Strip-FRAP analysis of GFP, GFP-UTF1, H2B-GFP, and Oct4-GFP. (A) FRAP analysis of EC cells expressing GFP (green line) or GFP-UTF1 (blue line). The graph shows the relative fluorescent recovery directly after bleaching. The prebleach level is normalized to 1. GFP displays a quick fluorescent recovery in the bleached region, whereas GFP-UTF1 shows only little recovery directly after photobleaching. (B) FRAP analysis of ES cells expressing GFP (green line) or GFP-UTF1 (blue line). GFP shows a quick recovery after bleaching, whereas GFP-UTF1 displays only a marginal recovery. (C) FRAP experiment of EC cells expressing either GFP-UTF1 (blue line) or histone H2B-GFP (red line). GFP-UTF1 and H2B-GFP both show only a small recovery directly after photobleaching. (D) FRAP experiment of EC cells expressing GFP (green line), GFP-UTF1 (blue line), or Oct4-GFP (red line). Oct4-GFP shows a quick recovery after fluorescence, although slower than GFP.

Cellular localization and subnuclear fractionation of GFP-UTF1 in EC and ES cells. (A) Western blot analysis of wild type (wt) EC cells and a clone stably expressing GFP-UTF1 (#1) using an antibody directed against UTF1. (B) Subnuclear fractionation of EC and ES cells, both expressing GFP-UTF1. Immunoblot analysis was performed with an antibody directed against UTF1 and the HA tag of the fusion protein. F, free-diffusing protein fraction; D, DNaseI fraction; AS, ammonium sulfate fraction; HS, high salt fraction; M, nuclear matrix fraction. (C) Confocal and transmission image of living EC cells expressing GFP-UTF1. Nucleoli are indicated by arrows. (D) Time-lapse imaging of a GFP-UTF1–expressing EC cell going through mitosis. (E) Confocal images of a mitotic GFP-UTF1–expressing cell treated with Hoechst. (F) Confocal and transmission images of GFP-UTF1–expressing ES cells grown on an STO cell feeder layer. The arrow indicates a nucleolus, and the arrowhead points to mitoticchromosomes. (G) Transmission image of AP staining of GFP-UTF1 ES cells. The underlying STO feeder cells are negative for AP activity. Bars, 15 μm.

Analysis of subcellular localization and mobility of wt and mutated GFP-UTF1. (A) A schematic representation and repressor activity of various GFP-UTF1 mutants. The Myb/SANT domain (aa 55–124) and conserved domain 2 (CD2) are indicated by black boxes and gray boxes, respectively. The W63G and E67K point mutations are indicated by asterisks. Repressor activity of the GFP-UTF1 fusion proteins was measured on a constitutively active UAS-TK-Luc reporter in transiently transfected HepG2 cells. Negative controls include pUC18 and a peGFP-C1 plasmid. Error bars represent SD. (B) Confocal images of living cells expressing GFP-UTF1 (1–339), GFP-UTF1 W63G E67K (W63G E67K), GFP-UTF1 1–300 (1–300), and GFP-UTF1 W63G E67K 1–300 (W63G E67K 1–300). (C) FRAP analysis of EC cells expressing either GFP (green line), GFP-UTF1 (1–339; blue line), or GFP-UTF1 W63G E67K (W63G E67K; red line). (D) FRAP experiment of EC cells expressing either GFP (green line), GFP-UTF1 (1–339; blue line), or GFP-UTF1 1–300 (1–300; red line). (E) FRAP experiment of EC cells expressing either GFP (green line), GFP-UTF1 (1–339; blue line), or GFP-UTF1 W63G E67K 1–300 (W63G E67K 1–300; red line). (F) Subnuclear fractionations of stable cell lines expressing GFP-UTF1 (1–339), GFP-UTF1 W63G E67K (W63G E67K), GFP-UTF1 1–300 (1–300), and GFP-UTF1 W63G E67K 1–300 (W63G E67K 1–300). Blots were developed with an antibody against HA. F, free-diffusing protein fraction; D, DNaseI fraction; AS, ammonium sulfate fraction; HS, high salt fraction; M, nuclear matrix fraction. Bar, 15 μM.

