Design and Characterization of a Cell-Penetrating Peptide Derived from the SOX2 Transcription Factor.

SOX2 is an oncogenic transcription factor overexpressed in nearly half of the basal-like triple-negative breast cancers associated with very poor outcomes. Targeting and inhibiting SOX2 is clinically relevant as high SOX2 mRNA levels are positively correlated with decreased overall survival and progression-free survival in patients affected with breast cancer. Given its key role as a master regulator of cell proliferation, SOX2 represents an important scaffold for the engineering of dominant-negative synthetic DNA-binding domains (DBDs) that act by blocking or interfering with the oncogenic activity of the endogenous transcription factor in cancer cells. We have synthesized an interference peptide (iPep) encompassing a truncated 24 amino acid long C-terminus of SOX2 containing a potential SOX-specific nuclear localization sequence, and the determinants of the binding of SOX2 to the DNA and to its transcription factor binding partners. We found that the resulting peptide (SOX2-iPep) possessed intrinsic cell penetration and promising nuclear localization into breast cancer cells, and decreased cellular proliferation of SOX2 overexpressing cell lines. The novel SOX2-iPep was found to exhibit a random coil conformation predominantly in solution. Molecular dynamics simulations were used to characterize the interactions of both the SOX2 transcription factor and the SOX2-iPep with FGF4-enhancer DNA in the presence of the POU domain of the partner transcription factor OCT4. Predictions of the free energy of binding revealed that the iPep largely retained the binding affinity for DNA of parental SOX2. This work will enable the future engineering of novel dominant interference peptides to transport different therapeutic cargo molecules such as anti-cancer drugs into cells.

Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions.

Liquid-liquid phase separation of proteins underpins the formation of membraneless compartments in living cells. Elucidating the molecular driving forces underlying protein phase transitions is therefore a key objective for understanding biological function and malfunction. Here we show that cellular proteins, which form condensates at low salt concentrations, including FUS, TDP-43, Brd4, Sox2, and Annexin A11, can reenter a phase-separated regime at high salt concentrations. By bringing together experiments and simulations, we demonstrate that this reentrant phase transition in the high-salt regime is driven by hydrophobic and non-ionic interactions, and is mechanistically distinct from the low-salt regime, where condensates are additionally stabilized by electrostatic forces. Our work thus sheds light on the cooperation of hydrophobic and non-ionic interactions as general driving forces in the condensation process, with important implications for aberrant function, druggability, and material properties of biomolecular condensates.

SOX2 is required independently in both stem and differentiated cells for pituitary tumorigenesis in p27-null mice.

P27, a cell cycle inhibitor, is also able to drive repression of Sox2 This interaction plays a crucial role during development of p27-/- pituitary tumors because loss of one copy of Sox2 impairs tumorigenesis [H. Li et al., Cell Stem Cell 11, 845-852 (2012)]. However, SOX2 is expressed in both endocrine and stem cells (SCs), and its contribution to tumorigenesis in either cell type is unknown. We have thus explored the cellular origin and mechanisms underlying endocrine tumorigenesis in p27-/- pituitaries. We found that pituitary hyperplasia is associated with reduced cellular differentiation, in parallel with increased levels of SOX2 in stem and endocrine cells. Using conditional loss-of-function and lineage tracing approaches, we show that SOX2 is required cell autonomously in p27-/- endocrine cells for these to give rise to tumors, and in SCs for promotion of tumorigenesis. This is supported by studies deleting the Sox2 regulatory region 2 (Srr2), the target of P27 repressive action. Single cell transcriptomic analysis further reveals that activation of a SOX2-dependent MAPK pathway in SCs is important for tumorigenesis. Altogether, our data highlight different aspects of the role of SOX2 following loss of p27, according to cellular context, and uncover an unexpected SOX2-dependent tumor-promoting role for SCs. Our results imply that targeting SCs, in addition to tumor cells, may represent an efficient antitumoral strategy in certain contexts.

Structural basis for nuclear import selectivity of pioneer transcription factor SOX2.

