Effect Of Promoter Methylation Research Articles

Effect of Promoter Methylation on the Expression of Porcine MUC2 Gene and Resistance to PEDV Infection.

Integrity of the intestinal mucosal barrier is closely related to the occurrence of diarrhea. As an important component protein of the intestinal mucosal barrier, Mucin 2 (MUC2) plays a critical role in preventing the invasion of pathogens, toxins, and foreign bodies. In the present study, we preliminary verified the function of the porcine MUC2 gene in resisting porcine epidemic diarrhea virus (PEDV) infection and investigated the effect of DNA methylation in the promoter region on MUC2 gene expression. The results showed that after PEDV infection, the intestinal mucosal barrier was damaged. Moreover, MUC2 expression was significantly higher in PEDV-infected piglets than in healthy piglets (P < 0.01). The mRNA expression of MUC2 was significantly higher in PEDV-infected IPEC-J2 cells than in non-infected IPEC-J2 cells (P < 0.05). Methylation of the mC-5 site in the MUC2 promoter inhibited the binding of Yin Yang 1 (YY1) to the promoter, down regulated the expression of MUC2 and increased the susceptibility of piglets to PEDV. In conclusion, this study suggests that MUC2 plays an essential regulatory role in PEDV infection. High MUC2 expression improves the resistance of pigs to PEDV infection. The binding of YY1 to the MUC2 promoter is hindered by the methylation of the mC-5 site, which downregulates MUC2 expression and ultimately affects the resistance of pigs to PEDV infection.

Association of Protein Expression and Methylation of DAPK1 with Clinicopathological Features in Invasive Ductal Carcinoma Patients from Kashmir

Aims:Death-associated protein kinase-1 (DAPK1) is a pro-apoptotic Ser/Thr kinase that participates in cell apoptosis and tumor suppression. DAPK1 is frequently lost in many different tumor types including breast cancer. The aim of this study was to evaluate the promoter methylation status of DAPK1 and a possible correlation with the expression of DAPK1 and standard clinicopathological features in invasive ductal breast carcinoma patients (IDC).Methods:Methylation Specific PCR (MSP) was carried out to investigate the promoter methylation status of DAPK1 from 128 breast cancer patients. The effect of promoter methylation on protein expression was evaluated by immunohistochemistry (n=128) and western blotting (n=56).Results:We found significant difference in DAPK1 promoter methylation frequency among breast tumors when compared with the corresponding normal tissues. Hypermethylation of DAPK1 is significantly correlated with the loss of DAPK1 protein expression (P < .001, rs= -0.361). The loss of DAPK1 protein was significantly associated with estrogen receptor (ER) negativity (p= 0.003), triple negative breast cancer (TNB) (p= 0.024) and advanced tumor stages (P = 0.001). Moreover, age at diagnosis (p= 0.041), tumor stage (p= 0.034), ER negativity (p= 0.004) and TNB cancers (p=0.003) correlated significantly with the hypermethylation of the DAPK1 promoter.Coclusion:This study indicates that DAPK1 is methylated in IDC and promoter hypermethylation could be attributed to silencing of DAPK1 gene expression in breast cancer. Thus, we consider DAPK1 inactivation by promoter hypermethylation likely plays a role in the development and progression of breast cancer.

The effect of promoter methylation on MdMYB1 expression determines the level of anthocyanin accumulation in skins of two non-red apple cultivars

BackgroundFruit color in apple (Malus domestica Borkh.) is ascribed mainly to the accumulation of anthocyanin pigments, and is an important trait for determining fruit market acceptance. Bagging is a commonly used treatment to enhance the red pigmentation in apple skin. The MdMYB1 transcription factor gene plays an important role in the biosynthesis of anthocyanin in apple after bag removal, but little is known about how MdMYB1 transcription is regulated.ResultsIn this study, we investigated pigmentation in the non-red skinned cultivars ‘Granny Smith’ and ‘Golden Delicious’ after bag removal. The fruit skins of the two cultivars showed red/pink pigmentation after bag treatment. Transcript levels of MdMYB1, the master regulator of anthocyanin biosynthesis in apple, increased, and showed a correlation with anthocyanin content in both cultivars after bag removal. The MdMYB1 genomic sequences were compared in the two cultivars, which showed that the green-fruited cultivar ‘Granny Smith’ harbors the MdMYB1–1 and MdMYB1–2 alleles, while the yellow-fruited cultivar ‘Golden Delicious’ harbors only MdMYB1–2. A comparison of methylation levels in the 2 kb region upstream of the MdMYB1 ATG between the bag-treated fruits after removal from the bags and the unbagged fruits showed a correlation between hypomethylation and the red-skin phenotype in ‘Granny Smith’. Moreover, ‘Granny Smith’ fruits responded to treatment with 5-aza-2′-deoxycytidine, an inducer of DNA demethylation. An investigation of the MdMYB1 promoter in ‘Granny Smith’ showed reduced methylation in the regions − 2026 to − 1870 bp, − 1898 to − 1633 bp, and − 541 to − 435 bp after bag removal and 5-aza-2′-deoxycytidine treatments.ConclusionsDifferences in anthocyanin levels between ‘Granny Smith’ and ‘Golden Delicious’ can be explained by differential accumulation of MdMYB1-specific mRNA. Different levels of MdMYB1 transcripts in the two cultivars are associated with methylation levels in the promoter region. Hypomethylation of the MdMYB1 promoter is correlated with the formation of red pigmentation in ‘Granny Smith’ fruit skins. As a result, red pigmentation in Granny Smith’ was more intense than in ‘Golden Delicious’ fruits after bag removal.

