Immunoglobulin mu-binding protein 2 (IGHMBP2/Smubp-2) is a helicase motif-containing DNA-binding protein that has been suggested to regulate various nuclear functions. Recent studies indicated that mutations in the IGHMBP2 gene are responsible for spinal muscular atrophy with respiratory distress type I (SMARD1). However, the mechanism of regulation of IGHMBP2 gene expression remains unclear. In the present study, a 2.0-kb fragment of the 5'-flanking (promoter) region of the human IGHMBP2 gene was isolated from the HL-60 genome by PCR and ligated into a luciferase (Luc) expression vector, pGL3, to generate the pSmu-Luc plasmid. Deletion analyses revealed that a 108-bp region is essential for basal promoter activity with a response to TPA in HL-60 cells. TF-SEARCH analysis showed that overlapping ets (GGAA) motifs are located upstream of the transcription start sites. Chromatin immunoprecipitation (ChIP) assay, electropheretic mobility shift assay (EMSA) and competition analyses indicated that PU.1 (Spi-1) recognizes and binds to the duplicated ets motifs in this 108-bp region. Moreover, co-transfection of the PU.1 expression plasmid and pSmu-Luc into HL-60 cells revealed that PU.1 modulates TPA-induced IGHMBP2 promoter activity. Taken together, these observations suggest that the duplicated GGAA motifs are essential for the IGHMBP2 promoter activity and its positive response to TPA in HL-60 cells.
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