Modulation of macrophage inflammatory function through selective inhibition of the epigenetic reader protein SP140

SP140 is a bromodomain-containing protein expressed predominantly in immune cells. Genetic polymorphisms and epigenetic modifications in the SP140 locus have been linked to Crohn’s disease (CD), suggesting a role in inflammation. We report the development of the first small molecule SP140 inhibitor (GSK761) and utilize this to elucidate SP140 function in macrophages. We show that SP140 is highly expressed in CD mucosal macrophages and in in vitro-generated inflammatory macrophages. SP140 inhibition through GSK761 reduced monocyte-to-inflammatory macrophage differentiation and lipopolysaccharide (LPS)-induced inflammatory activation, while inducing the generation of CD206+ regulatory macrophages that were shown to associate with a therapeutic response to anti-TNF in CD patients. SP140 preferentially occupies transcriptional start sites in inflammatory macrophages, with enrichment at gene loci encoding pro-inflammatory cytokines/chemokines and inflammatory pathways. GSK761 specifically reduces SP140 chromatin binding and thereby expression of SP140-regulated genes. GSK761 inhibits the expression of cytokines, including TNF, by CD14+ macrophages isolated from CD intestinal mucosa. This study identifies SP140 as a druggable epigenetic therapeutic target for CD.

macrophages to take on different functional properties in response to environmental stimuli. Epigenetic processes play an important role in the regulation of macrophage polarization/differentiation and gene expression, mediated by DNA methylation and histone post-translational modification [7,8]. As such, epigenetic mechanisms mediated through the activity of specific histone acetylases and deacetylases activity are implicated in CD pathogenesis [9][10][11].
Speckled 140 kDa (SP140) is a nuclear body protein [12,13] predominantly expressed in immune cells [14,15]. Genome-wide association studies identified an association between single-nucleotide polymorphisms in SP140 and CD [16] while rare SP140-associated variants have been identified in pediatric CD patients [17]. Further, we identified two differentially hyper-methylated positions at the SP140 locus in blood cells of CD patients [18], suggesting aberrant regulation of its expression. Although little is known about how SP140 may be functionally linked to CD, a recent study reported that SP140 knockdown in in vitro-generated inflammatory macrophages results a decrease in levels of pro-inflammatory cytokines [10]. Lower expression of SP140 in intestinal biopsies was correlated with an improved response to anti-TNF therapy in CD patients [10]. Conversely, in biopsies from those who showed resistance to anti-TNF therapy, SP140 was highly expressed [10]. Together, these previous findings suggest a role for SP140 in CD pathogenesis.
SP140 protein harbors both a bromodomain (Brd) and a plant homeodomain (PHD) finger [10,15] as illustrated in Fig. S1a. Since Brd and PHD domains are capable of binding to histones, this suggests that SP140 might function as an epigenetic reader. Readers are recruited to chromatin possessing specific epigenetic "marks" and regulate gene expression through mechanisms that modulate DNA accessibility to transcription factors (TFs) and the transcriptional machinery [19,20]. Brds are tractable to small molecule antagonists, with selective inhibitors of several Brd-containing proteins (BCPs) reported [20,21]. Such compounds have helped elucidate the function of their BCP targets; for instance, multiple inhibitors targeting BRD2, BRD3, and BRD4 have been used to demonstrate the role of these proteins in selectively regulating gene expression in the context of cancer and inflammation [9,22,23]. To date, no inhibitors targeting SP140 have been reported.
Here, we describe the discovery of the first specific SP140 inhibitor and utilize this compound to investigate the functional relevance of SP140 in CD pathogenesis, focusing on its role in macrophages. We show that SP140 functions as an epigenetic reader in controlling the generation of an inflammatory macrophage phenotype, and in regulating the expression of pro-inflammatory and CD-associated genes. We show that GSK761 inhibits SP140 binding to an array of inflammatory genes and demonstrate its efficacy in CD colon macrophages ex vivo, presenting evidence for SP140 as a target for regulating inflammation in CD. In addition, this study suggests that SP140 inhibition may also serve to support anti-TNF therapy in non-responder CD patients.

Elevated SP140 expression in inflammatory diseases and mucosal macrophages of CD patients
SP140 gene expression in a variety of cells and tissues was analyzed using an in-house GSK microarray profiler. SP140 was predominantly expressed in blood, CD8 + and CD4 + T cells, monocytes, and secondary immune organs such as lymph node and spleen (Fig. 1a). We then investigated SP140 expression in white blood cells (WBC) and intestinal tissue of IBD patients versus controls. While SP140 gene expression in WBC was comparable in IBD and healthy controls (Fig. 1b), SP140 gene and protein expression were found to be increased in inflamed colonic mucosa of both CD and UC patients (Fig. 1c, d). Notably, an increase in CD68 + SP140 + and HLA-DR + SP140 + but not CD68 + SP140 − or HLA-DR + SP140 − cells was observed in CD inflamed colonic mucosa compared to normal control mucosa or uninflamed tissue, respectively ( Fig. 1e-h). Elevated SP140 expression was also found , 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 in other inflammatory conditions including in WBC of systemic lupus erythematosus and rheumatoid arthritis patients (Fig. 1b), and in inflamed tissues of appendicitis, sarcoidosis, psoriatic arthritis, rheumatoid arthritis, Hashimoto's thyroiditis and Sjogren's syndrome patients (Additional file 1: Fig. S1b). High expression of SP140 was also observed in chronic lymphocytic leukemia (Fig. 1b). Next, we analyzed a publicly available single-cell RNA sequencing experiment [24] of inflamed and uninflamed ileal tissue (biopsies) obtained from CD patients. Unsupervised clustering analysis identified 22 cluster blocks (Additional file 1: Figs. S2a and b). Based on different cell markers, we identified cell types existing in each cluster (Fig. 2a, b). Expectedly, SP140 expression was predominantly observed in the immune cell compartment (Fig. 2c). Based on the expression of CD14, CD16 (FCGR3A), CD1C, CD64 (FCGR1A), CD68, CD163, CD206 (MRC1), HLA-DR, and CLEC4A ( Fig. 2a and Additional file 1: Fig. S2c), we identified cluster 6 as containing monocytes, macrophages, and dendritic cells (Additional file 1: Fig. S2c and Fig. 2b), altogether representing mononuclear phagocytes (MNP). As per the observations made by Martin et al. [24], the abundance of the MNPs was significantly higher in inflamed ileal biopsies (Additional file 1: Fig. S2d), which accordingly manifested as an increased percentage of SP140-expressing MNPs in inflamed ileal biopsies (Fig. 2d, e). Interestingly SP140 expression did not differ significantly in other immune cells (including T and B cells) between inflamed and uninflamed tissues (Fig. 2e). Through subclassification analysis of the MNPs, we mapped the monocytes (CD14 and FCGR3A) along their developmental trajectory towards macrophages (CD68, CD163, and CD206) or dendritic cells (CD1C and CLEC4A) (Additional file 1: Fig. S2e). Projecting the expression of SP140 along the inferred trajectory suggests significant association with the developmental process (Additional file 1: Fig. S2f ).

