Journal: bioRxiv

Article Title: Nonredundant roles of the HDAC3 corepressor complex subunits SMRT and NCOR in controlling inflammatory and metabolic macrophage pathways

doi: 10.1101/2025.05.13.653058

Figure Lengend Snippet: Depletion of NCOR vs. SMRT differentially alters gene expression (transcriptome). (A) Principal Component Analysis (PCA) illustrating the transcriptional outcomes in NCOR-vs. SMRT-depleted RAW cells ( n =3). (B) Heatmap displaying differentially expressed genes in NCOR-vs. SMRT-depleted RAW cells, categorized in six clusters to show the different regulation patterns. Representative genes from each gene cluster are highlighted. Data significance for gene expression was determined using DESeq2. (C) Network of the top enriched KEGG pathways for each of the six gene clusters. (D) Heatmap showing the transcriptome-based TF activity analysis for each gene cluster. (E) Heatmaps illustrating selected genes in NCOR-depleted (top panel) and in SMRT-depleted (bottom panel) RAW cells and BMDMs. (F) Barplots showing enriched up- and down-regulated KEGG pathways in shNCOR BMDMs and shSMRT BMDMs.

Article Snippet: We designed lentiviral shRNA sequences targeting NCOR ( Ncor1 ) (clone IDs: shNCOR-1 TRCN0000350169, shNCOR-2 TRCN0000310755) and SMRT ( Ncor2 ) (clone IDs: shSMRT-1 TRCN0000238140, shSMRT-2 TRCN0000238139) using the GPP Web Portal (Broad Institute).

Techniques: Gene Expression, Activity Assay

Journal: bioRxiv

Article Title: Nonredundant roles of the HDAC3 corepressor complex subunits SMRT and NCOR in controlling inflammatory and metabolic macrophage pathways

doi: 10.1101/2025.05.13.653058

Figure Lengend Snippet: Depletion of NCOR vs. SMRT differentially alters chromatin accessibility (epigenome, candidate cis-regulatory elements). (A) PCA plot of ATAC-seq for NCOR-vs. SMRT- depleted cells ( n =3). (B-C) Distribution of shSMRT- ( B ) shNCOR- ( C ) specific upregulated regions between enhancer and promoter regions. The distribution is presented along the distance from the transcription start site (TSS) of the annotated gene. (D-E) Scatter plots presenting the correlation of the RNA-seq expression and the promoter peaks from ATAC-seq data in SMRT- ( D ) and NCOR- ( E ) depleted cells. Data significance for gene expression was determined using DESeq2. (F) Heatmap of all differentially accessible genomic regions based on ATAC-seq data in NCOR- vs. SMRT- depleted cells, categorized in the same six clusters as in . Representative genomic regions from each cluster with differential accessibility are highlighted. ( G ) TF-binding site motif analysis of the SMRT-specific, NCOR- specific, and commonly repressed peaks. ( H ) IGV genome-browser tracks representing the NCOR, SMRT and H3K27ac ChIP-seq peaks in WT cells and the ATAC-seq changes in NCOR- vs. SMRT- depleted cells at the Pdcd1 (top panel) and Abca1 (bottom panel) loci. The statistically significantly changed peaks are highlighted with blue shadow.

Article Snippet: We designed lentiviral shRNA sequences targeting NCOR ( Ncor1 ) (clone IDs: shNCOR-1 TRCN0000350169, shNCOR-2 TRCN0000310755) and SMRT ( Ncor2 ) (clone IDs: shSMRT-1 TRCN0000238140, shSMRT-2 TRCN0000238139) using the GPP Web Portal (Broad Institute).

