GATA-3 is an important transcription factor for regulating human NKG2A gene expression.
CD94/NKG2A is an inhibitory receptor expressed by most human NK cells and a subset of T cells that recognizes HLA-E on potential target cells. To study the transcriptional regulation of the human NKG2A gene, we cloned a 3.9-kb genomic fragment that contains a 1.65-kb region upstream of the exon 1, as well as exon 1 (untranslated), intron 1 and exon 2. Using deletion mutants, we identified a region immediately upstream from the most upstream transcriptional initiation site that led to increased transcriptional activity from a luciferase reporter construct in YT-Indy (NKG2A positive) cells relative to Jurkat and K562 (both NKG2A negative) cells. We also localized a DNase I hypersensitivity site to this region. Within this 80-bp segment, we identified two GATA binding sites. Mutation of GATA binding site II (-2302 bp) but not GATA binding site I (-2332 bp) led to decreased transcriptional activity. Pull-down assays revealed that GATA-3 could bind oligonucleotide probes containing the wild type but not a mutated GATA site II. Using chromatin immunoprecipitation assays, we showed that GATA-3 specifically binds to the NKG2A promoter in situ in NKL and primary NK cells, but not in Jurkat T cells. Moreover, coexpression of human GATA-3 with an NKG2A promoter construct in K562 cells led to enhanced promoter activity, and transfection of NKL cells with small interfering RNA specific for GATA-3 reduced NKG2A cell surface expression. Taken together, our data indicate that GATA-3 is an important transcription factor for regulating NKG2A gene expression.
Marusina
Alina I
AI
Receptor Cell Biology Section, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA.
Kim
Dae-Ki
DK
Lieto
Louis D
LD
Borrego
Francisco
F
Coligan
John E
JE
D001483:Base Sequence; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D003850:Deoxyribonuclease I / Q000378:metabolism; D050990:GATA3 Transcription Factor; D005786:Gene Expression Regulation / Q000276:immunology*; D006801:Humans; D019169:Jurkat Cells; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000276:immunology* / Q000378:metabolism*; D008969:Molecular Sequence Data; D055654:NK Cell Lectin-Like Receptor Subfamily C; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D011971:Receptors, Immunologic / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D055607:Receptors, Natural Killer Cell; D015534:Trans-Activators / Q000378:metabolism / Q000502:physiology*; D015533:Transcriptional Activation; D014162:Transfection
D004268:DNA-Binding Proteins; D050990:GATA3 Transcription Factor; C494749:GATA3 protein, human; C527054:KLRC1 protein, human; D055654:NK Cell Lectin-Like Receptor Subfamily C; D034741:RNA, Small Interfering; D011971:Receptors, Immunologic; D055607:Receptors, Natural Killer Cell; D015534:Trans-Activators; D003850:Deoxyribonuclease I