis was carried out using FastStart Universal SYBR Green Master (Roche Diagnostics, Basel, Switzerland) on an ABI StepOne Plus (Applied Biosystems, Foster City, CA, USA). Chromatin immunoprecipitation assay Cells were cross-linked with 1% formaldehyde (37°C, 10 min), followed by addition of glycine to a final concentration of 200 mM for quenching. Chromatin was sheared into fragments ranging from 200 to 600 bp by sonication (Bioruptor UCD-250HSA, Cosmo Bio, Tokyo, Japan) in SDS lysis buffer (50 mM Tris/HCl pH 8.0, 10 mM EDTA, 1% SDS) supplemented with protease inhibitors. Precleared chromatin supernatants were immunoprecipitated with 2 µg of antibody coupled to 20 µl of salmon sperm DNA-blocked protein G sepharose for 2 h at 4°C. After the immunoprecipitates had been extensively washed with wash buffer 1 (twice) (50 mM Tris/HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate), wash buffer 2 (10 mM Tris/HCl pH 8.0, 0.5% NP-40, 0.5% sodium deoxycholate, 250 mM LiCl) and twice with TE buffer (10 mM Tris/HCl pH 8.0, 1 mM EDTA), the cross-links were reversed by incubating the beads in 200 mM NaCl at 65°C for 5 h. The immunoprecipitated DNA was purified and subjected to quantitative PCR using FastStart Universal SYBR Green Master on an ABI StepOne Plus. Oligonucleotide sequences used for amplification of the human C7orf24 promoter were as follows: −2 to +93, 5′-CCAGTCCCCTAACCCTGAG-3′ (sense) and 5′-AGCAAAGTGTAAGGAACGGC-3′ (antisense); −179 to −19, 5′-CGTGTCCAATGAGGAGCCA-3′ (sense) and 5′-GTCGAGTCAGGAGCGAGC-3′ (antisense); −251 to −171, 5′-CCTGTGAGGAGTGCCTGTC-3′ (sense) and 5′-TTGGACACGACCTACTGATGTC-3′ (antisense); −495 to −265, 5′-GAACCAGCAGAAAGGGTGAA-3′ (sense) and 5′-ATGGACACTGGCTGAAGGTC-3′ (antisense); −1218 to −1088, 5′-GAGTAAAGTTGGTGTAATAGTC-3′ (sense) and 5′-CAAACTGAAAAATGATTGGAA-3′ (antisense); −2792 to −2716, 5′-GGGGCCAAATAAGGGAATAA-3′ (sense) and 5′-AGCTTCCACAGCCTGGAG-3′ (antisense). DNA methylation analysis Genomic DNA was extracted from ARPE-19, IMR-90, HeLa and MCF-7 cells with a High Pure PCR Template Preparation Kit (Roche Diagnostics). Unmethylated cytosine residues were deaminated to uracil via bisulphate treatment (MethylEasy Xceed Rapid DNA Bisulphite Modification Kit, Human Genetic Signatures, Randwick, Australia). A 651-bp fragment of the human C7orf24 promoter gene (−587 to +64) was amplified by PCR from bisulphite-treated genomic DNA by using a sense primer (5′-GGGAAGGGAGTTTTAAGTATTTAGAG-3′) and an antisense primer (5′-CAACACTAAAACCCACTACCC-3′). The resultant fragment was cloned into the pGEM-3Zf(+) vector (Promega, Madison, WI, USA) using an In-Fusion HD Cloning kit (Clontech Laboratories, Mountain View, CA, USA) and sequenced (24 clones/cell line) in an automatic sequencer, ABI3130XL (Applied Biosystems). Results Elevation of human C7orf24 gene transcription in malignant cells We first quantified C7orf24 mRNA levels in human diploid epithelial ARPE-19 cells, human diploid fibroblast IMR-90 cells, human breast adenocarcinoma MCF-7 cells and human cervical carcinoma HeLa cells by quantitative RT-PCR (Fig. 1). Consistent with our previous data obtained by semi-quantitative RT-PCR analysis, a higher expression level of C7orf24 mRNA was detected in both cancer cell lines (HeLa cells, 5.9-fold; MCF-7 cells, 9.4-fold), compared to IMR-90 cells. Additionally, real-time RT-PCR analysis employing specific primers within intron 1 revealed abundant expression of primary heterogeneous nuclear RNA of the C7orf24 gene in cancer cell lines. These results suggest that transcription of the C7orf24 gene is activated in HeLa and MCF-7 cells but silenced in ARPE-19 and IMR-90 cells. Fig. 1 View largeDownload slide Levels of nascent and mature transcripts of the C7orf24 gene in non-malignant and malignant cell lines. Heterogenous nuclear RNA (open bar) and mRNA (closed bar) levels of C7orf24 were quantified by quantitative PCR and normalized with G3pdh mRNA levels. RNA levels are shown as a ratio against levels in ARPE-19 cells. Error bars are the standard errors of triplicate results. Similar results were obtained in three separate experiments. Fig. 1 