Engineering Critical Residues of SOX9 Discovers a Variant With Potent Capacity to Induce Chondrocytes.

Articular cartilage plays vital roles as a friction minimizer and shock absorber during joint movement but has a poor capacity to self-repair when damaged through trauma or disease. Cartilage tissue engineering is an innovative technique for cartilage regeneration, yet its therapeutic application requires chondrocytes in large numbers. Direct reprogramming of somatic cells to chondrocytes by expressing SOX9, KLF4, and c-MYC offers a promising option to generate chondrocytes in sufficient numbers; however, the low efficiency of the reprogramming system warrants further improvement. Here we referred to structural and functional features of SOX9 and performed alanine-scanning mutagenesis of functionally critical residues in the HMG box and at putative posttranslational modification (PTM) sites. We discovered that a SOX9 variant H131A/K398A, doubly mutated in the HMG box (H131) and at a PTM site (K398), significantly upregulated expression of chondrogenic genes and potently induced chondrocytes from mouse embryonic fibroblasts. The H131A/K398A variant remained unsumoylated in cells and exhibited a stronger DNA-binding activity than wild-type SOX9, especially when complexed with other proteins. Our results show that the novel SOX9 variant may be useful for efficient induction of chondrocytes and illuminate the strategic feasibility of mutating a transcription factor at functionally critical residues to expedite discovery of an optimized reprogramming factor.

Downregulation of Odd-Skipped Related 2, a Novel Regulator of Epithelial-Mesenchymal Transition, Enables Efficient Somatic Cell Reprogramming.

Somatic cell reprogramming proceeds through a series of events to generate induced pluripotent stem cells (iPSCs). The early stage of reprogramming of mouse embryonic fibroblasts is characterized by rapid cell proliferation and morphological changes, which are accompanied by downregulation of mesenchyme-associated genes. However, the functional relevance of their downregulation to reprogramming remains poorly defined. In this study, we have screened transcriptional regulators that are downregulated immediately upon reprogramming, presumably through direct targeting by reprogramming factors. To test if these transcriptional regulators impact reprogramming when expressed continuously, we generated an expression vector that harbors human cytomegalovirus upstream open reading frame 2 (uORF2), which reduces translation to minimize the detrimental effect of an expressed protein. Screening of transcriptional regulators with this expression vector revealed that downregulation of (odd-skipped related 2 [Osr2]) is crucial for efficient reprogramming. Using a cell-based model for epithelial-mesenchymal transition (EMT), we show that Osr2 is a novel EMT regulator that acts through induction of transforming growth factor-ß (TGF-ß) signaling. During reprogramming, Osr2 downregulation not only diminishes TGF-ß signaling but also allows activation of Wnt signaling, thus promoting mesenchymal-epithelial transition (MET) toward acquisition of pluripotency. Our results illuminate the functional significance of Osr2 downregulation in erasing the mesenchymal phenotype at an early stage of somatic cell reprogramming.

Locus-Specific Isolation of the Nanog Chromatin Identifies Regulators Relevant to Pluripotency of Mouse Embryonic Stem Cells and Reprogramming of Somatic Cells.

Pluripotency is a crucial feature of pluripotent stem cells, which are regulated by the core pluripotency network consisting of key transcription factors and signaling molecules. However, relatively less is known about the molecular mechanisms that modify the core pluripotency network. Here we used the CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements) to unbiasedly isolate proteins assembled on the Nanog promoter in mouse embryonic stem cells (mESCs), and then tested their functional relevance to the maintenance of mESCs and reprogramming of somatic cells. Gene ontology analysis revealed that the identified proteins, including many RNA-binding proteins (RBPs), are enriched in RNA-related functions and gene expression. ChIP-qPCR experiments confirmed that BCLAF1, FUBP1, MSH6, PARK7, PSIP1, and THRAP3 occupy the Nanog promoter region in mESCs. Knockdown experiments of these factors show that they play varying roles in self-renewal, pluripotency gene expression, and differentiation of mESCs as well as in the reprogramming of somatic cells. Our results show the utility of unbiased identification of chromatin-associated proteins on a pluripotency gene in mESCs and reveal the functional relevance of RBPs in ESC differentiation and somatic cell reprogramming.