The development of a selective inhibitor of SP140 (GSK761)
To identify selective SP140 binding compounds, we utilized encoded library technology to screen the GSK proprietary collection of DNA-linked small molecule libraries [31,32]. Affinity selection utilizing a recombinant protein construct spanning the PHD and Brd domains of SP140 was carried out, leading to the identification of an enriched building block combination in DNA Encoded Library 68 (DEL68) (Fig. 4a). Representative compounds of the identified three-cycle benzimidazole chemical series were synthesized, which yielded the small molecule, GSK761 (Fig. 4b, c). More details describing GSK761 development are added in the Fig. 2 Single-cell analyses of SP140 expression in inflamed and uninflamed CD ileum. Publicly available single-cell RNA sequencing [24] was used to illustrate SP140 expression in intestinal (ileum) macrophages in inflamed (n=11) and uninflamed (n=11) tissue CD patients. a Marker genes expression per cell type where the size of the dots represents the percentage of cells expressing the gene. Darker blue represents more reads per cell. b UMAP of all the cells annotated with their annotated cell type. c Visual illustration of SP140 expression in all cells. Darker blue represents more reads per cell. d Visual illustration of the actual counts of SP140 expression in cluster 6 (MNPs). Darker blue represents more reads per cell. e Comparative analyses of the percentage cells expressing SP140 when comparing inflamed with uninflamed in different immune cells "Methods" section. To quantify the interaction between GSK761 and SP140 (aa 687-867), the dissociation constant (K d ) was determined using a fluorescence polarization (FP)-binding approach. A fluorophore-conjugated version of GSK761, GSK064 (Fig. 4d), was prepared and subsequently used to determine a K d value of 41.07 ± 1.42 nM for the interaction with SP140 (aa 687-867) (Fig. 4e). To further validate this interaction, competition studies were carried out using GSK761 in a FP-binding assay configured using GSK064 and SP140 (aa 687-867). Competitive displacement of GSK064 from SP140 (aa 687-867) by GSK761 was observed, subsequently leading to the determination of an IC 50 value of 77.79 ± 8.27 nM (Fig. 4f ). Prior to utilizing GSK761 for cell-based binding studies to endogenous SP140, the cell penetration capacity of the compound was assessed using mass spectrometry and the methodology described by [33]. Concentration measurements showed that GSK761 had a pΔC total value of 1.45 ± 0.21, indicating that GSK761 permeates human cells and accumulates intracellularly by an order of magnitude when compared to GSK761 free in solution.
To confirm the binding of GSK761 to full-length SP140, immobilized GSK761 was used to probe for SP140 in nuclear extracts from HuT78 cells and HEK293 cells transfected with Halo-tagged SP140. Biotinylated beads only and untransfected HEK293 cells were utilized as controls. Both endogenous and Halo-tagged SP140 were pulled down with biotinylated GSK761 and visualized via Western Blotting (Fig. 4g). Endogenous SP140 is observed as a doublet containing the four largest isoforms (predicted 86-96 kDa) [10] while Halo-tagged SP140 exhibits a single band. No bands were detected with beads only or in untransfected HEK293 cells. The specificity of GSK761 for SP140 was profiled using the BROMOscan ® assay, in which DNA-tagged BCPs were incubated with an increasing concentration of GSK761 or DMSO. Binding is assessed by measuring the amount of bromodomain captured in GSK761 vs DMSO samples by using an ultra-sensitive qPCR. There was evidence of low affinity interaction between GSK761 and several tested BCPs, with binding detected at concentrations >21,000 nM (Additional file 4: Table S4). However, no binding was detected at concentrations ≤ 21,000 nM indicating a high degree of specificity of GSK761 for SP140 (Additional file 4: Table S4).

