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. 2014 Apr 1;16(2):130–139. doi: 10.1089/cell.2013.0070

FIG. 3.

FIG. 3.

Butyrate enhances transcriptional activity of Oct4 via transactivation domains. (A) Schematic structure of full-length Oct4 and VP16-tagged Oct4 DNA-binding domain. Human Oct4 consists of 360 amino acids. Amino-terminal domain (ND), DNA-binding domain (DBD), and carboxy-terminal domain (CD) are located in amino-terminal, middle, and carboxy-terminal region of Oct4, respectively. Numbers indicate those of amino acid residues from the amino-terminal portion. The Oct4 DBD was fused in-frame with VP16 (activation domain) by PCR, and the resulting construct was designated as pVP16-Oct4DBD. Numbers indicate amino acid positions of Oct4. (B) Effect of NaB on the DNA-binding activity of Oct4. A 100-ng amount of the (Oct4-RE)3-TATA-Luc reporter plasmid was transfected together with 350 ng of the pVP16-Oct4 DBD plasmid (or pVP16 vector) and 50 ng of pCMV-LacZ into 293T cells. Transfected cells were treated with or without 3-SM cocktail or NaB for 48 h before luciferase activity was measured and normalized by LacZ activity. Fold change in luciferase activity (right panel) was calculated (treatment vs. without treatment). (C) Schematic maps of Oct4 mutants fused to Gal4 DBD. Full-length Oct4 or Oct4 mutants containing ND, CD, or both of ND and CD were fused in-frame to the carboxyl terminus of the Gal4 DBD in the pM2 vector and designated as Gal4-Oct4, Gal4-Oct4-ND, Gal4-Oct4-CD, and Gal4-NCD, respectively. (D) Effect of small molecules on the transcriptional activity of Oct4 mutants. 293T cells were transfected with 350 ng of Gal4-Oct4 mutants (or pM2 vector), 100 ng of pFR-Luc consisting of five copies of Gal4-binding sites, and 50 ng of pCMV-LacZ. pCMV-LacZ was included in each transfection as an internal control. Relative luciferase activity was assayed 48 h after transfection and normalized by LacZ activity (left panel). Percentage increases in luciferase activity (right panel) were calculated by comparing relative luciferase activity for full-length Oct4 or each Oct4 mutant (treatment vs. without treatment). (*) p<0.05, (**) p<0.01.