A TLR4/TRAF6-dependent signaling pathway mediates NCoR coactivator complex formation for inflammatory gene activation

Significance The nuclear receptor corepressor NCoR is generally considered to mediate transcriptional repression dependent on its interaction with the histone deacetylase HDAC3. Here, we show that NCoR can be converted from a corepressor to a coactivator of NFκB in macrophages by specific signals that promote the interaction of NCoR and HDAC3 with PGC1β. These findings raise the possibility that many of the functions of NCoR in development, homeostasis and immunity are due to it serving as a coactivator, rather than as a corepressor. Because deacetylation of PBC1β by HDAC3 is required for this switch, these findings also suggest an additional mechanism by which HDAC3 inhibitors exert their biological effects.


Animal studies
All animal procedures were in accordance with University of California San Diego research guidelines for the care and use of laboratory animals.Mice were maintained under a 12 hr light/12 hr dark cycle at constant temperature (20-23°C) with free access to food and water.Animals were fed a normal chow diet (T8604, Envigo).Ncor f/f and Ncor f/f LysM-Cre mice were described previously (1).Pgc1b f/f mice were described previously (2).Ncor f/f and Pgc1b f/f mice were used as WT mice for Ncor f/f LysM-Cre and Pgc1b f/f LysM-Cre mice, respectively.
In Vivo LPS shock study 8-to 12-week-old C57BL/6 male mice were intraperitoneally injected with 6 mg/kg of LPS (Sigma-Aldrich) and observed continuously every 4 hrs.Intraperitoneal injection of 10 mg/kg RGFP966 (Selleckchem) or 10% DMSO as vehicle control was performed 6 hrs before LPS injection.Identities were blinded from the experimenter until the end of studies.All surviving animals were humanely euthanized at 96 hrs after LPS injection.

Antibodies
Mouse monoclonal antibody immunoglobulin G-Y8129 (IgG-Y8129) against mouse NCoR (amino acids 1817-1879) was produced by immunizing mice with gp64 fusion protein expressed by baculoviral system as described previously (3).A list of other antibodies used in this article is shown in Table S1.

Bone marrow-derived osteoclast culture
Bone marrow cells were obtained by flushing the tibia and femur from 8-to 12-week-old C57BL/6 mice with alpha MEM (Sigma-Aldrich) containing 10% FBS, 1% penicillin/streptomycin+Lglutamine and lysed using red blood cell lysis buffer.48 million cells per 15 cm tissue culture plates for ChIP-seq were cultured in alpha MEM containing 10% FBS, 1% penicillin/streptomycin+Lglutamine and 10 ng/ml M-CSF for 3 days to generate osteoclast precursor cells.After nonadherent cells were washed off with alpha MEM, adherent osteoclast precursor cells were cultured in alpha MEM containing 10% FBS, 1% penicillin/streptomycin+L-glutamine, 10 ng/ml M-CSF and 50 ng/ml RANKL for 4 days to differentiate to osteoclasts.The M-CSF plus RANKL containing culture media was replaced every 2 days.
Uncropped images of blots are shown in Fig. S7, S8 and S9.For immunoprecipitation, whole-cell lysates were immunoprecipitated in cell lysis buffer (50 mM HEPES-KOH (pH 7.9), 150 mM NaCl, 1.5 mM MgCl2, 1% NP-40, 1mM PMSF, 1X protease inhibitor cocktail) by wheel rotating overnight at 4°C in the presence of Dynabeads protein G (Thermo Fisher Scientific) and each antibody described in Table S1.After immunoprecipitation, beads were washed three times with cell lysis buffer and three times with PBS, and then eluted with sample buffer.The elution was subjected to immunoblotting as described above.

