The high mobility group I-C (HMGI-C) protein is an abundant component of rapidly proliferating undifferentiated cells. High level expression of this protein is characteristic for early embryonic tissue and diverse tumors. HMGI-C can function as an architectural factor enhancing the activity of transcription factor NF-B on the -interferon promoter. The protein has three minor groove DNA-binding domains (AT-hooks). Here, we describe the complex of HMGI-C with a fragment of the -interferon promoter. We show that the protein binds to NRDI and PRDII elements of the promoter with its first and second AT-hook, respectively. Phosphorylation by Cdc2 kinase leads to a partial derailing of the AThooks from the minor groove, affecting mainly the second binding domain. In contrast, binding to long AT stretches of DNA involves contacts with all three AThooks and is marginally sensitive to phosphorylation. Our data stress the importance of conformation of the DNA binding site and protein phosphorylation for its function.High mobility group proteins are abundant components of chromatin, which modulate DNA conformation and facilitate assembly of higher order structures (for a review, see Refs. 1 and 2). A subgroup of these proteins, the HMGI(Y) 1 family, comprises diverse proteins containing short DNA-binding domains (AT-hooks; Ref.3) and acidic C-terminal regions. They act as regulatory factors affecting indirectly the expression of genes (for a review see Ref. 4). High levels of the proteins were found in transcriptionally active chromatin (5) and in constitutive heterochromatin of metaphase chromosomes (6). In mammalian cells three proteins of this type were detected, the HMGI, HMGY, and HMGI-C (7, 8). They are abundantly expressed in embryonic, rapidly proliferating, and tumor cells. Rearrangements or impairing of the HMGI-C gene were found in a number of tumors of mesenchymal origin (9, 10) and lead to the pygmy phenotype (11), respectively. These observations emphasize involvement of this protein in cell growth and development.However, although different binding sites of HMGI(Y) proteins within gene enhancers and promoters have been described, the organization of this protein-DNA complex is poorly understood. The proteins have three putative AT-hooks that interact with the minor groove of DNA (12). Two of them are necessary for strong binding to DNA (13-15), and the involvement of particular AT-hooks depends on the DNA conformation (16). NMR studies of HMGI revealed that, in the protein-DNA complex, the central portion of the AT-hook, the tripeptide RGR, penetrates the minor groove of the AT-rich stretches, whereas residues on both sides of the peptide establish extensive contacts with the sugar-phosphate backbone (17). On the basis of the DNA-binding strength and extent of the contacts to the backbone, these DNA binding domains (DBD) were classified as type I DNA binding domains (I-DBD) and type II DBD with high and low binding affinity, respectively (17).The proteins of the HMGI(Y) family are phosphoproteins. They are ...