Identification of an upstream region that controls the transcription of the human autocrine motility factor receptor.

We have isolated from a human placenta cosmid library a 0.7 kb genomic clone that contains the 5' terminal portion of the autocrine motility factor receptor (hAMFR) coding region. Chloramphenicol Acetyl Transferase (CAT) reporter gene assays have identified this region as the promoter of the hAMFR gene. A single transcription initiation site (+1) has been mapped to 129 bp upstream of the ATG start codon by primer extension. DNA sequence analysis and CAT assay revealed a TATA element at the position -485/-468 which was able to conduct only a marginal transcription (less than 5% of the total activity). The majority of the hAMFR promoter's activity is contributed by a transcription initiator (Inr) element overlapping the initiation site (+1) which independently controls the transcription of the hAMFR gene. Gel mobility shift assays showed that DNA-binding proteins in HeLa cells nuclear extract can bind specifically to both promoter's elements. DNA-binding proteins were found to be differentially expressed by sparse and dense cultured normal fibroblasts. The nuclear-binding protein expressed by sparse NIH-3T3 cells induced a DNA mobility shift similarly to the nuclear protein of HeLa cells, while a different DNA-protein complex size was observed with nuclear proteins extracted from dense cultured NIH-3T3 cells. Also CAT-reporter gene analysis revealed a significant lower activity in dense NIH-3T3 cells as compared with the sparse-cultured counterparts. These results help to explain the previously observed cell-cell contact regulation of AMFR expression in normal cells and its consecutive expression in tumor cells.