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Abstract Background Platelet-rich plasma (PRP), which is prepared by concentrating platelets in autologous blood, shows efficacy in chronic skin wounds via multiple growth factors. However, it exhibits heterogeneity across patients, leading to unstable therapeutic efficacy. Human induced pluripotent stem cell (iPSC)-derived megakaryocytes and platelets (iMPs) are capable of providing a stable supply, holding promise as materials for novel platelet concentrate-based therapies. In this context, we evaluated the effect of iMPs on wound healing and validated lyophilization for clinical applications. Methods The growth factors released by activated iMPs were measured. The effect of the administration of iMPs on human fibroblasts and human umbilical vein endothelial cells (HUVECs) was investigated in vitro. iMPs were applied to dorsal skin defects of diabetic mice to assess the wound closure rate and quantify collagen deposition and angiogenesis. Following the storage of freeze-dried iMPs (FD-iMPs) for three months, the stability of growth factors and their efficacy in animal models were determined. Result Multiple growth factors that promote wound healing were detected in activated iMPs. iMPs specifically released FGF2 and exhibited a superior enhancement of HUVEC proliferation compared to PRP. Moreover, an RNA-seq analysis revealed that iMPs induce polarization to stalk cells and enhance ANGPTL4 gene expression in HUVECs. Animal studies demonstrated that iMPs promoted wound closure and angiogenesis in chronic wounds caused by diabetes. We also confirmed the long-term stability of growth factors in FD-iMPs and their comparable effects to those of original iMPs in the animal model. Conclusion Our study demonstrates that iMPs promote angiogenesis and wound healing through the activation of vascular endothelial cells. iMPs exhibited more effectiveness than PRP, an effect attributed to the exclusive presence of specific factors including FGF2. Lyophilization enabled the long-term maintenance of the composition of the growth factors and efficacy of the iMPs, therefore contributing to stable supply for clinical application. These findings suggest that iMPs provide a novel treatment for chronic wounds.

Abstract DNA hypomethylating agents (HMAs) are used for the treatment of myeloid malignancies, although their therapeutic effects have been unsatisfactory. Here we show that CRISPR-Cas9 screening reveals that knockout of topoisomerase 1-binding arginine/serine-rich protein (TOPORS), which encodes a ubiquitin/SUMO E3 ligase, augments the efficacy of HMAs on myeloid leukemic cells with little effect on normal hematopoiesis, suggesting that TOPORS is involved in resistance to HMAs. HMAs are incorporated into the DNA and trap DNA methyltransferase-1 (DNMT1) to form DNA-DNMT1 crosslinks, which undergo SUMOylation, followed by proteasomal degradation. Persistent crosslinking is cytotoxic. The TOPORS RING finger domain, which mediates ubiquitination, is responsible for HMA resistance. In TOPORS knockout cells, DNMT1 is stabilized by HMA treatment due to inefficient ubiquitination, resulting in the accumulation of unresolved SUMOylated DNMT1. This indicates that TOPORS ubiquitinates SUMOylated DNMT1, thereby promoting the resolution of DNA-DNMT1 crosslinks. Consistently, the ubiquitination inhibitor, TAK-243, and the SUMOylation inhibitor, TAK-981, show synergistic effects with HMAs through DNMT1 stabilization. Our study provides a novel HMA-based therapeutic strategy that interferes with the resolution of DNA-DNMT1 crosslinks.

Abstract The underlying mechanisms of atherosclerosis, the second leading cause of death among Werner syndrome (WS) patients, are not fully understood. Here, we establish an in vitro co-culture system using macrophages (iMφs), vascular endothelial cells (iVECs), and vascular smooth muscle cells (iVSMCs) derived from induced pluripotent stem cells. In co-culture, WS-iMφs induces endothelial dysfunction in WS-iVECs and characteristics of the synthetic phenotype in WS-iVSMCs. Transcriptomics and open chromatin analysis reveal accelerated activation of type I interferon signaling and reduced chromatin accessibility of several transcriptional binding sites required for cellular homeostasis in WS-iMφs. Furthermore, the H3K9me3 levels show an inverse correlation with retrotransposable elements, and retrotransposable element-derived double-stranded RNA activates the DExH-box helicase 58 (DHX58)-dependent cytoplasmic RNA sensing pathway in WS-iMφs. Conversely, silencing type I interferon signaling in WS-iMφs rescues cell proliferation and suppresses cellular senescence and inflammation. These findings suggest that Mφ-specific inhibition of type I interferon signaling could be targeted to treat atherosclerosis in WS patients.

Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in murine HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of ATP levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.