GSK761 reduces the inflammatory activation of macrophages and expression of pro-inflammatory cytokines
SP140 expression was selectively increased in "M1" compared to "M0" macrophages, prompting us to test whether SP140 is required for the polarization to an inflammatory macrophage phenotype. We first tested whether GSK761 demonstrated any toxicity in macrophages to determine the appropriate dose range. At ≤0.12 μM, GSK761 showed no cytotoxicity (Additional file 1: Figs. S5a and b). To investigate the effect of GSK761 on macrophage polarization to inflammatory phenotype, "M0" macrophages were treated with either DMSO or GSK761 for 3 days in presence of IFN-γ or IL-4 (during the polarization to "M1" and "M2" macrophages, respectively). GSK761 enhanced mRNA expression of the anti-inflammatory marker CD206 in both "M1" and "M2" macrophages and decreased the pro-inflammatory marker CD64 in "M1" macrophages ( Fig. 5a). FACS analysis showed that GSK761-treatment prior to IFN-γ ("M1") polarization reduced CD64 + cells and CD64 protein expression (Fig. 5b, c) and increased CD206 + cells and CD206 protein expression (Fig. 5d, e). Adding GSK761 to the cells during IFN-γ ("M1") polarization lowered TNF ("M1" polarization marker) gene expression in these cells compared to the control (Additional file 1: Fig. S5d). Altogether, these data suggest that SP140 inhibition during "M1" polarization biased differentiation towards a regulatory "M2" phenotype. Fig. 3 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 We next assessed the effect of SP140 inhibition on the response of "M1" polarized macrophages to inflammatory stimuli. LPS-stimulated "M1" macrophages pretreated with GSK761 showed a strong reduction in secretion of IL-6, TNF, IL-1β, and IL-12 ( Fig. 5g) at concentrations where cytotoxicity was not observed (0.04 and 0.12 μM). When employing RNA transcriptional profiling using a customized qPCR array, SP140 inhibition was found to reduce the expression of many other pro-inflammatory cytokines and chemokines including GM-CSF, CCL3, CCL5, and CCL1 (Fig. 5f ).
The marked anti-inflammatory effects of GSK761 on macrophages in vitro suggest that targeting SP140 may be an effective approach for IBD. Unfortunately, due to poor in vivo pharmokinetics (data not shown), GSK761 was not suitable to evaluate the effects of SP140 inhibition in in vivo animal models of colitis. To evaluate the impact of SP140 inhibition in human CD, CD14 + mucosal macrophages were isolated from CD anti-TNF refractory patients' colonic mucosa and then cultured in vitro with either DMSO or GSK761 for 4 h. Spontaneous gene expression of TNF, IL6, and IL10 was significantly decreased in GSK761-treated tissue macrophages, demonstrating that GSK761 inhibited their immune reactivity (Fig. 5h). No changes in CD64 expression were observed. Previous studies have demonstrated that successful anti-TNF therapy is associated with an increase in CD206 + regulatory macrophages [34] and a decrease in pro-inflammatory cytokines [10] and SP140 expression [10] in CD mucosa. Thus, our data suggests that SP140 inhibition could be also beneficial in supporting the anti-TNF therapy. Thus, combining SP140 inhibition with anti-TNF may potentiate the anti-inflammatory effects in macrophages and possibly in IBD in general. Consistent with this idea, M1 macrophages treated in vitro with both GSK761 and Infliximab (anti-TNF) demonstrated an additive reduction in TNF release in response to LPS compared to those treated with GSK761 or infliximab alone (Additional file 1: Fig. S5e).
To measure histone peptide binding in a more quantitative way, and to evaluate peptides for which binding is specifically dependent on the SP140 binding site of GSK761, we tested the ability of a range of peptides to compete for compound binding to SP140 PHD-Brd. We found that recombinant SP140 PHD-Brd protein bound to histone H3 peptides with sub-μM affinity (Additional file 1: Fig. S6b). The strongest binding was observed for an unmodified peptide corresponding to the N-terminal 21 aa, while methylation at lysine residue 4 (K4) led to substantially (~30-fold) reduced affinity (Additional file 1: Fig. S6c). K9 acetylation also resulted in lower affinity binding (4-5-fold), while SP140 binding was unaffected by either acetylation or methylation of K14 (Additional file 1: Fig. S6d). Similar binding affinity was observed for unmodified H3 1-21 vs H3 1-18 , while reduced (4-5-fold) but still significant SP140 binding was measured for H3 1-9 (Additional file 1: Fig. S6d). Taken together, these data suggest that the SP140 PHD-Brd module can function as a reader for unmodified histone H3, with binding (See figure on next page.) Fig. 4 The Development of the first small molecule inhibitor of SP140 (GSK761) and investigating its affinity and selectivity. a Scheme of the three-cycle benzimidazole library and b Spotfire cube view of the SP140 selection output from the benzimidazole library. BB1, cycle 1 building blocks; BB2, cycle 2 building blocks; and BB3, cycle 3 building blocks. Each individual dot in the cube represents discrete small molecule warheads after 3 rounds of affinity selection, while the size of the dot corresponds to the number of unique instances recorded by DNA sequencing . The dots are colored by the BB3s that compose the library molecules. Library members with a single copy were removed to simplify visualization revealing a prominent line defined by a specific BB1&BB3 combination (disynthon). The most enriched trisynthon (BB1, BB2, and BB3 combination) represents the biggest dot on the line. e Biochemical characterization of the interaction between c GSK761 and recombinant SP140 in a fluorescence polarization (FP) binding assay using d a fluorophore-conjugated version of GSK761: GSK064 (generated by fluorescent labelling of GSK761). The mean binding affinity for this interaction was a K d = 41.07 ± 1.42 nM (n = 5). f A FP-binding assay was configured using recombinant SP140 and GSK064, which was used to determine the potency of GSK761. Displacement of GSK064 from SP140 by GSK761 (circles) was achieved and determined to have a mean IC 50 of 77.79 ± 8.27 nM (n=3). No effect on GSK064 motion was observed in the presence of varying concentrations of GSK761 (triangles). The data presented in d and e are representative data from a single experimental replicate, affinities, and potencies are mean values determined from multiple test occasions. g Endogenous SP140 (HuT78 nuclear extracts) and Halo-tagged SP140 (transfected HEK29 cells) were pulled down using a biotinylated version of GSK761 (GSK675) and visualized by Western blotting and gel electrophoresis. Biotinylated beads only and SP140 untransfected cells were used as control Ghiboub et al. BMC Biology (2022) 20:182 focused around the N-terminal 9 aa, and that GSK761 competes for this binding.
To evaluate whether native SP140 is also capable of binding to histones, we used immobilized histone H3 peptides with varying modifications to precipitate proteins from nuclear extracts of anti-CD3/CD28-stimulated HuT78 T cells and probed via Western blotting for the presence of SP140. SP140 was efficiently pulled down by unmodified H3   (Fig. 6a). Notably, H3 1-21 peptides bearing certain methylation and acetylation marks (K4me3, K9me3, K9ac, and K14ac) were also capable of capturing native SP140 (Fig. 6a), despite the lower measured affinity of some of these for recombinant SP140 PHD-Brd (see above). To evaluate whether SP140 interacts with histone H3 in a cellular setting, we utilized a NanoBRET system (Additional file 1: Fig. S6e). Nano-BRET is a proximity-based assay that can detect protein interactions by measuring energy transfer from a bioluminescent protein donor NanoLuc ® (NL) to a fluorescent protein acceptor HaloTag ® (HT). This energy transfer was observed in the nuclei of HEK293 cells transfected with SP140-NL and Histone3.3-HT DNA, indicating close proximity of the two proteins (Additional file 1: Fig.  S6e).