Techniques: RNA Sequencing, Expressing, Gene Expression, Binding Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: Nonredundant roles of the HDAC3 corepressor complex subunits SMRT and NCOR in controlling inflammatory and metabolic macrophage pathways

doi: 10.1101/2025.05.13.653058

Figure Lengend Snippet: Depletion of NCOR vs. SMRT differentially alters H3K27ac (epigenome, enhancers). (A) PCA plot illustrating the H3K27ac ChIP-seq results for NCOR- vs. SMRT- depleted cells ( n =2). Two independent ChIP-seq experiments were performed, and all results were merged to eliminate batch effect. (B) Heatmap displaying the z-normalized counts of the leading 2000 H3K27ac peaks driving PC1. Representative genomic regions are highlighted. (C) Heatmap of all H3K27ac peaks in shGFP, shSMRT and shNCOR macrophages. Up- and downregulated peaks are plotted independently ( n =2). (D) Motif analysis of the upregulated H3K27ac peaks in NCOR- vs. SMRT- depleted cells, intersected with NCOR/SMRT peaks. (E) IGV genome browser tracks of H3K27ac ChIP-seq (basal condition) at Pdcd1 , Abca1 and Ptgs1 loci. NCOR, SMRT, PU.1, JunB, and CBP ChIP-seq data are used to annotate enhancer regions. Up- and downregulated H3K27ac peaks are highlighted. (F) RT-qPCR analysis of Pdcd1 and Abca1 expression in NCOR- vs. SMRT- depleted cells ( n =3). (G) IGV genome browser tracks of ATAC-seq and H3K27ac and H4K5ac ChIP-seq (basal condition) at the Rarb locus. Up- and downregulated peaks are highlighted with a blue shadow. (H) RNA-seq tag counts (-RPKM) of nuclear receptor gene expression in NCOR- vs. SMRT- depleted macrophages ( n =3). Unpaired t test was used to determine data significance for gene expression in the qPCRs. All data are represented as mean ± SEM. Data significance for gene expression in RNA-seq was determined using DESeq2. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: We designed lentiviral shRNA sequences targeting NCOR ( Ncor1 ) (clone IDs: shNCOR-1 TRCN0000350169, shNCOR-2 TRCN0000310755) and SMRT ( Ncor2 ) (clone IDs: shSMRT-1 TRCN0000238140, shSMRT-2 TRCN0000238139) using the GPP Web Portal (Broad Institute).

Techniques: ChIP-sequencing, Quantitative RT-PCR, Expressing, RNA Sequencing, Gene Expression

Journal: bioRxiv

Article Title: Nonredundant roles of the HDAC3 corepressor complex subunits SMRT and NCOR in controlling inflammatory and metabolic macrophage pathways

doi: 10.1101/2025.05.13.653058

Figure Lengend Snippet: Genome-wide chromatin co-occupancy of NCOR and SMRT (cistrome). (A) Genome-wide correlation analysis: peaks from NCOR and SMRT ChIP-seq experiments were merged for Pearson correlation test. (B) NCOR and SMRT peak distribution between enhancer and promoter regions. The distribution is presented along the distance from the TSS of the annotated gene. (C-D) Venn diagrams displaying the binding overlap between NCOR, SMRT and the TFs PU.1 ( C ) and JunB ( D ) in macrophages. (E-G) Peak coverage plots of NCOR/SMRT common or specific peaks and the corresponding motif enrichment in macrophages. (H-I) IGV genome browser tracks of H3K27ac, SMRT, NCOR and other related regulatory proteins ChIP-seq at common marked genes Ccl2 ( H ) and Abcg1 ( I ) loci. The representative superenhancer (SE) and promoter regions of both Ccl2 and Abcg1 genes are highlighted with blue shadow.

Article Snippet: We designed lentiviral shRNA sequences targeting NCOR ( Ncor1 ) (clone IDs: shNCOR-1 TRCN0000350169, shNCOR-2 TRCN0000310755) and SMRT ( Ncor2 ) (clone IDs: shSMRT-1 TRCN0000238140, shSMRT-2 TRCN0000238139) using the GPP Web Portal (Broad Institute).

