Transactivation of the estrogen receptor promoter by BRCA1

Abstract

Background: Absence of the estrogen receptor-α (ER) is perhaps the most distinctive pathological feature of breast cancers arising in women who inherit a mutation in BRCA1. Two hypotheses, not necessarily mutually exclusive, exist in the literature that describe mechanisms of ER transcriptional repression in breast cancer. One hypothesis suggests that methylation of cytosine-guanine dinucleotides (CpGs) primarily mediates repression, while the other maintains that transcriptional control is mediated by certain positive and negative promoter elements.

Methods: To determine if wild type BRCA1 could induce activity of the ER promoter, we performed a series of transient transfections with ER promoter segments linked to a luciferase reporter. The effect of BRCA1 on endogenous ER expression was evaluated by RNA analysis.

Results: Following cotransfection with a BRCA1 expression plasmid, we observed that ER promoter-driven luciferase activity was significantly increased in both MCF10A and IMEC cells (p < 0.005 and 0.0005 respectively, two-tailed t test). Specifically, the full length ER promoter construct showed approximately 5.6-fold (MCF10A) and tenfold (IMEC) increases in luciferase activity following BRCA1 transfection, compared with transfection with an empty expression plasmid (i.e. lacking BRCA1 sequence). We localized the ER promoter segment responsible for transactivation by BRCA1 to a 109 bp region containing an AP2γ homologous site.

Conclusions: The work described here, along with previously published work, indicates that activity of certain transcriptional regulatory elements and CpG methylation both represent important mechanisms by which the ER gene is typically inactive in breast cancers associated with BRCA1 mutations. The absence of ER in these breast cancers has significant implications for pathogenesis, prevention, and treatment.

Keywords: BRCA1; Estrogen receptor; Human mammary cell lines; Transcriptional activation.

Fig. 1

Topology of the ER promoter. The relative positions of the transcription initiation sites P1 and P0, as well as the documented regulatory elements are illustrated. The regulatory sites are indicated as follows: (a) ER-EH0 [18], (b) a negative regulatory element [17], (c) ERBF-1 [16], (d) ERUBF-1 [15], and (e) ERF-1 [13]

Fig. 2

Transcriptional activation of the ER promoter by BRCA1. Promoter activity for a series of ER promoter constructs with progressive 5′ deletions designed to sequentially remove the established regulatory sites. Fold induction in luciferase activity (x axis) is shown for each of the ER promoter constructs following transfection with either a BRCA1 expression plasmid (filled bars) or the empty pRK7 vector (open bars). The fold induction in luciferase activity is the number of photon units per unit time of data capture (RLU) divided by protein, normalized by the corresponding value from cells transfected with the empty pGL2 Basic vector. RLU/protein values from the empty pGL2 Basic vector ranged from ~35 to ~400, while those from the ER promoter constructs were >1500. Data shown represents the average of up to 7 experiments, with luciferase and protein measurements performed in triplicate for each experiment

Fig. 3

Transcriptional activation by BRCA1 with a series of ExoIII-generated ER promoter luciferase constructs. Luciferase activity for ExoIII-generated ER promoter constructs in MCF10A cells. Fold induction in luciferase activity (x axis) is shown for ER promoter constructs following co-transfection with either a BRCA1 expression plasmid (filled bars) or the empty pRK7 vector (open bars). Fold induction values were calculated as in Fig. 2. Data shown represents the average of 2–7 experiments, with luciferase and protein measurements performed in triplicate for each experiment

Fig. 4

Localization of the segment of ER promoter mediating transactivation by BRCA1. Mean fold induction in luciferase activity (y axis) by BRCA1 in MCF10A cells (top panel) and IMEC cells (bottom panel) is indicated for all ER promoter constructs (x axis), calculated as the ratio of promoter activity when co-transfected with BRCA1, divided by promoter activity measured with co-transfection of pRK7. Standard error of the mean is indicated

Fig. 5

Expression of ER mRNA following transfection with BRCA1 or pRK7. Cells were harvested 48 h after transfection and mRNA for RT-PCR analysis were prepared. PCR products from the RT-PCR analysis were run on an agarose gel for analysis of ER mRNA, with the products appearing at the expected size of 603 bp (arrow). Reverse transcriptase (RT) reactions were performed with or without RT to control for contamination, and mRNA prepared from untransfected MCF7 cells serving as positive control