Koji Hisatake

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.

Manipulation of KLF4 Expression Generates iPSCs Paused at Successive Stages of Reprogramming

Summary The detailed mechanism of reprogramming somatic cells into induced pluripotent stem cells (iPSCs) remains largely unknown. Partially reprogrammed iPSCs are informative and useful for understanding the mechanism of reprogramming but remain technically difficult to generate in a predictable and reproducible manner. Using replication-defective and persistent Sendai virus (SeVdp) vectors, we analyzed the effect of decreasing the expression levels of OCT4, SOX2, KLF4, and c-MYC and found that low KLF4 expression reproducibly gives rise to a homogeneous population of partially reprogrammed iPSCs. Upregulation of KLF4 allows these cells to resume reprogramming, indicating that they are paused iPSCs that remain on the path toward pluripotency. Paused iPSCs with different KLF4 expression levels remain at distinct intermediate stages of reprogramming. This SeVdp-based stage-specific reprogramming system (3S reprogramming system) is applicable for both mouse and human somatic cells and will facilitate the mechanistic analysis of reprogramming.

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

Background: Negative regulation of osteoclast differentiation is critical for suppression of pathological bone destruction. Results: Pax6 is induced by RANKL in osteoclasts and attenuates osteoclast differentiation via blocking TRAP gene expression. Conclusion: Pax6 functions together with its co-receptor to suppress TRAP gene expression and osteoclastogenesis. Significance: This study provides a new aspect for investigating the molecular targets linked to physiological bone resorption. 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-β (IFN-β), 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.

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

Significance The development of iPSCs provides unprecedented opportunities for life sciences, drug discovery, and regenerative medicine. The efficiency of iPSC generation is quite low: typically less than 1% of human primary somatic cells that have received reprogramming factors turn into iPSCs. Previous studies revealed that cellular senescence was a major barrier to iPSC generation. In this study using human FOP mutant cells, we provide evidence that the BMP-SMAD-ID signaling suppressed p16/INK4A-mediated cellular senescence during the early phase in iPSC generation. These results are unexpected because BMP-SMAD signaling has negative effects on the self-renewal of human iPSCs. Here, we show that a human natural mutation increases the efficiency of iPSC generation. 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.

Simple and effective generation of transgene-free induced pluripotent stem cells using an auto-erasable Sendai virus vector responding to microRNA-302

Transgene-free induced pluripotent stem cells (iPSCs) are valuable for both basic research and potential clinical applications. We previously reported that a replication-defective and persistent Sendai virus (SeVdp) vector harboring four reprogramming factors (SeVdp-iPS) can efficiently induce generation of transgene-free iPSCs. This vector can express all four factors stably and simultaneously without chromosomal integration and can be eliminated completely from reprogrammed cells by suppressing vector-derived RNA-dependent RNA polymerase. Here, we describe an improved SeVdp-iPS vector (SeVdp(KOSM)302L) that is automatically erased in response to microRNA-302 (miR-302), uniquely expressed in pluripotent stem cells (PSCs). Gene expression and genome replication of the SeVdp-302L vector, which contains miRNA-302a target sequences at the 3 untranslated region of L mRNA, are strongly suppressed in PSCs. Consequently, SeVdp(KOSM)302L induces expression of reprogramming factors in somatic cells, while it is automatically erased from cells successfully reprogrammed to express miR-302. As this vector can reprogram somatic cells into transgene-free iPSCs without the aid of exogenous short interfering RNA (siRNA), the results we present here demonstrate that this vector may become an invaluable tool for the generation of human iPSCs for future clinical applications.

The RNA-binding Complexes, NF45-NF90 and NF45-NF110, Associate Dynamically with the c-fos Gene and Function as Transcriptional Coactivators

Background: NF45-NF90 and NF45-NF110 possess double-stranded RNA binding domains. Results: NF45-NF90 and NF45-NF110 occupy the c-fos gene dynamically and enhance its transcriptional induction by extracellular stimuli. Conclusion: NF45-NF90 and NF45-NF110 function as coactivators and are involved in various steps of gene expression. Significance: Analysis of RNA binding coactivators is important for understanding the coordination among different steps of gene expression. 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.

A Role for KLF4 in Promoting the Metabolic Shift via TCL1 during Induced Pluripotent Stem Cell Generation

Summary Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is accompanied by morphological, functional, and metabolic alterations before acquisition of full pluripotency. Although the genome-wide effects of the reprogramming factors on gene expression are well documented, precise mechanisms by which gene expression changes evoke phenotypic responses remain to be determined. We used a Sendai virus-based system that permits reprogramming to progress in a strictly KLF4-dependent manner to screen for KLF4 target genes that are critical for the progression of reprogramming. The screening identified Tcl1 as a critical target gene that directs the metabolic shift from oxidative phosphorylation to glycolysis. KLF4-induced TCL1 employs a two-pronged mechanism, whereby TCL1 activates AKT to enhance glycolysis and counteracts PnPase to diminish oxidative phosphorylation. These regulatory mechanisms described here highlight a central role for a reprogramming factor in orchestrating the metabolic shift toward the acquisition of pluripotency during iPSC generation.

Heterogeneous nuclear ribonucleoprotein R cooperates with mediator to facilitate transcription reinitiation on the c-Fos gene.

The c-fos gene responds to extracellular stimuli and undergoes robust but transient transcriptional activation. Here we show that heterogeneous nuclear ribonucleoprotein R (hnRNP R) facilitates transcription reinitiation of the c-fos promoter in vitro in cooperation with Mediator. Consistently, hnRNP R interacts with the Scaffold components (Mediator, TBP, and TFIIH) as well as TFIIB, which recruits RNA polymerase II (Pol II) and TFIIF to Scaffold. The cooperative action of hnRNP R and Mediator is diminished by the cyclin-dependent kinase 8 (CDK8) module, which is comprised of CDK8, Cyclin C, MED12 and MED13 of the Mediator subunits. Interestingly, we find that the length of the G-free cassettes, and thereby their transcripts, influences the hnRNP R-mediated facilitation of reinitiation. Indeed, indicative of a possible role of the transcript in facilitating transcription reinitiation, the RNA transcript produced from the G-free cassette interacts with hnRNP R through its RNA recognition motifs (RRMs) and arginine-glycine-glycine (RGG) domain. Mutational analyses of hnRNP R indicate that facilitation of initiation and reinitiation requires distinct domains of hnRNP R. Knockdown of hnRNP R in mouse cells compromised rapid induction of the c-fos gene but did not affect transcription of constitutive genes. Together, these results suggest an important role for hnRNP R in regulating robust response of the c-fos gene.

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

In eukaryotes, the general transcription factor TFIIE consists of two subunits, α and β, 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 TFIIEβ. 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 TFIIEβ 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.

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.