Figure 4

TIF1β is involved in the regulation of expression of E2F downstream genes, cyclin A2, Cdc2 and Cdc25A . (A) Binding of TIF1β to the promoter of cyclin A2 gene is correlated with its repression. HeLa cells were synchronized to G1/S boundary by double thymidine block and subsequently released for 1 hour to the early S phase. ChIP was performed as described in the Materials and Methods section. Equal loadings of input derived from G1/S and S cells were used for ChIP experiments (upper panels), using 20A1, H3K4diMe, and H3K9diMe antibodies. PCR was performed with input samples (1/50) and ChIP eluates. Relative differences between G1/S and S phase of each indicated probe on cyclin A2 promoter are shown in the lower right panels (comparing the cyclin A2 signal of G1/S and S phase from each probe with the same total area, by ImageGauge software). The protein level of TIF1β (20A1), α-tubulin and phosphorylated TIF1β/Ser473 (S473) from formaldehyde cross-linked samples are shown in the lower left panel. PCR for cyclin E promoter was used as a control (upper panel). (B) HeLa cells were arrested at G1 phase by serum starvation and released into early S phase by addition of serum to the culture for 2 hours. ChIP was performed as in (A) with 20A1, anti-H3K4diMe and anti-H3K9diMe antibodies (upper panel). Relative differences between G1 and S phase of each indicated probe on cyclin A2 promoter are shown in the lower right panels (by ImageGauge, as in A), while the protein levels are shown in the lower left panel. (C) Flow cytometry analysis. FLAG-TIF1β/S473A, FLAG-TIF1β/S473E and wild-type FLAG-TIF1β were over-expressed in 293T cells, collected 36 hours post-transfection and analyzed by flow cytometry after staining with propidium iodide, and the FCM histograms are shown in the upper panels. Cell cycle phase distributions (%) of TIF1β over-expressing cells were analyzed with the CellQuest software and shown in the lower left panel, according to the gated region from the upper panel (M1, subG1; M2, G1; M3, S; and M4, G2/M, by CellQuest program), and the bar charts represented the cell cycle distribution (in total for 100%) are shown in the lower right panel (by Microsoft Excel). (D) The HP1β complex is preferentially associated with the promoters of Cdc2 and Cdc25A in TIF1β/S473A over-expressing interphase cells of HEK293T. FLAG-TIF1β, FLAG-TIF1β/S473A, FLAG-TIF1β/S473E and HA-HP-1β were ectopically expressed in HEK 293T cells for 36 hours and subjected to ChIP with the indicated antibodies (anti-tubulin, 10D8; anti-TIF1β, 20A1; anti-HA, HA). DNA from the immunoprecipitated chromatin was quantified by real-time PCR. Each result was normalized to input and the relative level to the transfected vector control. Results for the Cdc2 promoter (upper panels) and Cdc25A promoter (lower panels) are shown.