SOX (SRY-related HMG-box) transcription factors perform critical functions in development and cell differentiation. These roles depend on precise nuclear trafficking, with mutations in the nuclear targeting regions causing developmental diseases and a range of cancers. SOX protein nuclear localization is proposed to be mediated by two nuclear localization signals (NLSs) positioned within the extremities of the DNA-binding HMG-box domain and, although mutations within either cause disease, the mechanistic basis has remained unclear. Unexpectedly, we find here that these two distantly positioned NLSs of SOX2 contribute to a contiguous interface spanning 9 of the 10 ARM domains on the nuclear import adapter IMPα3. We identify key binding determinants and show this interface is critical for neural stem cell maintenance and for Drosophila development. Moreover, we identify a structural basis for the preference of SOX2 binding to IMPα3. In addition to defining the structural basis for SOX protein localization, these results provide a platform for understanding how mutations and post-translational modifications within these regions may modulate nuclear localization and result in clinical disease, and also how other proteins containing multiple NLSs may bind IMPα through an extended recognition interface.

In Silico Identification of SOX1 Post-Translational Modifications Highlights a Shared Protein Motif.

The transcription factor SOX1 is a key regulator of neural stem cell development, acting to keep neural stem cells (NSCs) in an undifferentiated state. Postnatal expression of Sox1 is typically confined to the central nervous system (CNS), however, its expression in non-neural tissues has recently been implicated in tumorigenesis. The mechanism through which SOX1 may exert its function is not fully understood, and studies have mainly focused on changes in SOX1 expression at a transcriptional level, while its post-translational regulation remains undetermined. To investigate this, data were extracted from different publicly available databases and analysed to search for putative SOX1 post-translational modifications (PTMs). Results were compared to PTMs associated with SOX2 in order to identify potentially key PTM motifs common to these SOXB1 proteins, and mapped on SOX1 domain structural models. This approach identified several putative acetylation, phosphorylation, glycosylation and sumoylation sites within known functional domains of SOX1. In particular, a novel SOXB1 motif (xKSExSxxP) was identified within the SOX1 protein, which was also found in other unrelated proteins, most of which were transcription factors. These results also highlighted potential phospho-sumoyl switches within this SOXB1 motif identified in SOX1, which could regulate its transcriptional activity. This analysis indicates different types of PTMs within SOX1, which may influence its regulatory role as a transcription factor, by bringing changes to its DNA binding capacities and its interactions with partner proteins. These results provide new research avenues for future investigations on the mechanisms regulating SOX1 activity, which could inform its roles in the contexts of neural stem cell development and cancer.

Investigation of the thermodynamic drivers of the interaction between the high mobility group box domain of Sox2 and bacterial lipopolysaccharide.

Gastric cancer is associated with high mortality and is preceded by an infection with Helicobacter pylori (H. pylori). H. pylori stimulates inflammation which involves the activation of Toll-like receptor 4 by lipopolysaccharide molecules from the H. pylori. This leads to chronic inflammation that can eventually lead to gastric cancer. Sox2 is a member of the high mobility group (HMG) box family of proteins, and recent studies have shown that HMG box proteins can modulate immune response by altering signaling to Toll-like receptors. Sox2 is overexpressed in most types of cancer with the exception of gastric cancer where expression of Sox2 is decreased. Here, we demonstrate that Sox2 can bind LPS and we investigated the thermodynamic drivers of the Sox2/LPS interaction.

The role of GLI-SOX2 signaling axis for gemcitabine resistance in pancreatic cancer.

