Biochem. J. (2012) 448, 21–33 (Printed in Great Britain) doi:10.1042/BJ20120725 21 DNA-dependent Oct4–Sox2 interaction and diffusion properties characteristic of the pluripotent cell state revealed by fluorescence spectroscopy Chen Sok LAM*, Tapan Kumar MISTRI†‡, Yong Hwee FOO†, Thankiah SUDHAHARAN*, Hui Theng GAN*, David RODDA‡, Leng Hiong LIM‡§, Chai CHOU‖, Paul ROBSON‡§, Thorsten WOHLAND† and Sohail AHMED* 1 *Neural Stem Cell Laboratory, Institute of Medical Biology, Singapore 138678, †Biophysical Fluorescence Laboratory, Department of Chemistry, National University of Singapore, Singapore 117543, ‡Developmental Cellomics Laboratory, Genome Institute of Singapore, Singapore 138672, §Department of Biological Sciences, National University of Singapore, Singapore 117543, and ‖Program in Neuroscience, DUKE-NUS Graduate Medical School, Singapore 169857 Oct4 and Sox2 are two essential transcription factors that co-regulate target genes for the maintenance of pluripotency. However, it is unclear whether they interact prior to DNA binding or how the target sites are accessed in the nucleus. By generating fluorescent protein fusions of Oct4 and Sox2 that are functionally capable of producing iPSCs (induced pluripotent stem cells), we show that their interaction is dependent on the presence of cognate DNA-binding elements, based on diffusion time, complex formation and lifetime measurements. Through fluorescence correlation spectroscopy, the levels of Oct4 and Sox2 in the iPSCs were quantified in live cells and two diffusion coefficients, corresponding to free and loosely bound forms of the protein, were distinguished. Notably, the fraction of slow- diffusing molecules in the iPSCs was found to be elevated, similar to the profile in embryonic stem cells, probably due to a change in the nuclear milieu during reprogramming. Taken together, these findings have defined quantitatively the amount of proteins pertinent to the pluripotent state and revealed increased accessibility to the underlying DNA as a mechanism for Oct4 and Sox2 to find their target binding sites and interact, without prior formation of heterodimer complexes. Key words: diffusion coefficient, fluorescence correlation spectroscopy (FCS), fluorescence lifetime microscopy (FLIM), F¨ orster resonance energy transfer (FRET), transcription factor, stem cell. INTRODUCTION Oct4 and Sox2 are two TFs (transcription factors) essential for maintaining the pluripotent cell state both in vitro in ESCs (embryonic stem cells) and in vivo within the developing mouse embryo [1,2]. By modulating the level of both of the TFs in ESCs using RNAi (RNA interference) or gene overexpression, it has been found that distinct cell fates pertinent to the germ layers could be acquired [3,4]. For instance, by increasing the expression level of Oct4 artificially above a certain threshold, ESCs were driven to endoderm and mesoderm lineages, whereas lowering the levels resulted in the loss of pluripotency and differentiation to trophectoderm [3]. Similarly, small increases in the level of Sox2 triggered the differentiation of ESCs into a wide range of differentiated cell types [4], whereas reducing the level of Sox2 promoted the differentiation of ESCs into trophectoderm- like cells [5]. Thus a narrow range for both Oct4 and Sox2 is required to maintain pluripotency, which otherwise will lead to the acquisition of divergent cell fates. The need for sustained ectopic expression of Oct4 and Sox2, together with other TFs, in somatic fibroblasts to obtain iPSCs (induced pluripotent stem cells) [6,7], further attest to a requirement for an appropriate expression level of the proteins to establish pluripotency. Intriguingly, little is known of the protein levels of Oct4 and Sox2 that constitutes the pluripotent cell state. The identification of several downstream molecular targets which are co-regulated by Oct4 and Sox2 underscores the importance of the interaction of these two proteins in establishing tightly regulated transcriptional networks associated with pluripotency [8]. Fgf4 (fibroblast growth factor 4) [9–11], Nanog [12–14] and Zfp206 [15] are salient examples of target genes that are directly regulated by Oct4 and Sox2. These genes possess consensus motifs that lie in close proximity for the binding of Oct4 and Sox2, leading to the idea that these two factors could interact with each other and synergize to control gene expression. In support of this idea, several thousand gene regulatory sites in the ESC genome were uncovered to be co-targeted by both of the TFs through ChIP (chromatin immunoprecipitation) studies [8,16]. Essentially, these target genes constitute one tightly controlled circuit, as the Oct4–Sox2 complex was found to activate Oct4 expression and Oct4 regulated Sox2 activity [5,17]. In turn, the level of Sox2 functioned as a molecular rheostat to control Oct4– Sox2 target genes [18]. Although the key function of the conserved POU and HMG (high-mobility group) domains in Oct4 and Sox2 respectively is to mediate DNA binding, these two domains are also known to selectively interact with one another so that a high- affinity ligand-binding complex is formed [19,20]. Moreover, specific residues on both Oct4 and Sox2 have been identified that affect its assembly and transcriptional activity [21,22]. Thus it is conceivable that Oct4–Sox2 heterodimerization could occur Abbreviations used: CCF, cross-correlation function; CHO, Chinese-hamster ovary; Cy5, indodicarbocyanine; EMSA, electrophoretic mobility-shift assay; ESC, embryonic stem cell; FBS, fetal bovine serum; FCCS, fluorescence cross-correlation spectroscopy; FCS, fluorescence correlation spectroscopy; Fgf4 /FGF4 , fibroblast growth factor 4; FLIM, fluorescence lifetime microscopy imaging; FP, fluorescent protein; FRET, F¨ orster resonance energy transfer; AP-FRET, acceptor-photobleaching FRET; GFP, green fluorescent protein; HMG, high-mobility group; iPSC, induced pluripotent stem cell; LIF, leukaemia- inhibitory factor; MEF, mouse embryonic fibroblast; mRFP, monomeric red FP; SW-FCCS, single-wavelength FCCS; TF, transcription factor. 1 To whom correspondence should be addressed (email sohail.ahmed@imb.a-star.edu.sg). c  The Authors Journal compilation c  2012 Biochemical Society