Coactivators and corepressors of NF-kappaB in IkappaB alpha gene promoter

Abstract

In this study, we investigated recruitment of coactivators (SRC-1, SRC-2, and SRC-3) and corepressors (HDAC1, HDAC2, HDAC3, SMRT, and NCoR) to the IkappaB alpha gene promoter after NF-kappaB activation by tumor necrosis factor-alpha. Our data from chromatin immunoprecipitation assay suggest that coactivators and corepressors are simultaneously recruited to the promoter, and their binding to the promoter DNA is oscillated in HEK293 cells. SRC-1, SRC-2, and SRC-3 all enhanced IkappaB alpha transcription. However, the interaction of each coactivator with the promoter exhibited different patterns. After tumor necrosis factor-alpha treatment, SRC-1 signal was increased gradually, but SRC-2 signal was reduced immediately, suggesting replacement of SRC-2 by SRC-1. SRC-3 signal was increased at 30 min, reduced at 60 min, and then increased again at 120 min, suggesting an oscillation of SRC-3. The corepressors were recruited to the promoter together with the coactivators. The binding pattern suggests that the corepressor proteins formed two types of corepressor complexes, SMRT-HDAC1 and NCoR-HDAC3. The two complexes exhibited a switch at 30 and 60 min. The functions of cofactors were confirmed by gene overexpression and RNA interference-mediated gene knockdown. These data suggest that gene transactivation by the transcription factor NF-kappaB is subject to the regulation of a dynamic balance between the coactivators and corepressors. This model may represent a mechanism for integration of extracellular signals into a precise control of gene transcription.

Fig 1. Interaction of SRC-1 with NF-kB.

(A) ChIP assay for SRC-1/NF-kB association. The signals for p65, p50 and Pol II indicate that NF-kB was activated and mRNA synthesis was initiated. Actin signal is a control of DNA input. (B) Cotransfection of SRC-1 with IkBα reporter. The IkBα reporter was activated by cotransfection of p65 and p50 expression vectors, which were used at 0.1 μg/point for each plasmid DNA. (C) Immunoblotting of IkBα protein in HEK293 cells transfected by SRC-1. SRC-1 expression is confirmed in the transfected cells. Actin protein is a control for protein loading. (D) Cotransfection of SRC-1 with NF-kB luciferase reporter. The same condition was used as described in the legend for panel 2 except the reporter DNA.

Fig 2. Substitution of SRC-2 by SRC-1.

(A) ChIP assay for SRC-2/NF-kB association. This result was obtained in the same condition as for ChIP assay of SRC-1. The signals for p65, p50 and Pol II is presented in Fig. 1A. (B) Nuclear exclusion of SRC-2. The cytoplasmic and nuclear proteins were extracted from HEK293 cells after TNF-treatment. SRC-2 protein was quantified in the extracts by immunoblotting. Actin and SP1 protein signals are used as controls for protein loading of the cytoplasmic and nuclear extracts, respectively. (C) Cotransfection of SRC-2 with IkBα reporter. The IkBα reporter was activated by cotransfection of p65 expression vectors at 0.1 μg/point. (D) Immunoblotting of IkBα protein in HEK293 cells transfected. SRC-2 protein level is confirmed in cells transfected for overexpression or knockdown.

Fig 3. Oscillation of SRC-3.

(A) ChIP assay for SRC-3/NF-kB association. This result was obtained in the same condition as for ChIP assay of SRC-1. The signals for p65 and p50 demonstrates activation of NF-kB by TNF-α. (B) Cotransfection of SRC-3 with IkBα reporter. The IkBα reporter was activated by cotransfection of p65 expression vector at 0.1 μg/point. The SRC-3 DNA (μg) is indicated. (C) Knockdown of SRC-3 by vector-based RNAi expression. The DNA (μg) of SRC-3 RNAi vector is indicated. (D) Immunoblotting of IkBα protein in HEK293 cells transfected. SRC-3 protein level is confirmed in cells transfected for knockdown.

Fig 4. Inhibition of NF-kB by HDAC1.

(A) ChIP assay for HDAC1/NF-kB association. This result was obtained in the same condition as for ChIP assay of SRC-1. (B) Cotransfection of HDAC1 with IkBα reporter. The IkBα reporter was activated by cotransfection of p65 expression vector at 0.1 μg/point. The HDAC1 DNA (μg) is indicated. (C) Knockdown of HDAC1 by vector-based RNAi expression. The DNA (μg) of HDAC1 RNAi (1RNAi) vector is indicated. (D) Immunoblotting of IkBα protein in HEK293 cells transfected. HDAC1 protein level is confirmed in cells transfected for overexpression knockdown.

Fig 5. HDAC2/NF-kB interaction.

(A) ChIP assay for HDAC2/NF-kB association. This result was obtained in the same condition as for ChIP assay of SRC-1. (B) Cotransfection of HDAC2 with IkBα reporter. The IkBα reporter was activated by cotransfection of p65 expression vector, which was used at 0.1 μg/point. The HDAC2 DNA (μg) is indicated. (C) Knockdown of HDAC2 by vector-based RNAi expression. The DNA (μg) of HDAC2 RNAi vector is indicated. 2RNAi is abbreviation of HDAC2 RNAi. (D) Immunoblotting of IkBα protein in HEK293 cells transfected. The change in HDAC2 protein is confirmed in cells transfected for overexpression knockdown.

Fig 6. HDAC3 oscillation.

(A) ChIP assay for HDAC3/NF-kB association. This result was obtained in the same condition as for ChIP assay of SRC-1. (B) Knockdown of HDAC3 by vector-based RNAi expression. The DNA (μg) of HDAC3 RNAi vector is indicated. (C) Cotransfection of HDAC3 with IkBα reporter. The IkBα reporter was activated by cotransfection of p65 expression vector at 0.1 μg/point. The HDAC3 DNA (μg) is indicated.

Fig 7. Switch between SMRT and NCoR.

(A) ChIP assay for SMRT and NCoR interaction with NF-kB association. This result was obtained in the same condition as for ChIP assay of SRC-1. (B) Cotransfection of SMRT or NCoR with IkBα reporter. The IkBα reporter was activated by cotransfection of p65 and p50 expression vectors, which was used at 0.1 μg/point for each vector. Plasmid DNA (μg) for SMRT or NCoR is indicated. (C) Knockdown of SMRT or NCoR by vector-based RNAi expression. Vector DNA (μg) of RNAi expressing plasmid is indicated.

Fig 8. Regulation of IkBα mRNA expression.

(A) The ChIP assay was conducted as stated in the method section. To help readers in understanding the complex switches among the coactivators and corepressors, and between the coactivator and corepressors, ChIP data presented in Figures 1 through 7 are combined together here. (B) IkBα mRNA expression was induced by TNF-treatment in 293 cells. IkBα mRNA was determined by Taqman RT-PCR as stated in methods. (C) Regulation of IkBα mRNA by the coactivators and corepressors.