Jun Nakabayashi

Essential role of the IRF8-KLF4 transcription factor cascade in murine monocyte differentiation

Monocytes regulate host defenses, inflammation, and tissue homeostasis. The transcription factor interferon regulatory factor-8 (IRF8) stimulates monocyte/macrophage differentiation, yet genome-wide understanding of the differentiation program initiated by IRF8 is lacking. By combining chromatin immunoprecipitation sequencing with gene expression profiling, we show that during IRF8-dependent monocyte differentiation, IRF8 binding occurs at both promoter-proximal and promotor-distal regions together with the transcription factor PU.1 and is associated with gene induction. Many of the promoter-distal IRF8 binding sites show an increase in histone H3 lysine 4 monomethylation, a signature for enhancers. However, about half the IRF8-induced genes were not bound by IRF8, suggesting the involvement of downstream transcription factors. Analysis of DNA motifs in cis-regulatory elements of these indirect IRF8 target genes predicted that Krüppel-like factor-4 (KLF4)-essential for Ly6C(+) monocyte development-is one such factor. Indeed, monocyte development in Irf8(-/-) mice is as defective as that in Klf4(-/-) chimeric mice. Moreover, Irf8(-/-) monocyte-dendritic cell progenitors do not express Klf4 messenger RNA. Introduction of KLF4 into an Irf8(-/-) myeloid progenitor cell line induced a subset of IRF8 target genes and caused partial monocyte differentiation. Taken together, our present results uncover genome-wide behavior of IRF8 and identify an IRF8-KLF4 axis that operates during monocyte differentiation.

In Vivo Imaging of Hierarchical Spatiotemporal Activation of Caspase-8 during Apoptosis

Background Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the dynamics of CASP8 activation are not fully understood. Methodology/Principal Findings We have established a biosensor based on fluorescence resonance energy transfer (FRET) for visualizing apoptotic signals associated with CASP8 activation at the single-cell level. Our dual FRET (dual-FRET) system, comprising a triple fusion fluorescent protein, enabled us to simultaneously monitor the activation of CASP8 and its downstream effector, caspase-3 (CASP3) in single live cells. With the dual-FRET-based biosensor, we detected distinct activation patterns of CASP8 and CASP3 in response to various apoptotic stimuli in mammalian cells, resulting in the positive feedback amplification of CASP8 activation. We reproduced these observations by in vitro reconstitution of the cascade, with a recombinant protein mixture that included procaspases. Furthermore, using a plasma membrane-bound FRET-based biosensor, we captured the spatiotemporal dynamics of CASP8 activation by the diffusion process, suggesting the focal activation of CASP8 is sufficient to propagate apoptotic signals through death receptors. Conclusions Our new FRET-based system visualized the activation process of both initiator and effector caspases in a single apoptotic cell and also elucidated the necessity of an amplification loop for full activation of CASP8.

The molecular mechanism of apoptosis upon caspase-8 activation: Quantitative experimental validation of a mathematical model

Caspase-8 (CASP8) is a cysteine protease that plays a pivotal role in the extrinsic apoptotic signaling pathway via death receptors. The kinetics, dynamics, and selectivity with which the pathway transmits apoptotic signals to downstream molecules upon CASP8 activation are not fully understood. We have developed a system for using high-sensitivity FRET-based biosensors to monitor the protease activity of CASP8 and its downstream effector, caspase-3, in living single cells. Using this system, we systematically investigated the caspase cascade by regulating the magnitude of extrinsic signals received by the cell. Furthermore, we determined the molar concentration of five caspases and Bid required for hierarchical transmission of apoptotic signals in a HeLa cell. Based on these quantitative experimental data, we validated a mathematical model suitable for estimation of the kinetics and dynamics of caspases, which predicts the minimal concentration of CASP8 required to act as an initiator. Consequently, we found that less than 1% of the total CASP8 proteins are sufficient to set the apoptotic program in motion if activated. Taken together, our findings demonstrate the precise cascade of CASP8-mediated apoptotic signals through the extrinsic pathway.

