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Volume 272, Number 44, Issue of October 31, 1997 pp. 27957-27965
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of an Upstream Enhancer within a Functional Promoter of the Human Leukemia Inhibitory Factor Receptor Gene and Its Alternative Promoter Usage

(Received for publication, May 30, 1997, and in revised form, August 21, 1997)

From the Cedars Sinai Research Institute-UCLA School of Medicine, Los Angeles, California 90048

Knockout of the leukemia inhibitory factor receptor (LIFR) gene results in disrupted placental architecture, imbalanced bone development, and losses of functional neurons. We here report the identification of an enhancer in a functional human LIFR gene promoter and alternative promoter usage by this gene. A single transcription start site was identified in placental JEG-3 cells and a genomic clone containing 4876-nucleotide upstream sequences was found to have promoter activity in JEG-3 cells. However, in osteogenic sarcoma U-2 OS cell, Northern blot using 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 region possessed promoter activity in U-2 OS cells. For the 4.8-kb promoter active in placental cells, a minimal promoter was localized within 162 nucleotides. Three regions increased and one inhibited promoter activity. Subcloning of an activation region (4876 to 3453 nucleotides) into SV40 promoter either upstream or downstream in either orientation to the luciferase reporter resulted in 10-35-fold luciferase induction, demonstrating the characteristics of an enhancer. Transfections into nine cell lines of different tissue origin indicated that the cloned promoter and enhancer in the 4.8-kb fragment was placental tissue-specific. A 226-base pair fragment (4625 to 4400 nucleotides) was further localized as the minimal enhancer region, in which deletion of either element A (4625 to 4581 nucleotides) or element B (4418 to 4400 nucleotides) resulted in the loss of enhancer activity. Electrophoretic mobility shift assay confirmed that these two elements bind to specific nuclear proteins individually. In the middle region between element A and B, disruption of enhancer integrity also led to a loss of enhancer activity, although two SP1 and three NF-B/c-Rel binding sites did not contribute to enhancer function. These results demonstrate a complex regulation of the human LIFR gene, including alternative promoter usage and tissue-specific elements at the transcription level.

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