Visualization of intracellular lipid metabolism in brown adipocytes by time-lapse ultra-multiplex CARS microspectroscopy with an onstage incubator.

We visualized a dynamic process of fatty acid uptake of brown adipocytes using a time-lapse ultra-broadband multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging system with an onstage incubator. Combined with the deuterium labeling technique, the intracellular uptake of saturated fatty acids was traced up to 9 h, a substantial advance over the initial multiplex CARS system, with an analysis time of 80 min. Characteristic metabolic activities of brown adipocytes, such as resistance to lipid saturation, were elucidated, supporting the utility of the newly developed system.

BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence.

Fibrodysplasia ossificans progressiva (FOP) patients carry a missense mutation in ACVR1 [617G > A (R206H)] that leads to hyperactivation of BMP-SMAD signaling. Contrary to a previous study, here we show that FOP fibroblasts showed an increased efficiency of induced pluripotent stem cell (iPSC) generation. This positive effect was attenuated by inhibitors of BMP-SMAD signaling (Dorsomorphin or LDN1931890) or transducing inhibitory SMADs (SMAD6 or SMAD7). In normal fibroblasts, the efficiency of iPSC generation was enhanced by transducing mutant ACVR1 (617G > A) or SMAD1 or adding BMP4 protein at early times during the reprogramming. In contrast, adding BMP4 at later times decreased iPSC generation. ID genes, transcriptional targets of BMP-SMAD signaling, were critical for iPSC generation. The BMP-SMAD-ID signaling axis suppressed p16/INK4A-mediated cell senescence, a major barrier to reprogramming. These results using patient cells carrying the ACVR1 R206H mutation reveal how cellular signaling and gene expression change during the reprogramming processes.

Mechanisms of the Metabolic Shift during Somatic Cell Reprogramming.

Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold a huge promise for regenerative medicine, drug development, and disease modeling. PSCs have unique metabolic features that are akin to those of cancer cells, in which glycolysis predominates to produce energy as well as building blocks for cellular components. Recent studies indicate that the unique metabolism in PSCs is not a mere consequence of their preference for a low oxygen environment, but is an active process for maintaining self-renewal and pluripotency, possibly in preparation for rapid response to the metabolic demands of differentiation. Understanding the regulatory mechanisms of this unique metabolism in PSCs is essential for proper derivation, generation, and maintenance of PSCs. In this review, we discuss the metabolic features of PSCs and describe the current understanding of the mechanisms of the metabolic shift during reprogramming from somatic cells to iPSCs, in which the metabolism switches from oxidative phosphorylation (OxPhos) to glycolysis.

DSIF, the Paf1 complex, and Tat-SF1 have nonredundant, cooperative roles in RNA polymerase II elongation.

Transcription elongation factor DSIF/Spt4-Spt5 is capable of promoting and inhibiting RNA polymerase II elongation and is involved in the expression of various genes. While it has been known for many years that DSIF inhibits elongation in collaboration with the negative elongation factor NELF, how DSIF promotes elongation is largely unknown. Here, an activity-based biochemical approach was taken to understand the mechanism of elongation activation by DSIF. We show that the Paf1 complex (Paf1C) and Tat-SF1, two factors implicated previously in elongation control, collaborate with DSIF to facilitate efficient elongation. In human cells, these factors are recruited to the FOS gene in a temporally coordinated manner and contribute to its high-level expression. We also show that elongation activation by these factors depends on P-TEFb-mediated phosphorylation of the Spt5 C-terminal region. A clear conclusion emerging from this study is that a set of elongation factors plays nonredundant, cooperative roles in elongation. This study also shows unambiguously that Paf1C, which is generally thought to have chromatin-related functions, is involve directlyd in elongation control.

The RNA binding complexes NF45-NF90 and NF45-NF110 associate dynamically with the c-fos gene and function as transcriptional coactivators.