View largeDownload slide Levels of nascent and mature transcripts of the C7orf24 gene in non-malignant and malignant cell lines. Heterogenous nuclear RNA (open bar) and mRNA (closed bar) levels of C7orf24 were quantified by quantitative PCR and normalized with G3pdh mRNA levels. RNA levels are shown as a ratio against levels in ARPE-19 cells. Error bars are the standard errors of triplicate results. Similar results were obtained in three separate experiments. Profiling of transcriptionally active epigenetic marks in the C7orf24 promoter To investigate the possible involvement of epigenetic control in C7orf24 gene transcription, we analysed the histone modification profiles in the promoter by using a chromatin immunoprecipitation (ChIP) assay. It is well known that H3K4me3 and H3K9ac are marks of actively transcribed chromatin (11). Therefore, we investigated the histone-modification status of the human C7orf24 promoter and compared it among the cell lines tested. As shown in Fig. 2, when the −2 to +93 region around the transcription start site of the C7orf24 promoter was used as the probe, enrichment of H3K4me3 and H3K9ac was observed in all cell lines tested. However, it was found that the modification levels in both HeLa and MCF-7 cells were clearly greater than those in ARPE-19 and IMR-90 cells. Significant elevation of H3K4me3 and H3K9ac levels in cancer cell lines was also detected in the proximal region of the promoter, when the −251 to −171 and −179 to −19 regions were used as probes. In contrast, no significant amplification of the non-genic upstream region of the C7orf24 gene (−2792 to −2716) was found from immunoprecipitates with antibodies against H3K4me3 and H3K9ac. The H3K4me3 modification is recognized by chromodomain helicase DNA binding protein-1. Histone acetyl transferases are then recruited, which results in the promotion of histone H3 Lys9 acetylation and subsequent chromatin structure alterations (12). Therefore, our data suggest that in human cancer cells, the C7orf24 promoter is in an open chromatin conformation allowing for active transcription of the gene. Fig. 2 View largeDownload slide Status of transcriptionally active histone modifications in the C7orf24 promoter. Cross-linked chromatin complexes were immunoprecipitated with antibodies against H3K4me3 or H3K9ac. Normal rabbit IgG was employed as a control antibody. The amount of precipitated DNA fragments derived from the C7orf24 promoter, i.e. −2792 to −2716, −1218 to −1088, −495 to −265, −251 to −171, −179 to −19 and −2 to +93, was measured by quantitative PCR. Data were calculated as a percentage of the input DNA, subtracted from that of control IgG, and shown with the standard error of quadruplicate results. Similar results were obtained in three separate experiments. Genomic DNA was prepared from ARPE-19 (open bar), IMR-90 (hatched bar), HeLa (gray bar) and MCF-7 (closed bar) cells. Fig. 2 View largeDownload slide Status of transcriptionally active histone modifications in the C7orf24 promoter. Cross-linked chromatin complexes were immunoprecipitated with antibodies against H3K4me3 or H3K9ac. Normal rabbit IgG was employed as a control antibody. The amount of precipitated DNA fragments derived from the C7orf24 promoter, i.e. −2792 to −2716, −1218 to −1088, −495 to −265, −251 to −171, −179 to −19 and −2 to +93, was measured by quantitative PCR. Data were calculated as a percentage of the input DNA, subtracted from that of control IgG, and shown with the standard error of quadruplicate results. Similar results were obtained in three separate experiments. Genomic DNA was prepared from ARPE-19 (open bar), IMR-90 (hatched bar), HeLa (gray bar) and MCF-7 (closed bar) cells. Profiling of transcriptionally repressive epigenetic marks in the C7orf24 promoter We next compared the gene-silencing histone modification status in the C7orf24 promoter of non-malignant cells with those of malignant cells. In striking contrast with the case of transcription associated-histone modification, H3K9me3 and H3K27me3 modification levels in normal diploid ARPE-19 and IMR-90 cells were higher than