To assess whether SP140 associates with chromatin in macrophages and how this might be regulated in inflammation, we initially conducted ChIP-qPCR experiments in unstimulated or LPS-stimulated "M1" macrophages. First, the specificity of the antibody used for ChIP experiments to precipitate SP140 protein was verified (Additional file 1: Fig. S7a). Binding of SP140 to the TSS of TNF and IL6 genes was observed in unstimulated cells, and this was increased following LPS stimulation (Fig. 6b). We then evaluated chromatin occupancy of SP140 on a genome-wide level using ChIP-seq and tested the effects of GSK761 on both SP140 binding and gene expression (RNA-seq) in the context of LPS stimulation. In DMSOtreated, LPS-stimulated macrophages, epigenome roadmap scan analysis revealed that the majority of SP140 occupancy was at strong transcription associated regions, enhancers, and TSS regions (Fig. 6c). However, this occupancy was decreased when the cells were pretreated with GSK761 at 1 h (Additional file 1: Fig. S7c) and 4 h (data not shown) post LPS stimulation. Heatmap rank ordering of SP140 occupancy in unstimulated and LPS-stimulated "M1" macrophages showed a strong SP140 enrichment at the TSS (Fig. 6d). The top 20 enriched genes belonged mostly to those involved in the innate immune response, including TNF, ICAM4, IRF1, LITAF, TNFAIP2, and NFKBIA (Fig. 6d). This was confirmed when Metagene analysis showed that SP140 preferentially binds near the TSS of immune innate genes, while this binding was minimal at the TSS of non-immune genes (Additional file 1: Fig. S7b). However, the most SP140-enriched gene FLOT1 (Fig. 6d) has been reported to be strongly involved in tumorigenesis [36] and anti-fungal immunity [37]. We found SP140 also to occupy active enhancers, as marked by H3K27Ac in human macrophages by overlapping the publicly available H3K27Ac ChIP-seq data (GSE54972) with our SP140 ChIP-seq dataset at 1 h of LPS stimulation (Additional file 1: Fig. S7d). ChIP-qPCR showed a strong reduction of SP140 occupancy at the TNF-TSS in GSK761-treated "M1" macrophages, which was reduced to that of unstimulated cells (Fig. 6e), correlating with the previously observed reduced expression of TNF in GSK761-treated macrophages.
Following LPS stimulation, we observed a marked increase in binding at the TSS of the top 1000 genes occupied by SP140, reaching its maximum at 4 h (Fig. 6f ). GSK761 treatment prior to LPS stimulation strongly reduced SP140 occupancy at the TSS (Fig. 6f ) associated with an altered LPS-induced gene expression (Fig. 6g). PCA of RNA-seq data revealed a large separation in global gene expression between DMSO-and GSK761treated macrophages at both time points post of LPS stimulation (4h and 8h) (Fig. 6g). Note that SP140 itself did not appear to be bound by SP140 protein, nor was its expression altered by GSK761 treatment (Additional Fig. 5 GSK761 affects macrophage polarization and cytokine production in peripheral blood and CD mucosal macrophages. Human primary CD14 + monocytes were differentiated with 20 ng/mL M-CSF for 3 days. The cells where then washed with PBS and treated with 0.1% DMSO or 0.04 μM GSK761 for 1 h prior to 3 days polarization to "M1" and "M2" macrophages phenotypes with 100 ng/mL INF-γ or 40 ng/mL IL-4, respectively (GSK761 and DMSO were not washed and kept in the culture during the 3 days of polarization). a Gene expression of the inflammatory marker CD64 ("M1" marker) and anti-inflammatory marker CD206 ("M2" marker) was measured by qPCR in IFN-γ ("M1") and IL-4 ("M2") polarized macrophages and b-e FACS analysis was performed in IFN-γ ("M1") polarized macrophages. b Scatter plots illustrating the frequency of CD64 + cells. c A graph bar summarizing the frequency of CD64 + macrophages in 3 donors (left) and CD64 protein expression intensity (right). d Scatter plots illustrating the frequency of CD206 + cells. e A graph bar summarizing the frequency of CD206 + macrophages in 3 donors (left) and CD206 protein expression intensity, n=3. f "M1" polarized macrophages were pretreated for 1 h with 1% DMSO (n=4) or with 0.04 μM GSK761 (n=5). The cells were then stimulated for 4 h with 100 ng/mL LPS and Customized RT 2 Profiler PCR Arrays was performed, the scatter plot illustrates the differentially expressed genes (2-fold change). g "M1" polarized macrophages were pretreated for 1 h with 1% DMSO or with an increasing concentration of GSK761 (0.01, 0.04, 0.12, 0.37, and 1.11 μM). The cells were then stimulated for 24 h with 100 ng/mL LPS. Protein levels of TNF, IL-6, IL-1β, IL-10, IL-8, and IL-12p70 were measured in the supernatant, n=4-7. The Y axis indicates the fold change in cytokine protein expression relative to DMSO control. h CD14 + cells were isolated from inflamed CD mucosa. The cells were then incubated ex vivo for 4 h with either 0.1% DMSO or 0.04 μM GSK761. Relative gene expression of TNF, IL6, IL10, and CD64 were measured using qPCR, n=4. The Y axis indicates the fold change in mRNA level relative to DMSO control. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001  Table S5-7 and Additional files 12 and 13:  Table S12-13). Gene Ontology analysis of SP140-bound genes showed enrichment of genes that participate in the processes of cytokine response and immune response that were inhibited through GSK761 pre-exposure (Fig. 6h). To elucidate the regulatory pathways enriched by SP140 and affected by GSK761, we carried out hallmark pathway analysis for SP140 differentially bound genes (DBGs) (Fig. 6i) and DEGs (Fig. 6j) upon DMSO or GSK761 treatment at each time point of LPS stimulation. In DMSO-treated "M1" macrophages, we found that SP140 binding is predominantly enriched at many pathways typically defined as inflammatory such as TNF signaling via NFKB, and inflammatory response (Fig. 6i). However, this enrichment was no longer seen in GSK761-treated macrophages (Fig. 6i). Similar pathways were observed for DEGs and were downregulated in GSK761-treated macrophages, especially at 4 h (Fig. 6j). In addition, we observed an upregulation of MYC targets and oxidative phosphorylation gene sets in GSK761-treated macrophages, whereas these normally are downregulated because of the induction of aerobic glycolysis upon LPS stimulation of macrophages (Additional file 1: Fig. S8a) [38]. These data suggest SP140 as a critical regulator of genes involved in the inflammatory response and that GSK761 can inhibit this response through displacing SP140 binding to TSS and enhancer regions.
In addition to SP140 binding to chromatin, we speculated that SP140 may also interact with other TFs proteins to regulate gene expression. To this end, we performed homer known motif enrichment analysis (HKMEA) at DMSO_1h LPS (Fig. 7g). We also conducted a homo de novo motif discovery to define new DNA sequence motifs that are bound by SP140 (Fig. 7g). Interestingly, HKMEA suggests that SP140 may bind the same DNA sequence motifs recognized by certain TFs defined as key proteins in TNF signaling via NFKB (ATF3, FOSL2, FOSL1, NFKB-p65-rel, JUNB, and JUN-AP1) and of cytokine-mediated signaling pathway (BATF, NFKB-p65-rel, JUNB, IRF8, and IRF3) (Fig. 7g).
(See figure on next page.) Fig. 6 LPS stimulation leads to SP140 protein recruitment to chromatin; GSK761 reduces this recruitment and dampens inflammatory pathways in inflammatory macrophages. a Nuclear extracts from αCD3/αCD28 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). LPS stimulation strongly recruits SP140 to multiple human leukocyte antigen (HLA) genes, including HLA-A, HLA-B, HLA-C, HLA-F, HLA-DPA, and HLA-DPB (Additional file 1: Fig. S8e), binding which was strongly reduced by GSK761. This indicates a role of SP140 in regulating antigen presentation-associated genes. Notably, the Brd-containing protein 2 (BRD2) gene was occupied by SP140, and GSK761 reduced this binding and lowered BRD2 gene expression (Additional file 1: Fig. S8e and Additional file 5: Table S5). BRD2 has been reported to play a key role in inflammatory response in murine macrophages and in inducing insulin resistance [39].

