A binding protein to the DNase I hypersensitive site II in HLA-DR alpha gene was identified as NF90

Abstract

We previously observed that IFN gamma-inducible expression of the human MHC class II, HLA-DR alpha, gene was enhanced by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) only in human monocytic leukemia THP-1 cells, but not in HeLa cells. In the HLA-DR alpha gene, three DNase I hypersensitive sites (DHS) are known to be present in the promoter region (DHS-I) and first intron (DHS-II and -III) and are assumed to be involved in HLA-DR alpha gene regulation. In this study, we found a binding factor which recognized a unique palindrome sequence (DHS-22) in the region of the DHS II site of the HLA-DR alpha gene in THP-1 cells and HeLa cells. The binding activity of this factor was decreased by TPA treatment in THP-1 cells, but not in HeLa cells. This binding activity was also detectable in nuclear extracts of bovine brains. Thus, we isolated the DHS-22 binding factor from bovine brain nuclear extracts and finally identified it as NF90 on the basis of molecular mass analysis of Lys-C-digested fragments and amino acid sequences of the two peptides of the trypsin-digested binding protein. The DHS-22 binding protein(s) in THP-1 cells is (are) further confirmed by reactivity to an antibody against NF90, and we have demonstrated that the GST fusion protein of NF90 interacts with DHS-22 by electrophoretic gel mobility shift assay (EMSA). The mRNA of NF90 was decreased by TPA treatment in THP-1 cells but not in HeLa cells. These results suggest that the binding of NF90 to the DNase I hypersensitive site II of HLA-DR alpha gene seems to negatively regulate HLA-DR alpha gene expression.