Generation and phenotypic characterization of p65–S276A (RRPA) knock-in mice. (A) Strategy to generate p65–S276A (RRPA) knock-in mice by homologous recombination. A PvuI restriction enzyme site was coupled to the mutation in the targeted allele for PCR analysis. Restriction enzyme sites for HindIII and XbaI and the probes used for Southern blot analysis were as indicated. (B) Sequencing of p65 cDNA obtained by RT–PCR from wild-type and RRPA homozygous mice. (C) Genotype analysis of offspring derived from RRPA heterozygous parents. The number of live embryos displaying an eye phenotype is indicated in parentheses. (D) Eye development abnormalities in RRPA homozygous embryos at 12.5 dpc. (E) Variegated developmental defects in RRPA homozygous embryos. Embryos at 13.5 dpc are shown, as well as wild-type and RRPA heterozygous control embryos. (F) Eye development abnormalities in RRPA homozygous embryos at 15.5 dpc. The embryo presented in the middle panels has a normal-sized eye on the right side and no discernible eye on the left side. The embryo in the right panels has two small eyes.

Impaired transcriptional activity of RRPA mutant protein and the underlying mechanism. (A) Transactivation by RRPA p65 detected by reporter gene assay in MEFs. MEFs were transfected with pBIIx-luc and control construct Renilla for 24 h; stimulated with TNFα, LPS, or IL-1 (10 ng/mL) for 6 h; and analyzed. (B) The ability of p65 to interact with CBP was determined by immunoprecipitation. Immunoblotting for IκBα and nuclear p65 was performed to confirm the stimulation by LPS. (C) The ability of p65 to interact with HDAC3 was detected by immunoprecipitation. MEFs were stimulated with TNFα. (D) ChIP assay was performed with untreated or TNFα-treated (2 h) MEFs using the indicated antibodies. Precipitated IL-6 κB site DNA and IκBα κB site DNA were assayed by semiquantitative PCR. (E) Differential expression of NF-κB-regulated genes in MEFs. Total RNA isolated from unstimulated MEFs or MEFs stimulated with TNFα for 1, 3, or 5 h was quantified by real-time PCR and normalized to the level of GAPDH.

RRPA mutant p65 affects Pax6 gene expression through epigenetic mechanisms. (A) κB-binding sites on/around the Pax6 gene on chromosome 2 in the mouse genome. (B) Binding of p65 to κB sites upstream of the Pax6 gene. ChIP assay was performed in MEFs using antibody against p65. (C) HDAC3 and acetylated H3K9 levels at the κB sites upstream of the Pax6 gene were determined by ChIP assay. (D) Impaired Pax6 expression in RRPA homozygous MEFs was rescued by TSA. MEFs were treated with TSA for 18 h and Pax6 expression was determined by semiquantitative RT–PCR. (E) Impaired Pax6 expression in p65-deficient 3T3 cells stably transfected with RRPA mutant was rescued by TSA. Cells were treated with TSA for 18 h and Pax6 expression was determined by semiquantitative RT–PCR. (F) Analysis of histone modifications on Pax6 promoters P0 and P1 using ChIP assays.

Characterization of p65–R274A–S276A (RAPA) knock-in mice. (A) p50 (lane 1), wild-type p65 (lane 2), p50 and wild-type p65 (lane 3), or p50 and RAPA p65 mutant (lane 4) were transiently transfected into rela^−/− 3T3 cells. EMSA using labeled κB probe was performed with nuclear extracts of transfected cells. (B) Sequencing of p65 cDNA obtained by RT–PCR from wild-type and RAPA homozygous mice. (C) Genotype analysis of offspring derived from RAPA heterozygous parents. (D) Wild-type and RAPA/RAPA embryos from 15.5 dpc are shown. The RAPA embryos develop normally and are in general of similar size as the wild-type embryos. (E) Haematoxylin and eosin staining for sections of liver from 15.5-dpc wild-type and RAPA/RAPA embryos is shown. The massive apoptosis in the RAPA/RAPA knock-in livers is apparent. (F) p65 translocation in MEFs upon LPS stimulation detected by immunoblotting of nuclear extracts. (G) EMSA using labeled κB probe and control NF-Y probe. (Top panels) Nuclear extracts (5 μg) from unstimulated MEFs and MEFs stimulated with TNFα or LPS were analyzed. (Bottom panels) Degradation and resynthesis of IκBα protein in MEFs following TNFα or LPS stimulation. (H) Supershift analysis of NF-κB–DNA-binding complexes was performed using indicated antibodies. Nuclear extracts (5 μg) from unstimulated MEFs and MEFs stimulated with TNFα were analyzed. In RAPA MEFs, p50:p65–RAPA complex failed to bind to DNA; instead, the major NF-κB complex on DNA is p50:c-Rel. (I) Transactivation by RAPA p65 detected by reporter gene assay in MEFs. MEFs were transfected with pBIIx-luc and control Renilla construct for 24 h and were stimulated with TNFα, IL-1, or LPS for 6 h, and luciferase activity was analyzed. (+/+) Wild-type; (+/m) +/RAPA; (m/m) RAPA/RAPA.

