ACTR depletion blocks cell entry into S phase. (A) IMR-90 cells were infected with RNAi adenovirus vectors targeting ACTR (ACTR-Ri) or GFP (GFP-Ri) or mock infected and harvested 12, 24, 36, and 48 h after infection. Fixed cells were stained with propidium iodide. Cell cycle profiles were determined by flow cytometry. (B and C) IMR-90 cells were infected with RNAi adenovirus vectors, deprived of serum for arrest at G₁/G₀, released to reenter the cell cycle, and then pulse-labeled with BrdU for 1 h before being harvested. Cells were first stained with anti-BrdU-FITC and then counterstained with DAPI. Results in panel B represent the 48-h time point, showing the same field of cells stained with DAPI or anti-BrdU-FITC. The percentage of BrdU-positive cells in panel C was determined by counting the BrdU-positive cells in five frames and dividing by the total number of DAPI-positive cells. Results represent averages of two independent experiments.

ACTR is recruited to the promoters of E2F target genes at G₁/S. T98G cells were synchronized by serum starvation for 3 days in DMEM containing 0.1% FBS and then released to the cell cycle by changing to medium containing 20% FBS. Cells were harvested for ChIP assay with the indicated antibodies at 0 (open bars), 12 (gray bars), and 18 (black bars) h after serum readdition. The relative occupancy at the specific promoters was determined by quantifying the ChIP PCR products obtained from three experiments. The location of the sequence analyzed at each promoter is indicated. IgG, immunoglobulin G.

ACTR controls its own promoter through E2F1. (A) HER cells were cotransfected with the indicated reporter plasmids, expression constructs for E2F1 or ACTR, and β-galactosidase. Luciferase activities were normalized with the β-galactosidase activities. The putative E2F binding sites were identified with the PROMO database (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) and are indicated by small bars on the schematics of the pGL3-ACTR-1.6kb and -0.6kb constructs. (B) Cells were cotransfected with the pGL3-ACTR-0.6kb reporter and an empty vector or an expression plasmid for mutant or wild-type ACTR. The structure-function domain of ACTR is shown in the schematic, which includes moderate sequence homology to bHLH and PAS domains, protein interaction domains for E2F (EID), nuclear hormone receptors (RID), and CBP/p300 (CID) and a region with histone acetyltransferase (HAT) activity. WT, wild type.

Transformation of human mammary epithelial cells by overexpressed ACTR requires its association with E2F. (A) Recruitment of ACTR and E2F1 to the ACTR promoter in MCF10A cells is stimulated by growth factors. Proliferating MCF10A cells maintained in DMEM/F12 with the full supplements (100%; details are in Materials and Methods) or changed to medium with 0.1% of the full supplements for 6 h (0.1%) were harvested for ChIP assay with anti-ACTR or -E2F1 antibodies. ChIP DNA was analyzed for ACTR and E2F1 occupancy at the ACTR promoter with PCR primers amplifying the F fragment as shown in Fig. 7B. (B) ACTR overexpression stimulates growth factor-independent cell proliferation. MCF10A cells maintained in six-well plates in DMEM/F12 with the full supplementation were infected with the ACTR- or GFP-adenovirus (Ad) vector at equal MOIs. Four hours later, the medium was changed to DMEM/F12 with 0.1% of the full supplements. On different days after infection, cells were harvested for proliferation analysis by cell enumeration in triplicate. (D to F) Wild-type ACTR, but not E2F association-defective mutant forms of ACTR, transforms MCF10A cells. MCF10A cells were infected with equal MOIs of adenovirus vectors for expression of GFP or different forms of ACTR as indicated in the diagram (C), harvested 24 h after infection, resuspended as individual cells mixed with soft agar, and plated for colony formation (D and F). Two weeks later, colonies (with diameters of 0.2 mm or larger) were counted and digital images of representative fields were captured. (Note that the one colony containing about 50 cells present in the images for del-EID and A38, respectively, was counted as a colony for the results shown in the bar graph in panel D.) The number of colonies presented was from 5 × 10⁴ plated cells infected by each of the adenovirus vectors. The experiment was repeated once, and essentially identical results were obtained. For protein analysis (E), infected cells were harvested 10 days after infection for Western blotting with anti-HA antibody or anti-ACTR antibody. WT, wild type; IP, immunoprecipitation.

