Human IL-12(p35) gene activation involves selective remodeling of a single nucleosome within a region of the promoter containing critical Sp1-binding sites.
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-gamma (IFN-gamma), 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-gamma-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-gamma 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.
Goriely
Stanislas
S
Laboratoire d'Immunologie Expérimentale, Université Libre de Bruxelles, Belgium.
Demonté
Dominique
D
Nizet
Séverine
S
De Wit
Dominique
D
Willems
Fabienne
F
Goldman
Michel
M
Van Lint
Carine
C
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D003713:Dendritic Cells / Q000648:ultrastructure; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D018664:Interleukin-12 / Q000235:genetics*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics; D008836:Micrococcal Nuclease / Q000378:metabolism; D008969:Molecular Sequence Data; D009000:Monocytes / Q000648:ultrastructure; D009707:Nucleosomes / Q000737:chemistry / Q000648:ultrastructure*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection
D008070:Lipopolysaccharides; D009707:Nucleosomes; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D018664:Interleukin-12; D007371:Interferon-gamma; D004247:DNA; D008156:Luciferases; D015246:Deoxyribonuclease EcoRI; D003850:Deoxyribonuclease I; D008836:Micrococcal Nuclease