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J Biol Chem, Vol. 275, Issue 12, 8959-8969, March 24, 2000

Structural and Functional Characterization of the Leukocyte Integrin Gene CD11d
ESSENTIAL ROLE OF Sp1 AND Sp3^*

John D. Noti, Andrew K. Johnson, and Jill D. Dillon

From the Guthrie Research Institute, Sayre, Pennsylvania 18840

CD11d encodes the latest -subunit of the leukocyte integrin family to be discovered, and it is expressed predominantly in myelomonocytic cells. We have isolated a genomic clone that contains CD11d and showed this gene to be 11,461 bp downstream and oriented in the same direction as the related CD11c gene. CD11d transcription begins 69-79 nucleotides upstream of the ATG codon. Transfection analysis of CD11d-luc reporter constructs revealed that the 173 to +74 region is sufficient to confer leukocyte-specific expression of luciferase in myelomonocytic cells (THP1 and HL60), B-cells (IM9), and T-cells (Jurkat). Transfection analysis showed that down-regulation of CD11d expression by phorbol ester was myelomonocyte-specific and is mediated by one or more cis-elements within the 173 to +74 region. In vitro DNase I footprint analysis and electrophoretic mobility shift analysis showed that Sp1 and Sp3 bind at 63 to 40. Deletion of the Sp-binding site significantly reduced CD11d promoter activity. Overexpression of either Sp1 or Sp3 in THP1 cells led to activation of the CD11d promoter even in the presence of phorbol ester, whereas down-regulation of either factor by antisense oligonucleotides decreased CD11d promoter activity. In contrast, overexpression of Sp3 in IM9 and Jurkat cells down-regulated CD11d promoter expression. In vivo genomic footprinting revealed that the 63 to 40 region is bound by a Sp protein in unstimulated HL60 cells but not in phorbol ester-stimulated HL60 cells. In contrast, this site is bound in both unstimulated and phorbol ester-stimulated IM9 and Jurkat cells. Together, these results show that myelomonocyte-specific phorbol ester down-regulation of CD11d is mediated through both Sp1 and Sp3.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF187881.

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