Pioneering role of MAF in the regulation of CCR1 SE. (A) IGV browser snapshot showing integration at the CCR1 putative SE and promoter of different data sets, as indicated; that is, in primary myeloma cells, myeloma cell lines MM.1S and JJN3, and in U266 cells expressing MAF. 1, 2, 3, and promoter peak (P) indicate accessibility peaks of interest. (B) CCR1 expression, as measured using qPCR, after CRISPR interference targeting of each of the enhancer peaks, 1, 2, and 3, and P, shown as red bars in the IGV plot. JJN3 cells were transduced with a dead Cas9-KRAB vector, with a guide targeting each 1 of the peaks, and RNA was harvested on day 4 after doxycycline induction. Two guides were used per peak and compared with a nontargeting gal4 control guide. Expression normalized to GAPDH and gal4 control. Bars represent mean + standard deviation, n = 3. ∗P < .05; ∗∗∗P < .001 using 1-way ANOVA with Dunnett post hoc multiple comparisons correction. C-D. ChIP-qPCR validation against the indicated TF (MAF, n = 2; IRF4, n = 3) and H3K4me1 (n = 3) at peak 2, the CCR1 promoter and chromatin region without binding. ∗P < .05; ns; not significant, 1-way ANOVA with Dunnett post hoc multiple comparisons correction. (F) Migration assay assessing the migration of EV and MAF overexpressing U266 cells in the presence of serum or the CCR1 chemokine ligand CCL3. Bars represent mean + standard error of the mean, n = 4. ∗∗P < .01; ∗∗∗P < .001 using 1-way ANOVA with Tukey post hoc multiple comparisons correction. MAF; MAF-overexpressing U266. (G) Microphotographs of Incucyte-based migration assays with EV and MAF-overexpressing (MAF) U266 myeloma cells. Magnification, ×10. HY, hyperdiploid; MAF; MAF-overexpressing U266.