Identification of ERF-1 as a member of the AP2 transcription factor family - PubMed (original) (raw)

Comparative Study

Identification of ERF-1 as a member of the AP2 transcription factor family

L A McPherson et al. Proc Natl Acad Sci U S A. 1997.

Abstract

The ERF-1 transcription factor was previously shown to be involved in the regulation of estrogen receptor (ER) gene transcription in hormonally responsive breast and endometrial carcinomas. In this study we sought to identify the gene for ERF-1. ERF-1 activates ER gene transcription by binding to the imperfect palindrome CCCTGCGGGG within the promoter of the ER gene. ERF-1 protein was purified from the ER-positive breast carcinoma cell line, MCF7, utilizing ion exchange and DNA affinity chromatography. Peptide sequence analysis was used to isolate a 2.7 kb cDNA clone from an MCF7 cDNA library. This cDNA encodes a protein of 48 kDa previously identified as the AP2gamma transcription factor. By gel-shift analysis, in vitro synthesized ERF-1 comigrates with MCF7 native ERF-1 complex and demonstrates identical sequence binding specificity as native ERF-1. In addition, AP2 polyclonal antisera supershifts both in vitro synthesized and native ERF-1 complexes. These results show that ERF-1 is a member of the AP2 family of developmentally regulated transcription factors. Given the central role of ER expression in breast carcinoma biology, ERF-1 is likely to regulate expression of a set of genes characteristic of the hormonally-responsive breast cancer phenotype.

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Figures

Figure 1

Figure 1

Mutational analysis of ERF-1 binding site. Gel shift analysis using nuclear extract prepared from MCF7 cells (lanes 2–19) or MDA-MB-231 (lane 20) with 30-bp wild-type oligonucleotide as probe. Competition with mutant oligonucleotides as listed.

Figure 2

Figure 2

Purification of ERF-1. Protocol for purification of ERF-1 protein using ion exchange and DNA affinity chromatography. See text for details.

Figure 3

Figure 3

Size determination of ERF-1. (A) ERF-1 complex was resolved by gel-shift analysis and the gel was subjected to UV crosslinking. The bound complex was excised and resolved on SDS/PAGE as shown. The location of molecular weight markers (kDa) is shown to the left of the gel lane. (B) Purified extract was electrophoresed on 8% SDS/PAGE and sectioned into 12 gel slices. The approximate location of gel slices 1–12 are shown to the right of the gel lane. Molecular weight markers (kDa) are listed to the left of the lane labeled markers. (C) Gel shift analysis of proteins renatured from gel slices in B. Fraction number 7 demonstrated ERF-1 activity.

Figure 4

Figure 4

ERF-1 cDNA. (A) Complete sequence of ERF-1 cDNA with predicted amino acid sequence. Boxes indicate peptide sequences obtained from protein sequencing of purified ERF-1. (B) Comparison of ERF-1 protein sequence with AP2α protein aligned to highlight similarities. ∗, Identical amino acids; ⋅ conserved substitution.

Figure 5

Figure 5

In vitro synthesized ERF-1. (A) In vitro transcription/translation from the ERF-1 cDNA using T3 polymerase (sense) (lane 1) or T7 polymerase (antisense) (lane 3). In vitro synthesized ERF-1 was immunoprecipitated using AP2 polyclonal antisera (lane 2). (B) Gel shift analysis of in vitro synthesized ERF-1 (lanes 2–13) with mutant competitors compared with MCF7 nuclear extract (lane 1). In vitro ERF-1 complex supershifted with AP2 antisera (lane 13).

Figure 6

Figure 6

Native ERF-1 complex supershift. MCF7 nuclear extract analyzed by gel shift with AP2 antisera (lanes 2–14) with competitors as shown compared with native ERF-1 complex (lane 1).

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