Abstract Background During mouse embryonic development, definitive hematopoiesis is first detected around embryonic day (E) 10.5 in the aorta-gonad-mesonephros (AGM) region. Hematopoietic stem cells (HSCs) arise in the dorsal aorta’s intra-aortic hematopoietic cell clusters (IAHCs). We have previously reported that a transcription factor Sox17 is expressed in IAHCs, and that, among them, CD45lowc-Kithigh cells have high hematopoietic activity. Furthermore, forced expression of Sox17 in this population of cells can maintain the formation of hematopoietic cell clusters. However, how Sox17 does so, particularly downstream signaling involved, remains poorly understood. The purpose of this study is to search for new Sox17 targets which contribute to cluster formation with hematopoietic activity. Methods RNA-sequencing (RNA-seq) analysis was done to identify genes that are upregulated in Sox17-expressing IAHCs as compared with Sox17-negative ones. Among the top 7 highly expressed genes, Rasip1 which had been reported to be a vascular-specific regulator was focused on in this study, and firstly, the whole-mount immunostaining was done. We conducted luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay to examine whether Sox17 regulates Rasip1 gene expression via binding to its enhancer element. We also analyzed the cluster formation and the multilineage colony-forming ability of Rasip1-transduced cells and Rasip1-knockdown Sox17-transduced cells. Results The increase of the Rasip1 expression level was observed in Sox17-positive CD45lowc-Kithigh cells as compared with the Sox17-nonexpressing control. Also, the expression level of the Rasip1 gene was increased by the Sox17-nuclear translocation. Rasip1 was expressed on the membrane of IAHCs, overlapping with the endothelial cell marker, CD31, and hematopoietic stem/progenitor marker (HSPC), c-Kit. Rasip1 expression was observed in most part of c-Kit+Sox17+ cells in IAHCs. Luciferase reporter assay and ChIP assay indicated that one of the five putative Sox17-binding sites in the Rasip1 enhancer region was important for Rasip1 expression via Sox17 binding. Rasip1 knockdown in Sox17-transduced cells decreased the cluster formation and diminished the colony-forming ability, while overexpression of Rasip1 in CD45lowc-Kithigh cells led to a significant but transient increase in hematopoietic activity. Conclusions Rasip1 knockdown in Sox17-transduced CD45lowc-Kithigh cells displayed a significant decrease in the multilineage colony-forming ability and the cluster size. Rasip1 overexpression in Sox17-untransduced CD45lowc-Kithigh cells led to a significant but transient increase in the multilineage colony-forming ability, suggesting the presence of a cooperating factor for sustained hematopoietic activity.

Polycomb repressive complex (PRC) 1 regulates stem cell fate by mediating mono-ubiquitination of histone H2A at lysine 119. While canonical PRC1 is critical for hematopoietic stem and progenitor cell (HSPC) maintenance, the role of non-canonical PRC1 in hematopoiesis remains elusive. PRC1.1, a non-canonical PRC1, consists of PCGF1, RING1B, KDM2B, and BCOR. We recently showed that PRC1.1 insufficiency induced by the loss of PCGF1 or BCOR causes myeloid-biased hematopoiesis and promotes transformation of hematopoietic cells in mice. Here we show that PRC1.1 serves as an epigenetic switch that coordinates homeostatic and emergency hematopoiesis. PRC1.1 maintains balanced output of steady-state hematopoiesis by restricting C/EBPα-dependent precocious myeloid differentiation of HSPCs and the HOXA9- and β-catenin-driven self-renewing network in myeloid progenitors. Upon regeneration, PRC1.1 is transiently inhibited to facilitate formation of granulocyte-macrophage progenitor (GMP) clusters, thereby promoting emergency myelopoiesis. Moreover, constitutive inactivation of PRC1.1 results in unchecked expansion of GMPs and eventual transformation. Collectively, our results define PRC1.1 as a novel critical regulator of emergency myelopoiesis, dysregulation of which leads to myeloid transformation.

Haematopoietic stem cells (HSCs) exhibit considerable cell-intrinsic changes with age. Here the authors demonstrate that differentially accessible regions in aged HSC chromatin are enriched for stress-responsive enhancers and act as an epigenetic hub to augment transcriptional responses of aged HSCs to external stimuli.