those in the HeLa and MCF-7 cancer cell lines over the entire promoter region that was tested (Fig. 3A). In particular, a significant difference of both H3 methylation levels between in non-malignant and malignant cells was observed in the −251 to −171 region. HP1α is known to bind to H3K9me3 through its chromodomain. HP1α is implicated in the spreading and maintenance of heterochromatin because of the recruitment of histone methyltransferases and formation of a homomulticomplex of the protein (11). EZH2 forms a heterochromatin complex—a component of polycomb repressive complex 2—and binds to H3K27me3 (13). Therefore, we analysed the accumulation of HP1α and EZH2 on the promoter region of the human C7orf24 gene (Fig. 3B). As expected, ChIP analysis revealed that both heterochromatin proteins were bound to the proximal region of the promoter in the ARPE-19 and IMR-90 cells but did not significantly accumulate in either the HeLa or the MCF-7 cells. In contrast, expression levels of HP1α and EZH2 in both non-malignant cells were much lower than those in malignant cells, indicating that cellular expression levels of both heterochromatin proteins were not associated with their accumulation levels in the proximal C7orf24 promoter in cells tested (Supplementary Data). Given that relative global histone modification levels among tested cell lines were also poorly correlated to their histone modification levels in the C7orf24 promoter, it is suggested that transcriptional repression of the C7orf24 gene in non-malignant cells is not attributed to global changes in the levels of epigenetic markers and heterochromatin proteins. Fig. 3 View largeDownload slide Status of silencing histone modifications and accumulation of heterochromatin proteins in the C7orf24 promoter. Cross-linked chromatin complexes were immunoprecipitated with antibodies against H3K9me3 or H3K27me (A) and HP1α or EZH2 (B). Normal rabbit IgG or mouse IgG2a was employed as the control antibody. The amount of precipitated DNA fragments derived from the C7orf24 promoter, i.e. −2792 to −2716, −1218 to −1088, −495 to −265, −251 to −171, −179 to −19 and −2 to +93, was measured by quantitative PCR. Data were calculated as a percentage of the input DNA, subtracted from that of control IgG, and shown with the standard error of quadruplicate results. Similar results were obtained in three separate experiments. Genomic DNA was prepared from ARPE-19 (open bar), IMR-90 (hatched bar), HeLa (gray bar) and MCF-7 (closed bar) cells. Fig. 3 View largeDownload slide Status of silencing histone modifications and accumulation of heterochromatin proteins in the C7orf24 promoter. Cross-linked chromatin complexes were immunoprecipitated with antibodies against H3K9me3 or H3K27me (A) and HP1α or EZH2 (B). Normal rabbit IgG or mouse IgG2a was employed as the control antibody. The amount of precipitated DNA fragments derived from the C7orf24 promoter, i.e. −2792 to −2716, −1218 to −1088, −495 to −265, −251 to −171, −179 to −19 and −2 to +93, was measured by quantitative PCR. Data were calculated as a percentage of the input DNA, subtracted from that of control IgG, and shown with the standard error of quadruplicate results. Similar results were obtained in three separate experiments. Genomic DNA was prepared from ARPE-19 (open bar), IMR-90 (hatched bar), HeLa (gray bar) and MCF-7 (closed bar) cells. DNA methylation pattern in the C7orf24 promoter Hypermethylation of the C-5 position of cytosine (DNA methylation) within gene regulatory regions contributes to gene silencing through the formation of stable heterochromatin structures (14, 15), in cooperation with transcriptionally repressive histone modifications and recruitment of heterochromatin proteins. In fact, as shown in Fig. 4, the proximal region of the human C7orf24 promoter (−580 to +50) contains 44 CpG dinucleotide sequences. Therefore, we examined the CpG dinucleotide methylation status of the human C7orf24 promoter. As bisulphide converts unmethylated cytosine to uracil but does not convert C-5-methylated cytosine, we determined the nucleotide sequences of the promoter