Characterization of localization, fractionation, and reporter activity of UTF1. (A) Immunofluorescence analysis of endogenous UTF1 in EC cells using an antibody directed against UTF1. (B) Subnuclear fractionation of EC cells: F, free-diffusing protein fraction; D, DNaseI fraction; AS, ammonium sulfate fraction; HS, high salt fraction; M, nuclear matrix fraction. Fractions were immunostained with antibodies recognizing UTF1, Oct4, HDAC1, and histone H2A. (C and D) Reporter analysis of HepG2 cells transfected with the SBE(inv)₅ reporter, (BRE)₂ reporter, Smad 3/4, Smad 1/4, and UTF1 as indicated. In all transfections, a LacZ expression plasmid, pDM2-LacZ, was included as an internal standard, and relative luciferase units (rlu) are depicted as the mean with SD (error bars). In all samples, equal amounts of expression plasmids were present by the addition of empty pcDNA3 plasmid when required. (E) Mapping the UTF1 repressor domains. A schematic representation of GAL4-UTF1 constructs used in this experiment; the Myb/SANT domain and CD2 are represented by black boxes and gray boxes, respectively. Different GAL4-UTF1 constructs and a constitutive active TK-luciferase reporter containing five GAL4-binding sites (UAS-TK-Luc) were transfected into HepG2 cells. The inhibitory effect of UTF1 on reporter activity is depicted as fold repression compared with GAL4 alone. In all transfections, a LacZ expression plasmid, pDM2-LacZ, was included as an internal standard, and normalized luciferase activity is depicted as the mean with SD. Bar, 15 μm.

UTF1 is involved in the differentiation of EC and ES cells. (A) UTF1 expression in P19CL6 EC cells (wt), 14-d DMSO-differentiated EC cells (wt d14), and four independent UTF1 EC KD clones (UTF1 #1–#4). The asterisk indicates a shorter variant of mUTF1 (aa 43–339) generated by transcription from an alternative start site (Nishimoto et al., 2001). (B) DMSO-induced differentiation of wt, Renilla luciferase KD (Renilla), and UTF1 KD (UTF1 #1 and #2) EC cells. Cell lysates were analyzed with antibodies against Oct4, UTF1, GATA4, and Troma1. Actin staining was performed as a loading control. (C) Western analysis of wt, Renilla luciferase KD (Renilla #1 and #2), and UTF1 KD (UTF1 #1 and #2) IB10 ES cells. Cell lysates were analyzed with antibodies against UTF1 and Oct4. Actin levels were determined to correct for gel loading. (D) Brightfield images of wt, Renilla luciferase KD (Renilla #1), and UTF1 KD (UTF1 #1) ES cells stained for AP activity. (E) Phase-contrast images of EBs from wt, Renilla luciferase KD (Renilla #1), and UTF1 KD (UTF1 #1 and #2) ES cells after 3, 5, and 7 d. (F) Phase-contrast and brightfield images of day 8 EBs from wt, Renilla luciferase KD (Renilla #1), and UTF1 KD (UTF1 #2) ES cells stained for AP activity. (G) Expression levels of markers for ES cells (UTF1, Oct4, REX1, and Nanog), ectoderm (FGF5 and GAP43), mesoderm (Brachyury and BMP5), and endoderm (GATA4 and GATA6) were measured by semiquantitative RT-PCR in undifferentiated ES cells and EBs cultured for 3, 5, and 10 d. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as a control. In the −RT lanes, reverse transcriptase was omitted from the reverse transcriptase reactions to control for genomic DNA contamination and amplified using glyceraldehyde-3-phosphate dehydrogenase primers. A representative experiment is depicted. The asterisk indicates that the 5- and 10-d GAP43 RT-PCR products were not loaded in adjacent lanes. Bars, 250 μm.