Techniques: Genome Wide, ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Nonredundant roles of the HDAC3 corepressor complex subunits SMRT and NCOR in controlling inflammatory and metabolic macrophage pathways

doi: 10.1101/2025.05.13.653058

Figure Lengend Snippet: Reciprocal influence of NCOR and SMRT at the cistrome level. (A-B) Heatmap showing NCOR (A) and SMRT (B) occupancy in NCOR- vs. SMRT- depleted cells. The peak center in each plot represents the SMRT or NCOR peaks for each individual plot ( n =2). (C) MA plot displaying SMRT peak changes in NCOR-depleted cells. Upregulated and downregulated peaks are highlighted ( n =2). Key inflammatory and metabolic target genes are labeled. Significantly altered peaks were identified using DESeq2. (D-E) Distribution of up (D) and down (E) -regulated SMRT binding to promoters and enhancers in NCOR-depleted cells. The distribution is presented along the distance from the TSS of the annotated gene. (F-G) Motif analysis of up (F) and down (G) -regulated SMRT peaks in NCOR-depleted cells. (H) Boxplots comparing the distribution of the ATAC-seq tags between shNCOR/shSMRT and control and the SMRT ChIP-seq tags between shNCOR and control. The comparison is done for SMRT upregulated peaks (left panel) and for SMRT downregulated peaks (right panel). (I) Boxplots presenting the distribution of the ATAC-seq tags between shNCOR/shSMRT and control and the NCOR ChIP-seq tags between shSMRT and control. (J-K) IGV genome browser tracks showing SMRT ChIP-seq at Abca1 and Ccl2 loci. The corresponding H3K27ac ChIP-seq is used to illustrate the epigenome alterations ( n =2). Wilcox test and DESeq2 were used to determine data significance for gene expression.

Article Snippet: We designed lentiviral shRNA sequences targeting NCOR ( Ncor1 ) (clone IDs: shNCOR-1 TRCN0000350169, shNCOR-2 TRCN0000310755) and SMRT ( Ncor2 ) (clone IDs: shSMRT-1 TRCN0000238140, shSMRT-2 TRCN0000238139) using the GPP Web Portal (Broad Institute).

Techniques: Labeling, Binding Assay, Control, ChIP-sequencing, Comparison, Gene Expression

Journal: bioRxiv

Article Title: Nonredundant roles of the HDAC3 corepressor complex subunits SMRT and NCOR in controlling inflammatory and metabolic macrophage pathways

doi: 10.1101/2025.05.13.653058

Figure Lengend Snippet: Influence of SMRT on NCOR subcellular localization. (A) Immunofluorescent (IF) staining showing the subcellular localization of SMRT in NCOR-depleted RAW cells; blue: DAPI, green: SMRT, scale bar: 2μm. (B) Immunofluorescent staining showing the subcellular localization of NCOR in SMRT-depleted RAW cells; blue: DAPI, green: NCOR, scale bar: 2μm. (C) Immunofluorescent double staining showing the subcellular localization of NCOR and SMRT in NCOR- and SMRT- depleted BMDMs; blue: DAPI, purple: NCOR, green: SMRT, scale bar: 2μm. (D) Subcellular fragmentation WB analysis of NCOR, SMRT, HDAC3 and GPS2 in NCOR- vs SMRT- depleted RAW cells. (E) Model of the compressor complex alterations upon NCOR vs. SMRT depletion in macrophages.

Article Snippet: We designed lentiviral shRNA sequences targeting NCOR ( Ncor1 ) (clone IDs: shNCOR-1 TRCN0000350169, shNCOR-2 TRCN0000310755) and SMRT ( Ncor2 ) (clone IDs: shSMRT-1 TRCN0000238140, shSMRT-2 TRCN0000238139) using the GPP Web Portal (Broad Institute).

Techniques: Staining, Double Staining

Journal: bioRxiv

Article Title: Nonredundant roles of the HDAC3 corepressor complex subunits SMRT and NCOR in controlling inflammatory and metabolic macrophage pathways

doi: 10.1101/2025.05.13.653058

Figure Lengend Snippet: Depletion of NCOR vs. SMRT differentially reprograms macrophage activation in response to multiple signals. (A-C) IGV genome browser tracks displaying H3K27ac increase in Pdcd1 , Arg1 , and Abca1 loci in response to LPS, IL4 or GW3965 treatment. Specific target epigenome regions are highlighted for each treatment. (D-F) Integration of CUT&Tag data for NCOR vs. SMRT depletion in LPS (D) , IL4 (E) or GW3965 (F) treated macrophages. The shNCOR-specific upregulated peaks are highlighted in orange and the shSMRT-specific upregulated peaks are highlighted in blue. (G-I) RT-qPCR analysis of related gene expression in LPS (G) , IL4 (H) and GW3965 (I) treatment in NCOR- vs. SMRT- depleted cells ( n =3). Unpaired t test was used to determine data significance for gene expression. All data were represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: We designed lentiviral shRNA sequences targeting NCOR ( Ncor1 ) (clone IDs: shNCOR-1 TRCN0000350169, shNCOR-2 TRCN0000310755) and SMRT ( Ncor2 ) (clone IDs: shSMRT-1 TRCN0000238140, shSMRT-2 TRCN0000238139) using the GPP Web Portal (Broad Institute).