amplified from the bisulphide-treated genome to identify CpG methylation sites. CpG sites located at −290 to −587 of the C7orf24 promoter in ARPE-19 and IMR-90 cells were highly methylated, whereas CpGs around the transcription start site were barely methylated. Notably, methylated CpG was found in the promoter region of all clones prepared from the ARPE-19 and IMR-90 cell genomes. As shown in Fig. 3, ChIP assay revealed that suppressive histone modifications and heterochromatin proteins were accumulated in the highly CpG methylated region in non-malignant cells, supporting a possibility that the C7orf24 promoter forms the repressive heterochromatin structure in these cells. Fig. 4 View largeDownload slide DNA methylation status of CpG dinucleotides in the C7orf24 promoter. Top: Schematic representation of the C7orf24 promoter. Numbers and vertical dashes represent positions of the CpG dinucleotide sequences. The arrowhead indicates the transcription start site (TSS). Bottom: Methylation status of each CpG dinucleotide was determined by DNA sequencing of 24 independent clones containing the DNA fragments obtained from the bisulphite-modified genomic DNA. Open and closed circles indicate unmethylated and methylated CpG sequences respectively. Fig. 4 View largeDownload slide DNA methylation status of CpG dinucleotides in the C7orf24 promoter. Top: Schematic representation of the C7orf24 promoter. Numbers and vertical dashes represent positions of the CpG dinucleotide sequences. The arrowhead indicates the transcription start site (TSS). Bottom: Methylation status of each CpG dinucleotide was determined by DNA sequencing of 24 independent clones containing the DNA fragments obtained from the bisulphite-modified genomic DNA. Open and closed circles indicate unmethylated and methylated CpG sequences respectively. On the other hand, no significant accumulation of CpG methylation was observed within either distal or proximal regions of the C7orf24 promoter in MCF-7 cells. Only three clones out of the 24 cloned sequences of the MCF-7 genome had methylated cytosines. Likewise, the proximal C7orf24 promoter of HT-29 cells (data not shown), which expressed C7orf24 at levels similar to levels in MCF-7 cells, barely contained methylated CpG dinucleotide sites (only three clones with one methylated CpG site). In HeLa cells, one-third of the sequenced clones contained a methylated CpG site only in the upstream region of the C7orf24 promoter. However, as no significant accumulation of heterochromatin proteins in this region was observed in HeLa cells (Fig. 3B), it is suggested that CpG methylation in this region does not contribute to the formation of heterochromatin around the C7orf24 gene in HeLa cells. Taken together, we hypothesize that transcription of the C7orf24 gene is suppressed in non-malignant cells through stable heterochromatin formation in its promoter region. Discussion In carcinogenesis, epigenetic regulation of tumour-suppressor gene expression has attracted considerable attention. Several tumour-suppressor genes, including the CDKN2A gene encoding a cyclin-dependent kinase-4/6 inhibitor, p16INK4A, and an MDM2 inhibitor, p15ARF, and the VHL gene encoding an E3 ligase, von Hippel-Lindau, are silenced by de novo methylation of CpG islands in their promoter regions in cancer cells (14, 16, 17). Many other genes negatively regulating malignant progression such as CDH1 (E-cadherin), MDR1 (multidrug resistance protein 1) and TIMP3 (tissue inhibitor of matrix metalloproteinase-3) were inactivated through epigenetic silencing in cancer cells (17). Thus, gene silencing through promoter hypermethylation has been widely recognized to promote tumourigenesis and malignant transformation. Moreover, errant promoter methylation is now considered a novel diagnostic and prognostic marker for tumours in the lung and prostate (17–19). In contrast, it is also well known that genome-wide DNA hypomethylation is frequently observed in cancer cells and occurs at various genomic sequences, including centromeric repeats, repetitive elements and gene-poor regions (14, 16). However, regulatory