The c-fos gene is rapidly induced to high levels by various extracellular stimuli. We used a defined in vitro transcription system that utilizes the c-fos promoter to purify a coactivator activity in an unbiased manner. We report here that NF45-NF90 and NF45-NF110, which possess archetypical double-stranded RNA binding motifs, have a direct function as transcriptional coactivators. The transcriptional activities of the nuclear factor (NF) complexes (NF45-NF90 and NF45-NF110) are mediated by both the upstream enhancer and core promoter regions of the c-fos gene and do not require their double-stranded RNA binding activities. The NF complexes cooperate with general coactivators, PC4 and Mediator, to elicit a high level of transcription and display multiple interactions with activators and the components of the general transcriptional machinery. Knockdown of the endogenous NF90/NF110 in mouse cells shows an important role for the NF complexes in inducing c-fos transcription. Chromatin immunoprecipitation assays demonstrate that the NF complexes occupy the c-fos enhancer/promoter region before and after serum induction and that their occupancies within the coding region of the c-fos gene increase in parallel to that of RNAPII upon serum induction. In light of their dynamic occupancy on the c-fos gene as well as direct functions in both transcription and posttranscriptional processes, the NF complexes appear to serve as multifunctional coactivators that coordinate different steps of gene expression to facilitate rapid response of inducible genes.

Association of the winged helix motif of the TFIIEα subunit of TFIIE with either the TFIIEß subunit or TFIIB distinguishes its functions in transcription.

In eukaryotes, the general transcription factor TFIIE consists of two subunits, α and ß, and plays essential roles in transcription. Structure-function studies indicate that TFIIE has three-winged helix (WH) motifs, with one in TFIIEα and two in TFIIEß. Recent studies suggested that, by binding to the clamp region of RNA polymerase II, TFIIEα-WH promotes the conformational change that transforms the promoter-bound inactive preinitiation complex to the active complex. Here, to elucidate its roles in transcription, functional analyses of point-mutated human TFIIEα-WH proteins were carried out. In vitro transcription analyses identified two classes of mutants. One class was defective in transcription initiation, and the other was defective in the transition from initiation to elongation. Analyses of the binding of this motif to other general transcription factors showed that the former class was defective in binding to the basic helix-loop-helix motif of TFIIEß and the latter class was defective in binding to the N-terminal cyclin homology region of TFIIB. Furthermore, TFIIEα-WH bound to the TFIIH XPB subunit at a third distinct region. Therefore, these results provide further insights into the mechanisms underlying RNA polymerase II activation at the initial stages of transcription.

Arginine methylation of FOXO transcription factors inhibits their phosphorylation by Akt.

Forkhead box O (FOXO) transcription factors, the key regulators of cell survival, are negatively controlled through the PI3K-Akt signaling pathway. Phosphorylation of FOXO by Akt leads to cytoplasmic localization and subsequent degradation via the ubiquitin-proteasome system. Here we show a paradigm of FOXO1 regulation by the protein arginine methyltransferase PRMT1. PRMT1 methylated FOXO1 at conserved Arg248 and Arg250 within a consensus motif for Akt phosphorylation; this methylation directly blocked Akt-mediated phosphorylation of FOXO1 at Ser253 in vitro and in vivo. Silencing of PRMT1 by small interfering RNA enhanced nuclear exclusion, polyubiquitination, and proteasomal degradation of FOXO1. PRMT1 knockdown led to a decrease in oxidative-stress-induced apoptosis depending on the PI3K-Akt signaling pathway. Furthermore, stable expression of enzymatic inactive PRMT1 mutant increased resistance to apoptosis, whereas this effect was reversed by expression of phosphorylation-deficient FOXO1. Our findings predict a role for arginine methylation as an inhibitory modification against Akt-mediated phosphorylation.

The paired-box homeodomain transcription factor Pax6 binds to the upstream region of the TRAP gene promoter and suppresses receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation.

