Fig 2: E2F1 transactivates the SLC16A1-AS1/SLC16A1 gene pair. (A) Identification of SLC16A1-AS1 homologs and phylogenomic analysis of the SLC16A1-AS1/SLC16A1 gene pair across vertebrates. LRIG2 functions as relative genomic anchor point. (B) qPCR to evaluate SLC16A1/MCT1 and SLC16A1-AS1 transcript levels in RT-4, RT-112 and T24 cells after 4-OHT-induced expression of E2F1, as compared to their ethanol-treated counterparts (n=3). Top right: Western blot of E2F1 and MCT1 after E2F1 knockdown. Actin was used as a loading control. (C) Top: Diagram of the promoter region shared between SLC16A1/MCT1 and SLC16A1-AS1. The predicted E2F1 binding site is indicated. Bottom: ChIP assay in T24.ER-E2F1 cells treated with 4-OHT (+) or EtOH control (-), in presence or absence of anti-E2F1 antibody. IgG was used as negative control. The input corresponds to 10% of the total amount of sonicated chromatin. (D) Relative luciferase activities after cotransfection of pGL3-SLC16A1-AS1-Prom luciferase vector with 1 or 2 µg of wild-type E2F1 in T24 cells. The pcDNA3.1-E2F1 (E132) and pcDNA3.1-E2F1 (ΔTA) plasmids which express transactivation-deficient forms of E2F1, as well as empty pcDNA3.1 were used as negative controls (n=3). Fold changes were calculated relative to controls (set as 1). E2F1 expression was confirmed by immunoblot with actin as loading control. (E) Relative SLC16A1/MCT1 and SLC16A1-AS1 mRNA levels in nuclear and cytoplasmic extracts of T24 cells. U2sn was used as nuclear localization control. Bar graphs are represented as means ± SD.

Fig 3: E2F1-mediated hPOMC regulation in human Cushing disease specimens. (a) Confocal images of human pituitary Cushing tumor specimen (Cush #1) immunostained with hPOMC (green), pS337-E2F1 (red), and 4′,6-diamidino-2-phenylindole (blue). To confirm POMC and pS337-E2F1 colocalization, a merged image is also depicted. (b) Primary cultures derived from two cultured pituitary Cushing tumor cells (Cush #1, 2) treated with HLM006474. hPOMC and human GH mRNA levels were analyzed by reverse transcription PCR and normalized with 18S ribosomal RNA. ACTH levels in culture medium were analyzed by RIA and reverse transcription PCR performed in triplicate. RIA values are from three sets of experiments, each assayed in duplicate tubes; values are mean ± SE. *P < 0.05; #no significant difference.

Fig 4: A mutant of E2F1 lacking a CY motif is not ubiquitylated by cyclin F and promotes DNA replication stress and cell death after Chk1 inhibitors HEK293T cells transfected with HA‐E2F1 wild type (WT) and HA‐E2F1 lacking a CY motif (ΔRxL) were harvested for immunoprecipitation using HA antibodies. Immunoprecipitates were immunoblotted as indicated.HEK293T cells were cotransfected with MYC‐tagged ubiquitin and HA‐E2F1 or HA‐E2F1 ΔRxL lacking the CY motif as indicated (+). HA‐E2F1 was immunoprecipitated with an anti‐HA resin under denaturing conditions and immunoblotted.HeLa cells stably expressing HA‐E2F1 WT and HA‐E2F1 ΔRxL under the control of a retroviral promoter were treated with cycloheximide (CHX) at 50 μg/ml for the indicated minutes (min).Quantification of E2F1 half‐life in (C).HeLa cells stably expressing an empty vector (EV), HA‐E2F1 WT and HA‐E2F1 ΔRxL under the control of a retroviral promoter were treated with Chk1i for the indicated hours (h). Indicated proteins were resolved by SDS–PAGE and detected by WB. H3 was used as a loading control.Relative survival (%) of HeLa cells stably expressing an empty vector (EV), HA‐E2F1 WT and HA‐E2F1 ΔRxL under the control of a retroviral promoter measured using resazurin and compared to EV treated with Chk1i at the indicated concentrations.Data information: Data are presented as mean ± SD, with at least three independent experiments. P‐values (*P < 0.05, **P < 0.005) were calculated by paired and two‐tailed t‐test.

Fig 5: E2F1 is a ubiquitylation substrate of SCFcyclin F degraded at the G2/M transition and after checkpoint inhibitors HeLa and CCNF K/O cells were harvested and lysed using SDS. The indicated proteins were resolved by SDS–PAGE and detected by WB. Ponceau S staining was used as a loading control.U‐2‐OS cells transfected with non‐targeting siRNA (siNC) or with an siRNA targeting Cyc F were lysed using SDS 48h after transfection. Whole cell lysates were analysed by immunoblotting. H3 was used as a loading control.Heatmap showing gene set variance analysis (GSVA) score for gene sets related to E2F targets (PID_E2F_PATHWAY, ISHIDA_E2F_TARGETS and HALLMARK_E2F_TARGETS).HEK293T cells transfected with empty vector (EV), FLAG‐cyclin F WT, FLAG‐cyclin F (ΔF) and FLAG‐cyclin F M309A were treated with MLN4924 for 4 h and harvested for immunoprecipitation using FLAG antibodies. Immunoprecipitates were immunoblotted as indicated.HEK293T cells were cotransfected with MYC‐tagged ubiquitin and HA‐E2F1 in the presence of FLAG‐cyclin F WT, FLAG‐cyclin F (ΔF) and FLAG‐cyclin F M309A as indicated (+). HA‐E2F1 was immunoprecipitated with an anti‐HA resin under denaturing conditions and immunoblotted.HeLa and CCNF K/O cells treated with 10 μM Chk1i (LY2603618) for the indicated time points (h = hours) were harvested and lysed using SDS. Indicated proteins were resolved by SDS–PAGE and detected by WB. H2B served as a loading control.HEK293 cells transfected with empty vector (EV) or HA‐E2F1 were treated with Chk1i for 20 h and harvested for immunoprecipitation using HA antibodies. Immunoprecipitates were immunoblotted as indicated.Quantification of immunoprecipitation results of (G) from three independent experiments with mean % ± SD. *< 0.05 P‐value and calculated by two‐tailed paired t‐test.HEK293T cells were cotransfected with MYC‐tagged ubiquitin and HA‐E2F1 and then treated with Chk1i for 20 h and siRNA of cyclin F as indicated (+). HA‐E2F1 was immunoprecipitated with an anti‐HA resin under denaturing conditions and immunoblotted.