Characterization of cis-acting elements of the gene for macrophage- stimulating protein from the human. The involvement of positive and negative regulatory elements

To analyze the promotor region of the human macrophage-stimulating protein (MSP) gene, the 5'-flanking region of this gene was cloned. The major initiation site was determined at T located 49 base pairs upstream of the translation initiation site by primer extention with mRNA from HepG2 and Hep3B cells. There was no TATA sequence in this region. Transient transfection assay with 5'-deletion constructs showed that the transcription of this gene was regulated by positive and negative regulatory elements (PRE and NRE). The PRE (-34 to +2) was essential for the maximal transcription of this gene, and the NRE (-141 to -34) appeared to be responsible for the tissue-specific expression of the gene. The PRE contained the CCAAT sequence and a mutation from CCAAT to CTGAT resulted in a significant loss of the transcriptional activity. Electrophoretic mobility shift assay suggested that two different proteins bound to the PRE (MSP-PRE-binding protein-1 (MSP- PREB1) and 2). MSP-PREB1 and 2 were detected in various cell types, and the CCAAT sequence was involved in these bindings. These findings indicate that MSP-PREB1 and 2 are positive regulators. Further characterization also revealed that MSP-PREB2 was identical to CCAAT binding transcription factor, also known as NF-Y.