Abstract Both EZH2 and its homolog EZH1 function as histone H3 Lysine 27 (H3K27) methyltransferases and repress the transcription of target genes. Dysregulation of H3K27 trimethylation (H3K27me3) plays an important role in the development and progression of cancers such as hepatocellular carcinoma (HCC). This study investigated the relationship between the expression of EZH1/2 and the level of H3K27me3 in HCC. Additionally, the role of EZH1/2 in cell growth, tumorigenicity, and resistance to sorafenib were also analyzed. Both the lentiviral knockdown and the pharmacological inhibition of EZH1/2 (UNC1999) diminished the level of H3K27me3 and suppressed cell growth in liver cancer cells, compared with EZH1 or EZH2 single knockdown. Although a significant association was observed between EZH2 expression and H3K27me3 levels in HCC samples, overexpression of EZH1 appeared to contribute to enhanced H3K27me3 levels in some EZH2lowH3K27me3high cases. Akt suppression following sorafenib treatment resulted in an increase of the H3K27me3 levels through a decrease in EZH2 phosphorylation at serine 21. The combined use of sorafenib and UNC1999 exhibited synergistic antitumor effects in vitro and in vivo. Combination treatment canceled the sorafenib-induced enhancement in H3K27me3 levels, indicating that activation of EZH2 function is one of the mechanisms of sorafenib-resistance in HCC. In conclusion, sorafenib plus EZH1/2 inhibitors may comprise a novel therapeutic approach in HCC.

Pulmonary hypertension is characterized by increased pulmonary arterial pressure, driven in part by inflammatory infiltrates. Here, the authors show that in mice, transgenic expression of mutant JAK2 leads to clonal hematopoiesis and lung accumulation of elastase- and cytokine-expressing neutrophils, and that the phenotype can be reversed by ALK1 inhibition.

POEMS syndrome is a rare monoclonal plasma cell disorder, with unique symptoms distinct from those of other plasma cell neoplasms, including high serum VEGF levels. Because the prospective isolation of POEMS clones has not yet been successful, their real nature remains unclear. Herein, we performed single-cell RNA-Seq of BM plasma cells from patients with POEMS syndrome and identified POEMS clones that had Ig λ light chain (IGL) sequences (IGLV1-36, -40, -44, and -47) with amino acid changes specific to POEMS syndrome. The proportions of POEMS clones in plasma cells were markedly smaller than in patients with multiple myeloma (MM) and patients with monoclonal gammopathy of undetermined significance (MGUS). Single-cell transcriptomes revealed that POEMS clones were CD19+, CD138+, and MHC class IIlo, which allowed for their prospective isolation. POEMS clones expressed significantly lower levels of c-MYC and CCND1 than MM clones, accounting for their small size. VEGF mRNA was not upregulated in POEMS clones, directly indicating that VEGF is not produced by POEMS clones. These results reveal unique features of POEMS clones and enhance our understanding of the pathogenesis of POEMS syndrome.

Abstract The novel small molecule PTC596 inhibits microtubule polymerization and its clinical development has been initiated for some solid cancers. We herein investigated the preclinical efficacy of PTC596 alone and in combination with proteasome inhibitors in the treatment of multiple myeloma (MM). PTC596 inhibited the proliferation of MM cell lines as well as primary MM samples in vitro, and this was confirmed with MM cell lines in vivo. PTC596 synergized with bortezomib or carfilzomib to inhibit the growth of MM cells in vitro. The combination treatment of PTC596 with bortezomib exerted synergistic effects in a xenograft model of human MM cell lines in immunodeficient mice and exhibited acceptable tolerability. Mechanistically, treatment with PTC596 induced cell cycle arrest at G2/M phase followed by apoptotic cell death, associated with the inhibition of microtubule polymerization. RNA sequence analysis also revealed that PTC596 and the combination with bortezomib affected the cell cycle and apoptosis in MM cells. Importantly, endoplasmic reticulum stress induced by bortezomib was enhanced by PTC596, providing an underlying mechanism of action of the combination therapy. Our results indicate that PTC596 alone and in combination with proteasome inhibition are potential novel therapeutic options to improve outcomes in patients with MM.

Abstract Background Left ventricular reverse remodeling (LVRR) has been detected in non-ischemic dilated cardiomyopathy (NIDCM) patients following optimal treatment. However, its prediction with only conventional modalities is often difficult. This study sought to examine whether RNA sequencing (RNA-seq) of myocardium tissue samples could predict LVRR in NIDCM. Methods A total of 17 advanced NIDCM patients with left ventricular ejection fraction (LVEF) below 30% who underwent cardiac biopsy from Left ventricle (LV) were prospectively recruited. They received optimal treatment and followed with echocardiogram every 6 months. Based on LVRR status after 12 months of treatment, patients were divided into the reverse remodeling (RR) or non-RR group. Tissue samples were analyzed by RNA-seq, and a functional analysis of differentially expressed genes was carried out. Results There were eight and nine patients in the RR and non-RR groups, respectively. No difference was found in age, sex, disease duration, LV end-diastolic diameter, and LVEF between the two groups. There were 155 genes that were differentially expressed between the two groups. Nicotinamide adenine dinucleotide ubiquinone oxidoreductase subunit (NDUF)S5 and Growth arrest and DNA-damage-inducible protein (GADD)45G, along with several genes related to the mitochondrial respiratory chain and ribosome, were significantly downregulated in the RR as compared to the non-RR group. Conclusion GADD45G and NDUFS5 are potential biomarkers for LVRR in patients with advanced NIDCM.