Pancreatic cancer, mostly pancreatic ductal adenocarcinomas (PDAC), is one of the most lethal cancers, with a dismal median survival around 8 months. PDAC is notoriously resistant to chemotherapy. Thus far, numerous attempts using novel targeted therapies and immunotherapies yielded limited clinical benefits for pancreatic cancer patients. It is hoped that delineating the molecular mechanisms underlying drug resistance in pancreatic cancer may provide novel therapeutic options. Using acquired gemcitabine resistant pancreatic cell lines, we revealed an important role of the GLI-SOX2 signaling axis for regulation of gemcitabine sensitivity in vitro and in animal models. Down-regulation of GLI transcriptional factors (GLI1 or GLI2), but not SMO signaling inhibition, reduces tumor sphere formation, a characteristics of tumor initiating cell (TIC). Down-regulation of GLI transcription factors also decreased expression of TIC marker CD24. Similarly, high SOX2 expression is associated with gemcitabine resistance whereas down-regulation of SOX2 sensitizes pancreatic cancer cells to gemcitabine treatment. We further revealed that elevated SOX2 expression is associated with an increase in GLI1 or GLI2 expression. Our ChIP assay revealed that GLI proteins are associated with a putative Gli binding site within the SOX2 promoter, suggesting a more direct regulation of SOX2 by GLI transcription factors. The relevance of our findings to human disease was revealed in human cancer specimens. We found that high SOX2 protein expression is associated with frequent tumor relapse and poor survival in stage II PDAC patients (all of them underwent gemcitabine treatment), indicating that reduced SOX2 expression or down-regulation of GLI transcription factors may be effective in sensitizing pancreatic cancer cells to gemcitabine treatment.

Increased transactivation and impaired repression of ß-catenin-mediated transcription associated with a novel SOX3 missense mutation in an X-linked hypopituitarism pedigree with modest growth failure.

SOX3, a transcription factor of the SRY-related high mobility group box family, has been implicated in the etiology of X-linked hypopituitarism. Here, we report a Chinese pedigree of X-linked hypopituitarism with variable phenotypes. Despite the complete growth hormone deficiency, the growth failure of the patients was relatively modest. A rare point variant of SOX3 (c.424C > A; p. P142T) was identified in the pedigree via target panel sequencing. An in vitro study showed that both the expression and nuclear targeting of SOX3 remained unaffected by the variant. However, increased transcriptional activation and impaired repression of ß-catenin-mediated transcription were noticed as a result of the SOX3 variant. This is the first study to report that the rare SOX3 missense variant associated with hypopituitarism possibly due to increased activation of SOX3 target genes and disregulation of ß-catenin target genes. In addition, we have expanded the phenotypic spectrum associated with SOX3 mutations.

LSD1 demethylase and the methyl-binding protein PHF20L1 prevent SET7 methyltransferase-dependent proteolysis of the stem-cell protein SOX2.

The pluripotency-controlling stem-cell protein SRY-box 2 (SOX2) plays a pivotal role in maintaining the self-renewal and pluripotency of embryonic stem cells and also of teratocarcinoma or embryonic carcinoma cells. SOX2 is monomethylated at lysine 119 (Lys-119) in mouse embryonic stem cells by the SET7 methyltransferase, and this methylation triggers ubiquitin-dependent SOX2 proteolysis. However, the molecular regulators and mechanisms controlling SET7-induced SOX2 proteolysis are unknown. Here, we report that in human ovarian teratocarcinoma PA-1 cells, methylation-dependent SOX2 proteolysis is dynamically regulated by the LSD1 lysine demethylase and a methyl-binding protein, PHD finger protein 20-like 1 (PHF20L1). We found that LSD1 not only removes the methyl group from monomethylated Lys-117 (equivalent to Lys-119 in mouse SOX2), but it also demethylates monomethylated Lys-42 in SOX2, a reaction that SET7 also regulated and that also triggered SOX2 proteolysis. Our studies further revealed that PHF20L1 binds both monomethylated Lys-42 and Lys-117 in SOX2 and thereby prevents SOX2 proteolysis. Down-regulation of either LSD1 or PHF20L1 promoted SOX2 proteolysis, which was prevented by SET7 inactivation in both PA-1 and mouse embryonic stem cells. Our studies also disclosed that LSD1 and PHF20L1 normally regulate the growth of pluripotent mouse embryonic stem cells and PA-1 cells by preventing methylation-dependent SOX2 proteolysis. In conclusion, our findings reveal an important mechanism by which the stability of the pluripotency-controlling stem-cell protein SOX2 is dynamically regulated by the activities of SET7, LSD1, and PHF20L1 in pluripotent stem cells.

KSHV SOX mediated host shutoff: the molecular mechanism underlying mRNA transcript processing.

