Fig. 5

Epigenetic regulation of the DUSP2 promoter by CTCF. a. Binding of CTCF at the DUSP2 promoter analyzed by quantitative ChIP. CTCF expression was induced in CTCF TREx293 cells by tetracycline (5 μg/ml) for 48 h and uninduced cells were used as control. The chromatin was prepared, precipitated with a CTCF-, histone H3- or control IgG-antibodies and amplified with gene specific primers for the DUSP2 promoter, a negative site and a bona fide positive site within the DUSP2 locus. CTCF binding was quantified by qPCR (triplicates from 2 independent experiments) and significance was calculated. Values of the precipitated sample were normalized to 1 % input (=1). b. Methyl-DNA immunoprecipitation (MeDIP) analysis of the DUSP2 promoter. MeDIP with 5mC- and 5hmC-antibodies was done with DNA from uninduced or induced CTCF TREx293 cells. The detection of the 5hmC and 5mC level was performed with semi-quantitative PCR of the DUSP2 promoter and a control locus. PCR products were separated together with a 100 bp marker (M) in 2 % agarose gel. c. Effect of CTCF on the DUSP2 promoter. A DUSP2 promoter fragment (454 bp) was cloned in the pRLnull vector and in vitro methylated (ivm). 2.7 μg DUSP2 promoter constructs (DUSP2-pr.) were transfected in HEK293 or CTCF TREx293 cells and GFP-CTCF or GFP vector (1 μg each) was co-transfected or CTCF was induced for 24 h, respectively. Expression of renilla luciferase was measured and normalized to the expression control vector or to the co-transfected firefly plasmid pGL3.1 (300 ng), respectively. Significance is indicated (t-test)