Discussion
SP140 has been implicated in CD and other autoimmune diseases through genetic and epigenetic association studies [16,40]. Here, we describe the first small molecule inhibitor of SP140 protein, which has been used to investigate its function. The novel SP140-binding small molecule GSK761 was shown to compete with the N-terminal tail of histone H3 for interactions with the SP140 PHD-Brd module. In human macrophages, SP140 associated with the regulatory regions of immune-related/ inflammatory genes in a stimulus-dependent manner. GSK761 inhibited SP140 binding to the TSS of many inflammatory genes, correlating with compound-induced decreased expression of these genes and reduced macrophage inflammatory function. The predominant SP140 expression in immune cells and in particular inflammatory macrophages, further identifies SP140 as an epigenetic reader protein that contributes to inflammatory gene expression.
Inflammatory macrophages generated in vitro or macrophages from CD inflamed tissue were shown to express high levels of SP140, and SP140 silencing in macrophages reduced the expression of a range of inflammatory genes. These anti-inflammatory effects were extended using GSK761, which was able to inhibit spontaneous cytokine expression from CD14 + macrophages isolated from CD anti-TNF refractory patients' colonic mucosa. LPS stimulation of macrophages was shown to cause SP140 recruitment to the TSS of a specific set of inflammatory genes, which was prevented by GSK761, dampening the induced expression of those genes. The strongest SP140 occupancy was at the TSS of a range of genes involved in TNF signaling. TNF signaling plays an integral role in CD as evidenced by the efficacy of chimeric anti-TNF mAbs therapy [41]. Notably, anti-TNF therapy response rests on TNF neutralization, but specifically in CD also on its potency to bind membrane-bound TNF on CD tissue infiltrated macrophages [42], which causes differentiation of macrophages to a more regulatory CD206 + "M2"-like phenotype [34,43]. This is for example evidenced by the clinical observation using etanercept (does not bind membranebound TNF), which is not effective in CD, while infliximab is [43]. A reduced expression of SP140 in CD biopsies was shown to be correlated with a better anti-TNF response [10]. In this study, we found that GSK761 increased CD206 expressing macrophages in in vitro. Furthermore, GSK761 reduced pro-inflammatory cytokine expression in CD mucosal macrophages of anti-TNF-resistant patients. Interestingly, the combination of GSK761 and infliximab was more effective at reducing TNF protein secretion in inflammatory macrophages in vitro than either treatment alone. Thus, we anticipate that SP140 inhibition would be useful in enhancing anti-TNF remission induction in CD patients. In this respect, SP140 was also shown by ChIPseq analysis to bind and regulate expression of some key CD-associated genes such as IL23A [44], TYK2 [27,28], JAK2 [27,28], NOD2 [45], and CARD9 [46]. In IBD, epigenetic mechanisms control macrophage inflammatory activation and polarization [47]. These macrophages are central components of the inflamed mucosa and contribute to disease pathology by producing inflammatory cytokines, which promote the differentiation and activation of Th1 and Th17 cells [48]. In this study, SP140 is shown to be critical for both the polarization of inflammatory macrophages and in the response of polarized macrophages to inflammatory stimuli. In particular, the TFs STAT1 and STAT2 are crucial for inflammatory macrophage polarization [5,49,50]. We found high levels of SP140 binding associated with the STAT1 and STAT2 genes; GSK761 disrupted SP140 binding to these genes and reduced their expression, providing a probable mechanistic basis for the inhibitory effects of the compound on "M1" polarization. Notably, GSK761 inhibited macrophage-induced cytokines and costimulatory molecules required for inflammatory T cell activation including IL23A [44], IL12 [51], IL18 [52], IL1β [52], CD40 [53], and CD80 [54]. Therefore, in addition to reducing the direct inflammatory function of macrophages, inhibition of SP140 may inhibit downstream activation/polarization of responding T cells.
While the mechanism by which SP140 is targeted to gene regulatory regions remains to be determined, motif analysis indicated a strong overlap between the DNA sequences enriched for SP140 binding and the binding motifs of some key TFs involved in regulating the inflammatory response as well as the differentiation and polarization to inflammatory macrophages, such as NFkB-p65-rel [55], IRF3 [56], and IRF8 [57]. Thus, it is possible that SP140 is recruited to chromatin via binding to stimulation-induced TFs-either before or after the TFs bind to DNA. Conversely, SP140 may associate with other proteins recruited to sites bound by these TFs. By conducting ChIP-seq at multiple time points (1 and 4 h), we observed differential binding kinetics of SP140 to different sets of genes; for example, SP140 is bound to the CCL5 TSS by 1 h after LPS stimulation but the TSS of CCL2 only after 4 h. The factors controlling the differential binding kinetics are currently unknown but could include differences in the initial chromatin environments of these genes in addition to distinct kinetics of TF activation and binding.
In a previous study, SP140 was found to be enriched at the HOXA genes in macrophages, and SP140 binding at these locations was proposed to play a role in repression of these lineage-inappropriate genes [10]. However, SP140 binding to this region was minimal in our study in both unstimulated and LPS-stimulated macrophages (Additional files 8, 9 and 10: Tables S8-10). In addition, GSK761 did not affect HOXA genes expression in M1 macrophages (Additional files 5, 6 and 7: Tables S5-7). Based on our findings and contrary to the observation of Mehta et al., HOXA genes do not appear to be bound by SP140, nor does their expression seem to be controlled by SP140. The reason for this discrepancy is unclear, but the use of different SP140 antibodies in the two studies is one possibility. The antibody used in our ChIP experiments was found to be the most SP140-specific among 5 antibodies that we tested. Furthermore, our ChIP-seq data has demonstrated a strong association between SP140 and several enhancer/strong transcription regions (Fig. 6c). GSK761 was able to reduce SP140 binding to those regions and as a consequence the global gene expression as represented in PCA in Fig. 6g. The displacement of SP140 binding by GSK761 provides strong support for the specificity of the SP140 binding identified in our ChIP-seq study. Altogether, we should conclude from our data that SP140 functions as a gene expression activator rather than a repressor.
Analysis of the gene sets identified in our SP140 ChIPseq study suggested a role for SP140 in immune defense against microbes (in accordance with [58] and [59]) and some other diseases, such as graft-versus-host disease, cancers, and rheumatoid arthritis. The intestinal microbiota is thought to play a central role in the pathogenesis of CD, and subsequent investigations could consider the role of SP140 in this respect as a previous study linked SP140 SNPs to microbial dysbiosis in human intestinal microbiota [60]. In this study, ChIP-seq analysis was mainly focused on investigating SP140 binding to coding genes. However, by including non-coding RNA molecules, we observed very strong SP140 binding to a limited set of microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) mainly involved in tumorigenesis, such as the miRNAs: MIR3687 [61], MIR3648 [62], and MIR663A [63], and the IncRNAs: MALAT1 [64] and NEAT1 [64]. Accordingly, previous studies have demonstrated that certain BCPs (such as BRD4) are capable of binding and modulating the transcription of some miRNAs and IncRNAs involved in tumorogenesis [65][66][67]. Contrary to the coding genes and the IncR-NAs, GSK761 was unable to affect SP140 binding to miRNAs. This suggests that SP140 binding in these locations is independent of the PHD-Brd region with which GSK761 interacts; binding of SP140 to DNA via the SAND domain is one possibility. While not the focus of this study, other potential links between SP140 and cancer were observed, including high expression of SP140 in chronic lymphocytic leukemia patients' cells (although reduced expression in acute myelogenous leukemia), and high binding of SP140 to the FLOT1 gene, which has been implicated in tumorigenesis [36]

