Fig 2: Co-translational formation of the extraordinarily stable FIH-OTUB1 HD. (A) Following ectopic expression of the indicated proteins, the FIH-OTUB1 heterodimer (HD) was allowed to form for 24 h prior to the analysis of the HD stability in hypoxia when FIH is inhibited and no additional HD can be formed. (B) HD formation kinetics and (C) HD formation during translation inhibition by cycloheximide (CHX) according to the experimental setups described in Fig. S3B. Re-Ox CHX, addition of CHX at the same time as re-oxygenation was started; Re-Ox CHX pre, pre-incubation of cells with CHX for 1 h prior to the start of re-oxygenation. (D) Bicistronic expression of the indicated His-OTUB1-MBP-FIH in E.coli followed by immunoblot analysis. Time points indicate the time after induction of protein production by addition of isopropyl-ß-D-thiogalactoside (IPTG). Nx, normoxia; M, monomer. Data are representative for three independent experiments throughout.

Fig 3: Denaturing condition-resistant FIH interactome composition. (A) Following ectopic expression of the indicated proteins, HEK293 cells were lysed under native or denaturing (boiling in 1% SDS) conditions followed by anti-FLAG IP and immunoblot analysis. HD, heterodimer; M, monomer; ab, antibody. (B) Following ectopic expression of either FIH-V5 or tandem EGFP, HEK293 cells were lysed as described in (A) followed by anti-V5 IP and immunoblot analysis. Arrow heads indicate protein signals that were only detected following IP from native samples (-SDS). *, protein signals detected in IPs from both conditions (native and denatured lysates); exp, exposure. (C) Following the same sample preparation as in (B), the samples were analysed by MS. Venn diagram displaying the overlap between FIH interactors under native (-SDS) and denaturing (+SDS) conditions. (D) Rank order of the 13 proteins interacting with FIH under both conditions shown in (C), according to the relative label free quantification (LFQ) intensity following IP in the presence of SDS and normalized to FIH pull-down. Data are shown as mean ± SEM. HD, heterodimer; M, monomer; ab, antibody; exp, exposure. Data are shown as mean ± SEM from (C, D) four biological replicates or are representative for (A) three or (B) one independent experiment. (B–D) Samples were processed in parallel and only differ in the analysis (immunoblot or MS).

Fig 4: FIH:FIH-OTUB1 heterotrimer formation. Analysis of the composition of the covalently linked FIH-OTUB1 complex by (A) blue native-PAGE (first dimension, 1D) following ectopic expression in HEK293 cells and by (B) SDS-PAGE as second dimension (2D). *, FIH-OTUB1 heterodimer; x, FIH homodimer; y, FIH monomer; arrows 1–3, monomeric FIH originating from FIH monomers [1], FIH:FIH homodimers [2] or the FIH:FIH-OTUB1 heterotrimer [3]. Data are representative for three independent experiments throughout.

Fig 5: Transcriptional cooperation between ETV4 and HIF-1 is disrupted by CITED2. (A) Schematic representation of HIF-1a and ETV4 domain structure and fusion constructs used in mammalian two-hybrid assays. PAS, PER-ARNT-SIM; bHLH, basic helix–loop–helix domain; ODD, oxygen-dependent degradation domain; NRR, negative regulatory region; NAD and CAD, amino-carboxy-terminal activation domain and CADs, respectively. A GAL4-DBD was fused to regions encompassing the HIF-1a NAD and CAD. Full-length ETV4 bearing two activation domains (AD, acidic domain; Ct, carboxy-terminal tail) flanking a central ETS domain was fused to a VP16 activation domain (VP16-AD). Numbers indicate the amino acids present in the respective constructs. (B) U2OS cells were cotransfected with a Gal4-responsive reporter plasmid and Gal4-HIF-1a (GH1a) constructs alone or in combination with VP16-ETV4. The GH1a fusion constructs are specified by the aminoterminal starting amino acid of the truncated HIF-1a regions (530, 740 and 786, respectively). Following transfection, cells were evenly split and incubated at 20 or 0.2% O2 before luciferase activities were determined 24 h later. Noninteracting Gal4 DBD-p53 and VP16-AD-CP1 served as negative control (neg. ctrl.), while the interactions between Gal4 DBD-PHD2 and VP16-AD-HIF-2a(ODD) or VP16-AD-FKBP38 were used as positive controls (pos. ctrl. 1 and pos. ctrl. 2, respectively). (C) Scheme of the potential interactions between HIF-1, p300/CBP and ETV4 as assessed by mammalian two-hybrid assays. Both CITED2 and FIH can block the interaction between HIF-1a and p300/CBP. (D) Cotransfection of the indicated amounts of a CITED2 expression construct together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). (E) Cotransfection of siRNA directed against p300 together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). The p300 knock down efficiency of different siP300 oligonucleotides was analyzed by immunoblotting (upper panel) and siP300#1 was chosen for further experiments. (F) Cotransfection of siRNA directed against FIH together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). The efficiency of the siFIH mediated FIH knock down was confirmed by immunoblotting as shown in the inset. (G) ChIP of normoxic or hypoxic PC3 cells using antibodies directed against HIF-1a or ETV4, or control serum. The amount of coprecipitated chromatin derived from the human P2P region (encoded by EGLN1) containing the HBS was determined by PCR followed by agarose gel electrophoresis.