IDENTIFICATION OF AN UPSTREAM ENHANCER WITHIN A FUNCTIONAL PROMOTER OF THE HUMAN LEUKEMIA INHIBITORY FACTOR-RECEPTOR GENE AND ITS ALTERNATIVE PROMOTER USAGE

Knockout of the leukemia inhibitory factor receptor (LIFR) gene result s in disrupted placental architecture, imbalanced bone development, an d losses of functional neurons. We here report the identification df a n enhancer in a functional human LIFR gene promoter and alternative pr omoter usage by this gene. A single transcription start site was ident ified in placental JEG-3 cells and a genomic clone containing 4876-nuc leotide upstream sequences was found to have promoter activity in JEG- 3 cells. However, in osteogenic sarcoma U-2 OS cell, Northern blot usi ng a probe of the first exon detected in JEG-3 cells failed to detect LIFR transcripts. 5'-Rapid amplification of cDNA ends (RACE) revealed an alternative first exon and a 0.6-kilobase pair (kb) 5'-flanking reg ion possessed promoter activity in U-2 OS cells. For the 4.8-kb promot er active in placental cells, a minimal promoter was localized within -162 nucleotides. Three regions increased and one inhibited promoter a ctivity. Subcloning of an activation region (-4876 to -3453 nucleotide s) into SV40 promoter either upstream or downstream in either orientat ion to the luciferase reporter resulted in 10-35-fold luciferase induc tion, demonstrating the characteristics of an enhancer. Transfections into nine cell lines of different tissue origin indicated that the clo ned promoter and enhancer in the 4.8-kb fragment was placental tissue- specific. A 226-base pair fragment (-4625 to -4400 nucleotides) was fu rther localized as the minimal enhancer region, in which deletion of e ither element A (-4625 to -4581 nucleotides) or element B (-4418 to -4 400 nucleotides) resulted in the loss of enhancer activity. Electropho retic mobility shift assay confirmed that these two elements bind to s pecific nuclear proteins individually. In the middle region between el ement A and B, disruption of enhancer integrity also led to a loss of enhancer activity, although two SP1 and three NF-kappa B/c-Rel binding sites did not contribute to enhancer function. These results demonstr ate a complex regulation of the human LIFR gene, including alternative promoter usage and tissue-specific elements at the transcription leve l.