HDAC3 activity assay
HDAC3 activity in BMDMs was measured using a HDAC3 Activity Assay Kit (Sigma-Aldrich) according to the manufacturer's protocol.The deacetylase activity of immunoprecipitated HDAC3 or PGC1β with anti-HDAC3 or PGC1β antibody in 300 µg of whole-cell lysate as described in Immunoblotting and immunoprecipitation.Briefly, HDAC3 assay buffer in the presence or absence of 10 µM GSK8612 was added to the HDAC3 or PGC1β antibody-Dynabeads Protein G complex and incubated for 10 min at 37°C.Then HDAC3 assay buffer containing HDAC3 substrate with a fluorophore [R-H-K-K(Ac)-AFC] were mixed with the complex and incubated for 30 min at 37°C.Once the incubation was complete, developing solution was added and incubated for 5 min at 37°C.The reaction solutions were transferred to a 96-well plate and the fluorescent signals were determined at 380 nm of excitation and 500 nm of emission.

Histone acetyltransferase (HAT) activity assay
HAT activity in BMDMs was measured using a HAT Assay Kit (Active Motif) according to the manufacturer's protocol.The HAT activity of immunoprecipitated NCoR or PGC1β with anti-NCoR or PGC1β antibody in 300 µg of whole-cell lysate as described in Immunoblotting and immunoprecipitation.Briefly, 50 µl of 1X assay buffer containing 50 µM acetyl-CoA and 50 µM Histone H3 was added to the NCoR or PGC1β antibody-Dynabeads Protein G complex and incubated for 30 min at room temperature.Once the incubation was complete, 50 µl of stop solution was mixed.Then 100 µl of developing solution was added and incubated for 15 min in the dark at room temperature.The reaction solutions were transferred to a 96-well plate and the fluorescent signals were determined at 380 nm of excitation and 450 nm of emission.ATAC-seq library preparation 70000 cultured cells were washed once with PBS and once with cold lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40).The cells were suspended in 50 µl of 1X Reaction Buffer (25 µl of Tagment DNA Buffer, 2.5 µl of Tagment DNA enzyme I, and 22.5 µl of water) (Nextera DNA Library Preparation Kit, Illumina) as previously described (5).Transposase reactions were carried out at 37°C for 30 min, and then DNA was purified using ChIP DNA Clean & Concentrator Kit (Zymo Research).DNA was amplified using the Nextera primer Ad1 and a unique Ad2.n barcoding primer using NEBNext High-Fidelity 2X PCR Master Mix (NEB) for 7 cycles.The amplified libraries were purified with 2 µl of SpeedBeads (GE Healthcare) in 20% PEG 8000/2.5 M NaCl (Final 13% PEG 8000), eluted with 15 µl of EB (Zymo Research), size selected using PAGE/TBE gel (Invitrogen) for 175-225 bp fragments by gel extraction, and single-end sequenced on HiSeq 4000 (Illumina).

RNA-seq analysis
FASTQ files were processed to assess quality by determining general sequencing bias, clonality and adapter sequence contamination.RNA sequencing reads were aligned to the mm10 mouse reference genome using STAR (8).Gene expression levels were calculated using HOMER (7) by counting all strand specific reads within exons.Only the most abundant transcripts, including multiple alternative variants, were selected for each gene, and the genes with a length smaller than 250 bp were removed.Transcripts per million (TPM) were used to evaluate the correlation among replicates.Differential gene expression was calculated using DESeq2 (9) to assess both biological and technical variability between experiments.Unsupervised hierarchical clustering was used to cluster the gene expression in the heatmaps.

ATAC-seq and ChIP-seq analysis
FASTQ files were mapped to the mm10 mouse reference genome with Bowtie2 (10).Peaks were called with HOMER (findPeaks) using parameters "-style factor -minDist 200 -size 200".After merging these peaks, correlations among replicates from the same cell subset/treatment were evaluated by correlation using tag counts.The two most highly correlated samples were used for identifying the most robust peaks using the irreproducible discovery rate (IDR) method (11).For this step, peaks were called with HOMER's findPeaks, using parameters "-L 0 -C 0 -fdr 0.9 -minDist 200 -size 200".IDR peaks from different conditions involved in a comparison merged with HOMER's mergePeaks and annotated with HOMER's annotatePeaks.pl.The raw tags of all samples which had reasonable correlation were quantified with HOMER (annotatePeaks.pl)using parameter "-noadj".Peaks which contained at least 4 tags in at least 1 sample were used to identify differentially bounded peaks (DBP) by DESeq2.Peaks were categorized as distal peaks which are 2 kb away from known TSS and promotor peaks which are located within 2 kb region of known TSS sites.Histone marks, such as H3K27ac single, were quantified by either ATAC IDR peaks under the same conditions or transcription factor ChIP IDR peaks.ATAC peak quantification was normalized to the total tags in peaks, while the ChIP peak single was normalized to the sequence depth.