A model of the mechanism through which p65 epigenetically affects gene expression. Upon stimulation, p50:p65 heterodimers containing phosphorylated p65 at Ser 276 enter the nucleus and recruit the transcription coactivator CBP/p300 to κB-binding sites on DNA, leading to normal gene transcription. In contrast, p50:p65–RRPA heterodimers lacking phosphorylation of Ser 276 cannot bind to CBP/p300 efficiently and instead recruit HDAC3 corepressor complexes to DNA, resulting in direct repression of a subset of NF-κB target genes and repression of non-NF-κB-regulated genes through epigenetic mechanisms. p50:p65–RAPA heterodimers cannot bind to DNA and thus cannot mediate the epigenetic effects induced by RRPA mutant p65.

Characterization of RRPA knock-in cells. (A) Immunoblot analysis of p65 expression levels in wild-type and RRPA homozygous MEFs generated from embryos at 12.5 dpc. (B) Interaction between p65 and IκBα or IκBβ in MEFs detected by immunoprecipitation. (C) Degradation and resynthesis of IκBα and IκBβ proteins in MEFs following TNFα stimulation (10 ng/mL). (D) p65 translocation in MEFs following TNFα stimulation. (E) EMSA using labeled κB probe and control NF-Y probe. Nuclear extracts (5 μg) from unstimulated MEFs and MEFs stimulated with TNFα or LPS (10 μg/mL) were analyzed. (F) Supershift analysis of NF-κB–DNA-binding complexes was performed using anti-p65 antibody. Nuclear extracts (5 μg) from unstimulated MEFs and MEFs stimulated with LPS for 60 min were analyzed.

Expression of Pax6 gene was diminished by RRPA mutant protein. (A) Pax6 expression in the eye. Embryonic eye cross-sections (13.5 dpc) were immunostained with the antibody recognizing Pax6 and then detected with secondary antibody coupled to Alexa 488. Nuclei were stained by DAPI. (B) Pax6 expression in untreated MEFs and in MEFs treated with TNFα. Gene expression levels were detected by semiquantitative RT–PCR. Cox-2 was used as a positive control for TNFα stimulation. αTN4 cells are mouse lens epithelial cells in which Pax6 is highly expressed, and were used as a positive control for Pax6 PCR analysis. (C) Pax6 was expressed at a high level in p65-deficient 3T3 cells detected by semiquantitative RT–PCR. p65 expression levels detected by immunoblotting are displayed in the bottom panels. (D) Pax6 expression was inhibited by RRPA mutant protein in stable cell lines. Wild-type p65 or p65-RRPA mutant was stably introduced into p65-deficient 3T3 cells. Pax6 levels were detected by semiquantitative RT–PCR. p65 expression levels detected by immunoblotting are displayed in the bottom panels.

Comparison of gene expression in wild-type versus RRPA knock-in cells using Affymetrix microarrays. Gene expression levels in untreated MEFs or in MEFs treated with TSA (300 nM, 18 h) were analyzed. Representative genes in each of three groups were hierarchically clustered and displayed, including 75 genes in Group I that were significantly repressed in RRPA/RRPA MEFs and could be rescued by TSA, 58 genes in Group II that were significantly repressed in RRPA/RRPA MEFs but not rescued by TSA, and 20 representative genes shown for Group III that were not significantly affected in RRPA MEFs. Each row represents a single gene, and each column represents an experimental sample. The ratio of the abundance of transcripts of each gene to the median abundance of the gene’s transcript among all of the samples is represented by the color in the matrix as indicated. Relative expression levels are shown at the bottom. Homeobox genes are marked with a pink box.

Rescue of p65 knock-in mice by TNFR1 knockout. (A) Genotype analysis of offspring derived from +/RRPA, TNFR1^+/− heterozygous parents. Among 161 pups from 26 litters, only three RRPA/RRPA homozygous mice were rescued by introducing the TNFR1 knockout. The three rescued mice had eye developmental defects, small body size, and lethargic behavior, and died ∼30 d after birth. (B) Knocking out TNFR1 could not rescue the phenotype induced by RRPA mutant protein in the majority of RRPA homozygous mice. (C) Eye developmental defect in RRPA/RRPA, TNFR1^−/− mice. (D) Survival curve of TNFR1 knockout rescued RAPA homozygous mice.