Immunogenetics (1999) 50 : 109–112 Q Springer-Verlag 1999 SEQUENCE REGISTER Ron Jennings 7 Teresita Mun ˜ oz-Antonia 7 Zhiwen Zhu Rosalind Jackson 7 Scott Antonia Identification of an enhancer element in the mouse B7-1 promoter Received: 10 January 1999 / Revised: 4 June 1999 R. Jennings 7 T. Mun ˜ oz-Antonia 7 R. Jackson Molecular Oncology Program, H. Lee Moffitt Cancer Center & Research Institute, University of South Florida School of Medicine, Tampa, FL 33612, USA Z. Zhu 7 S. Antonia (Y) Division of Medical Oncology and Hematology, H. Lee Moffitt Cancer Center & Research Institute, University of South Florida School of Medicine, Tampa, FL 33612, USA e-mail: antonias@moffitt.usf.edu, Tel.: c1-813-9793883, Fax: c1-813-9793893 Key words B7-1 7 Promoter 7 Enhancer 7 Mouse In order to become activated, T cells must receive two signals. The first signal is through the T-cell receptor after binding to a major histocompatibility complex (MHC) molecule/peptide complex, and the second sig- nal is provided by costimulatory molecules expressed on the surface of antigen presenting cells (Schwartz 1990). B7-1 is one such T-cell costimulatory molecule (Linsley et al. 1991). The expression of B7-1 is tightly regulated with expression normally limited to only a few cell types including B cells (Hathcock et al. 1994), monocytes (Freedman et al. 1991), macrophages (Ding et al. 1993), and dendritic cells (Larsen et al. 1992). B7- 1 expression is also regulated within these various cell types in response to a variety of stimuli including LPS (Verhasselt et al. 1997), CD40 ligand (Goldsteinet al. 1996), interferon-alpha (Hermann et al. 1998), and acti- vation of human Toll (Medzhitov et al. 1997). Some of the promoter elements responsible for the regulation of B7-1 gene expression have been de- scribed. Zhao and co-workers (1996) isolated a ge- nomic clone of the human B7-1 gene which contained approximately 14 kilobases (kb) of sequence 5b up- stream of the first exon. They identified a 183 base pair (bp) region within this sequence that is present approx- imately 3 kb 5b upstream of the transcription start site. This region contains several potential enhancer ele- ments, one of which is an NF-kB element. Fong and co-workers (1996) examined a region more proximal to the transcription start site of the human B7-1 promoter. They described a regulatory element present from P60 to P47 which had sequence similarity to the consensus NF-kB motif, but they were not able to identify the transcription factors that bind to this site. Several groups have characterized the mouse B7-1 promoter. Selvakumar and co-workers (1993) originally reported the genomic organization of the mouse B7-1 gene and determined the transcription start site to be 67 bp 5b upstream from the start of the cDNA clone. Borriello and co-workers (1994) reported the presence of a transcription start site that was approximately 1500 bp 5b upstream from the start of the cDNA. Zhang and co-workers (1996) confirmed the presence of the transcription start site reported by Selvakumar and co-workers (1993). They also cloned the region of DNA containing 3084 bp 5b upstream of this transcrip- tion start site into a reporter vector, and found that this sequence had significant promoter activity. Characteri- zation of this sequence revealed three regions which contained putative enhancer elements including the re- gions from P3597 to P1555, P130 to P110, and c25 to c269 (Zhang et al. 1996). We have also previously reported the cloning of 692 bp of sequence 5r upstream of the transcription start site originally reported by Sel- vakumar and co-workers (1993) that has promoter ac- tivity. A 5b deletion mutant analysis demonstrated a significant decrease in promoter activity when the re- gion from P692 to P480 was deleted, suggesting the presence of an enhancer element within this region (Antonia et al. 1996). To further narrow down the sequence which con- tains the putative enhancer element, we constructed an additional 5b deletion mutant. The region from P585 to c67 was amplified from a genomic clone containing this sequence using polymerase chain reaction (PCR) with oligonucleotides designed to contain restriction enzyme sites which allowed the directional cloning of the PCR product into the multiple cloning site of the