title: Deciphering the combinatorial DNA-binding code of the CCAAT-binding complex and the iron-regulatory basic region leucine zipper (bZIP) transcription factor HapX.
abstract: The heterotrimeric CCAAT-binding complex (CBC) is evolutionarily conserved in eukaryotic organisms, including fungi, plants, and mammals. The CBC consists of three subunits, which are named in the filamentous fungus Aspergillus nidulans HapB, HapC, and HapE. HapX, a fourth CBC subunit, was identified exclusively in fungi, except for Saccharomyces cerevisiae and the closely related Saccharomycotina species. The CBC-HapX complex acts as the master regulator of iron homeostasis. HapX belongs to the class of basic region leucine zipper transcription factors. We demonstrated that the CBC and HapX bind cooperatively to bipartite DNA motifs with a general HapX/CBC/DNA 2:1:1 stoichiometry in a class of genes that are repressed by HapX-CBC in A. nidulans during iron limitation. This combinatorial binding mode requires protein-protein interaction between the N-terminal domain of HapE and the N-terminal CBC binding domain of HapX as well as sequence-specific DNA binding of both the CBC and HapX. Initial binding of the CBC to CCAAT boxes is mandatory for DNA recognition of HapX. HapX specifically targets the minimal motif 5'-GAT-3', which is located at a distance of 11-12 bp downstream of the respective CCAAT box. Single nucleotide substitutions at the 5'- and 3'-end of the GAT motif as well as different spacing between the CBC and HapX DNA-binding sites revealed a remarkable promiscuous DNA-recognition mode of HapX. This flexible DNA-binding code may have evolved as a mechanism for fine-tuning the transcriptional activity of CBC-HapX at distinct target promoters.
authors.affiliation: From the Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, D-07745 Jena, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Hortschansky
authors.affiliation: the Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
authors.forename: Eriko
authors.initials: E
authors.lastname: Ando
authors.affiliation: From the Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, D-07745 Jena, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Tuppatsch
authors.affiliation: the Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Arikawa
authors.affiliation: the Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: the Faculty of Agriculture, Meijo University, Shiogama-guchi 1-501, Nagoya 468-8502, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Kato
authors.affiliation: the Division of Molecular Biology, Biocenter, Innsbruck Medical University, A-6020 Innsbruck, Austria, and.
authors.forename: Hubertus
authors.initials: H
authors.lastname: Haas
authors.affiliation: From the Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, D-07745 Jena, Germany, the Department of Microbiology and Molecular Biology, Institute for Microbiology, Friedrich Schiller University, D-07743 Jena, Germany axel.brakhage@hki-jena.de.
authors.forename: Axel A
authors.initials: AA
authors.lastname: Brakhage
mesh_terms: D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000235:genetics; D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics; D001665:Binding Sites; D023081:CCAAT-Binding Factor / Q000378:metabolism; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D007501:Iron / Q000378:metabolism*; D059372:Nucleotide Motifs / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D023081:CCAAT-Binding Factor; D022762:CCAAT-Enhancer-Binding Proteins; D014157:Transcription Factors; D007501:Iron
keywords: CCAAT-binding Complex (CBC); DNA-binding Protein; DNA-binding Site; HapX; Iron Metabolism; Surface Plasmon Resonance (SPR); Transcription Regulation; Transcription Repressor