Fig 2: PITX1 binds HIF-1β but not HIF-1α and it does not disrupt the HIF-1α/HIF-1β heterodimer in a general manner. (A) HeLa cells were treated or not with 1% O2 for 24 hours prior to whole cell lysis. 300 μg of protein were used to immunoprecipitate PITX1, using specific antibodies. Rabbit IgG was used as a negative control. Immunocomplexes were analyzed by western blot using the indicated antibodies. (B) Hek293 cells were transfected with 4 μg of empty vectors or GFP-HIF-1β and mCherry-PITX1 (mCh-PITX1) expression constructs for 48 hours prior to lysis. 300 μg of whole cell lysates were used to immunoprecipitate HIF-1β and normal rabbit IgG was used as a control. Immunocomplexes were analyzed by protein gel blot using the Figure 4 (See previous page). indicated antibodies. (C) HeLa cells were treated or not with 1% O2 for 24 hours prior to being fixed and stained for immunofluorescence using the indicated antibodies. Images were acquired using a DeltaVision microscope, deconvolved and processed using OMERO client (opensource software). (D) Hela cells were co-transfected with 1 μg of mCherry-PITX1 (mCh-PITX1) and 1 μg of GFP-HIF-1β, as indicated, for 48 hours prior to fixation. Images were acquired before and after acceptor photo-bleaching as indicated in the methods. (E) HeLa cells were transfected with control or PITX1 siRNA, prior to treatment with 1% O2 for 24 hours. Whole cell lysates were obtained and 300 μg of protein were used to immunoprecipitate HIF-1α, using specific antibodies. Mouse IgG was used as a negative control. Immunocomplexes were analyzed by western blot using the indicated antibodies. (F) HeLa cells were transfected with control or PITX1 siRNA, prior to treatment with 1% O2 for 24 hours prior to being fixed and stained for immunofluorescence using the indicated antibodies. Images were acquired using a DeltaVision microscope, deconvolved and processed using OMERO client.

Fig 3: PITX1 is a specificity determinant for HIF-1a-dependent target gene activation. (A) U2OS and HeLa cells were transfected with control or PITX1 siRNA, prior to treatment with 1% O2 for 24 hours. Whole cell lysates were obtained 48 hours post-transfection and analyzed by protein gel blot using the indicated antibodies. (B) HeLa cells were transfected with control or PITX1 siRNA, prior to treatment with 1% O2 for the indicated periods of time. Whole cell lysates were obtained 48 hours post-transfection and analyzed by western blot using the indicated antibodies. (C) HeLa cells were transfected, processed and analyzed as in B. (D) U2OS were co-transfected with 1 μg of GFP-HIF-1β and increasing amounts of PITX1 plasmid (0.1, 0.25 and 0.5 μg) prior to exposure to 1% O2 for 24 hours. Whole cell lysates were obtained 48 hours post-transfection and analyzed by protein gel blot using the indicated antibodies. (E) MDA-MB-231-HRE cells were transfected with control and PITX1 siRNA oligonucleotides prior to treatment with 1% O2 for 24 hours. Luciferase activity was measured 48 hours post-transfection. Graph depicts mean and standard deviation of a minimum of 3 independent experiments. Student's t-test was performed to calculate p values, and levels of significance are denoted as follows: *P < 0.05, **P < 0.01, and ***P < 0.001. (F) MDA-MB-231 cells were transfected with control and PITX1 siRNA oligonucleotides prior to treatment to 1% O2 for 24 hours. Whole cell lysates were obtained 48 hours post-transfection and analyzed by western blot using the indicated antibodies.

Fig 4: Experimental ChIP-seq data from the ovarian cancer cell line PEO4 illustrates transcription factors PITX1 and ERα co-occupying the promoter and gene bodies of three genes differentially expressed between epithelial ovarian cancer subtypes and stages. SLC7A2 and AFF2 were differentially expressed between serous borderline tumors (SBTs) and high-grade serous ovarian cancers (HGSOCs); TSPAN1 was differentially expressed between stage II and stage III HGSOCs. Binding data are displayed on tracks from the UCSC Human Genome Browser. Two experimental replicates were performed for each transcription factor, along with an input control. Blue asterisks indicate PITX1 binding and green asterisks ERα binding, where peaks displayed statistical significance above background for the relevant transcription factor. The vertical viewing axis was assigned the value of the highest peak and kept consistent for all panels at each locus

Fig 5: PITX1 levels control HIF transcriptional activity under hypoxic stress. (A) U2OS and HeLa cells stably expressing an HRE-luciferase reporter plasmid were transfected with control and PITX1 siRNA oligonucleotides prior to treatment with 1% O2 for 24 hours. Luciferase activity was measured 48 hours post-transfection. Graph depicts mean and standard deviation of a minimum of 3 independent experiments. Student's t-test was performed to calculate p values, and levels of significance are denoted as follows: *P < 0.05, **P < 0.01, and ***P < 0.001. (B) U2OS and HeLa-HRE luciferase cells were transfected with control and PITX1_B siRNA oligonucleotides prior to treatment with 1% O2 for 24 hours. Luciferase activity was measured 48 hours post-transfection. Graph depicts mean and standard deviation of a minimum of 3 independent experiments. Student's t-test was performed to calculate p values, and levels of significance are denoted as follows: *P < 0.05, **P < 0.01, and ***P < 0.001. (C) U2OS-HRE luciferase cells were transfected with Figure 1 (See previous page). empty vector or increasing concentration of PITX1 expression construct (0.1, 0.25, 0.5 1 and 2 μg) prior to exposure to 1% O2 for 24 hours. Luciferase activity was measured 48 hours post-transfection. For all graphs, data was normalized to control hypoxia treated sample. (D) U2OS and HeLs cells were transfected with control or PITX1 siRNA oligonucleotides for 48 hours prior to total RNA extraction. Levels of HIF-1α mRNA were analyzed by qPCR. Graph depicts mean and standard deviation of a minimum of 3 independent experiments. Data was normalized using actin and compared to control siRNA.