NF-Y regulates LIF-induced transcription of the signaling adaptor SKAP55R in myeloid cells.
Previously, we have shown that interleukin-6 (IL-6) or leukemia inhibitory factor (LIF)-induced differentiation of the myeloid cell line M1 was associated with a rapid increase in the level of mRNA encoding the signaling adaptor protein, SKAP55R. Furthermore, enforced over-expression of SKAP55R in primary bone marrow cells inhibited colony growth. In this study, we have identified the cis-acting elements that control SKAP55R transcription in myeloid cells. The 980 bp genomic sequence upstream of the transcriptional start site was cloned into a GFP reporter vector for transient (293 cells) or stable (M1 cells) transfection assays. This region contained cis-acting elements necessary for transcriptional activity in unstimulated 293 cells (10-fold higher levels than the control vector) or unstimulated M1 cells (two-fold higher levels). Significant LIF-induced transcription was observed in M1 (3.4-fold induction, P < 0.001), but not 293 cells. Deletion reporter constructs defined a promoter region (-317/-137) essential for the transcriptional activity in M1 cells. This region contained a CCAAT element recently implicated in IL-6/LIF-induced transcriptional regulation of junB in M1 cells. Mutation of the CCAAT element (-250/-246) significantly reduced both basal and LIF-induced transcription (P < 0.01). Electrophoretic mobility shift assays demonstrated that NF-Y bound to the CCAAT element of both SKAP55R and junB. These results suggest NF-Y binding may be a common mechanism of IL-6/LIF-regulated transcription in myeloid cells.
Curtis
D J
DJ
Genetics and Molecular Biology Branch, National Human Genome Research Institute, Bethesda, MD, USA.
Dougherty
L
L
Bodine
D M
DM
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D023081:CCAAT-Binding Factor / Q000494:pharmacology*; D006131:Growth Inhibitors / Q000494:pharmacology*; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D053605:Leukemia Inhibitory Factor; D008222:Lymphokines / Q000494:pharmacology*; D051379:Mice; D022423:Myeloid Cells / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection; D014407:Tumor Cells, Cultured
D023081:CCAAT-Binding Factor; D006131:Growth Inhibitors; D015850:Interleukin-6; D047908:Intracellular Signaling Peptides and Proteins; C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; C507838:Lif protein, mouse; D008222:Lymphokines; D010750:Phosphoproteins; C116431:src kinase associated phosphoprotein 2