Protein expression and methylation of MGMT, a DNA repair gene and their correlation with clinicopathological parameters in invasive ductal carcinoma of the breast.

Epigenetic mechanisms such as DNA methylation are being increasingly recognized to play an important role in cancer and may serve as a cancer biomarker. The aim of this study was to evaluate the promoter methylation status of MGMT (O6-methylguanine-DNA methyltransferase) and a possible correlation with the expression of MGMT and standard clinicopathological parameters in invasive ductal breast carcinoma patients (IDC) of Kashmir. Methylation-specific PCR was carried out to investigate the promoter methylation status of MGMT in breast tumors paired with the corresponding normal tissue samples from 128 breast cancer patients. The effect of promoter methylation on protein expression in the primary breast cancer and adjacent normal tissues was evaluated by immunohistochemistry (n = 128) and western blotting (n = 30). The frequency of tumor hypermethylation was 39.8 % and a significant difference in methylation frequency among breast tumors were found (p < 0.001) when compared with the corresponding normal tissue. Immunohistochemical analysis showed no detectable expression of MGMT in 68/128 (53.1 %) tumors. MGMT promoter methylation mediated gene silencing was associated with loss of its protein expression (rs = -0.285, p = 0.001, OR = 3.38, 95 % CI = 1.59-7.17). A significant correlation was seen between loss of MGMT and lymph node involvement (p = 0.030), tumor grade (p < 0.0001), loss of estrogen receptors (ER; p = 0.021) and progesterone receptors (PR) (p = 0.016). Also, MGMT methylation was found to be associated with tumor grade (p = 0.011), tumor stage (p = 0.009), and loss of ER (p = 0.003) and PR receptors (p = 0.009). To our knowledge, our findings, for the first time, in Kashmiri population, indicate that MGMT is aberrantly methylated in breast cancer and promoter hypermethylation could be attributed to silencing of MGMT gene expression in breast cancer. Our data suggests that MGMT promoter hypermethylation could have a potential function as molecular biomarker of breast oncogenesis. Also, based on their predictive value of response to therapy, the immunohistochemical evaluation and interpretation of MGMT may also help in future to establish therapeutic strategies for patients with breast cancer.

Abstract LB-85: Damage-inducible intragenic demethylation activates transcription of an alternative intronic promoter in TP53-wildtype but not mutant human cells and tumors

