Fig 6. EAST regulates the binding of insulator proteins to their sites in S2 cells.

(A) ChIP was performed with antibodies against Su(Hw), CP190, Mod(mdg4)-67.2 (the C-terminal region that corresponds to the specific isoform), and Mod-com (the region common to all Mod(mdg4) isoforms) in normal S2 cells (+) and in transfected S2 cells expressing FLAG×3-tagged EAST, EAST^1-1995, or EAST^933-2362. The quantitative PCR (qPCR) was performed on the intergenic and promoter regions bound by Su(Hw). Primers were positioned in the middle of the binding region identified in ModEncode by ChIP-seq. The ras64B coding region (Ras) was used as a control devoid of Su(Hw) binding sites. The percent recovery of immunoprecipitated DNA (Y axis) was calculated relative to the amount of input DNA. Error bars indicate standard deviation of four independent biological replicates. *P ≤ 0.05 (Student’s t-test), in other cases P ≤ 0.01. (B) Results of ChIP with antibodies against CP190, the region common to all Mod(mdg4) isoforms (Mod-com), and dCTCF in normal S2 cells (+) and in transfected S2 cells expressing FLAG×3-tagged EAST, EAST^1-1995 or EAST^933-2362. Quantitative qPCR was performed on the promoter regions of eight genes bound to by dCTCF and CP190 and on the 1A2 insulator. Primers were positioned in the middle of the binding region identified in ModEncode by ChIP-seq. Error bars indicate standard deviation of three independent biological replicates.