How regulatory information is encoded in the genome is poorly understood and poses a challenge when studying biological processes. We demonstrate here that genomic redistribution of Oct4 by alternative partnering with Sox2 and Sox17 is a fundamental regulatory event of endodermal specification. We show that Sox17 partners with Oct4 and binds to a unique ‘compressed’ Sox/Oct motif that earmarks endodermal genes. This is in contrast to the pluripotent state where Oct4 selectively partners with Sox2 at ‘canonical’ binding sites. The distinct selection of binding sites by alternative Sox/Oct partnering is underscored by our demonstration that rationally point‐mutated Sox17 partners with Oct4 on pluripotency genes earmarked by the canonical Sox/Oct motif. In an endodermal differentiation assay, we demonstrate that the compressed motif is required for proper expression of endodermal genes. Evidently, Oct4 drives alternative developmental programs by switching Sox partners that affects enhancer selection, leading to either an endodermal or pluripotent cell fate. This work provides insights in understanding cell fate transcriptional regulation by highlighting the direct link between the DNA sequence of an enhancer and a developmental outcome.

Leukemia inhibitory factor (LIF) activates the transcription factor signal transducer and activator of transcription 3 (STAT3), which results in the maintenance of mouse embryonic stem cells in the pluripotent state by inhibiting both mesodermal and endodermal differentiation. How the LIF/STAT3 pathway inhibits commitment to both mesoderm and endoderm lineages is presently unknown. Using a hormone-dependent STAT3 and with microarray analysis, we identified 58 targets of STAT3 including 20 unknown genes. Functional analysis showed that 22 among the 23 STAT3 target genes analyzed contribute to the maintenance of the undifferentiated state, as evidenced by an increase in the frequency of differentiated colonies in a self-renewal assay and a concomitant elevation of early differentiation markers upon knockdown. Fourteen of them, including Dact1, Klf4, Klf5, Rgs16, Smad7, Ccrn4l, Cnnm1, Ocln, Ier3, Pim1, Cyr61, and Sgk, were also regulated by Nanog. Analysis of lineage-specific markers showed that the STAT3 target genes fell into three distinct categories, depending on their capacity to inhibit either mesoderm or endoderm differentiation or both. The identification of genes that harness self-renewal and are downstream targets of both STAT3 and Nanog shed light on the mechanisms underlying functional redundancy between STAT3 and Nanog in mouse embryonic stem cells.

Leukemia inhibitory factor (LIF)/STAT3 signalling is a hallmark of naive pluripotency in rodent pluripotent stem cells (PSCs), whereas fibroblast growth factor (FGF)-2 and activin/nodal signalling is required to sustain self-renewal of human PSCs in a condition referred to as the primed state. It is unknown why LIF/STAT3 signalling alone fails to sustain pluripotency in human PSCs. Here we show that the forced expression of the hormone-dependent STAT3-ER (ER, ligand-binding domain of the human oestrogen receptor) in combination with 2i/LIF and tamoxifen allows human PSCs to escape from the primed state and enter a state characterized by the activation of STAT3 target genes and long-term self-renewal in FGF2- and feeder-free conditions. These cells acquire growth properties, a gene expression profile and an epigenetic landscape closer to those described in mouse naive PSCs. Together, these results show that temporarily increasing STAT3 activity is sufficient to reprogramme human PSCs to naive-like pluripotent cells.

Very few proteins are capable to induce pluripotent stem (iPS) cells and their biochemical uniqueness remains unexplained. For example, Sox2 cooperates with other transcription factors to generate iPS cells, but Sox17, despite binding to similar DNA sequences, cannot. Here, we show that Sox2 and Sox17 exhibit inverse heterodimerization preferences with Oct4 on the canonical versus a newly identified compressed sox/oct motif. We can swap the cooperativity profiles of Sox2 and Sox17 by exchanging single amino acids at the Oct4 interaction interface resulting in Sox2KE and Sox17EK proteins. The reengineered Sox17EK now promotes reprogramming of somatic cells to iPS, whereas Sox2KE has lost this potential. Consistently, when Sox2KE is overexpressed in embryonic stem cells it forces endoderm differentiation similar to wild-type Sox17. Together, we demonstrate that strategic point mutations that facilitate Sox/Oct4 dimer formation on variant DNA motifs lead to a dramatic swap of the bioactivities of Sox2 and Sox17. STEM CELLS 2011;29:940-951 Disclosure of potential conflicts of interest is found at the end of this article.