Abstract Unlike the well-known effects of promoter methylation, the functional significance of altered intragenic (gene body) CpG methylation remains unclear, particularly in genes such as the TP53 tumor suppressor which lack a 5′ CpG island. Here we show that CpG-specific demethylation of TP53 exon 5 in normal human and mouse cells is inducible by X-irradiation; this is accompanied by co-expression of a 5′-truncated TP53 isoform, raising the possibility that demethylation permits transcription from the known alternative promoter (P2) in intron 4. Consistent with this, in vivo expression of fully pre-methylated TP53 exonic constructs is associated with reduced basal and damage-inducible expression of both full-length and truncated TP53. In contrast, virally TP53-reconstituted Caco-2 and PC-3 human cancer cell lines respond to damage by upregulating expression of full-length TP53 and reducing expression of the truncated RNA isoform; CDKN1A expression varies directly with P2 isoform expression but inversely with the P1 transcript in these cells, suggesting that the isoform mediates downstream transactivation. 450K methylation analysis of adjacent normal and TP53-mutated human pancreatic cancer tissues suggests a mutation-specific decrease of intragenic TP53 methylation in intron 1 near P1 (p &amp;lt; 0.02 ) and a reciprocal increase in intron 4 near P2 (p &amp;lt; 0.01). Archival TP53 database analysis indicates that CpG-specific mutation frequency in human tumors is inversely associated with damage-inducible demethylation at the same sites in vitro (Spearman coefficient -7.1 ), consistent with methylation-dependent downregulation of transcription-coupled repair. We conclude that intragenic alterations of TP53 methylation are both physiologically inducible and functional, and hypothesise that tumor progression is drivable by either (i) intragenic hypermethylation affecting P1 as a field defect in TP53-wt tumor or adjacent normal tissues, or (ii) damage-inducible repression of P2 isoform expression associated with defective intron 4 demethylation, together with overexpression of full-length p53, in TP53-mutant tumors and cells. Citation Format: James Blackburn, Robert Ng, Daniel Roden, Jianmin Wu, Richard J. Epstein. Damage-inducible intragenic demethylation activates transcription of an alternative intronic promoter in TP53-wildtype but not mutant human cells and tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-85. doi:10.1158/1538-7445.AM2014-LB-85

Analysis of Methylation and Expression Profiles of CPT1-A and APOE Genes in Patients with NAFLD

Analysis of Methylation and Expression Profiles of CPT1-A and APOE Genes in Patients with NAFLD Non-alcoholic fatty liver disease (NAFLD) is the most common cause of abnormal hepatic steatosis in the absence of alcohol abuse worldwide. The aim of this study was to investigate the effect of promoter methylation of carnitine palmitoyltransferase I (CPT1) and apolipoprotein E (APOE) genes on the risk of non-alcoholic fatty liver disease (NAFLD). Methods: The promoter methylation of APOE and CPT-1A were analyzed using a methylation-specific polymerase chain reaction (MS-PCR) in blood samples taken from 80 NAFLD individuals and 100 healthy controls. The expression levels of APOE and CPT- 1A were also assessed in 10 blood mRNA samples from NAFLD patients. These cases were compared to the blood samplesof healthy controls (n=10) with real-time quantitative reverse transcriptase PCR. Results: The percentage of methylation for CPT-1A gene was significant between normal individuals and patients. The APOE gene methylation was not significant between cases and controls. A statistically significant relationship was found for methylation of CPT-1A between cases and controls (p<0.001). The relative expression of CPT-1A and APOE mRNA in NAFLD blood was no significantly different in comparison of blood samples between healthy controls and cases. The present outcomes indicate that the methylation status of the CPT-1A gene has a significant function in the process of NAFLD and suggest further study with a large sample size for this reason.

Effects of promoter methylation on increased expression of polyamine biosynthetic genes in suicide

Suicide is among the leading causes of death worldwide. The polyamine system has been increasingly implicated in the neurobiology of suicide. Previous research has indicated that epigenetic mechanisms play a role in explaining dysregulation of polyamine genes in suicide completers. Nevertheless, regulatory mechanisms explaining polyamine biosynthetic genes displaying dysregulated expression in suicide completers, including ornithine decarboxylase antizymes 1 and 2 (OAZ1 and OAZ2), S-adenosylmethionine decarboxylase (AMD1), and arginase 2 (ARG2), have yet to be elucidated. In this study, we investigated methylation patterns in the promoter region of OAZ1, OAZ2, AMD1, and ARG2 in Brodmann area 44 from a group of 33 suicide completers and 31 non-suicide controls. We found significant site-specific differences in methylation in the promoter of ARG2 and AMD1 that were also significantly negatively correlated with gene expression. These findings provide further support for a role for the involvement of epigenetic modifications in the regulation of genes associated with polyamine biosynthesis, and which may contribute to the complexity of suicidal behaviors.

The Effects of Promoter Methylation on Downregulation of DAZAP2 in Multiple Myeloma Cell Lines