regions of several tumour-related genes, including PAX2, cyclin D2 and membrane type-1 matrix metalloprotease, were de-methylated in cancer cells, resulting in aberrant gene expression (20–22). In this article, we demonstrated for the first time that the C7orf24 gene is a novel malignant-associated de-repression gene. It is important to clarify the trigger and molecular mechanism for the de-methylation of the C7orf24 gene promoter in malignant alterations. In addition, although no obvious correlation between the positive rate of C7orf24 and the histological grading of urothelial carcinoma was reported in a previous immunohistochemical analysis (2), a correlation study between the epigenetic status of the C7orf24 gene and tumour grading in clinical samples may provide valuable insights into the practical application of C7orf24 as a cancer biomarker. C7orf24 acts as a γ-glutamyl cyclotransferase by cleaving γ-glutamyl-l-amino acid into 5-oxoproline and l-amino acid; it is proposed to be involved in the γ-glutamyl cycle of glutathione homeostasis (8). γ-Glutamyl transpeptidase (GGT), another enzyme involved in glutathione degradation, is expressed in a tissue- or developmental phase-specific manner, and it is also detected in many cancers, including hepatocarcinoma, at high levels (23–25). The human and rat GGT genes have multiple tandemly positioned promoters, each of which contains a different composition of transcription factor binding sites. The tissue- or developmental phase-specific expression of GGT is largely attributed to unique promoter activation patterns, whereas DNA hypermethylation of the GGT promoter influences gene transcription to some extent (23, 26, 27). In hepatoma, GGT expression is induced by carcinogens via activation of the redox-signalling pathway (23). Hypomethylation of CpG islands in the GGT gene promoter in patients with hepatocellular carcinoma was also reported, implying that the aberrant expression of GGT is attributed to epigenetic status alterations, similar to the C7orf24 gene shown in this article (25). On the other hand, in contrast to up-regulation of GGT gene transcription by oxidant stressors and other enzyme genes comprising the glutathione antioxidant defence system, the expression of C7orf24 gene was not affected by the oxidant stress exposure, whereas its promoter contains an antioxidant responsive element-like sequence (10). Although the pathophysiological function of C7orf24 remains poorly understood, C7orf24 may have a function other than as a γ-glutamyl-l-amino acid degrading enzyme. We have demonstrated that NF-Y is recruited to the proximal promoter of the C7orf24 gene and that it plays an essential role in transcription (10). In addition to acting as a transcription factor, NF-Y may contribute to the open chromatin structure formation of the C7orf24 gene through regulation of histone covalent modifications. Recently, it has been reported that NF-Y co-localizes with histone marks indicating active chromatin such as H3K4me3 and H3K9ac; overexpression of a dominant negative mutant or elimination of NF-YA caused a decrease in H3K4me3 levels in CCAAT promoters of cell cycle regulating genes, and histone methylation complexes were recruited to CCAAT promoters in an NF-Y-dependent fashion (28, 29). These data strongly suggest that NF-Y regulates histone mark status on CCAAT promoters. Consistent with these data, we observed accumulation of euchromatin marks (i.e. H3K4me3 and H3K9ac) around the transcription start site of the C7orf24 gene in C7orf24-expressing MCF-7 and HeLa cells. It is possible that NF-Y bound on CCAAT boxes in the C7orf24 promoter serves as a spatial landmark for the recruitment of histone methylation complexes, which results in chromatin structure alterations at the promoter. To further investigate this hypothesis, we determined DNA methylation sites within the C7orf24 promoter. Using bisulphide sequence analysis, we identified CpG methylation sites within the promoter; we observed for the first time that the proximal region of the C7orf24 promoter in non-malignant cells was highly