Conclusions
We conclude that SP140 is an epigenetic reader protein that regulates gene expression in macrophages in response to inflammatory stimuli, including those present in the CD intestinal mucosa of patients failing therapy. Targeting SP140 with a selective inhibitor was shown to displace SP140 from chromatin and decrease inflammatory gene expression in macrophages. This study suggests that SP140 inhibition could represent a promising approach for CD, which might include in addition to remission induction, the potential for combination with anti-TNF therapy.

In-house GSK microarray profiler
Human gene expression data, along with accompanying sample descriptions, was purchased from GeneLogic (GeneLogic Division, Ocimum Biosolutions, Inc.) in 2006, and later organized by sample type. Gene expression data for each sample had been determined using mRNA amplification protocols as recommended by Affymetrix (Affymetrix, Inc.) and subsequently hybridized to the Affymetrix U133_plus2 chip. Purchased data was subject to reported quality control measures including ratios for ACTB and GAPDH, as well as maximal scale factors as reported by Affymetrix MAS 5.0. Expression data was normalized using MAS5.0 with a target intensity of 150.

Immunohistochemistry
An anti-SP140 antibody produced in rabbit (HPA006162; Sigma Prestige Antibody) was used to visualize SP140 protein in a Cambridge Bioscience 69571061 Normal Human tissue microarray, a Cambridge Bioscience 4013301 Human Autoimmune Array, a Cambridge Bioscience 4013101 Human Colitis Array, and on in-house rheumatoid arthritis synovial samples. Anti-SP140 antibody was detected with a Leica polymer secondary antibody. Sections were de-waxed using proprietary ER1, low pH 6 and ER2, high pH 8 buffers for 20 min at 98 °C. Sites of antibody binding were visualized with peroxidase and DAB. Staining was performed on a Leica Bondman immunostaining instrument, using protocol IHC F.

SP140 expression in ileal macrophages
Publicly available single-cell RNA sequencing data from 11 involved (inflamed) and 11 paired uninvolved (uninflamed) ileal biopsies was downloaded from the sequence read archive accession number SRP216273 [24]. Raw reads were aligned against GRCh38 using Cellranger (v3.1.0). The resulting unique molecular identifier (UMI) count matrices were then imported into the R statistical environment (v3. 6 To further disentangle the MNPs, we redid the clustering and classification analysis on cluster 6 alone, which yielded 10 subclusters. Clusters 6.2, 6.4, and 6.8 were annotated as monocytes (CD14+FCGR3A+). Cluster 6.0 was annotated as the dendritic cells (DC) (CD1C+CLEC4A+HLA− DQA1+). Clusters 6.5 and 6.6 were annotated as macrophages (CD168+FCER1A+MRC1+), which were notably inflammatory (TNF+IL6+). Cluster 6.1 and 6.3 were dead or dying DCs and macrophages, respectively as they presented a high percentage of mitochondrial reads and a low number of unique genes per cell. Cluster 6.9 was a dead or dying cell population as well but could not be annotated to any of the MNPs. Similarly, cluster 6.7 did not fit any of the known MNPs. The monocyte development trajectories were inferred using slingshot (v1.6.1) [70]. Differential abundance analyses were performed using the Wald test as implemented in DESeq2 (v1.24.0). Comparative percentage non-zero cells were performed using t-tests.

siRNA-mediated SP140 knockdown
Human "M1" macrophages were generated in vitro from human primary CD14 + monocytes as described above. "M1" macrophages were transfected with siGENOME human smartpool SP140 siRNA or non-targeting scrambled siRNA for 48 h with DharmaFECT ™ transfection reagents according to manufacturer's protocol (Dharmacon). The cells were left unstimulated or stimulated with 100 ng/mL LPS (E. coli 0111:B4; Sigma) for 4 h (for qPCR) or 24 h (for Elisa). The supernatant was harvested for cytokine measurement and the cells were lysed (ISOLATE II RNA Lysis Buffer RLY-Bioline) for RNA extraction.

Microarray
RNA from human "M1" macrophages transfected with SP140 siRNA or scrambled siRNA was extracted using a RNeasy mini kit (Qiagen). One hundred fifty nanograms total RNA was labelled using the cRNA labelling kit for Illumina BeadArrays (Ambion) and hybridized with Ref8v3 BeadArrays (Illumina). Arrays were scanned on a BeadArray 500GX scanner, and data were normalized using quantile normalization with background subtraction (GenomeStudio software; Illumina). Genes with negative values were removed from the analysis. Differentially expressed genes (DEGs) had a P-value <0.05 (analysis of variance). The data were analyzed in R (v3.6.3) and R2 Genomics Analysis and Visualization Platform-UMC (r2.amc.nl). Gene ontology overrepresentation analyses were performed in ShinyGO v0.60 [72].

Discovery and synthesis of the compounds The development of a selective inhibitor of SP140 (GSK761)
To identify selective SP140 binding compounds, we utilized encoded library technology to screen the GSK proprietary collection of DNA-encoded small molecule libraries (DELs) of more than 1 billion unique molecules [31,32]. Affinity selection utilizing a recombinant protein construct spanning the PHD and Brd domains of SP140 was carried out. The SP140 (687-867) protein was first immobilized on an Anti-Flag resin tip (Phynexus), a pool of DEL molecules was passed over the bound protein for 1 h and the non-binders were washed away. The protein was denatured at 80 °C, and the bound library molecules were recovered. Two additional rounds of selection were performed, using fresh protein at each round. In addition, a parallel selection with Anti-Flag resins only was carried as a no-target control to eliminate matrix binders. After three rounds of selection, the DNA tags of the eluted population were PCR amplified and sequenced, and the sequences were translated to identify structures of putative SP140 binders. One disynton series (combination of two building blocks) of putative SP140 binders was identified from a three-cycle benzimidazole library (Fig. 4a). The library was synthesized by first installing the DNA head piece to two BB1 nitrobenzoic acids (i.e., 3-fluoro-4-nitrobenzoic acid and 4-fluoro-3-nitrobenzoic acid) by SNAr reactions. After reduction of the nitro group to an amine group, 1456 BB2 aldehydes were incorporated to the DNA-linked anilines through reductive alkylation. Finally, 3157 BB3 amines were used to cap the free acids via the amide bond formation. This library contains 9.19 million different compounds, each of which is encoded by a unique DNA tag. The analysis of the affinity selection output was performed in a Spotfire cube view with each axis represents a cycle of diversity in the library. In Fig. 4b, individual points, corresponding to discrete small molecule warheads enriched from this benzimidazole library, are shown and sized according to the number of unique instances recorded by DNA sequencing. Families of related compounds are of particular interest because they contain one or more building blocks in common and are easily recognized in these views as lines or planes. A family of small molecule warheads defined by a combination of 4-fluoro-3-nitrobenzoic acid (BB1) and 3-(2-(tertbutoxy)ethyl) aniline (BB3) was identified through a highly populated line in the cube view plot (Fig. 4b).
The most enriched warhead from this line is defined by a trisynthon combination of 4-fluoro-3-nitrobenzoic acid (BB1), 4-formylbenzoic acid (BB2), and 3-(2-(tertbutoxy)ethyl)aniline (BB3) and shown significant higher copy numbers than other warheads on the line. This most enriched warhead was chosen for hit confirmation by off-DNA synthesis which yielded the small molecule, GSK761 (Fig. 4b, c). More details about the development of a selective inhibitor of SP140 (GSK761) are added in Additional file 16.
The text describing the discovery and the synthesis of different other compounds (GSK064, GSK675, and GSK306) is added as Additional file 16.

