Fig 2: GPS2 restricts chromatin accessibility at IL4 target gene loci. (A) Volcano plot showing log2-fold changes of ATAC-seq peaks between control and IL4-treated RAW cells. The significantly up-regulated or down-regulated peaks are highlighted in blue. Peaks at marker gene loci are highlighted in red (n = 3). (B) IGV genome browser tracks of ATAC-seq at the Ptgs1 locus in control and IL4-treated RAW cells. (C) Peak coverage representing genome-wide ATAC-seq changes in WT versus Gps2 KO RAW cells under basal condition (n = 3). (D–F) Volcano plot showing log2-fold changes of ATAC-seq peaks between depletion of GPS2 (D), SMRT (E) and NCOR (F) vs. control cells. The significantly up-regulated or down-regulated peaks are highlighted in blue. Peaks at marker gene loci are highlighted in red. (G) Motif analysis of the significantly up- and down-regulated ATAC-seq peaks in Gps2 KO versus WT RAW cells. (H-I) IGV genome browser tracks representing the ATAC-seq peaks in WT and Gps2 KO RAW cells at the Ptgs1 (H) and Flt1 (I) loci. Enhancers and promoters are highlighted. (J, K) IGV genome browser tracks representing the H3K27ac mark and Pol II recruitment at the Ptgs1 and Flt1 loci in WT and Gps2 KO cells under basal condition versus IL4 treatment. Unpaired t-test was used to determine data significance. All data are represented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: (A) IGV genome browser tracks representing the GPS2, NCOR, SMRT and H3K27ac ChIP-seq peaks at the Ptgs1 locus in both BMDMs and RAW cells. Enhancer (E1, E2) and promoter (P) regions are highlighted. (B–D) RT-qPCR showing the mRNA expression of Ptgs1 in lentivirus shRNA-mediated GPS2, SMRT and NCOR knockdown RAW cells (n = 3). Unpaired t test was used to determine data significance. (E, F) IGV genome browser tracks representing the H3K27ac ChIP-seq peaks at the Ptgs1 and Flt1 loci in control (shGFP) and GPS2, SMRT and NCOR knockdown cells (n = 2). The peak changes within enhancer (E1, E2) and promoter (P) regions are highlighted in percentage. (G–I) MA plot showing the significant H3K27ac peak changes in shGPS2 (G), shSMRT (H) and shNCOR (I) RAW cells under basal conditions (n = 2). Significantly up-regulated and down-regulated peaks are highlighted in purple or blue. Data were re-analyzed from GSM4848501, GSM4848502, GSM4848505, GSM4848506. (J) IGV genome browser tracks representing the GPS2 ChIP-seq peaks at the Ptgs1 locus in NCOR and SMRT knockdown cells (n = 2). (K) RT-qPCR analysis of Ptgs1 expression upon GPS2/NCOR or GPS2/SMRT double depletion (n = 3). One-way ANOVA test was used for multiple comparisons. All data are represented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: (A) Venn diagram showing the overlap of GPS2, STAT6, and KDM1A occupancy (obtained by ChIP-seq) in RAW cells (left panel), along with the enriched TF motifs in the common peaks. (B) RNA-seq tag counts (-rpkm) representing the relative TF gene expression in RAW cells upon 6 h IL4 treatment versus control. (C) Co-immunoprecipitation of HA-GPS2 with the indicated FLAG-tagged TFs and coregulators in HEK293 cells. Previously reported interactions with JUN, NCOR and SMRT served as positive controls (36) (D) Co-immunoprecipitation of HA-KDM1A with the indicated FLAG-tagged TFs and coregulators in HEK293 cells. Previously reported interaction with p65 served as positive control (74). (E, F) ChIP-qPCR of GPS2 and KDM1A at Ptgs1 enhancer (E1) and promoter region (Pro) upon PU.1 inhibition (PU.1i) in RAW cells (n = 3). (G) Model of GPS2-KDM1A antagonism in regulating IL4 target gene expression during M2 macrophage activation. Unpaired t test was used to determine data significance. All data are represented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: Depletion of NCOR versus 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. Integration of CUT&Tag data for NCOR versus 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. RT-qPCR analysis of related gene expression in LPS (G), IL4 (H), and GW3965 (I) treatment in NCOR- versus SMRT-depleted cells (n= 3). One-way ANOVA was used to determine data significance for gene expression. All data were represented as mean ± SEM. *P < .05, **P < .01, ***P< .001, ****P< .0001.