Fig. 6: The cyclin D1 Glutamic acid (E)-rich region is required for Top2A promoter occupancy.

A Schematic representation of cyclin D1^wt and cyclin D1^ΔE mutant with internal deletion of the E-rich region (red), FLAG tag (gray), C terminal region (pink), and remaining (orange) region. B Western blot detection of the FLAG-tagged expression vectors and (C) densitometric analysis of cyclin D1 proteins shown as mean ± SEM for N = 5 separate experiments (P < 0.05). D Immunofluorescence staining of the FLAG epitope. GFP (Green) from the vector IRES, DAPI (Blue), and FLAG (Red). E Mammalian 2-hybrid interaction of cyclin D1^wt and cyclin D1^ΔE. shown as mean ± SEM for N > 5 separate transfections conducted in MCF7 cells. F ChIP assays of cyclin D1^-/- 3T3 cells rescued with the indicated FLAG-tagged cyclin D1 proteins conducted with the Top2A promoter with oligonucleotide pairs that were either specific (S) to the region identified in ChIP-seq or non-specific (NS) targeted to a region not identified in ChIP-seq as binding to cyclin D1. PCR and (G) detected occupancy. Quantitation of PCR products was shown as mean ± SEM for N = 3. H, I Representative ChIP analysis and quantitation of the PCR products derived from cyclin D1 proteins binding Zw10 or (J, K) Mlf1 regulatory region.