CTCF Mediates the Cell-Type Specific Spatial Organization of the Kcnq5 Locus and the Local Gene Regulation (original) (raw)
Figure 4
The dynamic spatial organization of Kcnq5 locus and local gene regulation.
(A) Reverse transcriptional quantitative PCR (RT-qPCR) analysis of KCNQ5 expression levels in MCF-7 and Jurkat cells. (B) RT-qPCR analysis of KCNQ5 relative transcription levels in MCF-7 cells transfected with CTCF shRNA, Rad21 shRNA, or shRNA control. The relative transcription levels in the cells were normalized with respect to the endogenous control GAPDH. Error bars represent mean ± s.d. (n = 3). (C) The quantitative PCR of the FAIRE assay with MCF-7 cells. The independent quantitative PCR amplification efficiency was normalized with the input template. Error bars represent mean ± s.d. (n = 3). (D) The cis regulatory element assays were performed in MCF-7 cells using the Luciferase Reporter System (Promega). Three DNA fragments of No. 225 (sense), No. 319 (sense), and CT6 (sense and antisense) were directly cloned into the Bam HI/Sal I site of the pGL3-promoter. The property of each target chromatin fragment was determined by the relative activity of the luciferase reporter. The values of the fluorescence units of firefly luciferase activities were normalized with Renilla luciferase activities (Firefly/Renilla). The values represent the average of independent quadruple repetition experiments and the error bars represent the standard deviation (n = 4); *: P<0.05; **: P<0.01. (E) A schematic representation of dynamic spatial organization at the Kcnq5 locus. A schematic model representation of the CTCF-mediated dynamic spatial organization of the kcnq5 locus, which regulates the local gene activity. CTCF mediates the contact of the repressive chromatin fragments within the inner gene locus and confines them within the hub with the assistance of cohesin.