Demethylation of promoter C region of estrogen receptor alpha gene is correlated with its enhanced expression in estrogen-ablation resistant MCF-7 cells

Abstract

Long-term estrogen deprivation (LTED) MCF-7 cells showing estrogen-independent growth, express estrogen receptor (ER) alpha at a much higher level than wild-type MCF-7 cells. Enhanced expression of ERalpha associated with partial localization of ERalpha to the plasma membranes in LTED cells is thought to be an important step for acquisition of estrogen-ablation resistance. In this study, we compared the regulation of ERalpha gene expression between wild type and LTED cells, examining the usage of the promoters A and C as well as their methylation status. We found that transcription from the promoter C was drastically enhanced in LTED cells, compared with that in wild-type cells. Furthermore, the promoter C region was highly unmethylated in LTED cells, but partially methylated in wild-type cells. Our findings imply that demethylation of promoter C region in the ERalpha gene is in part responsible for the enhanced expression of ERalpha gene in LTED cells.

Fig. 1

Relative expression of total ERα mRNA in wild type and LTED cells. Real-time RT-PCR was conducted for quantification of total ERα and β-actin mRNA as described in Section 2. Expression levels of total ERα mRNA were normalized using β-actin mRNA.

Fig. 2

Expression of ERα mRNA transcribed from promoters A and C in wild-type and LTED cells. Schematic representation of a part of ERα gene organization is shown above. The transcription start site of promoter A is defined as +1. Relative expression of ERα mRNA from promoters A and C in wild-type and LTED cells is shown. Expression levels of ERα mRNA from promoters A and C were quantified by real-time RT-PCR as described in Section 2 being normalized by β-actin mRNA.

Fig. 3

Reporter activities of promoters A and C in wild-type and LTED cells. Wild-type and LTED cells were transiently transfected with a reporter gene construct pGL3-ProA 1.3K or pGL3-ProC together with control vector pRL-TK as described in Section 2. The measured luciferase activities were normalized using the control Renilla luciferase activity.

Fig. 4

Methylation-specific PCR analysis of ERα gene promoters A and C in wild-type and LTED cells. Location of primers used in methylation-specific PCR for promoters A and C is shown in the left. Photos of electrophoresis of PCR products of methylated (M) or unmethylated (UM) DNA fragments are shown in the right.

Fig. 5

Direct sequencing of PCR products of ERα gene promoter C using bisulfite-converted DNA from wild-type and LTED cells. First PCR products of bisulfite-converted DNA from wild-type or LTED cells using primers ERPROCBSF and ERPROCBSR were subjected to direct sequencing with ERPROCBSF primer as described in Section 2. Three CpG sites within the amplified region are indicated by rectangles with numbering over the rectangle. Cytosine residues that will be shown as thymine in the sequence of PCR product of bisulfite converted DNA are marked with vertical arrows in black. Thick arrows indicate the positions of three CpG sites.

Fig. 6

COBRA assay of ERα gene promoter C in wild-type and LTED cells. First PCR products of bisulfite converted DNA from wild-type or LTED cells using primers ERPROCBSF and ERPROCBSR were digested with restriction enzyme Hpy188III as described in Section 2. Rectangle indicates the position of the CpG site to be tested. Underline indicates the recognition site of Hpy188III that will be generated by bisulfite conversion of methylated DNA, but not unmethylated one. An image of the polyacrylamide gel electrophoresis is shown below. U, untreated. D, digested with Hpy188III.

Fig. 7

Bisulfite-real-time PCR analysis of ERα gene promoter C in wild-type and LTED cells. Bisulfite-real-time PCR analysis based on SYBR-Green chemistry was conducted using methylation-specific primers ERPROCM1F and ERPROCM1R or unmethylation-specific primers ERPROCUM1F and ERPROCUM1R as described in Section 2. Representative results of bisulfite-real-time PCR are shown above. Comparison of methylated or unmethylated DNA amounts in tested samples from wild-type and LTED cells are shown below. S.D.: standard deviation.