Molecular characterization of Celtix-1, a bromodomain protein interacting with the transcription factor interferon regulatory factor 2

Transcriptional control at the G1/S-phase transition of the cell cycle requ ires functional interactions of multimeric promoter regulatory complexes th at contain DNA binding proteins, transcriptional cofactors, and/or chromati n modifying enzymes. Transcriptional regulation of the human histone H4/n g ene (FO108) is mediated by Interferon Regulatory Factor-2 (IRF-2), as well as other histone gene promoter factors. To identify proteins that interact with cell-cycle regulatory factors, we performed yeast two-hybrid analysis with IRF-2 and identified a novel human protein termed Celtix-1 which binds to IRF-2. Celtix-1 contains several phylogenetically conserved domains, in cluding a bromodomain, which is found in a number of transcriptional cofact ors. Using a panel of IRF-2 deletion mutants in yeast two-hybrid assays, we established that Celtix-1 contacts the C-terminus of IRF-2. Celtix-1 direc tly interacts with IRF-2 based on binding studies with glutathione S-transf erase (GST)/IRF-2 fusion proteins, and immunofluorescence studies suggest t hat Celtix-1 and IRF-2 associate in situ. Celtix-1 is distributed throughou t the nucleus in a heterodisperse pattern. A subset of Celtix-1 colocalizes with the hyperacetylated forms of histones H3 and H4, as well as with the hyperphosphorylated, transcriptionally active form of RNA polymerase II. We conclude that the bromodomain protein Celtix-1 is a novel IRF-2 interactin g protein that associates with transcriptionally active chromatin in situ. (C) 2000 Wiley-Liss, Inc.