The Transcription Factor IRF8 Counteracts BCR-ABL to Rescue Dendritic Cell Development in Chronic Myelogenous Leukemia

BCR-ABL tyrosine kinase inhibitors (TKI) have dramatically improved therapy for chronic myelogenous leukemia (CML). However, several problems leading to TKI resistance still impede a complete cure of this disease. IFN regulatory factor-8 (IRF8) is a transcription factor essential for the development and functions of immune cells, including dendritic cells. Irf8(-/-) mice develop a CML-like disease and IRF8 expression is downregulated in patients with CML, suggesting that IRF8 is involved in the pathogenesis of CML. In this study, by using a murine CML model, we show that BCR-ABL strongly inhibits a generation of dendritic cells from an early stage of their differentiation in vivo, concomitant with suppression of Irf8 expression. Forced expression of IRF8 overrode BCR-ABL (both wild-type and T315I-mutated) to rescue dendritic cell development in vitro, indicating that the suppression of Irf8 causes dendritic cell deficiency. Gene expression profiling revealed that IRF8 restored the expression of a significant portion of BCR-ABL-dysregulated genes and predicted that BCR-ABL has immune-stimulatory potential. Indeed, IRF8-rescued BCR-ABL-expressing dendritic cells were capable of inducing CTLs more efficiently than control dendritic cells. Altogether, our findings suggest that IRF8 is an attractive target in next-generation therapies for CML.

N-Myristoylation of the Rpt2 subunit of the yeast 26S proteasome is implicated in the subcellular compartment-specific protein quality control system.

Ubiquitination is the posttranslational modification of a protein by covalent attachment of ubiquitin. Controlled proteolysis via the ubiquitin-proteasome system (\UPS) alleviates cellular stress by clearing misfolded proteins. In budding yeast, UPS within the nucleus degrades the nuclear proteins as well as proteins imported from the cytoplasm. While the predominantly nuclear localization of the yeast proteasome is maintained by the importin-mediated transport, N-myristoylation of the proteasome subunit Rpt2 was indicated to cause dynamic nucleo-cytoplasmic localization of proteasomes. Here, we quantitatively analyzed the ubiquitinated peptides using anti-K-ε-GG antibody in yeast cell lines with or without a mutation in the N-myristoylation site of Rpt2 and detected upregulated ubiquitination of proteins with nucleo-cytoplasmic localizations in the mutant strains. Moreover, both the protein and ubiquitinated peptide levels of two Hsp70 family chaperones involved in the nuclear import of misfolded proteins, Ssa and Sse1, were elevated in the mutant strains, whereas levels of an Hsp70 family chaperone involved in the nuclear export, Ssb, were reduced. Taken together, our results indicate that N-myristoylation of Rpt2 is involved in controlled proteolysis via regulation of the nucleo-cytoplasmic localization of the yeast proteasome.

A mathematical model of the stoichiometric control of Smad complex formation in TGF-β signal transduction pathway

Cell fate in multicellular organism is regulated by the diffusible factor from surrounding cells in concentration-dependent manner. TGF-beta is a large protein family of the diffusible proteins secreted from a localized source. The signal of TGF-beta is transduced by Smad family transcription factor. Though it is well known that the stoichiometry of Smads in the transcriptional complex regulates the specificity of target genes of TGF-beta signal, little is known what the stoichiometry of Smads in the transcriptional complex is determined in TGF-beta signal transduction in concentration-dependent manner. To investigate the dynamics of Smad complex formation, we construct a two-compartment model for Smad complex formation in TGF-beta signal transduction. A simplified one-way oligomerization model, which ignores dissociation and well appropriate the full model under high expression levels of R- and Co-Smad, is constructed to analytically investigate the effect of the oligomerization of Smad. Our one-way model reveals that not only shuttling of the Smad from the cytoplasm to the nucleus but also the preferential accumulation of the heteromeric complex in oligomerization can contribute to the predominant production of the heteromeric complex of Smad including both R- and Co-Smad. It is also shown that oligomerization of Smad can contribute to the specificity of signal transduction. In endothelial cells, both Smad-1/5/8 and -2/3 pathways are activated by TGF-beta. The difference of the activity between the two pathways is amplified by trimerization but not by dimerization, suggesting possible importance of trimerization in maintaining the specificity of signal transduction.

