Purification of a novel MHC class I element binding activity from thymus nuclear extracts reveals that thymic RBP-Jkappa/CBF1 binds to NF-kappaB-like elements.
We purified a DNA binding protein that recognizes a portion of the MHC class I regulatory element region 1/NF-kappaB binding site whose expression correlates with the expression of a MHC class I transgene in the thymus. The N-terminal amino acid sequence and the molecular size matched the RBP-Jkappa protein, also known as the EBV C-promoter binding factor, CBF1. Anti-peptide sera reactive with RBP-Jkappa/CBF1 also reacted with this protein in gel mobility shift assays. Although RBP-Jkappa/CBF1 is ubiquitously expressed, binding to the MHC class Ia NF-kappaB site was limited to the thymus. Comparison of the DNA binding specificities of RBP-Jkappa/CBF1 in thymic and splenic nuclear extracts revealed strong binding from both extracts to an IFN-beta kappaB site containing the RBP-Jkappa/CBF1 consensus sequence (CGTGGGAA). In contrast, only the thymic nuclear extract showed strong DNA binding activity with probes containing the NF-kappaB recognition sequences present in the MHC class Ia, IL-2Ralpha, and granulocyte-macrophage CSF promoters. Thus, RBP-Jkappa/CBF1 in thymic extracts demonstrates a clearly distinguishable DNA binding specificity that correlates with tissue-specific expression of a class I transgene. This, coupled with the fact that our previous study showed enhanced expression of the transgene in CD4+CD8+ thymocytes, suggests that RBP-Jkappa/CBF1 may play a role in the development of the immune system.
Shirakata
Y
Y
Laboratory of Molecular Structure, National Institute of Allergy and Infectious Diseases, National Institute of Health, Rockville, MD 20852, USA.
Shuman
J D
JD
Coligan
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005805:Genes, MHC Class I; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012045:Regulatory Sequences, Nucleic Acid; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013154:Spleen / Q000378:metabolism; D013950:Thymus Gland / Q000737:chemistry* / Q000378:metabolism
D004268:DNA-Binding Proteins; D016328:NF-kappa B; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides