Figure 2.

A-MYB regulates Cyclin B2 by indirectly binding to its CHR promoter element through MuvB. (A) siRNA knockdown was performed in HCT116 (n = 5) and U2OS (n = 4) cells. Twenty-four hours after knockdown, cells were transfected with luciferase reporter constructs carrying the wild-type (wt) or CHR-mutated (CHRmut) Cyclin B2 promoter. Forty-eight hours after knockdown, luciferase activity was quantified from cell lysates. Differences between B-MYB KD and A-MYB + B-MYB dKD are statistically not significant. (B) To rescue HCT116 cells from siRNA knockdown of A-MYB and B-MYB, cells were transfected with the mouse A-Myb-overexpressing plasmid mA-Myb-GFP or the empty vector pEGFP as well as luciferase reporter constructs carrying the wild-type (wt) or CHR-mutated (CHRmut) Cyclin B2 promoter in parallel to the siRNA transfections. Forty-eight hours after knockdown, luciferase activity was quantified from cell lysates (n = 3). (C) For luciferase assays after mouse A-Myb overexpression, HCT116, RPE-1, Hep3B or HeLa cells were transfected with the mAmyb-GFP overexpression vector as well as luciferase reporter constructs carrying the wild-type (wt) or CHR-mutated (CHRmut) Cyclin B2 promoter. Forty-eight hours after knockdown, luciferase activity was quantified from cell lysates (n = 12). (D) Binding of A-MYB to LIN37 was tested in CoIP assays with HCT116 native protein extracts. As controls, E2F4 representing the DREAM complex and B-MYB as MMB component were precipitated (representative experiment of n = 4). (E) Binding of A-MYB to the MuvB core components LIN9, LIN37 and LIN54 was tested in CoIP assays with RPE-1 native protein extracts. As controls, LIN9 and LIN37 representing the MuvB core complex and B-MYB as an MMB component were precipitated (representative experiment of n = 3). (F) Binding of the MuvB core to A-MYB was tested in CoIP assays with RPE-1 native protein extracts. As negative control, p130 representing the DREAM complex was precipitated (representative experiment of n = 3). (G) T98G cells were synchronized in the cell cycle by density arrest and released for 15 h or 24 h into the cell cycle to obtain cell populations enriched in G₀, S and G₂/M phase, respectively. From each time point, native protein extracts were isolated and subjected to a LIN37 CoIP. Binding of DREAM and MMB components B-MYB, E2F4, and LIN37 as well as A-MYB was analyzed by Western blot (representative from n = 3). (H) Binding of A-MYB to wild-type (wt) and CHR mutant (CHRmut) Cyclin B2 promoter probes in DNA-affinity purification was analyzed by Western blot (representative of n = 3). Mean ± SD are given, and significances were calculated by two-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001).