Techniques: Activation Assay, Quantitative RT-PCR, Gene Expression

Journal: Nature neuroscience

Article Title: NCOR1/2 loss of function impairs memory through a novel GABAergic hypothalamus–CA3 projection

doi: 10.1038/s41593-018-0311-1

Figure Lengend Snippet: . (a) Patients carrying copy number variants (CNV) or single nucleotide variants (SNV) in NCOR1 , NCOR2 , or HDAC3 . Genomic coordinates are shown in hg19. DDD_SNV, single nucleotide variants retrieved from Deciphering Developmental Disorders (DDD) website (United Kingdom); kb, kilobase. (b-d) Schematic representations for the deletions and point mutations affecting NCOR1 , NCOR2 , or HDAC3 , respectively, observed in patients with neurodevelopmental disorders. The locations of deletions are depicted in red, and the point mutations in pink. (e) Western blot of HEK-293 cells transfected with plasmids expressing wild-type (WT) HDAC3 with or without mutant L266S. The experiment was repeated independently once with similar results. The blot images have been cropped. (f) Fluorescence-based HDAC enzyme assay after anti-HDAC3 immunoprecipitates from cell lysates overexpressing the indicated HDAC3 proteins. Box plots center line, median; box limits, upper and lower quartiles; whiskers, minimal and maximum values. Data were analyzed by two-tailed unpaired t test. n=4 biological independent samples for each group. (g) Western blot of HEK-293 cells transfected with plasmids expressing WT HDAC3, WT NCOR1, with or without the NCOR1 deletion mutant (Del). The experiment was repeated independently once with similar results. Data were analyzed by two-tailed unpaired t test. n=3 biological independent samples for each group. The blot images have been cropped. (h) Chromatin immunoprecipitation (ChIP) with anti-HDAC3 antibodies followed by qPCR using primers targeting promoters of the indicated genes ARNTL and CDKN1A . RPLP0 serves as a negative control. Data is expressed as mean ± S.E.M. For detailed statistics results, see . * P ≤ 0.05 is set as significance.

Article Snippet: Human full-length cDNA for NCOR1 was obtained from transOMIC and was sub-cloned into the pcDNA3.1-Zeo+ plasmid with C-terminal Flag-tag using standard PCR method for both WT and the 1-666 truncation. pcDNA3-based Flag-tagged HDAC3 cDNA construct was described before and the L266S point mutation was introduced using standard PCR method.

Techniques: Western Blot, Transfection, Expressing, Mutagenesis, Fluorescence, Enzymatic Assay, Two Tailed Test, Chromatin Immunoprecipitation, Negative Control

Journal: eLife

Article Title: Unanticipated mechanisms of covalent inhibitor and synthetic ligand cobinding to PPARγ

doi: 10.7554/eLife.99782

Figure Lengend Snippet: The active LBD (PDB 6ONJ) is stabilized by agonist (rosiglitazone) and coactivator peptide (TRAP220/MED1), whereas the repressive LBD (PDB 6ONI) is stabilized by covalent inverse agonist (T0070907) and corepressor peptide (NCoR1).

Article Snippet: Peptide, recombinant protein , NCoR1 , LifeTein , synthesized , residues 2256–2,278 (DPASNLGLEDIIRKALMGSFDDK) synthesized with or without a N-terminal FITC label with a six-carbon linker (Ahx) and an amidated C-terminus for stability.

