A promoter characterisation of the Ets transcription factor gene, Ese-2/Elf-5

E26 transformation specific (Ets) proteins are a versatile class of transcription factors. They act both up and downstream of signal transduction cascades, are causative in cancer generation and are able to control the expression of other transcription factors. Thus they play an important role in cellular growth, proliferation and differentiation. The ets gene family is characterised by an 85 amino acid DNA binding domain and currently consists of around 30 members. They have been shown to be proto-oncogenes and it is this feature that has elicited much research into their regulation and function in both normal and neoplasic tissue. The high level of conservation within the ETS domain of the family identifies all Ets proteins as sequence-specific DNA-binding proteins. Many have been identified as having a role in the regulation of gene expression. The retention of many ets genes throughout evolution implies there would be few redundant genes (Thomas, 1993) and therefore each gene would have a unique biological role. Because of the common DNA binding domain, the regulation of the ets genes must be complex to provide specificity of action throughout the family (Laudet et al., 1999). A cDNA sequence was isolated from mammary gland tissue and characterised as belonging to the Ets family of transcription factors. The gene was designated BrETS for Breast ETS. Recent publications have described the gene as Elf-5 (Zhou et al., 1998) and ESE-2 (Oettgen et al., 1999). BrEts showed a restricted pattern of spatial expression, being strongly expressed in the mammary gland. Its expression in the mammary gland also exhibits a tightly controlled temporal pattern, which implicates it in mammary processes such as development and differentiation. As BrETS is highly expressed in the mammary gland (where intense extracellular remodelling occurs), and has been localised to the epithelial tissue, it may play a role in the regulation of matrix metalloproteinases or their inhibitors. Genomic DNA spanning the BrETS locus was isolated in this study. It was found to encompass approximately 3kb of upstream sequence, the first two exons and intron 1. Multiple transcription start sites have been identified, indicative of a TATA-less, tissue- specific gene. Deletion constructs of the proximal promoter (850bp) were examined in cell culture. The mammary gland derived cell lines HC11 and Comma1D were used, in conjunction with a chloramphenicol acetyltransferase reporter system. A region of sequence between -70 and -200bp was identified as having up-regulated transcriptional activity above an empty expression vector in cell culture. Analysis of more distal promoter constructs resulted in decreased activity, to a level representative of basal activity. The first intron (6kb) was also examined in vitro as a complete construct and as smaller fragments. Low levels of reporter activity were seen with these constructs in cell culture. DNasel hypersensitive analysis on the immediate promoter region revealed the presence of a hypersensitive site within the first intron. This implies a regulatory element of potential importance is located here, though further research is required to determine the element(s) and significance. The identification of regulatory regions may allow for the determination of tissue-specific elements and regions that control the expression of the gene. This information may be later extrapolated and manipulated in vivo as a means of controlling the expression of the BrETS gene