Genome-wide DNA methylation profiling identifies primary central nervous system lymphoma as a distinct entity different from systemic diffuse large B-cell lymphoma

Taishi Nakamura1,2 · Satoshi Yamashita3 · Kazutaka Fukumura4 · Jun Nakabayashi5 · Kazuhiro Tanaka6 · Kaoru Tamura7 · Kensuke Tateishi2 · Manabu Kinoshita8 · Shintaro Fukushima1 · Hirokazu Takami1 · Kohei Fukuoka1 · Kai Yamazaki1 · Yuko Matsushita9 · Makoto Ohno9 · Yasuji Miyakita9 · Soichiro Shibui10 · Atsuhiko Kubo11 · Takashi Shuto12 · Sylvia Kocialkowski13 · Shoji Yamanaka14 · Akitake Mukasa15 · Takashi Sasayama6 · Kazuhiko Mishima16 · Taketoshi Maehara7 · Nobutaka Kawahara2 · Motoo Nagane17 · Yoshitaka Narita9 · Hiroyuki Mano4 · Toshikazu Ushijima3 · Koichi Ichimura2

Proteome and behavioral alternations in phosphorylation-deficient mutant Collapsin Response Mediator Protein2 knock-in mice

ABSTRACT CRMP2, alternatively designated as DPYSL2, was the first CRMP family member to be identified as an intracellular molecule mediating the signaling of the axon guidance molecule Semaphorin 3A (Sema3A). In Sema3A signaling, cyclin‐dependent kinase 5 (Cdk5) primarily phosphorylates CRMP2 at Ser522. Glycogen synthase kinase‐3&bgr; (GSK‐3&bgr;) subsequently phosphorylates the residues of Thr509 and Thr514 of CRMP2. Previous studies showed that CRMP2 is involved in pathogenesis of neurological disorders such as Alzheimers disease. In Alzheimers disease, hyper‐phosphorylated forms of CRMP2 are accumulated in the paired helical filaments. To get insight into the possible involvement of the phosphorylation of CRMP2 in pathogenesis of neurological disorders, we previously created CRMP2 S522A knock‐in (crmp2ki/ki) mice and demonstrated that the phosphorylation of CRMP2 at Ser522 is involved in normal dendrite patterning in cortical neurons. However, the behavioral impact and in vivo signaling network of the CRMP2 phosphorylation are not fully understood. In this study, we performed behavioral and proteomics analysis of crmp2ki/ki mice. The crmp2ki/ki mice appeared healthy and showed no obvious differences in physical characteristics compared to wild‐type mice, but they showed impaired emotional behavior, reduced sociality, and low sensitivity to pain stimulation. Through mass‐spectrometry‐based proteomic analysis, we found that 59 proteins were increased and 77 proteins were decreased in the prefrontal cortex of crmp2ki/ki mice. Notably, CRMP3, CRMP4, and CRMP5, the other CRMP family proteins, were increased in crmp2ki/ki mice. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses identified 14 pathways in increased total proteins and 13 pathways in decreased total proteins which are associated with the pathogenesis of Parkinsons, Alzheimers, and Huntingtons diseases. We also detected 20 pathways in increased phosphopeptides and 16 pathways in decreased phosphopeptides including “inflammatory mediator regulation of TRP channels” in crmp2ki/ki mice. Our study suggests that the phosphorylation of CRMP2 at Ser522 is involved in the signaling pathways that may be related to neuropsychiatric and neurodegenerative diseases and pain. HighlightsCRMP2S522A mice showed impaired emotional behavior and lowered sociality and sensitivity to pain.Proteomics analyses of the prefrontal cortex revealed upregulation of other CRMPs.The enriched pathways are those in neurodegenerative and psychiatric disorders.