Techniques:

Journal: eLife

Article Title: Unanticipated mechanisms of covalent inhibitor and synthetic ligand cobinding to PPARγ

doi: 10.7554/eLife.99782

Figure Lengend Snippet: ( A ) TR-FRET coregulator interaction assays performed using PPARγ LBD protein with or without preincubation of GW9662 or T0070907 to determine how the non-covalent synthetic ligands influence recruitment of peptides derived from NCoR1 corepressor protein and TRAP220/MED1 coactivator protein fit to a sigmoidal dose response equation or biphasic dose response equation for select cases where a biphasic response is observed (n=3; mean ± s.d.). ( B ) IC 50 and EC 50 values extracted from the TR-FRET coregulator interaction data. For curves showing a biphasic response, the higher affinity value is displayed; no value is displayed in cases where the dose response is flat. Error bars when present represent the fitted errors; some fits did not converge to a well-fitted error. See . Figure 3—source data 1. TR-FRET coregulator interaction assay data.

Article Snippet: Peptide, recombinant protein , NCoR1 , LifeTein , synthesized , residues 2256–2,278 (DPASNLGLEDIIRKALMGSFDDK) synthesized with or without a N-terminal FITC label with a six-carbon linker (Ahx) and an amidated C-terminus for stability.

Techniques: Derivative Assay

Journal: eLife

Article Title: Unanticipated mechanisms of covalent inhibitor and synthetic ligand cobinding to PPARγ

doi: 10.7554/eLife.99782

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , NCoR1 , LifeTein , synthesized , residues 2256–2,278 (DPASNLGLEDIIRKALMGSFDDK) synthesized with or without a N-terminal FITC label with a six-carbon linker (Ahx) and an amidated C-terminus for stability.

Techniques: Sequencing, Recombinant, Expressing, Plasmid Preparation, Synthesized, Software

Journal: Nature Communications

Article Title: Defining a conformational ensemble that directs activation of PPARγ

doi: 10.1038/s41467-018-04176-x

Figure Lengend Snippet: Nuclear receptor co-regulator interaction differentiates pharmacologically distinct PPARγ ligands. TR-FRET biochemical assay shows the effect of the compounds on the interaction between PPARγ LBD and peptides derived from the a MED1 coactivator and b NCoR corepressor, plotted as TR-FRET ratio (665 nm/620 nm) vs. ligand concentration ( n = 2, standard deviation). The data shown represents technical replicates from a single experiment and the experiment was repeated four times with similar results. The window of efficacy in these data is representative of ligand-induced changes in co-regulator affinity for PPARγ. An increase in TR-FRET ratio indicates a strengthening of affinity for MED1/NCoR compared to apo while a decrease indicates a ligand-induced weakening of affinity for the co-regulator. The effect of vehicle (DMSO) is negligible (Supplementary Fig. ); furthermore, the DMSO concentration is constant across the titration both in this figure and all other TR-FRET data presented

Article Snippet: Peptides were synthesized by Lifetein LLC (Somerset, NJ, USA) for the for MED1 peptide, sequence: NTKNHPMLMNLLKDNPAQD; and the NCoR peptide, sequence: GHSFADPASNLGLEDIIRKALMG (2251–2273).

Techniques: Derivative Assay, Concentration Assay, Standard Deviation, Titration

Journal: Nature Communications

Article Title: Defining a conformational ensemble that directs activation of PPARγ

doi: 10.1038/s41467-018-04176-x

Figure Lengend Snippet: Ligand-directed helix 12 ensemble dictates PPARγ–co-regulator interaction. a Plot of mean 19 F NMR chemical shift values (PPARγ K502C -BTFA) vs. TR-FRET endpoint data for the recruitment of MED1, NCoR, and CBP peptides to PPARγ K502C -BTFA (top panels) and wt PPARγ LBD (bottom panels) for the set of 16 pharmacologically distinct synthetic PPARγ ligands and apoprotein (select ligands for CBP). Error bars are relatively small for endpoint TR-FRET values (Fig. and Supplementary Figure ) and are excluded for clarity. b Plot of mean 19 F NMR chemical shift values (PPARγ K502C -BTFA) vs. MED1, NCoR, and SMRT peptide dissociation constant ( K d ) for PPARγ K502C -BTFA (top panels) and wt PPARγ LBD (bottom panels) as measured by fluorescence polarization for a subset of the ligands in a . Linear regression fit is shown as a solid line and the correlation coefficient ( R 2 ) for the fitted line is indicated. The ligands with the highest efficacy for MED1 and NCoR recruitment for PPARγ K502C -BTFA are highlighted. Error bars represent standard deviation of two (K502C-BTFA K d and wt SMRT K d ) or three (wt MED1 and NCoR K d ) independent experiments

