Fig 2: Effects of RJW-58 in IL-10 expression via OLF-1. A, The cluster heat map reveals the results of microarray analysis. CTSS knockdown increases the mRNA levels of the olfactory receptor family. B, Western blot analysis of OR5B3, OR5I1, and OR5M3 proteins when SIM-A9 cells were treated RJW-58 (5 µM) for 24 h. C, OLF-1 disassociated with CBP by RJW-58 for 24 h treatment in SIM-A9 cells. D, The effect of RJW-58 for 24 h on OLF-1 binding to the IL-10 promoter region in SIM-A9 cells was analyzed using ChIP. E, RJW-58 treatment facilitated IL-10 promoter activity in SIM-A9. IL-10 promoters were identified using the promoter assay. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. The statistical significance was determined using a two-tailed Student's t-test. Data are presented as the mean ± SEM.

Fig 3: Additional support for Ikaros and EBF1 binding at the Igll1 promoter in B cell progenitors.V5 epitope tag-specific EBF1 ChIP and input (top, Schwickert et al., 2014), ChIP for endogenous Ikaros using the C-terminal Ikaros antibody employed in this study (Bossen et al., 2015; Ferreiros-Vidal et al., 2013), HA-tagged Ikaros using a HA epitope tag-specific reagent (Ferreiros-Vidal et al., 2013) and input (Ferreiros-Vidal et al., 2013) show binding of EBF1 and Ikaros to the Igll1 promoter.DOI: http://dx.doi.org/10.7554/eLife.22767.029

Fig 4: Ikaros binding and EBF eviction have different kinetics.(A) Time course of nuclear translocation of HA-Ikaros-ERt2 induced by 4-OHT. The fraction of nuclear Ikaros-ERt2 was estimated by immunofluorescence staining for the HA tag (red) as <5%, 20%, 50%, 80% and 95% at 0, 5, 15, 30 and 60 min after 4-OHT in 3 independent biological replicates. (B) ChIP-PCR time course of Ikaros and EBF1 binding at the Igll1 promoter after 4-OHT. ChIP was done with antisera against the Ikaros C-terminus that detect Ikaros-ERt2 as well as pre-bound endogenous Ikaros. Log2 of mean ± SE, 3 independent biological replicates. Increased binding of Ikaros was significant by 5 min (p<0.05, Students' T test). Decreased binding of EBF1 was significant by 15 min. (C) The arrangement of binding sites for Ikaros (red) and EBF1 (green) at the Igll1 promoter. (D) Two-state model with DNA binding sites occupied either by EBF1 or by Ikaros (EBF-DNA <=> Ikaros-DNA). Increase in Ikaros binding (red) comes at the expense of EBF binding (green). (E) Three-state model where a fraction of DNA binding sites is unbound at any moment in time (turquoise).DOI: http://dx.doi.org/10.7554/eLife.22767.00210.7554/eLife.22767.003Figure 1—source data 1.Numerical data used to generate Figure 1B.DOI: http://dx.doi.org/10.7554/eLife.22767.003

Fig 5: Interdependence of silencing mechanisms leveraged by Ikaros.(A) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to ?-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with ?-satellite DNA is shown as mean ± SE. Where indicated, control or shChd4 cells were treated over night with 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of Chd4 knockdown was statistically significant across replicates (p=5.54×10-38 GLM binomial logit). (B) ChIP kinetics of Ikaros and EBF binding to the Igll1 promoter in control (left) and shChd4 cells (right). Increased binding of Ikaros to the Igll1 promoter was significant for both control and shChd4 cells, decreased binding of EBF1 was significant in control, but not in shChd4 cells. Mean ± SE, 3 independent biological replicates. Ikaros and EBF1 binding at 15, 30 and 120 min were significantly higher in shChd4 than control cells. (C) MNase-seq data from 3 independent biological replicates were integrated with Ikaros ChIP-seq data to show nucleosome occupancy at Ikaros binding peaks before and 6 hr after nuclear translocation of Ikaros. (D) Dynamics of Ikaros binding, RNAP2 eviction, loss of primary transcripts, nucleosome invasion, and histone deacetylation.DOI: http://dx.doi.org/10.7554/eLife.22767.02310.7554/eLife.22767.024Figure 7—source data 1.Numerical data used to generate Figure 7A and B.DOI: http://dx.doi.org/10.7554/eLife.22767.024