Osteoclast formation is regulated by balancing between the receptor activator of nuclear factor-κB ligand (RANKL) expressed in osteoblasts and extracellular negative regulatory cytokines such as interferon-γ (IFN-γ) and interferon-ß (IFN-ß), which can suppress excessive bone destruction. However, relatively little is known about intrinsic negative regulatory factors in RANKL-mediated osteoclast differentiation. Here, we show the paired-box homeodomain transcription factor Pax6 acts as a negative regulator of RANKL-mediated osteoclast differentiation. Electrophoretic mobility shift and reporter assays found that Pax6 binds endogenously to the proximal region of the tartrate acid phosphatase (TRAP) gene promoter and suppresses nuclear factor of activated T cells c1 (NFATc1)-induced TRAP gene expression. Introduction of Pax6 retrovirally into bone marrow macrophages attenuates RANKL-induced osteoclast formation. Moreover, we found that the Groucho family member co-repressor Grg6 contributes to Pax6-mediated suppression of the TRAP gene expression induced by NFATc1. These results suggest that Pax6 interferes with RANKL-mediated osteoclast differentiation together with Grg6. Our results demonstrate that the Pax6 pathway constitutes a new aspect of the negative regulatory circuit of RANKL-RANK signaling in osteoclastogenesis and that the augmentation of Pax6 might therefore represent a novel target to block pathological bone resorption.

An engineered ligand-responsive Csy4 endoribonuclease controls transgene expression from Sendai virus vectors.

BACKGROUND: Viral vectors are attractive gene delivery vehicles because of their broad tropism, high transduction efficiency, and durable expression. With no risk of integration into the host genome, the vectors developed from RNA viruses such as Sendai virus (SeV) are especially promising. However, RNA-based vectors have limited applicability because they lack a convenient method to control transgene expression by an external inducer. RESULTS: We engineered a Csy4 switch in Sendai virus-based vectors by combining Csy4 endoribonuclease with mutant FKBP12 (DD: destabilizing domain) that becomes stabilized when a small chemical Shield1 is supplied. In this Shield1-responsive Csy4 (SrC) switch, Shield1 increases Csy4 fused with DD (DD-Csy4), which then cleaves and downregulates the transgene mRNA containing the Csy4 recognition sequence (Csy4RS). Moreover, when Csy4RS is inserted in the viral L gene, the SrC switch suppresses replication and transcription of the SeV vector in infected cells in a Shield1-dependent manner, thus enabling complete elimination of the vector from the cells. By temporally controlling BRN4 expression, a BRN4-expressing SeV vector equipped with the SrC switch achieves efficient, stepwise differentiation of embryonic stem cells into neural stem cells, and then into astrocytes. CONCLUSION: SeV-based vectors with the SrC switch should find wide applications in stem cell research, regenerative medicine, and gene therapy, especially when precise control of reprogramming factor expression is desirable.

Structurally-discovered KLF4 variants accelerate and stabilize reprogramming to pluripotency.

Non-genetically modified somatic cells can only be inefficiently and stochastically reprogrammed to pluripotency by exogenous expression of reprogramming factors. Low competence of natural reprogramming factors may prevent the majority of cells to successfully and synchronously reprogram. Here we screened DNA-interacting amino acid residues in the zinc-finger domain of KLF4 for enhanced reprogramming efficiency using alanine-substitution scanning methods. Identified KLF4 L507A mutant accelerated and stabilized reprogramming to pluripotency in both mouse and human somatic cells. By testing all the variants of L507 position, variants with smaller amino acid residues in the KLF4 L507 position showed higher reprogramming efficiency. L507A bound more to promoters or enhancers of pluripotency genes, such as KLF5, and drove gene expression of these genes during reprogramming. Molecular dynamics simulations predicted that L507A formed additional interactions with DNA. Our study demonstrates how modifications in amino acid residues of DNA-binding domains enable next-generation reprogramming technology with engineered reprogramming factors.

Early reactivation of clustered genes on the inactive X chromosome during somatic cell reprogramming.

