Dissecting pluripotency through the different OCT4 and SOX2 assembly configurations

The transcription factors OCT4 and SOX2 are required for generating induced pluripotent stem cells (iPSCs) and for maintaining embryonic stem cells (ESCs). To this end, OCT4 and SOX2 associate and bind to DNA in different configurations depending on the composite DNA element present in their target genes. Here, we have investigated the role of the different OCT4-SOX2 conformational arrangements in regulating and inducing pluripotency. To this end, we have generated SOX2 mutants that interfere with specific OCT4-SOX2 heterodimer configurations and we have assessed their abilities to generate iPSCs and to rescue ESC self-renewal. Our results demonstrate that the OCT4-SOX2 configuration that dimerizes on a Hoxb1-like composite, a canonical element with juxtaposed individual binding sites, plays a more critical role in the induction and maintenance of pluripotency than other OCT4-SOX2 configurations. Overall, the results of this study provide a new insight into the protein interactions required to establish a de novo pluripotent network and to maintain a true pluripotent cell fate. Sox2-null ESCs containing a doxycycline-inducible Sox2 transgene, a tet transactivator and a constitutive red-fluorescent protein transgene (DsRed) have been previously described. The different SOX2 mutants were cloned in the hygromycin-resistant PiggyBac plasmid pPBCAG-cHA-IH that is a modification of the previously reported pGG131 plasmid. 500 ng of each plasmid together with 500 ng of pCAG-pBase plasmid were co-transfected into Sox2-null ESCs using Lipofectamine 2000. Next, transfected cells were treated with doxycycline (1 µg/ml) and hygromycin (300 µg/ml). One week later, individual clones were picked and expanded. Finally, RNA was extracted for microarray analysis. 16 samples were analyzed: S113-4, Sox2-/- ES plus human Sox2 113 mutant clone 4 . in Piggy Bac, plus doxycycline, 2 replicates; S113-5, Sox2-/- ES plus human Sox2 113 mutant clone 5 overexpressed in Piggy Bac, plus doxycycline, 2 replicates; SWT-4, Sox2-/- ES plus human Sox2 WT mutant clone 4 overexpressed in Piggy Bac, plus doxycycline, 2 replicates; SWT-5, Sox2-/- ES plus human Sox2 WT mutant clone 5 overexpressed in Piggy Bac, plus doxycycline, 2 replicates; S95-1, Sox2-/- ES plus human Sox2 95 mutant clone 1 overexpressed in Piggy Bac, plus doxycycline, 2 replicates; S95-2, Sox2-/- ES plus human Sox2 95 mutant clone 2 overexpressed in Piggy Bac, plus doxycycline, 2 replicates; S95-4, Sox2-/- ES plus human Sox2 95 mutant clone 4 overexpressed in Piggy Bac, plus doxycycline, 2 replicates; TSS, Sox2-/- ES without doxycycline, so mouse Sox2 is expressed, 2 replicates.