miR-17 and miR-20a temper an E2F1-induced G1 checkpoint to regulate cell cycle progression

Abstract

The stringent regulation of cell cycle progression helps to maintain genetic stability in cells. MicroRNAs (miRNAs) are critical regulators of gene expression in diverse cellular pathways, including developmental patterning, hematopoietic differentiation and antiviral defense. Here, we show that two c-Myc-regulated miRNAs, miR-17 and miR-20a, govern the transition through G1 in normal diploid human cells. Inhibition of these miRNAs leads to a G1 checkpoint due to an accumulation of DNA double-strand breaks, resulting from premature temporal accumulation of the E2F1 transcription factor. Surprisingly, gross changes in E2F1 levels were not required to initiate the DNA damage response and checkpoint, as these responses could occur with a less than twofold change in E2F1 protein levels. Instead, our findings indicate that the precise timing of E2F1 expression dictates S-phase entry and that accurate timing of E2F1 accumulation requires converging signals from the Rb/E2F pathway and the c-Myc-regulated miR-17 and miR-20a miRNAs to circumvent a G1 checkpoint arising from the untimely accumulation of E2F1. These data provide a mechanistic view of miRNA-based regulation of E2F1 in the context of the emerging model that miRNAs coordinate the timing of cell cycle progression.

Figure 1. Inhibition of miR-17 or miR-20a results in a G1 checkpoint

(a) Mid-passage normal human embryonic lung fibroblasts were serum-starved in media containing 0.1% serum for 96h and then mock transfected (0.1%, 10%) or transfected with 2′-O-methyl oligonucleotides complementary to miR-17 (17), miR-20a (20a), or luciferase (luc) using Oligofectamine (Invitrogen, Carlsbad, CA, USA) at a final concentration of 20 nM. After 6h, transfection media was replaced with media containing 0.1% serum and 10uM BrdU (0.1%), or media containing 10% serum and 10uM BrdU (10%, Luc, 17, or 20a). Cells were fixed and immunostained for BrdU incorporation at the indicated time points and the percentage of BrdU positive cells is graphed. At least 200 cells per treatment were counted positive if they had 3 or more foci per cell. Error bars indicate the s.d. for at least three independent experiments. (b–c) Fibroblasts were transfected with the indicated siRNAs using Lipofectamine 2000 (Invitrogen) at a final concentration of 60 nM prior to serum-starvation for 48h. During the Oligofectamine: RNA complex reaction, serum-starved cells were trypsinized and soybean trypsin inhibitor was added to inactivate the trypsin. Cells were pelleted and resuspended in DMEM containing 0.1% FBS. The transfection complex was mixed with cells and plated. After 6h, transfection media was replaced with medium containing BrdU until the indicated time points. Protein depletion by siRNA transfection is quantified using Multianalyst software (BioRad, Hercules, CA) of immunoblot analysis.

Figure 2. Inhibition of miR-17 or miR-20a leads to a DNA damage response

(a) Cycling fibroblasts were transfected with 2′oMe-miR17, 2′oMe-miR20a or 2′oMe-Luc and immunostained for γH2AX foci (Pickering & Kowalik, 2006). (b) Quantification of panel a. (c–f) Cells were treated as in Fig 1a and the percentage of cells positive for γH2AX foci (c) 53BP1 foci (Frame et al., 2006; Pickering & Kowalik, 2006) (d) neutral comet assay tail formation (Pickering & Kowalik, 2006) (e) is shown. (f) The total percentage of cells positive for γH2AX foci is shown at 21h post serum addition. The percentage of cells that costain (red) and do not costain with BrdU (green) is shown. The percentage of cells in S phase is shown on the inset graph.

Figure 3. Inhibition of miR-17 or miR-20a leads to an E2F1-associated DDR and G1 checkpoint

(a) The levels of E2F1 protein in random cycling fibroblasts 24 h post-transfection with 2'oMe-miR17, 2'oMe-miR20a or 2'oMe-Luc. The fold change in E2F1 levels is indicated. (b) E2F1 levels were examined by immunoblot analysis of serum starved, G0 fibroblasts that were released into complete media (top) and of cells treated as shown in Figure 1a (middle). The relative intensity of E2F1 signal in 2'oMe-Luc-transfected cells compared to 2'oMe-miR17 (bottom left) or 2'oMe-miR20a (bottom right) is shown. (c) Fibroblasts were synchronized and treated as shown in Figure 1b. At 18 h, cells were harvested and the level of protein depletion is shown for the indicated protein. (d and e) Cells were treated as shown in Figure 1b, immunostained, and the percentage of cells positive for (d) gH2AX foci or (e) BrdU incorporation is shown.