Krüppel-like factors (Klf) 4 and 5 are two closely related members of the Klf family, known to play key roles in cell cycle regulation, somatic cell reprogramming and pluripotency. Here we focus on the functional divergence between Klf4 and Klf5 in the inhibition of mouse embryonic stem (ES) cell differentiation. Using microarrays and chromatin immunoprecipitation coupled to ultra-high-throughput DNA sequencing, we show that Klf4 negatively regulates the expression of endodermal markers in the undifferentiated ES cells, including transcription factors involved in the commitment of pluripotent stem cells to endoderm differentiation. Knockdown of Klf4 enhances differentiation towards visceral and definitive endoderm. In contrast, Klf5 negatively regulates the expression of mesodermal markers, some of which control commitment to the mesoderm lineage, and knockdown of Klf5 specifically enhances differentiation towards mesoderm. We conclude that Klf4 and Klf5 differentially inhibit mesoderm and endoderm differentiation in murine ES cells.

ABSTRACTpim-1 and pim-3 encode serine/threonine kinases involved in the regulation of cell proliferation and apoptosis in response to cytokine stimulation. We analyzed the regulation of pim-1 and pim-3 by the leukemia inhibitory factor (LIF)/gp130/signal transducer and activator of transcription-3 (STAT3) pathway and the role of Pim-1 and Pim-3 kinases in mouse embryonic stem (ES) cell self-renewal. Making use of ES cells expressing a granulocyte colony-stimulating factor:gp130 chimeric receptor and a hormone-dependent signal transducer and activator of transcription-3 estrogen receptor (STAT3-ER T2 ), we showed that expression of pim-1 and pim-3 was upregulated by LIF/gp130-dependent signaling and the STAT3 transcription factor. ES cells overexpressing pim-1 and pim-3 had a greater capacity to self-renew and displayed a greater resistance to LIF starvation based on a clonal assay. In contrast, knockdown of pim-1 and pim-3 increased the rate of spontaneous differentiation in a self-renewal assay. Knockdown of pim-1 and pim-3 was also detrimental to the growth of undifferentiated ES cell colonies and increased the rate of apoptosis. These findings provide a novel role of Pim-1 and Pim-3 kinases in the control of self-renewal of ES cells. STEM CELLS 2007;25:2996 -3004 Disclosure of potential conflicts of interest is found at the end of this article.

The unique ability of Sox2 to cooperate with Oct4 at selective binding sites in the genome is critical for reprogramming somatic cells into induced pluripotent stem cells (iPSCs). We have recently demonstrated that Sox17 can be converted into a reprogramming factor by alteration of a single amino acid (Sox17EK) within its DNA binding HMG domain. Here we expanded this study by introducing analogous mutations to 10 other Sox proteins and interrogated the role of N-and C-termini on the reprogramming efficiency. We found that point-mutated Sox7 and Sox17 can convert human and mouse fibroblasts into iPSCs, but Sox4, Sox5, Sox6, Sox8, Sox9, Sox11, Sox12, Sox13, and Sox18 cannot. Next we studied regions outside the HMG domain and found that the C-terminal transactivation domain of Sox17 and Sox7 enhances the potency of Sox2 in iPSC assays and confers weak reprogramming potential to the otherwise inactive Sox4EK and Sox18EK proteins. These results suggest that the glutamate (E) to lysine (K) mutation in the HMG domain is necessary but insufficient to swap the function of Sox factors. Moreover, the HMG domain alone fused to the VP16 transactivation domain is able to induce reprogramming, albeit at low efficiency. By molecular dissection of the C-terminus of Sox17, we found that the b-catenin interaction region contributes to the enhanced reprogramming efficiency of Sox17EK. To mechanistically understand the enhanced reprogramming potential of Sox17EK, we analyzed ChIPsequencing and expression data and identified a subset of candidate genes specifically regulated by Sox17EK and not by Sox2.

Delineating candidate genes at the chromosomal breakpoint regions in the apparently balanced chromosome rearrangements (ABCR) has been shown to be more effective with the emergence of next-generation sequencing (NGS) technologies. We employed a large-insert (7–11 kb) paired-end tag sequencing technology (DNA-PET) to systematically analyze genome of four patients harbouring cytogenetically defined ABCR with neurodevelopmental symptoms, including developmental delay (DD) and speech disorders. We characterized structural variants (SVs) specific to each individual, including those matching the chromosomal breakpoints. Refinement of these regions by Sanger sequencing resulted in the identification of five disrupted genes in three individuals: guanine nucleotide binding protein, q polypeptide (GNAQ), RNA-binding protein, fox-1 homolog (RBFOX3), unc-5 homolog D (C.elegans) (UNC5D), transmembrane protein 47 (TMEM47), and X-linked inhibitor of apoptosis (XIAP). Among them, XIAP is the causative gene for the immunodeficiency phenotype seen in the patient. The remaining genes displayed specific expression in the fetal brain and have known biologically relevant functions in brain development, suggesting putative candidate genes for neurodevelopmental phenotypes. This study demonstrates the application of NGS technologies in mapping individual gene disruptions in ABCR as a resource for deciphering candidate genes in human neurodevelopmental disorders (NDDs).