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. 2004 Oct 28;23(23):4615–4626. doi: 10.1038/sj.emboj.7600459

Figure 7.

Figure 7

A role for B-Myb in the control of G2/M gene expression. (A) The binding of B-Myb to endogenous G2-regulated promoters (cdc2, CycA2, and cyclin B1) is delayed compared to the binding of the activator E2F (E2F3) to these promoters. T98G cells were released from serum starvation and samples were harvested at quiescence (Q), or 9, 12, 15, 18, 21, and 24 h after serum addition for ChIP analysis using the indicated antibodies. Two E2F-regulated G1/S genes (p68 and DHFR) and a non-E2F-regulated gene (albumin) were used as controls. (B) Relative kinetics of B-Myb and E2F3 protein accumulation. Western analysis of E2F3 and B-Myb using 40 μg of nuclear extract of T98G cells harvested at quiescence (Q) or at the indicated times following serum addition. NS: nonspecific band. (C) The binding of B-Myb to endogenous G2-regulated promoters (cdc2, cyclin B1, and cyclin A2) is prolonged (9 h time point) compared to the binding of activator E2F3 to these promoters. T98G cells were made quiescent (Q), blocked with HU (0 h), released from HU for 6, 9, and 18 h, and then harvested for ChIP analysis using the indicated antibodies (α-B-MybN: N-19; α-B-MybC: H115). Two E2F-regulated G1/S genes (cdc6 and PCNA) were used as controls. (D) Mutation in the MYB binding site abolishes the binding of B-Myb to human cdc2 promoter. REF52 cells transiently transfected with wild-type (WT), the distal E2F mutant (dE2Fm), and the MYB site mutant (MYBm) reporter constructs were made quiescent and then restimulated with serum for 18 h. The luciferase activities for these constructs are shown in Figure 3B. The presence of the cdc2 promoter or control sequence (Renilla) from the transfected constructs in immunoprecipitates using anti-E2F3, anti-B-Myb, or control antibodies (NR) was detected by PCR as described in Figure 5. (E) B-Myb is required for G2/M target gene expression. Western analysis of several G2/M genes (cdc2, cyclin A2, and cyclin B1) and G1/S genes (E2F3, DHFR, and cdc6) and C-Myb using cell extracts from asynchronously growing T98G cells with mock transfected as a control (con), or transfected with an effective B-MYB RNAi duplex 1 (#1) and a noneffective B-MYB RNAi duplex 2 (#2) as another control. Cell lysates were prepared 48 h after siRNA transfection. NS=nonspecific band.