Reprogramming of murine female somatic cells to induced pluripotent stem cells (iPSCs) is accompanied by X chromosome reactivation (XCR), by which the inactive X chromosome (Xi) in female somatic cells becomes reactivated. However, how Xi initiates reactivation during reprogramming remains poorly defined. Here, we used a Sendai virus-based reprogramming system to generate partially reprogrammed iPSCs that appear to be undergoing the initial phase of XCR. Allele-specific RNA-seq of these iPSCs revealed that XCR initiates at a subset of genes clustered near the centromere region. The initial phase of XCR occurs when the cells transit through mesenchymal-epithelial transition (MET) before complete shutoff of Xist expression. Moreover, regulatory regions of these genes display dynamic changes in lysine-demethylase 1a (KDM1A) occupancy. Our results identified clustered genes on the Xi that show reactivation in the initial phase of XCR during reprogramming and suggest a possible role for histone demethylation in this process.

cAMP-response element-binding protein (CREB) controls MSK1-mediated phosphorylation of histone H3 at the c-fos promoter in vitro.

The rapid induction of the c-fos gene correlates with phosphorylations of histone H3 and HMGN1 by mitogen- and stress-activated protein kinases. We have used a cell-free system to dissect the mechanism by which MSK1 phosphorylates histone H3 within the c-fos chromatin. Here, we show that the reconstituted c-fos chromatin presents a strong barrier to histone H3 phosphorylation by MSK1; however, the activators (serum response factor, Elk-1, cAMP-response element-binding protein (CREB), and ATF1) bound on their cognate sites recruit MSK1 to phosphorylate histone H3 at Ser-10 within the chromatin. This activator-dependent phosphorylation of histone H3 is enhanced by HMGN1 and occurs preferentially near the promoter region. Among the four activators, CREB plays a predominant role in MSK1-mediated phosphorylation of histone H3, and the phosphorylation of Ser-133 in CREB is essential for this process. Mutational analyses of MSK1 show that its N-terminal inhibition domain is critical for the kinase to phosphorylate chromatin-embedded histone H3 in a CREB-dependent manner, indicating the presence of an intricate regulatory network for MSK1-mediated phosphorylation of histone H3.

Utilization of a novel Sendai virus vector in ex vivo gene therapy for hemophilia A.

Sendai virus (SeV) vectors are being recognized as a superior tool for gene transfer. Here, we report the transfection efficacy of a novel, high-performance, replication-defective, and persistent Sendai virus (SeVdp) vector in cultured cells and in mice using a near-infrared fluorescent protein (iRFP)-mediated in vivo imaging system. The novel SeVdp vector established persistent infection, and strong expression of inserted genes was sustained indefinitely in vitro. Analysis of iRFP-expressing cells transplanted subcutaneously into NOG, nude, and ICR mice suggests that innate immunity was involved in the exclusion of the transplanted cells. We also evaluated the feasibility of this novel SeVdp vector for hemophilia A gene therapy. This system enabled insertion of full-length FVIII genes, and transduced cells secreted FVIII into the culture medium. Transient FVIII activity was detected in the plasma of mice after intraperitoneal transplantation of these FVIII-secreting cells. Further improvement in methods to evade immunity, such as simultaneous expression of immunomodulatory genes, would make this novel vector a very useful tool in regenerative medicine.

Live-cell imaging of subcellular structures for quantitative evaluation of pluripotent stem cells.

Pluripotent stem cells (PSCs) have various degrees of pluripotency, which necessitates selection of PSCs with high pluripotency before their application to regenerative medicine. However, the quality control processes for PSCs are costly and time-consuming, and it is essential to develop inexpensive and less laborious selection methods for translation of PSCs into clinical applications. Here we developed an imaging system, termed Phase Distribution (PD) imaging system, which visualizes subcellular structures quantitatively in unstained and unlabeled cells. The PD image and its derived PD index reflected the mitochondrial content, enabling quantitative evaluation of the degrees of somatic cell reprogramming and PSC differentiation. Moreover, the PD index allowed unbiased grouping of PSC colonies into those with high or low pluripotency without the aid of invasive methods. Finally, the PD imaging system produced three-dimensional images of PSC colonies, providing further criteria to evaluate pluripotency of PSCs. Thus, the PD imaging system may be utilized for screening of live PSCs with potentially high pluripotency prior to more rigorous quality control processes.

