Molecular and Functional Characterization of the Mouse PEA3 Promoter

Abstract

PEA3 is a member of the expanding Ets family of transcription factors. In the adult mouse, PEA3 mRNA is expressed at highest levels in the brain, epididymis and at lower levels in the mammary gland, testes, ovary and uterus. PEA3 mRNA is expressed differentially during mouse embryogenesis and is down-regulated following retinoic acid induced differentiation in mouse embryonal carcinoma cell lines. PEA3 is overexpressed at the transcriptional level in 93% of all HER2/neu positive human breast tumors. The molecular basis for differential transcription of the PEA3 gene is not known. Sequence analysis revealed that the upstream region of the PEA3 gene has characteristics of a CpG island and does not possess a recognizable "TATA" element. Rapid amplification of 5' eDNA ends (5'RACE) reveals that transcription initiates from multiple sites, consistent with the absence of TATA elements. To localize cis-acting sequences required for PEA3 expression, deletions of the putative promoter were placed upstream of a luciferase reporter gene and tested for activity in the FM3A cell line. FM3A cells express substantial levels of PEA3 mRNA and protein, which suggests that all of the factors required for transcription are present in the cells. Transient transfections of 5' and 3' deletion mutants of the PEA3 promoter indicated that the efficiency of the PEA3 promoter depended on both negative and positive cis-elements, located upstream and downstream of the transcription start sites. A DNA fragment containing a region from -3 to +676, relative to the major start site of transcription, was sufficient for maximal promoter activity. Luciferase reporter plasmids containing more 5' flanking sequence had lower activity indicating the presence of silencer elements. To aid the identification of critical sequence elements within the minimal PEA3 promoter, we cloned and sequenced the putative human PEA3 promoter. Comparison of the mouse and human PEA3 DNAs revealed that sequences required for maximal promoter activity in the mouse were highly conserved in the human gene. Furthermore, these conserved sequences corresponded to a variety of consensus binding sites: 6 Sp1, 8 c-ets-1, 3 PEA3, 3 AP-2, 3 MZF-1, 2 MyoD, 2 Ik-1, 2 c/EBPB, 2 oEF-1/USF, 2 HSFI and one of each of the following: AP-4, Ik-2, SRY, CP2, HEN-I, CREB andE47.