Characterizing the effect of SUMOylation-deficient WASp on the promoters of NF-κB response genes in the presence or absence of panHDAC inhibitors. (A) MNase-ChIP assays performed as described in the legend to Figure 4D in the absence (no Tx) or presence of panHDACi (pHDACi-Tx). The displayed ChIP values (mean ± SEM) are percentage of total nuclear input chromatin and were derived after subtracting the background values obtained with isotype IgG antibody; the latter is not shown. IFNG (TH1-signature cytokine), STAT1 (TH1-transcription factor), and TLR1 were selected as representative genes downregulated by SUMOylation-deficient WASp, whereas CSF2 (TH17-proinflammatory cytokine) and TNFAIP2 (proinflammatory cytokine) were representative genes ectopically upregulated by SUMOylation-deficient WASp. UT, untransfected WASp-null TH-cells; FL, same TH cells transfected with full-length WASp; PSM, transfected with penta-SUMO mutant; V75M, transfected with V75M disease-causing mutant. (B) MNase-ChIP assay performed on primary TH1-skewed cells from normal donor depleted of endogenous SUMO1 (by SUMO1-shRNA) or its control (scrambled shRNA) (see supplemental Figure 7 for chromatin-shearing efficiency and supplemental Figure 2B for temporal progression of SUMO1 depletion in these TH1 cells over 6 days following shRNA transfection). (C) MNase-ChIP assay performed on primary Th0 nonskewed and Th1-skewed cells with indicated antibodies at the indicated gene loci. (D) A working model of WASp*SUMO1 chromatin signaling at WASp target gene promoters proposing differential, activating vs repressive outputs of chromatin-located WASp that is informed by its SUMOylaton state. NEM+/−, treated/nontreated with NEM.
