Figure 5.

Regulation of expression of the CCND2, CDKN2B and SUMO1 genes. (A) Promoter occupancies with BCL-6 and STAT5 at the CCND2, CDKN2B and SUMO1 gene promoters was determined by ChIP with the indicated antibodies using lysates of the three indicated cell lines (see legend to Figure 2D for details). A representative semi-qPCR of the precipitated promoter fragments and a graphical representation of the respective band intensities, *P < 0.05, are shown. A control genomic fragment from the MUTYH gene was amplified to confirm the specificity of the precipitated target gene promoters. Note that BCL-6 binds predominantly in the PAK1-lacking SW480 cells and whereas a switch to STAT5 occurs in HT29 cells with active Rac1/PAK1 signalling. (B) Gene expression data corresponding to the ChIP analysis of the three genes in the three cell lines. Left panel shows representative semi-quantitative reverse transcriptase-PCRs (RT-PCRs), whereas graph at the right shows the result of qPCR analysis of cDNAs collected from the three cell lines at three different splitting times. Genes encoding RNA polymerase II (pol 2) and the glycolytic enzyme phosphoglycerate kinase (PGK1) were amplified as control housekeeping genes and a pool of cDNAs mixed at equal parts from the three cell lines was used as reference for qPCR. Serial dilutions served to assure semi-quantitative conditions in the conventional RT-PCR reactions.