Our previous studies had shown that DAZAP2 was profoundly downregulated in bone marrow mononuclear cells from multiple myeloma patients. In this report, we analyzed epigenetic changes in multiple myeloma cell lines to understand the molecular mechanisms underlying the downregulation of DAZAP2. Four multiple myeloma cell lines, KM3, MM.1S, OPM-2 and ARH-77, were studied. The results of methylation specific PCR (MSP) showed that the promoter of DAZAP2 was methylated for KM3, MM.1S, OPM-2 and unmethylated for ARH-77. The DAZAP2 promoter region was amplified to obtain a series of different length sequences. All of the amplified sequences were inserted to luciferase reporter vector. The constructs were transfected into COS-7 cells and the luciferase activities were measured to search for the core region of DAZAP2 promoter. Two CpG islands were found in DAZAP2 promoter region. The results of luciferase assay showed that CpG island 1 displayed weak transcriptional activity, whereas CpG island 2 exhibited strong transcriptional activity (273 folds) compared to the control. The sequence that covered both CpG islands 1 and 2 showed higher activity (1,734 folds) compared to the control, suggesting that the two islands had synergistic effect on regulating DAZAP2 expression. We also found that M. Sss I methylase could inhibit the luciferase activity, whereas demethylation using 5-aza-2′-deoxycytidine treatment rescued the expression of DAZAP2 for multiple myeloma cell lines. These data revealed that methylation of DAZAP2 promoter was involved in downregulation of DAZAP2 in multiple myeloma cells.

Clinical significance of promoter hypermethylation of DNA repair genes in tumor and serum DNA in invasive ductal breast carcinoma patients

Aims The clinical relevance of frequent methylation of CpG islands of key cancer genes in breast cancer is being increasingly recognized. Our study aimed to evaluate the promoter methylation status of DNA repair genes— BRCA1MGMT and GSTP1 in tumor and circulating DNA of invasive ductal breast carcinoma patients. Main methods Methylation-specific PCR was carried out to investigate the promoter methylation status of genes in tumor and circulating DNA of 100 breast cancer patients in a prospective study. The effect of promoter methylation on protein expression was evaluated by immunohistochemistry. Key findings The frequency of tumor hypermethylation was 27% in BRCA1, 32% in MGMT and 25% in GSTP1 and correlated with methylation of these genes in paired serum DNA . Immunohistochemical analysis showed no detectable expression of BRCA1 and MGMT in 51/89 (57%) and 35/89 (39%) tumors, respectively. MGMT promoter methylation mediated gene silencing was associated with loss of its protein expression ( p = 0.002, O.R. = 4.5, 95% C.I. = 1.7–12.0). BRCA1 promoter methylation was not associated with loss of its protein expression, indicating that methylation is not the sole mechanism accounting for the loss/reduced BRCA1 protein expression. Importantly, GSTP1 and BRCA1 hypermethylation were found to be independent of other prognostic factors in predicting disease recurrence ( p = 0.02, HR = 7.6, 95% C.I. = 1.4–44.1; p = 0.04, HR = 6.2, 95% C.I. = 1.1–35.7). Significance Our study underscores the potential utility of DNA methylation of these genes in serum as a promising biomarker and can serve as a surrogate for tumor DNA methylation for diagnosis and prognosis of breast cancer.

Abstract 834: IGF-II promoter specific methylation patterns are associated with its expression and disease progression in epithelial ovarian cancer

Abstract Purpose: High insulin-like growth factor-II (IGF-II) expression in epithelial ovarian cancer is associated with aggressive disease and poor prognosis. IGF-II expression is regulated by promoter methylation; aberrant methylation is common in cancer. The effect of promoter methylation on IGF-II expression and the role of methylation in ovarian cancer are investigated in a clinical study. Patients and Methods: The study enrolled 211 epithelial ovarian cancer patients who were followed after surgery for disease outcomes. Fresh tumor samples were collected from the patients and were analyzed for IGF-II promoter methylation using methylation-specific PCR, as well as for IGF-II mRNA expression with qRT-PCR. IGF-II peptide concentrations were measured with an ELISA kit in tumor tissues. Results: The study found different levels of methylation in 3 IGF-II promoters (P2-P4) which are located in a large CpG island, high methylation in P2 (64.2%) and P3 (52.1%) and low in P4 (9.8%). Promoter-specific mRNA expression was lower in methylated than in unmethylated tumors. P3 methylation and expression appeared to play a major role for IGF-II in ovarian cancer. While methylation in individual promoter was not related to the disease, a methylation pattern between P2 and P3 was significantly associated with clinical and pathological features of ovarian cancer, including tumor grade, debulking results, residual tumor size and treatment response. The methylation pattern was also linked to disease progression, but not overall survival. Conclusions: Our findings suggest that IGF-II expression affects tumor progression and the expression is regulated by methylation which may have clinical implications in disease management. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 834.

