Identification of a ccaat motif essential for basal, but not leukemia inhibitory factor-stimulated, expression of the SKAP55R gene in myeloid cells

David J. Curtis, C. Glenn Begley, David M. Bodine

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Abstract

The adaptor protein, SKAP55R is a substrate of the src-family of protein tyrosine kinases (Liu J et al. PNAS 95:8779, 1998). In hematopoietic cells, SKAP55R mRNA levels are most abundant in myeloid and erythroid lineages. Furthermore, interleukin (IL)6 or leukemia inhibitory factor (LIF)-induced differentiation of the myeloid cell line M l is associated with a rapid increase in SKAP55R mRNA levels (Curtis et al. Exp Hematol in press, 2000). We have characterized the promoter of murine SKAP55R to identify cisacting elements controlling basal and cytokine-induced transcription of SKAP55R in myeloid cells. 5' RACE and RNase protection assay revealed 2 major transcription start sites 99 and 85 nucleotides upstream of the initiation ATG. The putative proximal promoter region was OC rich (67%) and contained potential binding sites for Spl, AP-1, STAT and MZF1, a transcription factor implicated in myeloid differentiation. There was no TATA box. The promoter sequence (-980/+78) was cloned into a GFP reporter vector for transient (293 cells) or stable (Ml cells) transfection assays. This region contained cis-acting elements necessary for transcriptional activity in unstimulated 293 cells (14-fold induction) or unstimulated M l cells (2.0-fold induction). However, LIF-induced transcription was observed in Ml (1.8-fold greater than basal activity) but not 293 cells. The LIF response in Ml cells was not mediated by the STAT (-34S/-337) binding site because a control STAT-responsive reporter construct was LIF-responsive in 293 cells. Deletion reporter constructs defined a promoter region (-317/-114) essential for both basal transcription and LIF response in M l cells. This region contained a CCAAT motif recently implicated in IL-6 mediated transcriptional regulation of JunB in M1 cells (Sjin et al. JBC 274:28697, 1999). Mutation of the CCAAT motif (-2S4/-245) completely abolished basal transcriptional activity of the SKAP55R promoter in Ml cells but did not affect LIFinduced transcription. Mutation of 2 Spl/MZFI sites had no effect on basal or LIFinduced transcription. Electrophoretic mobility shift assays demonstrated binding of a nuclear protein complex to the CCAAT motif of the SKAP55R promoter. The nuclear complex was inhibited by a 50-fold molar excess of wild type oligonucleotide. In summary, we have identified a cis-acting element critical for basal transcriptional activity of the SKAP55R promoter. Further, the cytokine-induced transcriptional activity was cell-type specific and mediated by a cis-acting element in the -317/-114 region distinct from the control of basal transcription.

Original languageEnglish
Number of pages1
JournalBlood
Volume96
Issue number11 PART II
Publication statusPublished - 1 Dec 2000
Externally publishedYes

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