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. Author manuscript; available in PMC: 2016 Jun 1.
Published in final edited form as: Stem Cells. 2015 Jun;33(6):1705–1718. doi: 10.1002/stem.1993

Figure 4. YAP1 regulates Sox2 gene expression through Oct4 transcription factor.

Figure 4

(A) A schematic representation of Sox2 proximal core promoter and enhancer region 1 showing potential TEAD2 binding sites represented as clear ellipses and Oct4 binding sites as filled ellipses. The arrows represent the position of primers spanning the TEAD2 and Oct4 binding sites tested for ChIP assays. (B) ChIP assays conducted on asynchronously growing A549 and H1650 cells with the indicated antibodies show presence of YAP1 on SRR1 region. Acetylated Histone H3 (Lys9) was used as positive control and non-specific IgG was used as the negative control for immunoprecipitation. (C) Transient transfection experiments in A549 and H1650 cells with co-transfection with YAP1 and Oct4 expression vectors showed an additive effect on SRR1 enhancer-luc activity. Control lanes had the reporter with empty vector. (D) Presence of YAP1 on Sox2 core promoter and SRR1 enhancer region through Oct4 binding site is confirmed by ChIP assays conducted on asynchronously growing A549 and H1650 cells using the indicated antibodies. (E) Transient transfection assays in A549 and H1650 cells transfected with YAP1 or Oct4 expression vectors showed an increase in Sox2 core promoter-luc activity. An additive effect on Sox2 core promoter-luc activity is observed when YAP1 and Oct4 are co-expressed. (F) Sox2 core promoter-luc activity is reduced in both A549 and H1650 cells upon depletion of Oct4 using siRNA in the presence of exogenous YAP1; transfection of TEAD2, TEAD3 and TEAD4 siRNA had no significant effect. (G) Transient transfections in YAP1 depleted cells with siRNA treatment show reduction in Sox2 core promoter-luc activity with Oct4 overexpression as compared to non-target control siRNA treated A549 and H1650 cells. (H) Transient transfections of Sox2 core promoter-luc with mutated Oct4 binding (mutSox2-luc) site exhibit significantly reduced promoter activity even with increased exogenous YAP1 and Oct4 expression. The above data is expressed as mean ± SD of three independent experiments. * represents p < 0.05, ** represents p < 0.01.