Abstract
The leukocyte integrin p150,95 (CD11c/CD18) is involved in a number of cell-cell and cell-extracellular matrix interactions and mediates signal transduction into the cytoplasm. p150,95 is expressed on cells of the myeloid lineage as well as on certain activated T and B lymphocytes, and its expression is regulated during cell activation and differentiation. Since CD18 is expressed on all leukocyte lineages, the restricted expression of p150,95 must be controlled at the level of CD11c gene transcription. To understand the mechanisms that direct the constitutive and regulated leukocyte expression of p150,95 we have structurally characterized the CD11c promoter region and initiated its functional dissection. The CD11c promoter lacks TATA- and CCAAT-boxes, directs the synthesis of transcripts with heterogeneous 5'-ends, and contains an initiator-like sequence at the major transcription initiation site. Several putative binding sequences for ubiquitous (Sp1, AP-1, AP-2, and NF-kB) and leukocyte-specific (PU.1) transcription factors have been identified in the proximal region of the CD11c promoter which may participate in the regulation of the expression of p150,95. Transient expression of CD11c-based reporter gene constructs indicates that the CD11c promoter dictates the tissue-specific expression of p150,95 and that sequences contained within 160 base pairs 5' from the major transcriptional start site are involved in the tissue-specific and regulated expression of p150,95. DNase I protection analysis on the promoter region spanning from -160 to +40 revealed four regions of DNA-protein interactions (FPI-FPIV), two of which (FPII and FPIV) correlate with the cell type-specific and regulated expression of the CD11c gene.
Highlights
The leukocyte integrinp150,95(CDllc/CD18)is glycoproteins involvedin both intercellular adhesion acnedllinvolved in a number of cell-cell and cell-extracellular extracellular matrix interactions [1, 2]
Several putative bindingsequences for kemia [7] and other B-cell chronic lymphoproliferative disubiquitous (Spl, AP- 1,AP-2, and NF-kB) and leuko- orders [8].The expression of p150,95 is regulated during cell cyte-specific (PU.1) transcription factors have been identified in the proximal region of the CDllc promoter which may participate in the regulation of the expression of p150,95T.ransient expression of CDl lc-based reporter gene constructs indicates that the CD1ICpromoter dictates thtiessue-specific expression of p150,95 and that sequences contained within 160 base pairs 5’ from themajor transcriptional start site are involved in the tissue-specific and regulated expression of p150,95
To determine the transcriptional start site of the CDllc gene, primer extension analysis were performed on RNA from phorbol myristate acetate (PMA)-induced HL60 promyelocytic cells (CDllc+) using three different anti-sense oligonucleotides derived from the reportedcDNA sequence
Summary
Mapping of the Transcription Initiation Site of the CDllc Gene-The genomic structure of the CDllc gene has been previously described [19]. To determine the transcriptional start site of the CDllc gene, primer extension analysis were performed on RNA from PMA-induced HL60 promyelocytic cells (CDllc+) using three different anti-sense oligonucleotides derived from the reportedcDNA sequence. Ident,ical extension products were obtained upon analysis of PMA-treated U937 cells (data not shown) These results demonstrate the existenceof multiple transcription initiation sites in the CDllc gene, withtwo major sites located and 67 bp upstream from the initiation methionine,whichwereassignedthe+2and+1position, respectively (Fig. 1). +1 nucleotide (Fig. 1).T h e init.iator sequence has heen previously described in a number of developmentally regulated and R-cell-specific genes [28], anad similar sequence appears at the major transcription initiation site of theCDllb The size of the extended fragments was determined by sequencing of the CDllc gene using the same oligonucleotide as primer (lanes A, C, G, and 7’). C, nuclease S1 pro- 6 ) .or yeast tRNA (/anrs and 5 1, and the mixture w a s digrstrd with tection assay. p15On PE:l-(/anes 4 - 7 ) and p15On PE5-(lanes I-.?)- nuclease S1 and analvzed on denaturing polyncr?.lnmitlr pis. l’roprimed antisense single-stranded probes (lanes 1 and 4 ) were an- tectetl fragments are highlighted hy hraciwfr
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