Human IL-12(p35) gene activation involves selective remodeling of a single nucleosome within a region of the promoter containing critical Sp1-binding sites

• Published in: Blood 2003-06, Vol.101 (12), p.4894-4902
• Main Authors: Goriely, Stanislas, Demonté, Dominique, Nizet, Séverine, De Wit, Dominique, Willems, Fabienne, Goldman, Michel, Van Lint, Carine
• Format: Article
• Language: English
• Subjects: Base Sequence; Binding Sites; Biological and medical sciences; Cell Line; Dendritic Cells - ultrastructure; Deoxyribonuclease EcoRI - metabolism; Deoxyribonuclease I - metabolism; DNA - chemistry; DNA - metabolism; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Gene Expression Regulation - drug effects; Humans; Immunobiology; Interferon-gamma - pharmacology; Interleukin-12 - genetics; Lipopolysaccharides - pharmacology; Luciferases - genetics; Micrococcal Nuclease - metabolism; Modulation of the immune response (stimulation, suppression); Molecular Sequence Data; Monocytes - ultrastructure; Nucleosomes - chemistry; Nucleosomes - ultrastructure; Promoter Regions, Genetic - genetics; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - biosynthesis; Sp1 Transcription Factor - metabolism; Transcriptional Activation; Transfection
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2002-09-2851

To get insight into the regulation of human interleukin-12 (IL-12) synthesis, we determined the chromatin organization of the IL-12(p35) promoter region. First, we determined positioning of nucleosomes within the IL-12(p35) promoter using the indirect end-labeling technique in the THP-1 monocytic cell line. On stimulation with bacterial lipopolysaccharide (LPS) and interferon-γ (IFN-γ), hypersensitivity to digestion with DNase I, micrococcal nuclease, and specific restriction enzymes was detected in the region encompassing nucleotide (nt) –310 to –160, indicating selective inducible chromatin remodeling involving disruption of a single nucleosome (named nuc-2). Using p35 promoter deletion mutants and reporter gene assays, we demonstrated that the –396/–241 region contained critical cis-acting elements. Within this latter region, we characterized physically and functionally 2 Sp1-binding sites, which were acting as key regulatory elements for both basal and LPS/IFN-γ–inducible p35 gene expression: Sp1#1 lies within the remodeled nuc-2 region and Sp1#2 is located in the nucleosome-free region immediately upstream of nuc-2. Finally, we extended the chromatin structure analysis to dendritic cells (DCs) derived from human monocytes and observed the same nucleosomal organization and remodeling as in the THP-1 cell line. Moreover, we found that in DCs, LPS and IFN-γ synergized in the induction of nucleosomal remodeling and that chromatin remodeling at the p35 locus immediately preceded IL-12(p35) mRNA synthesis. Taken together, our results demonstrate that IL-12(p35) gene activation in the course of DC maturation involves selective and rapid remodeling of a single positioned nucleosome within a region of the promoter containing critical Sp1-binding sites.