Phenotypic Complementation Establishes Requirements for Specific POU Domain and Generic Transactivation Function of Oct-3/4 in Embryonic Stem Cells

Stem Cell Regulation Research, Area of Molecular Therapeutics, Course of Advanced Medicine, Osaka University Graduate School of Medicine, Suita C, Osaka 565-0871, Japan.
Molecular and Cellular Biology (Impact Factor: 4.78). 04/2002; 22(5):1526-36. DOI: 10.1128/MCB.22.5.1526-1536.2002
Source: PubMed


Transcription factors of the POU family govern cell fate through combinatorial interactions with coactivators and corepressors.
The POU factor Oct-3/4 can define differentiation, dedifferentation, or self-renewal of pluripotent embryonic stem (ES) cells
in a sensitive, dose-dependent manner (H. Niwa, J.-I. Miyazali, and A. G. Smith, Nat. Genet. 24:372-376, 2000). Here we have
developed a complementation assay based on the ability of Oct-3/4 transgenes to rescue self-renewal in conditionally null
ES cells and used this to define which domains of Oct-3/4 are required to sustain the undifferentiated stem cell phenotype.
Surprisingly, we found that molecules lacking either the N-terminal or C-terminal transactivation domain, though not both,
can effectively replace full-length Oct-3/4. Furthermore, a fusion of the heterologous transactivation domain of Oct-2 to
the Oct-3/4 POU domain can also sustain self-renewal. Thus, the unique function of Oct-3/4 in ES cell propagation resides
in combination of the specific POU domain with a generic proline-rich transactivation domain. Interestingly, however, Oct-3/4
target gene expression elicited by the N- and C-terminal transactivation domains is not identical, indicating that at least
one class of genes activated by Oct-3/4 is not required for ES cell propagation.

Download full-text


Available from: Jun-ichi Miyazaki, Jan 10, 2014
  • Source
    • "The pcDNA3-Sohlh2-Myc and pcDNA3-Sohlh1-Myc vectors were obtained by inserting mouse Sohlh2 and Sohlh1 cDNA, respectively, into the pcDNA3-Myc-His vector (Invitrogen, Carlsbad, CA; Cat#V855-20). Mouse Sp1 cDNA obtained from testis RNA by reverse transcription followed by PCR was inserted into a pCAG-IP plasmid vector [19]. All the PCR-amplified fragments were confirmed by sequencing. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Tissue-specific basic helix-loop-helix (bHLH) transcription factor proteins often play essential roles in cellular differentiation. The bHLH proteins SOHLH2 and SOHLH1 are expressed specifically in spermatogonia and oocytes and are required for early spermatogonial and oocyte differentiation. We previously reported that knocking out Sohlh2 causes defects in spermatogenesis and oogenesis similar to those in Sohlh1-null mice, and that Sohlh1 is downregulated in the gonads of Sohlh2-null mice. We also demonstrated that SOHLH2 and SOHLH1 can form a heterodimer. These observations led us to hypothesize that the SOHLH2/SOHLH1 heterodimer regulates the Sohlh1 promoter. Here, we show that SOHLH2 and SOHLH1 synergistically upregulate the Sohlh1 gene through E-boxes upstream of the Sohlh1 promoter. Interestingly, we identified an SP1-binding sequence, called a GC-box, adjacent to these E-boxes, and found that SOHLH1 could bind to SP1. Furthermore, chromatin-immunoprecipitation analysis using testes from mice on postnatal day 8 showed that SOHLH1 and SP1 bind to the Sohlh1 promoter region in vivo. Our findings suggest that an SOHLH2/SOHLH1/SP1 ternary complex autonomously and cooperatively regulates Sohlh1 gene transcription through juxtaposed E- and GC-boxes during early spermatogenesis and oogenesis.
    PLoS ONE 07/2014; 9(7):e101681. DOI:10.1371/journal.pone.0101681 · 3.23 Impact Factor
  • Source
    • "Immunophenotypic characterization of both cell types demonstrated the presence of the common, well-defined human mesenchymal stem cell markers CD90, CD44, CD73, CD166, CD105, CD29, CD13, CD49e, CD54, as well as the embryonic stem-cell markers TRA-1-60, TRA-1-81, SSEA-3 and -4 and STRO-1 (Bilic et al. 2008; Díaz- Prado et al. 2010; Stadler et al. 2008). In addition, amniotic epithelial cells express Oct-4 and Nanog, two transcription factors known to be required for selfrenewal and pluripotency (Niwa et al. 2002; Chambers et al. 2003). Unlike human embryonic stem cells, amnion epithelial cells do not express telomerase and are nontumorigenic upon transplantation. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Amniotic membrane (AM) is the innermost, multilayered part of the placenta. When harvested, processed and stored properly, its properties, stemming from AM biological composition, make it a useful tissue for ophthalmic surgery. AM was shown to have several beneficial effects: it promotes epithelization, has antimicrobial effects, decreases inflammation, fibrosis and neovascularization. Many case reports and case series as well as practical experience (e.g. reconstruction of conjunctival and corneal defects, treatment of corneal ulcers) demonstrated the beneficial effect of AM for different ophthalmological indications. The combination of the above mentioned beneficial effects and reasonable mechanical properties are also the reason why AM is used as a substrate for ex vivo expansion of epithelial progenitor cells. Recently, amnion-derived cells, which also have stem cell characteristics, have been proposed as potential contributors to cell-based treatment of ocular surface disease. However, the use of AM remains one of the least standardized methods in ophthalmic surgery. In this review, the various properties of AM and its current clinical use in ophthalmology in Slovenia are discussed.
    Cell and Tissue Banking 12/2013; 15(2). DOI:10.1007/s10561-013-9417-6 · 1.25 Impact Factor
  • Source
    • "Initial Methionine of all drug resistance genes in Egfp expression vectors were fused in frame to ATG sequence of the NcoI site in 3′ terminus of EMCV-IRES sequence derived from pCITE-1 (Novagen). pCAG-IP and pCAG-IZ plasmid was constructed for puromycin and zeocin selection as described [9,28]. pCAG-IB was constructed by replacing the NcoI-XbaI fragment in pCAG-IZ with the BsaI-SpeI fragment containing the bsd gene derived from pUC-SV-BSD (Funakoshi). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Stable expression of transgenes is an important technique to analyze gene function. Various drug resistance genes, such as neo, pac, hph, zeo, bsd, and hisD, have been equally used as selection markers to isolate a transfectant without considering their dose-dependent characters. We quantitatively measured the variation of transgene expression levels in mouse embryonic stem (mES) cells, using a series of bi-cistronic expression vectors that contain Egfp expression cassette linked to each drug resistant gene via IRES with titration of the selective drugs, and found that the transgene expression levels achieved in each system with this vector design are in order, in which pac and zeo show sharp selection of transfectants with homogenously high expression levels. We also showed the importance of the choice of the drug selection system in gene-trap or gene targeting according to this order. The results of the present study clearly demonstrated that an appropriate choice of the drug resistance gene(s) is critical for a proper design of the experimental strategy.
    BMC Biotechnology 08/2013; 13(1):64. DOI:10.1186/1472-6750-13-64 · 2.03 Impact Factor
Show more