A nuclear receptor corepressor transcriptional checkpoint controlling activator protein 1-dependent gene networks required for macrophage activation

Abstract

The nuclear receptor corepressor (NCoR) and the related factor known as silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) are essential components of multiprotein complexes that mediate active repression by unliganded nuclear receptors. Recent studies suggest that NCoR and SMRT can interact with and exert repressive effects on several other classes of DNA-binding transcription factors, but the physiological importance of these interactions has not been established. Here, investigation of endogenous transcriptional programs regulated by NCoR in macrophages reveals that NCoR acts as a transcriptional checkpoint for activator protein (AP)-1-dependent gene networks that regulate diverse biological processes including inflammation, cell migration, and collagen catabolism, with loss of NCoR, resulting in derepression of AP-1 target genes. The NCoR corepressor complex imposes an active block of exchange of c-Jun for c-Jun/c-Fos heterodimers, with targeted deletion of the c-Jun locus, resulting in loss of NCoR complexes from AP-1 target genes under basal conditions. The checkpoint function of NCoR is relieved by signal-dependent phosphorylation of c-Jun, which directs removal of NCoR/HDAC3/TBL1/TBLR1 complexes through recruitment of a specific ubiquitylation complex, as a prerequisite to the default binding of c-Jun/c-Fos heterodimers and transcriptional activation. The requirement for a checkpoint function to achieve the appropriate dynamic range of transcriptional responses to inflammatory signals is likely to be used by other signal-dependent transcription factors that regulate diverse homeostatic and developmental processes.

Fig. 1.

NCoR regulates programs of gene expression involved in inflammation and cell-cycle control. (A) Significance analysis of microarrays plot illustrating significant changes in gene expression in NCoR^-/- macrophages as compared with wild-type macrophages. Expression data from four independent experiments were used for analysis. Points above the upper diagonal line represent significantly overexpressed genes and points below the diagonal line represent significantly underexpressed genes. Red and green points represent genes that are at least 1.5-fold over- or underexpressed in NCoR^-/- macrophages, respectively. (B) Subsets of genes up-regulated in NCoR^-/- macrophages with associated GO biological process annotations related to responses to external stimuli (including inflammatory response/chemotaxis annotations), cell proliferation (including M-phase annotations), and collagen catabolism. (C) Enhanced invasive activities of NCoR^-/- macrophages. Wild-type and NCoR^-/- macrophages were placed in the upper cell of a Boyden chamber that was separated from the lower cell by a porous filter and a collagen (Matrigel) matrix. Transmigration of macrophages through the matrix in response to murine monocyte chemotactic protein 1 was quantified 96 h later. hpf, high-power field. (Upper) Macrophages that are adherent to the undersides of filters.

Fig. 2.

NCoR is required for active repression of a subset of TPA-inducible genes in macrophages. (A) Venn diagram indicating relationships between genes derepressed in NCoR^-/- macrophages and genes induced by LPS and/or TPA. (B) Expression profiles for the 30 most highly induced TPA-responsive genes. Each column in the heat map represents the ratio of normalized expression for the two indicated conditions. Genes that exhibited increases in basal activity are red. (C) Expression profiles of target genes activated by TPA only (Mmp12), LPS only (Usp18), TPA or LPS (Cxcl1), or retinoic acid (Rarb). (D) ChIP assays of NCoR on target genes (Mmp12, Usp18, Cxcl1, or Rarb) in cells treated with TPA or LPS.

Fig. 3.

c-Jun is a required molecular beacon for recruitment of NCoR to a subset of inflammatory response genes. (A) Activity of the wild-type Mmp12 promoter and an analogous Mmp12 promoter containing a mutated AP-1 site in RAW264.7 macrophages. Activities of a simple AP-1-responsive reporter gene (3xAP-1-Luc) and a β-actin-luciferase reporter gene in RAW264.7 macrophages were also demonstrated. Cells were cotransfected with an NCoR expression vector and treated with TPA as indicated. (B) Development of c-Jun^-/- macrophages. Breeding of c-Jun^f/f mice and MxCre transgenic mice was performed to obtain mice with c-Jun^f/f × MxCre^- or c-Jun^f/f × MxCre⁺ genotypes. Both genotypes were treated with polyinosinic/polycytidylic acid, with induction of Cre in MxCre⁺ mice, resulting in quantitative recombination of the c-Jun^f/f locus in bone marrow-derived macrophages. Western blotting indicates absence of c-Jun protein c-Jun^f/f × MxCre⁺ macrophages. Macrophages from three mice of each genotype were analyzed. (C) ChIP assays of three TPA-inducible NCoR target genes (Mmp9, Mmp12, and Mmp13) and two control genes (Csf3 and Alcam) in wild-type and c-Jun^-/- macrophages. Specific antibodies against c-Jun, NCoR, or normal IgG were used to immunoprecipitate cross-linked chromatin derived from wild-type or c-Jun^-/- macrophages. (D) ChIP assays of distal and promoter-proximal regions of the Mmp12 gene for NCoR, SMRT, c-Jun, and c-Fos in RAW264.7 cells before and 1 h after TPA treatment. (E) ChIP assays for c-Jun, c-Fos, NCoR, and HDAC3 on the Mmp12 promoter in wild-type and NCoR^-/- macrophages. (F) ChIP assay documenting presence of NCoR, TBL1, TBLR1, and HCAC3 on Mmp12, Usp18, Cxcl1, and Rarb promoters under basal conditions in RAW264.7 macrophages.

Fig. 4.

Mechanism of signal-specific derepression of AP-1 target genes. (A) Gal-c-Jun represses transcription from a UAS-TK-LacZ reporter gene in HS68 cells. Cells were microinjected with the UAS-TK-lacZ reporter gene, GAL4-c-Jun expression plasmid, and NCoR siRNA, and treated with TPA as indicated. The percent of LacZ-positive cells was determined 12 h later. (B) Gal-c-Jun and Gal-c-Jun^S63/73A are equivalently derepressed after siRNA-mediated knockdown of NCoR expression. RAW264.7 cells were transfected with the indicated plasmids and NCoR siRNA and luciferase activity was measured 16 h later. (C) TPA-dependent dismissal of NCoR from the Mmp12 promoter requires phosphorylation of c-Jun S63/73. RAW264.7 macrophages were transfected with HA-c-Jun or HA-c-Jun^S63/73A and treated with TPA or control solvent. ChIP was performed by using anti-HA antibody followed by a second round of precipitation using anti NCoR IgG or control IgG. (D) ChIP assay indicates that UbcH5 is recruited to the Mmp12 promoter and NCoR and HDAC3 are dismissed within minutes of TPA treatment of RAW264.7 cells. (E) TPA induction of the Mmp12 promoter gene requires TBLR1. HS68 cells were microinjected with the Mmp12 promoter linked to lacZ and the indicated siRNAs. Cells were treated with control solvent or TPA and assayed for lacZ expression 12 h later. (F) TPA-dependent recruitment of UbcH5 to the Mmp12 promoter requires phosphorylation of c-Jun S63/73. RAW264.7 macrophages were transfected with HA-c-Jun or HA-c-Jun^S63/73A and treated with TPA or control solvent. ChIP was performed by using anti-HA antibody followed by a second round of precipitation using anti-UbcH5 IgG or control IgG.

Fig. 5.

Model for relief of the NCoR-dependent checkpoint on AP-1 target genes. Signal-dependent phosphorylation of c-Jun results in TBLR1-dependent recruitment of UbcH5 and proteolytic removal of the NCoR/HDAC3 complex. This procedure allows binding of c-Jun/c-Fos heterodimers and transcriptional activation.