A functional screen for regulators of CKDN2A reveals MEOX2 as a transcriptional activator of INK4a

Abstract

The CDKN2A locus encodes two important tumor suppressors, INK4a and ARF, which respond to oncogenic stresses by inducing cellular senescence. We conducted a genome-scale cDNA overexpression screen using a reporter containing INK4a regulatory sequences to identify novel transcriptional activators of this locus. This screen revealed 285 cDNAs that putatively regulate the transcriptional activation of INK4a. Of these, 56 are annotated as transcription factors, including two previously reported activators of the locus, ETS2 and JUNB. Fourteen genes were further validated for activity and specificity, including several homeodomain proteins. We found that the transcription of one of these, the homeodomain protein MEOX2 (GAX) is enhanced in primary cells during the induction of senescence, and forced expression of this protein results in the induction of premature senescence. We further demonstrate that MEOX2-induced senescence is dependent upon INK4a activity, and chromatin immunoprecipitation studies indicate that MEOX2 directly binds the INK4a promoter. These results support a role for this homeodomain protein as a direct regulator of INK4a transcription and senescence in human cells.

Figure 1. Transcriptional activators of INK4A identified by a functional genomics screening approach.

(A) Screening strategy. Numbering indicates portion of the INK4a regulatory region, relative to the natural translational start site, inserted into the pGL3 luciferase reporter. Validation of the reporter was performed by cotransfection in U2OS cells under screening conditions. Mean luciferase values from four replicates are shown; error bars represent one standard deviation. (B) Screen results. Luciferase values from duplicate wells were normalized by plate, transformed and scaled to generate relative activity values (see Materials and Methods), then plotted according to rank.

Figure 2. Confirmation of 14 INK4a activators.

Results from four independent cotranfections of the indicated reporter with the indicated cDNA construct into U2OS cells are shown. Fold activation values were calculated for each experiment by normalizing luciferase levels of the indicated cDNA to the empty vector control (indicated as Vector). These values were then ranked by ratio of activation of the INK4a construct to activation of the empty luciferase reporter vector pGL3. Activators with relative activation values of two or more are shown. Homeodomain family members are in bold; error bars represent one standard deviation.

Figure 3. MEOX2 is a regulator of senescence in human cells.

(A) MEOX2 is upregulated during senescence. Results of semiquantitative RT-PCR analysis on mRNA derived from primary human keratinocytes at the indicated number of population doublings are shown. (B) Dose-response for p16INK4a reporter activation by MEOX2. Results are shown from three independent cotranfections of U2OS with the indicated reporter and cDNA constructs as in Fig. 2. Error bars represent one standard deviation; p53 serves as a negative control.

Figure 4. MEOX2 reduces proliferative capacity of primary human fibroblasts.

Early passage WI38 fibroblasts were infected with retroviral particles expressing the indicated cDNAs, selected on puromycin, and assessed for proliferative capacity by periodic trypsinization and cell counting.

Figure 5. MEOX2 induces senescence phenotypes in primary human fibroblasts.

Senescence-associated beta-galactosidase assays were performed on day 13 postselection. FACS analysis reveals accumulation of cells in the G1 phase of the cell cycle in response to MEOX2 expression.

Figure 6. Structure/function analysis of the histidine/glutamine (H/Q) rich and homeobox domains of MEOX2.

Retroviral constructs expressing the fragments of MEOX2 indicated by bars in the schematic (top panel) were assessed for induction of senescence as in Fig. 4. Results of three independent infections are depicted as mean values; error bars represent one standard deviation. Senescence-inducing constructs are shown in color.

Figure 7. MEOX2 induces senescence through transcriptional activation of p16INK4a.

(A) MEOX2 activates p16INK4a expression. Fibroblasts infected with retroviral constructs expressing the indicated cDNA (top) were harvested 9 days postinfection, and whole cell extracts assayed by western blot with the indicated antibodies (left). Bid serves as a loading control. Band intensities relative to the EGFP control are indicated. (B) MEOX2 binds to the p16INK4a promoter. Fibroblasts stably infected with a retroviral construct expressing hemagglutinin (HA)-tagged MEOX2 were harvested 15 days postinfection and subjected to chromatin immunoprecipitation with HA, PolII, or preimmune IgG antibodies. The resulting chromatin was assayed for enrichment of the indicated sequences by semiquantitative PCR with increasing cycle numbers. Band intensities relative to the input are indicated for the higher PCR cycle number; (0) indicates values at or below background intensity. The p16 primers amplified a fragment located 970 to 621 bp upstream of the INK4a ATG site, which is selectively enriched by MEOX2-HA immunoprecipitation. GAPDH and an amplicon located 5 kilobases upstream of the INK4a coding sequence (“p16 upstr”) serve as controls.

Figure 8. MEOX2-induced senescence requires p16INK4a.

Early passage p16INK4a −/− (Leiden) or +/− (Foo3) fibroblasts were infected with retroviral particles and assayed for proliferation as in Figure 4. Mean values of three replicates are shown; error bars represent one standard deviation.