CHARACTERIZATION OF 2 CIS-REGULATORY REGIONS IN THE MURINE BETA-1,4-GALACTOSYLTRANSFERASE GENE - EVIDENCE FOR A NEGATIVE REGULATORY ELEMENTTHAT CONTROLS INITIATION AT THE PROXIMAL SITE

The beta1,4-galactosyltransferase (beta1,4-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 d ifferent sets of start sites that are separated by approximately 200 b ase pairs. Translation of each mRNA results in the predicted synthesis of two related protein isoforms that differ only in the length of the ir NH2-terminal cytoplasmic domain (Russo, R. N., Shaper, N. L., and S haper, J. H. (1990) J. Biol. Chem. 265, 3324-3331). In this study we s how that the cellular requirement for beta1,4-GT correlate with the tr anscriptional start site used. In cells and tissues that express low t ranscript levels, the 4.1-kb transcriptional start site is apparently used exclusively. Increased transcription from the 4.1-kb start site p lus low levels of transcription from the 3.9-kb start site result in t he intermediate, beta1,4-GT transcript levels that are found in almost all somatic cell types. However, in mid- to late pregnant and lactati ng mammary glands very high transcript levels are observed, which corr elate with the predominant use of the 3.9-kb transcriptional start sit e. To identify the cis-acting elements that regulate the use of the tw o transcriptional start sites, we constructed a series of beta1,4-GT/C AT 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 negativ e cis-acting regulatory region was identified that represses transcrip tion from the proximal site. These results suggest a model of transcri ptional regulation in which the distal region functions as a housekeep ing promoter while the proximal region functions as a mammary cell-spe cific promoter. Differential initiation from the two promoters is a me chanism for regulation of beta1,4-GT enzyme levels. The predictions fr om this model are consistent with the conclusion that both protein iso forms are functionally equivalent resident trans-Golgi membrane-bound enzymes.