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). 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