Identification of upstream cis-acting regulatory elements controlling lineage-specific expression of the mouse NK...

Mouse NKR-P1C (NK1.1) is a homodimeric type II transmembrane protein expressed on natural killer (NK) cells, NKT cells, and on CD117+ progenitor thymocytes capable of giving rise to cells of the T and NK lineages. Although its physiological ligands remain unknown, NKR-P1C engagement with a monoclonal antibody (mAb) leads to interferon-gamma (IFN-gamma) production and the directed release of cytotoxic granules from NK cells. We have cloned and sequenced a approximately 10-kb genomic fragment corresponding to the 5'-flanking region of the C57Bl/6 mouse NKR-P1C gene. A transcriptional initiation site has been mapped in NK cells and an NK1.1+ T cell line by primer extension and rapid amplification of 5'-cDNA ends (5'-RACE) techniques. Although the 5'-flanking region of NKR-P1C is TATA-less, we have identified an initiator region and a downstream promoter element, which together constitute the principal minimal functional promoter. Computational analysis of the 10-kb 5'-flanking region revealed potential regulatory factor binding sites. DNaseI hypersensitivity assays identified a single hypersensitive site (HS) about a 9-kb upstream of the transcriptional initiation site. This site, termed HS1, was able to act as a transcriptional enhancer element in an NK cell line, while minimally affecting transcription in non-NK cell lines. Moreover, the HS1 element was shown to function as a promoter, with a transcript detected only in fetal NK1.1+ cells. An additional promoter and two non-coding exons were also characterized. These results identify the minimal upstream cis-acting elements, and point to a complex regulatory mechanism involved in the lineage-specific control of NKR-P1C expression in NK lymphocytes.