ACTR is required for the proliferation of normal and cancerous human cells. (A and B) Depletion of ACTR inhibits the proliferation of diploid human fibroblast cells. IMR-90 cells were infected 24 h after plating with equal MOIs of adenoviral vectors expressing ACTR siRNA (Ai) or GFP siRNA (Gi) or mock infected (M). Cells were harvested 2, 4, and 6 days after infection for Western blotting (A) or cell proliferation (B). Four days after infection, cells were fixed with 70% ethanol and stained with crystal violet before digital images of representative fields of cells were taken. (C) Depletion of ACTR inhibits the proliferation of HeLa and T98G cells. Cells growing in six-well plates were transfected with synthetic siRNA targeting ACTR (ACTR-Ri) or control siRNA (Control-Ri). On different days after transfection, cells were harvested and counted in triplicates. Western analysis was performed with cell lysates harvested 2 days after transfection.

Depletion of ACTR inhibits the expression of cdc25A, cdc6, and MCMs, as well as cyclins and Cdk. (A) IMR-90 cells were infected with RNAi adenovirus vectors as in Fig. 1A and harvested at different times after infection for Western blotting (part a) or semiquantitative RT-PCR analysis (part b). The relative transcript levels are presented as arbitrary units of PCR products quantified by the Fluorchem software for cells treated with adenovirus vectors of ACTR-RNAi (open bars) or GFP-RNAi (filled bars). (B) HeLa cells were transfected with synthetic siRNA targeting either ACTR (ACTR-Ri) or a control RNAi oligonucleotide (Control-Ri) and harvested 48 later for Western blotting (part a) or 48 and 72 h later for quantitative RT-PCR, with the value from control RNAi set as 100 (part b). No significant difference in expression is illustrated by the gel image of semiquantitative RT-PCR products.

Expression of ACTR is induced at late G₁ and attenuated during S phase. (A) T98G cells were synchronized by serum deprivation as for Fig. 4 and harvested at the indicated times after serum stimulation for flow cytometry, semiquantitative RT-PCR, and Western blot analysis. (B) T98G cells were synchronized in mitosis by treatment with hydroxyurea and then with nocodazole. Cells were released to reenter the cell cycle by switching to nocodazole-free growth medium. Cells were harvested at the indicated times after release for analyses as in panel A. As, asynchronous proliferating cells.

The ACTR-E2F complex is recruited to the ACTR promoter at late G₁ to stimulate its expression. (A) T98G cells were synchronized by serum derivation and harvested at the indicated times after serum readdition for FACS and ChIP analyses. The relative occupancy at the indicated region of the ACTR promoter was determined by quantifying the PCR products of coprecipitated genomic DNA by E2F1 and ACTR antibodies. (B, left part) A ChIP assay was performed as for panel A with T98G cells. Coprecipitated genomic DNA from cells harvested at 18 h after serum readdition was analyzed for occupancy by E2F1, ACTR, and RNA Pol II over the 10-kb region containing the ACTR promoter. The location of each amplicon (A to H) is shown in the diagram above, with the number indicating the position relative to the transcription initiation site of the 5′ primers used. (B, right part) A ChIP assay was performed with asynchronously proliferating MCF7 cells, and precipitated genomic DNA was analyzed as in the left part of panel B. (C) T98G cells were infected with adenovirus vectors to mediate the ectopic expression of ACTR (lanes A) or GFP (lanes G). Cells were harvested at the indicated times after infection and analyzed by semiquantitative RT-PCR for ectopic or endogenous ACTR expression with specific primers (for ectopic expression, a primer annealed to the 3× HA tag sequence 3′ of the ACTR cDNA coding sequence, together with a primer to the coding region, was used; for endogenous expression, a primer pair annealed to the cDNA sequence of the 3′ untranslated region of the endogenous ACTR transcripts was used). The relative increase shown in the right part of panel C was determined by quantifying the RT-PCR products of endogenous ACTR with the digital number of the product from adenovirus-GFP-infected cells at 24 h set as 1 U.