Article Snippet: Peptides were synthesized by Lifetein LLC (Somerset, NJ, USA) for the for MED1 peptide, sequence: NTKNHPMLMNLLKDNPAQD; and the NCoR peptide, sequence: GHSFADPASNLGLEDIIRKALMG (2251–2273).

Techniques: Fluorescence, Standard Deviation

Journal: Nature Communications

Article Title: Defining a conformational ensemble that directs activation of PPARγ

doi: 10.1038/s41467-018-04176-x

Figure Lengend Snippet: Co-regulator-binding shifts the helix 12 conformational ensemble. a Deconvoluted 19 F NMR spectra of apo PPARγ C313A,K502C -BTFA and PPARγ K502C -BTFA bound to GW1929 or T0070907 in the absence and presence of MED1 coactivator or NCoR corepressor peptides. The percent of total signal area found in the left sharp peak is shown for T0070907 and GW9662 spectra. The small sharp peak at ~−83.3 ppm is free BTFA. b Mean-weighted chemical shift values from the plots in a . c Fraction of the total peak areas in the four colored boxed regions in a , which roughly correspond to the clustered spectral regions from Fig. ; with the two middle regions corresponding to the two peaks observed for apoprotein. Agonist-bound PPARγ is changed little by MED1 binding, whereas NCoR binding changes the spectrum drastically and vice versa for inverse-agonist (T0070907)-bound PPARγ. *SMRT-induced mean chemical shift in GW1929-bound PPARγ K502C -BTFA is the same as NCoR. These experiments were performed once

Article Snippet: Peptides were synthesized by Lifetein LLC (Somerset, NJ, USA) for the for MED1 peptide, sequence: NTKNHPMLMNLLKDNPAQD; and the NCoR peptide, sequence: GHSFADPASNLGLEDIIRKALMG (2251–2273).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Defining a conformational ensemble that directs activation of PPARγ

doi: 10.1038/s41467-018-04176-x

Figure Lengend Snippet: Heterodimerization of PPARγ LBD with RXRα LBD favors coactivator binding. a 19 F NMR of PPARγ K502C -BTFA bound to T0070907 and PPARγ C313A,502C -BTFA bound to MRL24 or GW1929 or with no ligand bound was performed in the presence (orange) or absence (black) of RXRα LBD. These experiments were performed once. d The 19 F NMR signal from the MRL24 ligand. Broadening and consequent reduction in signal intensity is expected as a consequence of the increased rotational correlation time of the heterodimer complex. b , c TR-FRET was used to measure interaction between wt PPARγ LBD and MED1 or NCoR in the presence or absence of equimolar concentrations of RXRα. Error bars represent standard deviation of two technical replicates within a single experiment. The experiment was repeated twice and gave similar results each time. e Heterodimerization favors MED1 binding ( p = 0.0017) and disfavors NCoR binding ( p = 0.0076) to apo PPARγ. In addition, visual and statistical comparison of NCoR and MED1 recruitment to PPARγ LBD saturated with ligand (four highest concentrations of ligands) indicates that RXRα affects co-regulator recruitment to T0070907-bound PPARγ (NCoR, p = 0.0042; MED1, p = 0.0027) more than GW1929 (NCoR, p = 0.44; MED1, p = 0.34) or MRL24 (NCoR, p = 0.0141; MED1, p = 0.061). All p values are derived from a two-tailed t test

Article Snippet: Peptides were synthesized by Lifetein LLC (Somerset, NJ, USA) for the for MED1 peptide, sequence: NTKNHPMLMNLLKDNPAQD; and the NCoR peptide, sequence: GHSFADPASNLGLEDIIRKALMG (2251–2273).

Techniques: Binding Assay, Standard Deviation, Comparison, Derivative Assay, Two Tailed Test