Abstract: KDM2B together with RING1B, PCGF1, and BCOR or BCORL1 comprise polycomb repressive complex 1.1 (PRC1.1), a noncanonical PRC1 that catalyzes H2AK119ub1. It binds to nonmethylated CpG islands through its zinc finger-CxxC DNA binding domain and recruits the complex to target gene loci. Recent studies identified the loss of function mutations in the PRC1.1 gene, BCOR and BCORL1 in human T-cell acute lymphoblastic leukemia (T-ALL). We previously reported that Bcor insufficiency induces T-ALL in mice, supporting a tumor suppressor role for BCOR. However, the function of BCOR responsible for tumor suppression, either its corepressor function for BCL6 or that as a component of PRC1.1, remains unclear. We herein examined mice specifically lacking the zinc finger-CxxC domain of KDM2B in hematopoietic cells. Similar to Bcor-deficient mice, Kdm2b-deficient mice developed lethal T-ALL mostly in a NOTCH1-dependent manner. A chromatin immunoprecipitation sequence analysis of thymocytes revealed the binding of KDM2B at promoter regions, at which BCOR and EZH2 colocalized. KDM2B target genes markedly overlapped with those of NOTCH1 in human T-ALL cells, suggesting that noncanonical PRC1.1 antagonizes NOTCH1-mediated gene activation. KDM2B target genes were expressed at higher levels than the others and were marked with high levels of H2AK119ub1 and H3K4me3, but low levels of H3K27me3, suggesting that KDM2B target genes are transcriptionally active or primed for activation. These results indicate that PRC1.1 plays a key role in restricting excessive transcriptional activation by active NOTCH1, thereby acting as a tumor suppressor in the initiation of T-cell leukemogenesis.

Translocation of (6;8)(p21;q24), a recurrent abnormality of blastic plasmacytoid dendritic cell neoplasm, involves adjacent MYC and RUNX2 regions. Here, the authors show that the RUNX2 super-enhancer is hijacked to activate MYC in addition to RUNX2 expression, promoting the development of this cancer.

The recent development of next-generation sequencing (NGS) technologies has contributed to research into various biological processes. These novel NGS technologies have revealed the involvement of epigenetic memories in trained immunity, which are responses to transient stimulation and result in better responses to secondary challenges. Not only innate system cells, such as macrophages, monocytes, and natural killer cells, but also bone marrow hematopoietic stem cells (HSCs) have been found to gain memories upon transient stimulation, leading to the enhancement of responses to secondary challenges. Various stimuli, including microbial infection, can induce the epigenetic reprogramming of innate immune cells and HSCs, which can result in an augmented response to secondary stimulation. In this review, we introduce novel NGS technologies and their application to unraveling epigenetic memories that are key in trained immunity and summarize the recent findings in trained immunity. We also discuss our most recent finding regarding epigenetic memory in aged HSCs, which may be associated with the exposure of HSCs to aging-related stresses.

Summary: Polycomb repressive complex (PRC) 2 represses transcription through histone H3K27 trimethylation (H3K27me3). We previously reported that the hematopoietic-cell-specific deletion of Ezh2, encoding a PRC2 enzyme, induced myelodysplastic syndrome (MDS) in mice, whereas the concurrent Ezh1 deletion depleted hematopoietic stem and progenitor cells (HSPCs). We herein demonstrated that mice with only one Ezh1 allele (Ezh1+/-Ezh2Δ/Δ) maintained HSPCs. A chromatin immunopreciptation sequence analysis revealed that residual PRC2 preferentially targeted genes with high levels of H3K27me3 and H2AK119 monoubiquitination (H2AK119ub1) in HSPCs (designated as Ezh1 core target genes), which were mostly developmental regulators, and maintained H3K27me3 levels in Ezh1+/-Ezh2Δ/Δ HSPCs. Even upon the complete depletion of Ezh1 and Ezh2, H2AK119ub1 levels were largely retained, and only a minimal number of Ezh1 core targets were de-repressed. These results indicate that genes marked with high levels of H3K27me3 and H2AK119ub1 are the core targets of polycomb complexes in HSPCs as well as MDS stem cells. : Biological Sciences; Genetics; Molecular Biology; Immunology; Cell Biology; Developmental Biology Subject Areas: Biological Sciences, Genetics, Molecular Biology, Immunology, Cell Biology, Developmental Biology