Onset of the lytic phase in the KSHV life cycle is accompanied by the rapid, global degradation of host (and viral) mRNA transcripts in a process termed host shutoff. Key to this destruction is the virally encoded alkaline exonuclease SOX. While SOX has been shown to possess an intrinsic RNase activity and a potential consensus sequence for endonucleolytic cleavage identified, the structures of the RNA substrates targeted remained unclear. Based on an analysis of three reported target transcripts, we were able to identify common structures and confirm that these are indeed degraded by SOX in vitro as well as predict the presence of such elements in the KSHV pre-microRNA transcript K12-2. From these studies, we were able to determine the crystal structure of SOX productively bound to a 31 nucleotide K12-2 fragment. This complex not only reveals the structural determinants required for RNA recognition and degradation but, together with biochemical and biophysical studies, reveals distinct roles for residues implicated in host shutoff. Our results further confirm that SOX and the host exoribonuclease Xrn1 act in concert to elicit the rapid degradation of mRNA substrates observed in vivo, and that the activities of the two ribonucleases are co-ordinated.

Mitotic phosphorylation of SOX2 mediated by Aurora kinase A is critical for the stem-cell like cell maintenance in PA-1 cells.

Transcription factor SOX2 is multiple phosphorylated. However, the kinase and the timing regulating SOX2 phosphorylation remains poorly understood. Here we reported mitotic phosphorylation of SOX2 by Aurora kinase A (AURKA). AURKA inhibitors (VX680, Aurora kinase Inhibitor I) but not PLK1 inhibitors (BI2536, CBB2001) eliminate the mitotic phosphorylation of SOX2. Consistently, siRNA inhibition of AURKA can eliminate mitotic SOX2 phosphorylation. Ser220 and Ser251 are two sites that identified for mitotic phosphorylation on SOX2. Moreover, SOX2 mutants (S220A and S251A) can promote SOX2 induced OCT4 re-expression in differentiated cells. These findings reveal a novel regulation mechanism of SOX2 phosphorylation mediated by AURKA in mitosis and its function in stem cell pluripotency maintenance in cancer cells.

Interrogating Endogenous Protein Phosphatase Activity with Rationally Designed Chemosensors.

We introduce a versatile approach for repurposing protein kinase chemosensors, containing the phosphorylation-sensitive sulfonamido-oxine fluorophore termed Sox, for the specific determination of endogenous protein phosphatase activity from whole cell lysates and tissue homogenates. As a demonstration of this approach, we design and evaluate a direct chemosensor for protein tyrosine phosphatase-1B (PTP1B), an established signaling node in human disease. The optimal sensor design is capable of detecting as little as 6 pM (12 pg) full-length recombinant PTP1B and is remarkably selective for PTP1B among a panel of highly homologous tyrosine phosphatases. Coupling this robust activity probe with the specificity of antibodies allowed for the temporal analysis of endogenous PTP1B activity dynamics in lysates generated from HepG2 cells after stimulation with insulin. Lastly, we leveraged this assay format to profile PTP1B activity perturbations in a rat model of nonalcoholic fatty liver disease (NAFLD), providing direct evidence for elevated PTP1B catalytic activity in this disease state. Given the modular nature of this assay, we anticipate that this approach will have broad utility in monitoring phosphatase activity dynamics in human disease states.

Novel AKT phosphorylation sites identified in the pluripotency factors OCT4, SOX2 and KLF4.

The four OSKM factors OCT4, SOX2, KLF4 and c-MYC are key transcription factors modulating pluripotency, self-renewal and tumorigenesis in stem cells. However, although their transcriptional targets have been extensively studied, little is known about how these factors are regulated at the posttranslational level. In this study, we established an in vitro system to identify phosphorylation patterns of the OSKM factors by AKT kinase. OCT4, SOX2, KLF4 and c-MYC were expressed in Sf9 insect cells employing the baculoviral expression system. OCT4, SOX2 and KLF4 were localized in the nucleus of insect cells, allowing their easy purification to near homogeneity upon nuclear fractionation. All transcription factors were isolated as biologically active DNA-binding proteins. Using in vitro phosphorylation and mass spectrometry-based phosphoproteome analyses several novel and known AKT phosphorylation sites could be identified in OCT4, SOX2 and KLF4.

