Regulation of gene expression in mammary epithelial cells by cellular confluence and sequence-specific DNA binding factors.

Milk protein gene expression in mammary epithelial cells is regulated by interactions of the cells with each other and with extracellular-matrix components, and by the lactogenic hormones. Cell-cell and cell-extracellular-matrix interactions confer a state of competence to HC11 mammary epithelial cells. Cellular confluence and matrix deposition are prerequisites for the lactogenic hormone induction of, for example, beta-casein synthesis. We have studied how these cellular interactions influence transcription factor activity. Proximal and distal regulatory elements have been identified in the DNA of the beta-casein gene promoter that confer transcriptional induction to the lactogenic hormones in competent cells. A region located between positions -221 and -170 of the rat beta-casein promoter contains overlapping binding sites for DNA binding factors with positive and negative regulatory activity. A construct containing 221 nt of 5' promoter sequences linked to a chloramphenicol acetyltransferase (CAT) reporter gene and transfected into HC11 cells has low constitutive expression and is strongly inducible. Deletion of the sequences to -183 results in an increase in both constitutive and induced expression. Mutations in or deletion of the region from -183 to -170 abolish promoter activity. A sequence-specific single-stranded DNA binding transcriptional repressor (STR), composed of two proteins, binds to the upper strand of the -194 to -163 fragment and negatively regulates transcription. STR also recognizes the 5' untranslated region of the beta-casein mRNA and is sequestered into the cytoplasm by RNA after lactogenic hormone induction. Sequestration by RNA allows an activator to bind to the fragment -183 to -170. This activator has been identified as SARP, a sequence-specific single-stranded DNA activator region binding protein. The binding site of SARP is found both in the upper and the lower strands of this fragment. SARP has no affinity for RNA. It enhances transcription of a promoter construct containing rat beta-casein promoter sequences from -183 to -1 and of a heterologous promoter containing multimerized copies of the -194 to -163 fragment in a lactogenic-hormone-independent manner. Mutations between positions -183 and -170, which result in a loss of promoter activity, also prevent SARP from binding to the DNA. Confluence of HC11 cells up-regulates the DNA binding activity of SARP. High SARP activity is also detected in mammary gland cells of lactating mice and is regulated by suckling. Withdrawal of pups from their lactating mothers results in a rapid decrease of SARP activity. We have purified SARP from the lactating mammary tissue of sheep and have identified proteins of 28 and 35 kDa.