Fig. 4

SMC3 binding to Rab27a gene is crucial for gene expression. A Hi-C dataset analysis for the study of the binding of SMC3 protein in the Rab27a gene using the H1-hESC cell line. Dashed lines highlight the region of the Rab27a gene. B ChIP-qPCR of SMC3 for different binding sites in the Rab27a gene (Promoter, Enhancer and Next to Enhancer regions) in MIA PaCa-2 cells (n = 4). IgG antibody was used as control so that we could identify regions of specific binding for Rab27a. Negative region for SMC3 binding was used to confirm specificity of the enrichment (Neg). One-way anova **p < 0.01, ****p < 0.0001. C Luminescent imaging (left) and quantification (right) of MIA PaCa-2 cells non-transfected (1), transfected with pGL3-Promoter empty vector (2), pGL3-Promoter vector with a control sequence (sense, 3), pGL3-Promoter vector with a control sequence (anti-sense, 4), pGL3-Promoter vector with SMC3 binding site in the enhancer region of Rab27a gene (sense, 5) or pGL3-Promoter vector with SMC3 binding site in the enhancer region of Rab27a gene (anti-sense, 6). N = 6, two-way anova, *p < 0.05, ****p < 0.0001. D Genome browser panel highlighting regions in the Rab27a gene targeted by sgE1, sgE2, sgE3 and sg NTE1. Sg1 and 3 target regions of the Rab27a enhancer. SgE2 targets SMC3 binding site in the Rab27a enhancer region and sgNTE1 targets SMC3 binding site in a region next to the Rab27a enhancer. Dashed lines identify the enhancer region in the Rab27a gene. E Western blot of Rab27a (top) and quantification (bottom) in MIA PaCa-2 cells transduced with a control sg (intron region of Rab27a gene), a sg for Rab27a enhancer region 1, a sg for Rab27a enhancer region 2, a sg for Rab27a enhancer region 3, a combination of sgs for different regions of the Rab27a enhancer (1 + 2 + 3) or a sg for next to enhancer region 1. β-actin was used as loading control. F Schematic representation of the proposed mechanism of SMC3 binding to the Rab27a gene. Data are Mean ± SEM