Novel Function of Lysine Methyltransferase G9a in the Regulation of Sox2 Protein Stability.

G9a is a lysine methyltransferase (KMTase) for histone H3 lysine 9 that plays critical roles in a number of biological processes. Emerging evidence suggests that aberrant expression of G9a contributes to tumor metastasis and maintenance of a malignant phenotype in cancer by inducing epigenetic silencing of tumor suppressor genes. Here, we show that G9a regulates Sox2 protein stability in breast cancer cells. When G9a lysine methyltransferase activity was chemically inhibited in the ER(+) breast cancer cell line MCF7, Sox2 protein levels were decreased. In addition, ectopic overexpression of G9a induced accumulation of Sox2. Changes in cell migration, invasion, and mammosphere formation by MCF7 cells were correlated with the activity or expression level of G9a. Ectopic expression of G9a also increased Sox2 protein levels in another ER(+) breast cancer cell line, ZR-75-1, whereas it did not affect Sox2 expression in MDA-MB-231 cells, an ER(-) breast cancer cell line, or in glioblastoma cell lines. Furthermore, treatment of mouse embryonic stem cells with a KMT inhibitor, BIX-01294, resulted in a rapid reduction in Sox2 protein expression despite increased Sox2 transcript levels. This finding suggests that G9a has a novel function in the regulation of Sox2 protein stability in a cell type-dependent manner.

Impact of protein/protein interactions on global intermolecular translocation rates of the transcription factors Sox2 and Oct1 between DNA cognate sites analyzed by z-exchange NMR spectroscopy.

Oct1 and Sox2 synergistically regulate developmental genes by binding to adjacent sites within promoters. We have investigated the kinetics of global intermolecular translocation of Sox2 and Oct1 between cognate sites located on different DNA molecules by z-exchange NMR spectroscopy. In the Hoxb1 promoter, the Sox2 and Oct1 sites are immediately adjacent to one another, and the intermolecular translocation rates are too slow to be measured by z-exchange spectroscopy. By introducing a 3-bp insertion between the Sox2 and Oct1 sites to mimic the spacing in the FGF4 enhancer, the interprotein contact surface is reduced, and the translocation rates are increased. Interaction between Sox2 and the POU-specific domain (POU(S)) of Oct1 does not affect the translocation mechanism but modulates the rates. Translocation involves only jumping (dissociation and reassociation) for Sox2, but both jumping and direct intersegment transfer (no dissociation into free solution) for Oct1. The dissociation (k(off) ∼1.5 s(-1)) and association (k(on) ∼5.1 × 10(9) m(-1)s(-1)) rate constants for Sox2 are reduced 4-fold and increased 5-fold, respectively, in the presence of Oct1. k(off) (∼3.5 s(-1)) for Oct1 is unaffected by Sox2, whereas k(on) (∼1.3 × 10(9) m(-1)s(-1)) is increased ∼13-fold. The direct intermolecular translocation rate (k(inter) ∼1.8 × 10(4) m(-1)s(-1)) for the POU(S) domain of Oct1 is reduced 2-fold by Sox2, whereas that for the POU homeodomain (POU(HD)) of Oct1 (k(inter) ∼ 1.7 × 10(4) m(-1)s(-1)) remains unaltered, consistent with the absence of contacts between Sox2 and POU(HD). The data suggest a model for the sequence of binding events involved in synergistic gene regulation by Sox2 and Oct1.

Selection of reprogramming factors of induced pluripotent stem cells based on the protein interaction network and functional profiles.

Induced pluripotent stem cells have displayed great potential in disease investigation and drug development applications. However, selection of reprogramming factors in each cell type or disease state is both expensive and time consuming. To deal with this kind of situation, a fast computational framework was developed by optimize the reprogramming factors via the protein interaction network and gene functional profiles. It can be used to select reprogramming factors from millions of possibilities. It is anticipated that the novel approach will become a very useful tool for both basic research and drug development.