Fluorescence polarization (FP) binding affinity studies
6HisFLAGTEVSP140 (687-867) was serially diluted in the presence of 3 nM GSK064 in assay buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 0.05% Pluronic F-127, 1 mM DTT) in a total assay volume of 10 μl. Following incubation for 60 min, FP was measured on a Perkin Elmer Envision multi-mode plate reader, by exciting the Alexa647 fluorophore of GSK064 at a wavelength of 620 nm and then measuring emission at 688 nm in both parallel and perpendicular planes. The FP measurement, expressed as milliP (mP), was then calculated using the following equation; These data were then used to calculate an apparent K d value by application of the following Langmuir-Hill equation; AB = (A0 + B)/ (Kd + B) where AB is the concentration of the bound complex, A 0 is the total amount of one binding molecule added, B is the free concentration of the second binding molecule, and K d is the dissociation complex. For IC 50 determination, GSK761 was serially diluted in DMSO (1% final assay concentration) and tested in the presence of 50 nM SP140 and 3 nM GSK064 in the same assay buffer and volume as above. Reactions were incubated for 60 min, and IC 50 values calculated using a four-parameter logistic equation; y = x/ 1 + I IC50 nH + D where y is the mP signal in the presence of the inhibitor at concentration I, x is the mP = parallel fluorescence intensity − perpendicular fluorescence intensity parallel fluorescence intensity + perpendicular fluorescence intensity mP signal without the inhibitor, IC 50 is the concentration of the inhibitor that gives 50% inhibition, nH is the Hill coefficient, and D is the assay background.

Pull-down of endogenous SP140 and Halotag-transfected SP140 with GSK761
HuT78 cells were stimulated with 100 ng/mL anti-CD3 antibody (in-house reagent) and 3 mg/mL anti-CD28 antibody (in-house reagent) for 96 h. Nuclear extract from HuT78 cells (endogenous SP140) was prepared following the manufacturer's protocol (Active Motif nuclear extract and co-IP kit

Cell penetration assessment assay of GSK761
Intracellular GSK761 compound concentration was measured by RapidFire Mass Spectrometry utilizing the methodology described by [33].

Cell viability and cytotoxicity assays
"M1" macrophages were generated in vitro from human primary CD14 + monocytes as described above. "M1" macrophages were plated into an opaque-walled 96-well plate at 10 × 10 5 cells per well and incubated with a concentration gradient of GSK761 (0.04-1.11 μM) for 1 h (0.1% DMSO was used as control). The cells were left unstimulated or stimulated with 100 ng/mL LPS for 24 h. Cell viability was assessed using CellTiter-Glo ® Luminescent Cell Viability Assay kit (Promega) according to the manufacturer's protocol. This assay quantifies ATP, an indicator of metabolically active cells. An equal volume of freshly prepared CellTiter-Glo ® reagent was added to each well, the plate was shaken for 10 min at RT, and luminescent signals were recorded using a plate reader (SpectraMax M5). The index of cellular viability was calculated as the fold change of luminescence with respect to untreated control cells.
Cell viability was also determined using Muse ® Count & Viability Kit (Luminex Corp) according to the manufacturer's protocol and measured on the Guava ® Muse ® Cell Analyzer (Luminex Corp).

Histone H3 peptide displacement
Various H3 peptides (Anaspec library (https:// www. anasp ec. com/)) were serially diluted in DMSO (1% final assay concentration) and tested in the presence of 10 nM GSK306 (FAM-labelled version of GSK761) and 80 nM 6HisFLAGTEVSP140 (687-867) (2x apparent K d for this ligand) in 50 mM HEPES (Sigma-Aldrich), pH 7.5, 50 mM NaCl (Sigma-Aldrich), 1 mM CHAPS (Sigma-Aldrich), and 1 mM DTT (Sigma-Aldrich) in a total volume of 10 μl. Reactions were incubated for 30 min, and FP was measured on a Perkin Elmer Envision multi-mode plate reader (PerkinElmer), by exciting the FAM fluorophore of GSK306 at a wavelength of 485 nm and then measuring emission at 535 nm in both parallel and perpendicular planes. The FP measurement, expressed as milliP (mP), was then calculated as described previously, and normalized to free and bound ligand controls to determine % response. IC 50 values were calculated using the described four-parameter logistic equation.

Modified histone peptide array with SP140
Modified histone peptide arrays (Active Motif ) were used to screen the interaction of SP140 PHD-Brd with 59 acetylation, methylation, phosphorylation, and citrullination modifications on the N-terminal tails of histones H2A, H2B, H3, and H4. Each peptide array contains 384 unique histone modification combinations in duplicate. In brief, the array slides were incubated with 3% BSA (Sigma) to block non-specific binding sites, followed by 10 mg/mL of 6His-Flag-Tev-SP140-PHD-Brd (687-867) protein or with no protein (negative control). The slides were then washed and incubated with α-6His Tag antibody (ab9108, Abcam) and α-rabbit IgG HRP antibody (A4914, Sigma) and stained with Super signal West femto kit (Thermo Scientific) according to the manufacturer's instructions. The data were processed on Fujifilm LAS-3000 imager. All positive and negative signals and the corresponding peptide location can be tracked in Additional file 15: Table S15.

SP140/histone nano-bioluminescence resonance energy transfer ™ (NanoBRET ™ ) assay testing
The NanoBRET ™ System is a proximity-based assay that can detect protein interactions by measuring energy transfer from a bioluminescent protein donor Nano-Luc ® (NL) to a fluorescent protein acceptor HaloTag ® (HT). Briefly, SP140-NL and Hitone3.3-HT DNA were transfected into HEK293 cells using the following ratios: 1:1, 1:10, and 1:100, respectively. Signal window was determined by relative NL/HT-fused protein expression. Minus HT controls were used as the baseline in this experiment for each condition. The data is presented as NanoBRET response (mBU) which is dependent on the presence of the HT Ligand, and by microscopic imaging of NL/HT-fused protein signal. For more information about NanoBRET assay protocol, check the following: https:// www. prome ga. com/-/ media/ files/ resou rces/ proto cols/ techn ical-manua ls/ 101/ nanob retprote inpro tein-inter action-system-proto col. pdf? la= en.

