Fig 2: SP140 preferentially controls the expression of specific gene sets involved in the innate immune response. a Heatmap of the top 100 DEGs and b volcano plots of the all genes comparing 0.1% DMSO- with 0.04 µM GSK761-treated “M1” macrophages after 4 h of 100 ng/mL LPS stimulation. c R2 TSS plot comparing a global differentially SP140-bound genes (DBGs) after 1 h of 100 ng/mL LPS stimulation of 0.1% DMSO- and 0.04 µM GSK761-treated “M1” macrophages. d SP140 ChIP-seq genome browser view of some of the most affected DBGs; TNF, TRAF1, IRF1, and TRAFAIP2. Y axis represents a signal score of recovered sequences in 0.1% DMSO and 0.04 µM GSK761 treated macrophages after 0, 1, and 4 h of 100 ng/mL LPS stimulation. e RNA sequencing-derived gene expression of some of the most DBGs. f Comparative analyses of the top 1000 DBGs (signal) with their gene expression (Wald statistic). Gene expression at 4 h and ChIP at 1 h (left) and gene expression at 4 h and ChIP at 4 h (right), Y axis represents the SP140 differential binding signal (BD). g Homer Known Motif Enrichment using TF motifs and their respective p-value scoring (top) and Homer de novo Motif results with best match TFs (bottom) in 0.1% DMSO-treated “M1” macrophages after 1 h of 100 ng/mL LPS stimulation. h An enrichment analysis targeted chemokine activity for SP140 ChIP-seq (top) and RNA-seq (bottom) comparing 0.1% DMSO- with 0.04 µM GSK761-treated “M1” macrophages at 0, 1, and 4 h of 100 ng/mL LPS stimulation

Fig 3: SP140 expression associates with inflammatory diseases and mucosal macrophages of CD patients. a SP140 gene expression in human tissues and cell types as indicated. Expression is given as count normalized with MAS5.0. b SP140 gene expression in white blood cells of normal healthy controls (N) (n=33), Crohn’s disease (CD) (n=6), ulcerative colitis (UC) (n=6), systemic lupus erythematosus (SLE) (n=64), chronic lymphocytic leukemia (CCL) (n=21), acute myeloid leukemia (AML) (n=7), and rheumatoid arthritis (RA) (n=32) patients. c SP140 gene expression in human colon tissue obtained from N (n=18), non-inflamed and inflamed CD (n=5 and 13, respectively), and UC (n=3 and 17, respectively) colonic tissues. Data was collected from in-house GSK microarray profiler. Expression is given as count normalized with MAS5.0. d Immunohistochemistry of SP140 protein in colon tissue obtained from N and inflamed CD and inflamed UC tissue, scale bar: 100 µm. e Immunofluorescence staining of DAPI (blue), CD68 (green), and SP140 (red) in N or inflamed CD colon tissue, scale bar: 100 µm. f The average of mucosal cell count per 3 visual fields of total CD68+ macrophages, SP140+ CD68+ macrophages and SP140- CD68+ macrophages in N and inflamed CD tissue (n = 3 patients per group). g Immunofluorescence staining of DAPI (blue), HLA-DR (green), and SP140 (red) in uninflamed or inflamed CD colon tissue, scale bar: 100 µm. h The average of mucosal cell count of 3 visual fields per tissue of total HLA-DR+ macrophages, SP140+ HLA-DR+ macrophages, and SP140- HLA-DR+ macrophages in N and inflamed CD tissue (n=3 patients per group). Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ****P < 0.0001

Fig 4: LPS stimulation leads to SP140 protein recruitment to chromatin; GSK761 reduces this recruitment and dampens inflammatory pathways in inflammatory macrophages. a Nuclear extracts from aCD3/aCD28 stimulated HuT78 cells were incubated with unmodified or modified (acetylated and methylated) histone H3 peptides as indicated. SP140 was then pulled down to visualize its interaction with H3 peptides. b ChIP-qPCR of SP140 occupancy at TSS of TNF and IL6 in “M1” macrophages stimulated with 100 ng/mL LPS for 4 h or without LPS stimulation, n=3 donors (DN). c Epigenome roadmap scan illustrating proportions of SP140 genome-wide occupancy after SP140 ChIP-seq. d Heatmap (1000 top SP140-bound genes) of SP140 ChIP-seq reads ranked on 1 h LPS-stimulated (left) or 4 h LPS-stimulated (right) “M1” macrophages rank-ordered from high to low occupancy centered on TSS. Top 20 genes with high SP140 occupancy are listed. e ChIP-qPCR of SP140 occupancy at TSS of TNF in “M1” macrophages pretreated with 0.1% DMSO or 0.04 µM GSK761 for 1 h and then stimulated with 100 ng/mL LPS for 1 or 4 h or kept unstimulated (0 h LPS). f Metagene created from normalized genome-wide average reads for SP140 centered on TSS. g PCA of RNA-seq comparing 0.1% DMSO to 0.04 µM GSK761 treated “M1” macrophages after 4 h of LPS stimulation (left) or after 8 h of LPS stimulation (right). h SP140 ChIP-seq gene ontology analysis of the most enriched molecular function and biological process after 1 h of LPS stimulation, comparing 0.1% DMSO with 0.04 µM GSK761 treated “M1” macrophages. i Hallmarks pathway enrichment analysis at 0, 1, and 4 h of 100 ng/mL LPS stimulation for ChIP-seq and at j 0, 4, and 8 h of 100 ng/mL LPS stimulation for RNA-seq. j The direction and color of the arrow indicate the direction and size of the enrichment score, the size of the arrow is proportional to the -log10 (p-value), and non-transparent arrows represent significantly affected pathways

Fig 5: SP140 knockdown reduces the activity of the inflammatory macrophages. a Scheme of polarization protocol of human CD14+ monocytes to “M0,” “M1,” and “M2” macrophage phenotypes, with indicated cytokines as described in the “Methods” section. b Relative gene expression of SP140 in “M0,” “M1,” and “M2” macrophages, n=6. c Immunofluorescence staining of SP140 speckles in “M1” and “M2” macrophages imaged by microscopy (left) and quantified per nuclei as nuclear bodies count (right). Images were counted in 150 cells selected randomly from 3 different staining per condition (Total number of SP140 speckled in 150 cells/150 = average per cell), scale bar: 30 µm. d Scheme of SP140 silencing protocol as described in “Methods” section. e The efficiency of SP140 silencing was assessed by measuring relative gene expression (qPCR) of SP140 (n=6) and f immunofluorescence staining of SP140 speckles nuclear bodies (left) and SP140 speckled nuclear bodies count (counted as described above) (right), scale bar: 3 µm. g Relative gene expression after LPS stimulation: (top, n=6), and LPS-induced protein levels (bottom, n=3) n=3, of TNF, IL-6, and IL-8. h PCA of gene expression dataset of LPS induced genes in scrambled- or SP140 siRNA-treated “M1” macrophages (unstimulated or stimulated with 4 h 100 ng/mL LPS) assessed through microarray; PC1 represents most variance associated with the data (LPS stimulation) and PC2 represents second most variance (siRNA), n=3. i Heatmap of top 50 DEGs from microarray gene expression dataset of LPS induced genes in scrambled- or SP140 siRNA-treated “M1” macrophages (unstimulated or stimulated with 4 h 100 ng/mL LPS) (non-annotated genes were not included). j Hallmark pathways analysis and k Reactome pathway analysis were carried out using ShinyGO v0.60 illustrating the most impacted pathways by SP140 siRNA in unstimulated “M1” macrophages (bottom) or after 4 h of 100 ng/mL LPS stimulation (bottom). In all assays, statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001