methylated, which was not observed in malignant cell lines (MCF-7, HT-29 and HeLa cells). These data strongly suggest that the C7orf24 promoter is included in a heterochromatin structure of the genome in non-malignant cells, which leads to transcriptional repression of the C7orf24 gene. However, we cannot rule out the possible involvement of other suppressors such as miRNAs and a lack of activation machinery because C7orf24 mRNA levels were barely affected by treatment of IMR-90 cells with a DNA methyltransferase inhibitor, 5-aza-2′ deoxycytidine, up to 72 h (data not shown). On the other hand, demethylation-mediated chromatin structure alterations of the C7orf24 promoter may allow active transcription of the gene in malignant cells. Similar to covalent histone modification of the promoter, elucidation of a generation mechanism underlying malignant-associated alterations of CpG dinucleotide methylation is required in future studies. Several immunohistochemical studies have shown that C7orf24 is marginally expressed in several normal epithelial tissues, including endometrial epithelium and renal tubules (5, 30). It may be interesting to investigate the possible involvement of epigenetic regulation and its molecular basis in tissue-specific C7orf24 gene expression. In conclusion, we found a striking contrast in the DNA methylation and histone H3 modification status of the C7orf24 promoter between non-malignant and malignant cells in this article. These results suggest that the C7orf24 gene is suppressed in non-malignant cells through stable heterochromatin formation and the aberrant expression of C7orf24 in malignant cells caused by chromatin structural alterations in the C7orf24 promoter (Fig. 5). These studies may provide valuable information for promoting the practical application of C7orf24 as a cancer marker for diagnosis, classification, prognosis and monitoring the recurrence of cancer. Fig. 5 View largeDownload slide Chromatin dynamics of the C7orf24 promoter in non-malignant and malignant cells. HP1, heterochromatin protein 1; EZH2, enhancer of zeste homolog 2; PRC2, polycomb repressor complex-2. Fig. 5 View largeDownload slide Chromatin dynamics of the C7orf24 promoter in non-malignant and malignant cells. HP1, heterochromatin protein 1; EZH2, enhancer of zeste homolog 2; PRC2, polycomb repressor complex-2. Acknowledgements This work was supported in part by research grants from the Japan Society for the Promotion of Science (JSPS), the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) and the 3rd Term Comprehensive Control Research for Cancer from the Ministry of Health, Labor and Welfare of Japan (MHLW). Conflict of Interest None declared. Abbreviations Abbreviations C7orf24 chromosome 7 open reading frame 24 ChIP chromatin immunoprecipitation EZH2 enhancer of zeste homolog 2 GGT γ-glutamyl transpeptidase H3K4me3 histone H3 Lys4 trimethylation H3K9ac histone H3 Lys9 acetylation H3K9me3 histone H3 Lys9 trimethylation H3K27me3 histone H3 Lys27 trimethylation HP1α heterochromatin protein 1α References 1 Kageyama S,  Isono T,  Iwaki H,  Wakabayashi Y,  Okada Y,  Kontani K,  Yoshimura K,  Terai A,  Arai Y,  Yoshiki T.  Identification by proteomic analysis of calreticulin as a marker for bladder cancer and evaluation of the diagnostic accuracy of its detection in urine,  Clin. Chem. ,  2004, vol.  50 (pg.  857- 866) Google Scholar CrossRef Search ADS PubMed  2 Kageyama S,  Iwaki H,  Inoue H,  Isono T,  Yuasa T,  Nogawa M,  Maekawa T,  Ueda M,  Kajita Y,  Ogawa O,  Toguchida J,  Yoshiki T.  A novel tumor-related protein, C7orf24, identified by proteome differential display of bladder urothelial carcinoma,  Proteomics Clin. 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TI - Association of epigenetic alterations in the human C7orf24 gene with the aberrant gene expression in malignant cells
JF - The Journal of Biochemistry
DO - 10.1093/jb/mvt063
DA - 2013-07-12
UR - https://www.deepdyve.com/lp/oxford-university-press/association-of-epigenetic-alterations-in-the-human-c7orf24-gene-with-0mYQgZ266C
SP - 355
EP - 362
VL - 154
IS - 4
DP - DeepDyve
ER -