Non-invasive in vivo imaging of UCP1 expression in live mice via near-infrared fluorescent protein iRFP720.

Uncoupling protein 1 (UCP1) is a mitochondrial protein that is expressed in both brown and beige adipocytes. UCP1 uncouples the mitochondrial electron transport chain from ATP synthesis to produce heat via non-shivering thermogenesis. Due to their ability to dissipate energy as heat and ameliorate metabolic disorders, UCP1-expressing adipocytes are considered as a potential target for anti-obesity treatment. To monitor the expression of UCP1 in live mice in a non-invasive manner, we generated the Ucp1-iRFP720 knock-in (Ucp1-iRFP720 KI) mice, in which the gene encoding a near-infrared fluorescent protein iRFP720 is inserted into the Ucp1 gene locus. Using the heterozygous Ucp1-iRFP720 KI mice, we observed robust iRFP fluorescence in the interscapular region where brown adipose tissue is located. Moreover, the iRFP fluorescence was clearly observable in inguinal white adipose tissues in live mice administered with ß3-adrenergic receptor agonist CL316,243. We also found that the homozygous Ucp1-iRFP720 KI mice, which are deficient in UCP1, displayed prominent iRFP fluorescence in the inguinal regions at the standard housing temperature. Consistent with this, the mice exhibited expanded populations of beige-like adipocytes in inguinal white adipose tissue, in which the Ucp1 promoter was dramatically activated. Thus, the Ucp1-iRFP720 KI mice provide a convenient model for non-invasive in vivo imaging of UCP1 expression in both brown and beige adipocytes in live mice.

Template Activating Factor-I α Regulates Retroviral Silencing during Reprogramming.

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) is accompanied by dramatic changes in epigenetic programs, including silencing of endogenous and exogenous retroviruses. Here, we utilized replication-defective and persistent Sendai virus (SeVdp)-based vectors to monitor retroviral silencing during reprogramming. We observed that retroviral silencing occurred at an early reprogramming stage without a requirement for KLF4 or the YY1-binding site in the retroviral genome. Insertional chromatin immunoprecipitation (iChIP) enabled us to isolate factors assembled on the silenced provirus, including components of inhibitor of histone acetyltransferase (INHAT), which includes the SET/TAF-I oncoprotein. Knockdown of SET/TAF-I in mouse embryonic fibroblasts (MEFs) diminished retroviral silencing during reprogramming, and overexpression of template activating factor-I α (TAF-Iα), a SET/TAF-I isoform predominant in embryonic stem cells (ESCs), reinforced retroviral silencing by an SeVdp-based vector that is otherwise defective in retroviral silencing. Our results indicate an important role for TAF-Iα in retroviral silencing during reprogramming.

Live cell imaging of X chromosome reactivation during somatic cell reprogramming.

Generation of induced pluripotent stem cells (iPSCs) with naive pluripotency is important for their applications in regenerative medicine. In female iPSCs, acquisition of naive pluripotency is coupled to X chromosome reactivation (XCR) during somatic cell reprogramming, and live cell monitoring of XCR is potentially useful for analyzing how iPSCs acquire naive pluripotency. Here we generated female mouse embryonic stem cells (ESCs) that carry the enhanced green fluorescent protein (EGFP) and humanized Kusabira-Orange (hKO) genes inserted into an intergenic site near either the Syap1 or Taf1 gene on both X chromosomes. The ESC clones, which initially expressed both EGFP and hKO, inactivated one of the fluorescent protein genes upon differentiation, indicating that the EGFP and hKO genes are subject to X chromosome inactivation (XCI). When the derived somatic cells carrying the EGFP gene on the inactive X chromosome (Xi) were reprogrammed into iPSCs, the EGFP gene on the Xi was reactivated when pluripotency marker genes were induced. Thus, the fluorescent protein genes inserted into an intergenic locus on both X chromosomes enable live cell monitoring of XCI during ESC differentiation and XCR during reprogramming. This is the first study that succeeded live cell imaging of XCR during reprogramming.