Motif analysis
To identify motifs enriched in peak regions over the background, HOMER's motif analysis "findMotifsGenome.pl" including known default motifs and de novo motifs was used (7).The background peaks used either from random genome sequences or from peaks in comparing condition were indicated throughout the main text and in the figure legends.

Data visualization
ChIP-seq data were visualized in the UCSC genome browser (12).

Statistical analysis
The significance of differences in the experimental data were determined using GraphPad Prism 8.0 software.All data involving statistics are presented as mean ± s.d.The number of replicates and the statistical test used are described in the figure legends.

Figures
Figures Fig. S1 (A) Bar plots for expression of Abca1 and Scd2 in BMDMs from WT and NKO mice.The significance symbols indicate statistical significance, **p-adj < 0.01 reported by DESeq2 using the Benjamini-Hochberg method for the multiple-testing correction.(B) BMDMs at Day7 after differentiation were treated with or without each ligand, and then subjected to experiments.(C, D, E, F) Scatter plots of RNA-seq data showing RANKL (C), Poly I:C (D), IFNβ (E) or IL4 (F)regulated gene expression and NKO-regulated gene expression in the presence of RANKL, Poly I:C, IFNβ or IL4 (light blue dots in left panels: significantly RANKL, Poly I:C, IFNβ or IL4-suppressed genes, dark blue dots in left panels: significantly RANKL, Poly I:C, IFNβ or IL4-induced genes, dark red dots in right panels: significantly NKO-induced genes, dark blue dots in right panels: significantly NKO-suppressed genes, FDR < 0.05, FC > 1.5).
Fig. S2 (A, B) The overlap between IDR-defined NCoR (A) or HDAC3 (B) ChIP-seq peaks at Veh and KLA is shown by Venn diagram.(C) Scatter plot of normalized H3K27ac ChIP-seq tags having at least 16 tags associated with ATAC-seq IDR peaks at Veh in a 1000 bp window.KLA-induced H3K27ac peaks (FDR < 0.05, FC > 2) are color-coded (light blue dots: significantly lost H3K27ac by KLA treatment, dark blue dots: significantly gained H3K27ac by KLA treatment).(D) The overlaps between KLA-or LPS-induced NCoR, Fosl2 and ATF3 (13) ChIP-seq peaks (FC > 2) are shown by Venn diagram.
Fig. S3(A, B) Bar plots for expression of Ptgs2 and Cp in peritoneal macrophages from LPS-injected WT and NSDAD mice (A) and HDAC3 WT or Y298F-rescued HDAC3-deficient BMDMs treated with or without LPS for 4 hours (B)(13).The significance symbols indicate statistical significance, *p-adj < 0.05, ***p-adj < 0.001 reported by DESeq2 using the Benjamini-Hochberg method for the multiple-testing correction.
Fig. S6 (A) The overlap between IDR-defined ERK1 ChIP-seq peaks at Day0 and Day4 after RANKL treatment is shown by Venn diagram.(B) De novo motif enrichment analysis of ERK1 peaks under Day4 after RANKL treatment (n=12467 in Fig. S6A) using a GC-matched genomic background.(C) The overlap between IDR-defined ERK1, PGC1β, p65 and Fosl2 ChIP-seq peaks under Day4 after RANKL treatment (14) is shown by Venn diagram.(D) The overlaps of ATAC-defined gained H3K27ac peaks in the presence of KLA (n=2247 in Fig. S2C) with NCoR, HDAC3, PGC1β and/or ERK1 ChIP-seq peaks are shown by pie chart.(E) The overlaps of ATAC-defined gained H3K27ac peaks in the presence of RANKL (n=1525 in Figure S2E from (14)) with NCoR, HDAC3, PGC1β and/or ERK1 ChIP-seq peaks are shown by pie chart.

Fig. S9
Fig. S9Representative original images of immunoblot analysis for Fig.7B and S4A