Clinical significance of Stratifin, ERα and PR promoter methylation in tumor and serum DNA in Indian breast cancer patients

Objectives: The objective of this study was to determine the concordance of promoter methylation of stratifin, ERα and PR in tumor and circulating DNA in breast cancer patients and their association with clinicopathological parameters and disease prognosis . Design and methods: Methylation specific PCR were carried out to investigate the promoter methylation status of stratifin, ERα and PR in tumor and circulating DNA in 100 breast cancer patients in a prospective study. The effect of promoter methylation on protein expression was evaluated by immunohistochemistry. Results: Significant association was observed between promoter methylation of stratifin in tumors (61%) and paired sera (56%) ( r = 0.78; p ≤ 0.001). Loss of stratifin expression was observed in 47% tumors and was associated with poor overall survival ( p = 0.05). Significant correlation was observed between methylation status of ERα with PRB ( p < 0.0001, OR = 20.8, 95% CI = 7.4–58.0) and stratifin ( p = 0.003, OR = 2.0, 95% CI = 0.8–4.4). Conclusion: This study underscores the potential utility of serum DNA methylation of these genes as surrogate for tumor DNA methylation as a promising tool for cancer diagnosis.

CpG hypomethylation of MDR1 gene in tumor and serum of invasive ductal breast carcinoma patients

Objectives: Multidrug resistance 1 ( MDR1) gene encodes P-glycoprotein (P-gp), a transmembrane calcium-dependent efflux pump, implicated in drug resistance. In this prospective study, methylation status of MDR1 promoter and its correlation with clinicopathological parameters were evaluated in tumor and serum of breast cancer patients. Design and methods: Methylation-specific PCR was carried out to investigate the promoter methylation status of MDR1 in tumor and serum of 100 patients with invasive ductal carcinomas of breast (IDCs). The effect of promoter methylation on protein expression was evaluated by immunohistochemistry. Results: MDR1 was hypomethylated in 47% tumors and 44% paired sera of IDC patients and correlated significantly with increased tumor size and advanced tumor stage. Promoter hypomethylation of MDR1 in serum DNA showed 98% specificity and 50% sensitivity. Conclusions: Hypomethylation of MDR1 promoter in IDCs accounted for P-gp overexpression and aggressive biologic behavior in a subset of patients. Detection of these epigenetic changes in circulating DNA may not only enhance insight into the biological behavior of the primary tumor of an individual but may also provide valuable information regarding prognosis that can be readily monitored throughout the disease course.

W1875 Effect of Promoter Methylation of Multidrug Resistance 1 (MDR1) Gene in Gastric Carcinogenesis

W1875 Effect of Promoter Methylation of Multidrug Resistance 1 (MDR1) Gene in Gastric Carcinogenesis

Epigenetic silencing of the interferon regulatory factor ICSBP/IRF8 in human multiple myeloma

Multiple myeloma (MM) is presently an incurable malignant plasma cell tumor. The objective of this study was to investigate expression of the interferon regulatory factor family (IRF1-9) and the potential role of DNA methylation in silencing IRF genes in MM cell lines and purified MM cells from patients. Using a panel of 13 human MM cell lines and purified CD138+ cells from nine MM patients, expression of IRF genes was investigated by quantitative reverse transcriptase polymerase chain reaction and Western blot. DNA methylation of the interferon consensus sequence-binding protein (ICSBP/IRF8) gene was measured using pyrosequencing, and the effect of promoter methylation on expression was analyzed by in vitro methylation of a cloned ICSBP/IRF8 promoter, and treatment of MM cells with 5-aza-2'-deoxycytidine (DAC). Eight of thirteen of the MM cell lines were found to lack ICSBP/IRF8 expression, associated with hypermethylation of the CpG island in the ICSBP/IRF8 promoter. We also found that ICSBP/IRF8 was significantly underexpressed in primary MM cells, whereas the ICSBP/IRF8 promoter was methylated in only one of nine of primary purified CD138+ MM samples. DAC-mediated demethylation restored endogenous ICSBP/IRF8 expression, whereas in vitro methylation silenced the promoter. Expression of the ICSBP/IRF8 gene is silenced in a majority of MM cell lines and primary CD138+ MM cells. DNA methylation of the ICSBP/IRF8 gene is a frequent event in MM cell lines, but silencing is also observed in the absence of methylation. These results suggest that silencing of ICSBP/IRF8 expression, by DNA methylation or other epigenetic mechanisms, may be associated with the malignant phenotype of MM.

