Characterization of two cis-regulatory regions in the murine beta 1,4-galactosyltransferase gene. Evidence for a negative regulatory element that controls initiation at the proximal site.

The beta 1,4-galactosyltransferase (beta 1,4-GT) gene is unusual in that it specifies two mRNAs in somatic cells of 3.9 and 4.1 kilobases (kb). These two transcripts arise as a consequence of initiation at two different sets of start sites that are separated by approximately 200 base pairs. Translation of each mRNA results in the predicted synthesis of two related protein isoforms that differ only in the length of their NH2-terminal cytoplasmic domain (Russo, R.N., Shaper, N. L., and Shaper, J.H. (1990) J. Biol. Chem. 265, 3324-3331). In this study we show that the cellular requirements for beta 1,4-GT correlate with the transcriptional start site used. In cells and tissues that express low transcript levels, the 4.1-kb transcriptional start site is apparently used exclusively. Increased transcription from the 4.1-kb start site plus low levels of transcription from the 3.9-kb start site result in the intermediate beta 1,4-GT transcript levels that are found in almost all somatic cell types. However, in mid- to late pregnant and lactating mammary glands very high transcript levels are observed, which correlate with the predominant use of the 3.9-kb transcriptional start site. To identify the cis-acting elements that regulate the use of the two transcriptional start sites, we constructed a series of beta 1,4-GT/CAT hybrids and carried out transient transfection assays using mouse L cells and Drosophila SL2 cells. These studies have delineated both a distal and proximal regulatory region just upstream of the 4.1- and 3.9-kb transcriptional start sites, respectively. In addition, a negative cis-acting regulatory region was identified that represses transcription from the proximal site. These results suggest a model of transcriptional regulation in which the distal region functions as a housekeeping promoter while the proximal region functions as a mammary cell-specific promoter. Differential initiation from the two promoters is a mechanism for regulation of beta 1,4-GT enzyme levels. The predictions from this model are consistent with the conclusion that both protein isoforms are functionally equivalent resident trans-Golgi membrane-bound enzymes.