RUNX1 distal binding regions exert differential effects on Hmga2 expression. (A) Schematic of distal element-promoter luciferase constructs. The distal elements being tested are the RUNX1-binding regions that are downstream, upstream, and in the intron of the Hmga2 locus. Distal element sequences were cloned downstream of the Hmga2 promoter in the promoter-luciferase constructs. (B) These constructs were cotransfected with empty vector (Control) or with RUNX1 and CBFβ (designated as RUNX1) expression constructs into NIH3T3 cells. Luciferase activity was performed 24 hours after transfection, and error bars indicate standard deviation from 4 biological replicates. (C) The same experiment as described in (B) was performed using U937 cells. Luciferase activity was performed 24 hours after transfection, and error bars indicate standard deviation from 3 biological replicates. For (B) and (C), Promoter alone is designated as “P,” Promoter + Upstream is “P + U,” Promoter + Downstream is “P + D,” and Promoter + Intron is “P + I.” (D) Diagram and chart showing enrichment of interaction between the promoter and upstream element (located at approximately −139 kb upstream) as demonstrated by 3C-qPCR assay, which was performed using EML cells. Data shown are average of 2 independent 3C assays and error bars indicate standard deviation. *P < .05.