Rescue of stem cell self-renewal by wild-type Oct-3/4 and the 267V/P mutant lacking DNA-binding ability. (A) Diagram of wild-type and mutant gene products. N-TD, C-TD, and the POU domain are represented as boxes. The point mutation in the HA267V/P mutant is positioned in the POU homeodomain. Both cDNAs have an in-frame HA tag upstream of the start codon. Rescue index is scored as in Table 3. (B) Western blot and EMSA analysis of nuclear extracts prepared from BMT10 cells transfected with the HAwt or HA267V/P expression vector. Production of comparable amounts of transgene products was confirmed in each transfectant by immunoblotting with anti-HA antibody (upper panel). EMSA analysis of the same nuclear extracts with the DIG-labeled probe confirmed that HA267V/P binds to octamer motif with much lower efficiency than Oct3/4wt (lower panel). Equal loading of nuclear extracts was confirmed by the intensity of shift bands corresponding to endogenous Oct-1. (C) Western blot analysis of nuclear extracts prepared from the rescued and unrescued ES cells transfected with HAwt or HA267V/P. Similar amounts of transgene product with HA tag were detected by anti-HA antibody.

Rescue of stem cell renewal by EGFP fusion constructs. (A) Diagram of EGFP fusion constructs with wild-type and mutant gene products. The rescue index was determined as in Table 3. (B) Western blot analysis of nuclear extracts prepared from rescued transfectants with EGFPwt and EGFPΔN grown in the presence of Tc and unrescued transfectants with EGFPΔNΔC grown in the absence of Tc. Anti-Oct3/4N antibody detects wild-type Oct-3/4 in the latter but not the former, confirming the rescue events in EGFPwt and EGFPΔN transfectants. (C) Western blot analysis of the blot used in panel B with anti-GFP antibody. Proper production of EGFP fusion proteins was confirmed. (D) Photomicrographs of phase-contrast and fluorescent views of a stem cell colony rescued by the expression of EGFPwt (top) and of nucleus-localized EGFPΔNΔC in a stem cell colony grown in the absence of Tc (bottom). Nuclear fluorescence from EGFPwt is retained in differentiated cells produced after withdrawal of LIF (middle).

Comparison of gene expression pattern in rescued ES cells. (A) Expression of Oct-3/4 target genes in ZHBTc4 ES cells and rescued ES cells with wild-type, ΔN, or ΔC. Total RNAs prepared from these ES cells were blotted on nylon membranes, and replicate filters were analyzed by nonradioactive filter hybridization with the indicated cDNA probes. Gap was used as a loading control. (B) Expression of Ebaf/Lefty1 in ES cells rescued by Δ2-62ΔC+Oct2CTD and Δ2-62ΔC+1-62 mutant. Sox2 was used as a loading control.

Episomal expression of various mutants of Oct-3/4 in MG1.19 ES cells. The bar chart shows the proportion of undifferentiated colonies obtained after supertransfection of episomal vectors for expression of the indicated mutants and selection for 8 days in puromycin. Supertransfection of Oct-3/4wt and most mutants gave predominantly differentiated colonies. Mutants lacking both transactivation domains failed to induce significant differentiation.

Oct-3/4 complementation assay. (A) Schematic of complementation assay system. ZHBTc4 ES cells (29) contain a Tc-regulated Oct-3/4 transgene and both alleles of endogenous Oct-3/4 have been inactivated by targeted integration of IRESzeopA and IRESBSDpA cassettes. The Oct-3/4 transgene is repressed by addition of Tc, so stem cell colonies can form in the presence of Tc only when an introduced transgene in the pCAG-IP vector can replicate the function of Oct-3/4 and sustain stem cell renewal. Abbreviations: BSD, Aspergillus blasticidin S deaminase; zeo, zeocin resistance gene; IRES, encephalomyocarditis virus internal ribosome entry site; hph, hygromycin phosphotransferase; CAG, CAG expression unit; tTA, Tc-dependent transactivator; β-geo, fusion gene between the Escherichia coli genes for β-galactosidase and neomycin phosphotransferase; pac, puromycin acetyltransferase; Ori, DNA replication origin. (B) Photomicrographs of ZHBTc4 ES cells cultured in the absence of Tc (left upper, undifferentiated), ZHBTc4 cells cultured in the presence of Tc (right upper, differentiated), ZHBTc4 cells transfected with the Oct3/4wt expression vector cultured in the presence of Tc (left lower, undifferentiated), and the rescued ZHBTc4 Oct-3/4wt transfectants transferred to medium lacking Tc (right lower, differentiated). (C) Expression of Oct-3/4 transgenes in ZHBTc4 ES cells transfected with the wild-type (wt) expression vector. ZHBTc4 cells maintained in the absence of Tc express full-length and truncated mRNAs derived from the Oct-3/4βgeo transgene (29) and lack endogenous Oct-3/4 transcripts. Oct-3/4wt transfectants grown with Tc lack transcripts derived from the Oct-3/4βgeo transgene and express the Oct-3/4IP transgene only. Oct-3/4wt transfectants grown without Tc express transcripts derived from both transgenes. EB3 was maintained by the endogenous Oct-3/4.

Rescue of stem cell renewal by deletion mutants. (A) Diagram of wild-type and mutant gene products. ΔN and ΔC lack aa 3 to 129 and 283 to 352, respectively, and ΔNΔC consists of aa 130 to 282. The rescue index was determined as in Table 3. (B) Western blot analysis of nuclear extracts prepared from rescued transfectants with wild-type, ΔN, and ΔC grown in the presence of Tc and unrescued transfectants with ΔNΔC grown in the absence of Tc with anti-Oct3/4N antibody. The rescued cells with ΔN or ΔC lack wild-type Oct-3/4 protein. Mutant protein of ΔC but not ΔN and ΔNΔC can be detected by anti-Oct3/4N antibody. Two different sizes of bands are occasionally detected with anti-Oct3/4N antibody. The upper band appears to represent the phosphorylated form, because this signal can be intensified by preparation of nuclear extracts in the presence of phosphatase inhibitors (PI). We always detected a single ΔC band, because the phosphorylation sites of Oct-3/4 are located in the C terminus (6, 35). (C) EMSA analysis of nuclear extracts analyzed in panel B with the Cy5-labeled probe. All mutants can bind to the octamer motif with comparable intensity to the wild-type Oct-3/4. The signal for ΔNΔC is weaker in lane 4, but it expressed an amount comparable to the others in unrescued cells selected with 9 μg of puromycin per ml in lane 5.

Rescue of stem cell renewal by combination of the POU domain with transactivation domains. Diagram of mutant gene products and the rescue index for them are shown. The rescue index was determined as in Table 3.

Transactivation ability of mutant proteins. (A) Oct-3/4-dependent activation of reporter constructs. Luciferase reporter plasmids carrying various regulatory elements were transfected into ZHBTc4 ES cells in the presence or absence of Tc. The data were calibrated by both pRL-CMV and PGK-luc, and the ratio for PGK was set at 1.0. Fgf4-5", Fgf4-3", 6W, 052, and Rex1 showed severalfold-higher ratios, confirming their Oct-3/4-dependent activation in ES cells. (B) Expression of luciferase reporters in rescued ES cells. Reporter plasmids tested in A were transfected into ES cells rescued by wild-type, ΔN, or ΔC, and their activities were estimated. The data were calibrated by both pRL-CMV and PGK-luc, and the ratio for PGK was set at 1.0 as above. Note the lower magnitude of activation of 6W in ΔN than in the others.