Flow cytometry (FACS)
Primary human CD14 + monocytes were positively selected from PBMCs using CD14 Microbeads according to the manufacturer's instructions (Miltenyi Biotec) and differentiated for 3 days with 20 ng/mL M-CSF. The monocytes were then washed with PBS and treated with 0.1% DMSO or 0.04 μM GSK761. After 1 h, 100 ng/mL IFN-γ (R&D systems) was added to the cells to generate "M1" macrophages. After 3 days of incubation, the cells were harvested and subsequently permeabilized using 1% Saponin (Sigma-Aldrich) for 10 min on ice. Cells were then stained using the conjugated antibodies; PerCP-Cy5 mouse anti-human CD64 (dilution 1:50) (305023, Biolegend) and PE mouse anti-human CD206 (dilution 1:20) (2205525, Sony Biotechnology). The analysis was performed by flow cytometry (FACS) using the LSRFortessa and FACSCalibur (both BD Biosciences). FlowJo (BD LSRFortessa ™ cell analyzer) was used for data analysis.

Customized qPCR array
Human "M1" macrophages were generated in vitro from human primary CD14 + monocytes as described above (from 5 independent donors). "M1" macrophages were treated either with 0.1% DMSO or 0.04 μM GSK761 for 1 h. The cells were then washed with PBS and stimulated with LPS for 4 h. Total RNA was isolated using RNeasy Mini Kit (Qiagen) and treated with DNaseI (Qiagen) according to the manufacturer's instructions. RNA was reverse transcribed using the First-Strand Synthesis Kit (Qiagen) and loaded onto a customized RT [2] profiler array for selected 89 genes according to the manufacturer's instructions (Qiagen) and run on QuantStudio 7 Flex (software v1.0). Qiagen's online GeneGlobe Data Analysis Center (https:// geneg lobe. qiagen. com/ us/ analy ze/) was used to determine the DEGs. The data was presented as a scatter plot. All data were normalized to the geometric mean of two reference genes (RPL37A and ACTB). The list of genes included in this experiment was selected from DEG in SP140 silenced "M1" macrophages in [10] and from our MSD, qPCR, and microarray datasets of SP140 silenced "M1" macrophages.

Chromatin immunoprecipitation (ChIP)
"M1" macrophages were generated in vitro from human primary CD14 + monocytes as described above (10 7 cells were used for each condition). "M1" macrophages were either incubated for 1 h with 0.1% DMSO or with 0.04 μM GSK761 and left unstimulated or simulated with 100 ng/mL LPS for 1 or 4 h. The cells were cross-linked with 1% formaldehyde for 10 min at RT and quenched with 2.5 M glycine (Diagenode) for 5 min at RT. The ChIP assay was performed using the iDeal ChIP kit for Transcription Factors (Diagenode) and sonication was performed using the Picoruptor TM (Diagenode) according to the manufacturer's protocols. Chromatin shearing was verified by migration on a 1% agarose gel (E-Gel, Thermo Fisher) and visualized using E-Gel imager (Thermo Fisher). Immunoprecipitation was performed with a polyclonal SP140 antibody (H00011262-M07, Abnova). DNA was purified using IPure kit (Diagenode) according to the manufacturer's protocol. For validation, quantitative real-time ChIP-qPCR was performed on DNA isolated from input (unprecipitated) chromatin and SP140 ChIP DNA with primer pairs specific for the TSS of TNF and IL6 genes. For detailed PCR primer sequences, please see Additional file 4: Table S3. Quantitative PCR was performed using SYBR Green (Applied Biosystems) and StepOnePLus (Applied Biosystems). Results were quantitated using the delta-delta CT (ΔΔCT) method. Libraries of input DNA and ChIP DNA were prepared from gel-purified >300-base pair DNA.

Global profiling of chromatin binding sites
The DNA was used to generate sequencing libraries according to the manufacturer's procedure (Life Technologies). The DNA was end polished and dA tailed, and adaptors with barcodes were ligated. The fragments were amplified (eight cycles) and quantified with a Bioanalyzer (Agilent). Libraries were sequenced using the HiSeq PE cluster kit v4 (Illumina) with the HiSeq 2500 platform (Illumina), resulting in 125-bp reads. FastQ files were adapter clipped and quality trimmed using BBDukF (java -cp BBTools.jar jgi.BBDukF -Xmx1g in=fastqbef.fastq.gz out=fastq.fastq minlen=25 qtrim=rl trimq=10 ktrim=r k=25 mink=11 hdist=1 ref=adapters.fa). Subsequently, the reads were mapped to NCBI37/HG19 using Bowtie (bowtie2 -p 8 -x hg19 fastq.fastq > sample.sam) and sorted and converted into bam files using samtools. Peaks were called using MACS2, with the reads being extended to 200 bp (callpeak --tempdir /data/tmp -g hs -B -t sample.bam -c control.bam -f BAM -n result/ --nomodel --extsize 149) and duplicates removed. BDG files were binned in 25-bp regions and loaded in the R2 platform (r2.amc.nl) for subsequent analyses and visualization. For motif analyses, Summits obtained from the MACS2 analyses were extended by 250 bases and subsequently analyzed on a repeat masked hg19 genome with HOMER to identify over-represented sequences (both known as well as de novo). The data was uploaded and analyzed in R2 Genomics Analysis and Visualization Platform-UMC (r2. amc.nl).

SP140 inhibition in isolated human mucosal macrophages
Colon tissues were obtained during surgery procedures from patients with CD. Patients' characteristics are added as Additional file 14: Table S14. The mucosa was stripped and dissociated in GentleMACS tubes in digestion medium (DM; complete medium (RPMI 1640 with PGA/L-glutamine/10% FCS) with 1 mg/mL Collagenase D (Roche), 1 mg/mL soybean Trypsin inhibitor (Sigma), 50 μg/mL DNase I (Roche)), and then mechanically dissociate on GentleMACS using program B01. The mucosa was then incubated in DM for 1 h at 37 °C while shaking. During digestion, the dissociation was repeated two times. The tissue suspension was passed through a cell strainer (200-300 um) and centrifuged at 1500 rpm for 10 min. The dissociated cells were resuspended in cold MACS Buffer, and macrophages were isolated using CD14 MicroBeads according to the manufacturer's instructions (MiltenyiBiotec). The macrophages were incubated with either 0.1% DMSO or 0.04 μM GSK761 for 4 h. RNA was extracted as described above, and gene expression of IL6, TNF, IL10, and CD64 was measured by qPCR. All data were normalized to the reference gene ACTB.