Phosphorylation of Sox2 cooperates in reprogramming to pluripotent stem cells.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by transduction of reprogramming factors, including Oct4, Sox2, Klf4, and c-Myc. A coordinated network of these factors was suggested to confer a pluripotency of iPSCs. Together with Oct4, Sox2 plays a major role as a master regulator in ESCs. However, the underlying mechanisms by which Sox2 contributes to self-renewal or reprogramming processes remain to be determined. Here, we provide new evidence for a phosphorylation-based regulation of Sox2 activity. Akt directly interacts with Sox2 and promotes its stabilization through phosphorylation at Thr118, which enhances the transcriptional activity of Sox2 in ESCs. Moreover, phosphorylation of Sox2 cooperates in the reprogramming of mouse embryonic fibroblasts by enabling more efficient induction of iPSCs. Overall, our studies provide new insights into the regulatory mechanism of Sox2 in ESCs and also provide a direct link between phosphorylation events and somatic cell reprogramming.

Comparative analysis of SOX3 protein orthologs: Expansion of homopolymeric amino acid tracts during vertebrate evolution.

To understand more fully the structure and evolution of the SOX3 protein, we comparatively analyzed its orthologs in vertebrates. Since complex disorders are associated with human SOX3 polyalanine expansions, our investigation focused on both compositional and evolutionary analysis of various homopolymeric amino acid tracts observed in SOX3 orthologs. Our analysis revealed that the observed homopolymeric alanine, glycine, and proline tracts are mammal-specific, except for one polyglycine tract present in birds. Since it is likely that the SOX3 protein acquired additional roles in brain development in Eutheria, we might speculate that development of novel brain functions during the course of evolution was affected, at least in part, by such structural-functional changes in the SOX3 protein.

Regulated fluctuations in nanog expression mediate cell fate decisions in embryonic stem cells.

There is evidence that pluripotency of mouse embryonic stem (ES) cells is associated with the activity of a network of transcription factors with Sox2, Oct4, and Nanog at the core. Using fluorescent reporters for the expression of Nanog, we observed that a population of ES cells is best described by a dynamic distribution of Nanog expression characterized by two peaks defined by high (HN) and low (LN) Nanog expression. Typically, the LN state is 5%-20% of the total population, depending on the culture conditions. Modelling of the activity of Nanog reveals that a simple network of Oct4/Sox2 and Nanog activity can account for the observed distribution and its properties as long as the transcriptional activity is tuned by transcriptional noise. The model also predicts that the LN state is unstable, something that is born out experimentally. While in this state, cells can differentiate. We suggest that transcriptional fluctuations in Nanog expression are an essential element of the pluripotent state and that the function of Sox2, Oct4, and Nanog is to act as a network that promotes and maintains transcriptional noise to interfere with the differentiation signals.

Quantitative mRNA expression of sox3 and DMRT1 during sex reversal, and expression profiles after GnRHa administration in black porgy, Acanthopagrus schlegeli.

We cloned full-length sox3 cDNA from testis of black porgy, Acanthopagrus schlegeli. Black porgy sox3 cDNA consists of 897 base pairs (bp) and encodes a protein of 298 amino acids. We have investigated the expression pattern of sox3 and DMRT1 mRNA during the sex-reverse process from male to female (immature testis, mature testis, testicular portion of mostly testis, ovarian portion of mostly testis, testicular portion of mostly ovary, ovarian portion of mostly ovary and ovary). The expression of sox3 and DMRT1 mRNA was high in mature testis of black porgy during sex-reverse process. In a histological analysis, testicular portion of gonad was degenerated and the ovary portion was increased during sex reversal from male to female, and then oocytes were increased in ovary. Also we examined the expression of sox3 and DMRT1 mRNA after gonadotropin-releasing hormone analogue (GnRHa) treatment in immature black porgy. The expression of sox3 and DMRT1 mRNA was increased after GnRHa treatment (in vivo and in vitro experiment) in immature black porgy. Therefore, we concluded that sox3 and DMRT1 were involved in the development of testis than ovary in black porgy.