Involvement of TSC genes and differential expression of other members of the mTOR signaling pathway in oral squamous cell carcinoma

BackgroundDespite extensive research, the five-year survival rate of oral squamous cell carcinoma (OSCC) patients has not improved. Effective treatment of OSCC requires the identification of molecular targets and signaling pathways to design appropriate therapeutic strategies. Several genes from the mTOR signaling pathway are known to be dysregulated in a wide spectrum of cancers. However, not much is known about the involvement of this pathway in tumorigenesis of OSCC. We therefore investigated the role of the tumor suppressor genes, TSC1 and TSC2, and other members of this pathway in tumorigenesis of OSCC.MethodsExpression of genes at the RNA and protein levels was examined by semi-quantitative RT-PCR and western blot analyses, respectively. Loss of heterozygosity was studied using matched blood and tumor DNA samples and microsatellite markers from the TSC1, TSC2 and PTEN candidate regions. The effect of promoter methylation on TSC gene expression was studied by treating cells with methyltransferase inhibitor 5-azacytidine. Methylation status of the TSC2 promoter in tissue samples was examined by combined bisulfite restriction analysis (COBRA).ResultsThe semi-quantitative RT-PCR analysis showed downregulation of TSC1, TSC2, EIF4EBP1 and PTEN, and upregulation of PIK3C2A, AKT1, PDPK1, RHEB, FRAP1, RPS6KB1, EIF4E and RPS6 in tumors. A similar observation was made for AKT1 and RPS6KB1 expression in tumors at the protein level. Investigation of the mechanism of downregulation of TSC genes identified LOH in 36.96% and 39.13% of the tumors at the TSC1 and TSC2 loci, respectively. No mutation was found in TSC genes. A low LOH rate of 13% was observed at the PTEN locus. Treatment of an OSCC cell line with the methyltransferase inhibitor 5-azacytidine showed a significant increase in the expression of TSC genes, suggesting methylation of their promoters. However, the 5-azacytidine treatment of non-OSCC HeLa cells showed a significant increase in the expression of the TSC2 gene only. In order to confirm the results in patient tumor samples, the methylation status of the TSC2 gene promoter was examined by COBRA. The results suggested promoter hypermethylation as an important mechanism for its downregulation. No correlation was found between the presence or absence of LOH at the TSC1 and TSC2 loci in 50 primary tumors to their clinicopathological variables such as age, sex, T classification, stage, grade, histology, tobacco habits and lymph node metastasis.ConclusionOur study suggests the involvement of TSC genes and other members of the mTOR signaling pathway in the pathogenesis of OSCC. LOH and promoter methylation are two important mechanisms for downregulation of TSC genes. We suggest that known inhibitors of this pathway could be evaluated for the treatment of OSCC.

Expression of the chloramphenicol acetyltransferase gene in mammalian cells under the control of adenovirus type 12 promoters: effect of promoter methylation on gene expression.

The effect of DNA methylation at specific promoter sites on gene expression was tested by using a sensitive and quantitative assay system. The plasmid pSVO CAT contains the prokaryotic gene chloramphenicol acetyltransferase (CAT) and a HindIII site in front of it for experimental promoter insertion. Upon insertion into pSVO CAT, the E1a and protein IX gene promoters from adenovirus type 12 (Ad12) DNA were capable of mediating CAT expression upon transfection in mouse cells. In many viral and nonviral eukaryotic genes, DNA methylation at highly specific sites in the promoter region can attain a regulatory function in gene expression. One of the important sites is the 5' C-C-G-G 3' sequence. The CAT-promoting activity of the early simian virus 40 promoter in plasmid pSV2 CAT is refractory to methylation by the Hpa II or Hha I DNA methyltransferase at 5' C-C-G-G 3' or 5' G-C-G-C 3' sequences, respectively, because this promoter lacks such sites. The CAT coding sequence of this plasmid carries four Hpa II and no Hha I sites. Methylation of the Hpa II sites in the coding region does not affect expression. The E1a promoter of Ad12 DNA comprising the leftmost 525 base pairs of the viral genome carries two 5' C-C-G-G 3' and three 5' G-C-G-C 3' sites upstream from the leftmost "TATA" signal. Methylation of the Hpa II or Hha I sites incapacitates this promoter. The promoter of protein IX gene of Ad12 DNA contains one 5' C-C-G-G 3' and one 5' G-C-G-C 3' site downstream and two 5' G-C-G-C 3' sites greater than 300 base pairs upstream from the TATA motif and probably outside the promoter. The protein IX promoter is not inactivated by methylation of these sites. These data demonstrate that critical 5' methylations in the promoter region decrease or eliminate transcription; methylations of sites too far upstream or probably any sites downstream from the TATA site do not affect expression.