Abstract Epstein-Barr virus (EBV) infection is associated with tumours such as Burkitt lymphoma, nasopharyngeal carcinoma, and gastric cancer. We previously showed that EBV(+) gastric cancer presents an extremely high-methylation epigenotype and this aberrant DNA methylation causes silencing of multiple tumour suppressor genes. However, the mechanisms that drive EBV infection-mediated tumorigenesis, including other epigenomic alteration, remain unclear. We analysed epigenetic alterations induced by EBV infection especially at enhancer regions, to elucidate their contribution to tumorigenesis. We performed ChIP sequencing on H3K4me3, H3K4me1, H3K27ac, H3K27me3, and H3K9me3 in gastric epithelial cells infected or not with EBV. We showed that repressive marks were redistributed after EBV infection, resulting in aberrant enhancer activation and repression. Enhancer dysfunction led to the activation of pathways related to cancer hallmarks (e.g., resisting cell death, disrupting cellular energetics, inducing invasion, evading growth suppressors, sustaining proliferative signalling, angiogenesis, and tumour-promoting inflammation) and inactivation of tumour suppressive pathways. Deregulation of cancer-related genes in EBV-infected gastric epithelial cells was also observed in clinical EBV(+) gastric cancer specimens. Our analysis showed that epigenetic alteration associated with EBV-infection may contribute to tumorigenesis through enhancer activation and repression.

Abstract Bmal1 (encoded by Arntl gene) is a core circadian clock gene that regulates various genes involved in circadian rhythm. Although Bmal1 is expressed rhythmically in macrophages, the role of Bmal1 in the regulation of their cellular function remains insufficiently understood. Here, we report that Bmal1 regulates time-dependent inflammatory responses following Toll-like receptor 4 (TLR4) activation by modulating enhancer activity. Global transcriptome analysis indicated that deletion of Arntl perturbed the time-dependent inflammatory responses elicited by TLR4 activation by Kdo2-lipid A (KLA). Although the recruitment of NF-κB p65 was unaffected, the acetylation status of lysine 27 of histone 3, which correlates positively with enhancer activity, was globally increased at PU.1-containing enhancers in Arntl −/− macrophages as compared to wild-type cells. Expression of Nr1d1 and Nr1d2, encoding RevErb transcription factors, which repress enhancer RNA expression, was significantly decreased in Arntl −/− macrophages. Moreover, the level of H3K27 acetylation was increased by Arntl deletion at RevErb-dependent eRNA-expressing enhancers. These results suggest that Bmal1 controls KLA-responsive enhancers, in part by regulating RevErb-directed eRNA transcription. Taken together, the results of this study show that the clock transcription factor network containing Bmal1 controls the inflammatory responses of macrophages by regulating the epigenetic states of enhancers.

Abstract: High-mobility group AT-hook 2 (HMGA2) is crucial for the self-renewal of fetal hematopoietic stem cells (HSCs) but is downregulated in adult HSCs via repression by MIRlet-7 and the polycomb-recessive complex 2 (PRC2) including EZH2. The HMGA2 messenger RNA (mRNA) level is often elevated in patients with myelofibrosis that exhibits an advanced myeloproliferative neoplasm (MPN) subtype, and deletion of Ezh2 promotes the progression of severe myelofibrosis in JAK2V617F mice with upregulation of several oncogenes such as Hmga2. However, the direct role of HMGA2 in the pathogenesis of MPNs remains unknown. To clarify the impact of HMGA2 on MPNs carrying the driver mutation, we generated ΔHmga2/JAK2V617F mice overexpressing Hmga2 due to deletion of the 3′ untranslated region. Compared with JAK2V617F mice, ΔHmga2/JAK2V617F mice exhibited more severe leukocytosis, anemia and splenomegaly, and shortened survival, whereas severity of myelofibrosis was comparable. ΔHmga2/JAK2V617F cells showed a greater repopulating ability that reproduced the severe MPN compared with JAK2V617F cells in serial bone marrow transplants, indicating that Hmga2 promotes MPN progression at the HSC level. Hmga2 also enhanced apoptosis of JAK2V617F erythroblasts that may worsen anemia. Relative to JAK2V617F hematopoietic stem and progenitor cells (HSPCs), over 30% of genes upregulated in ΔHmga2/JAK2V617F HSPCs overlapped with those derepressed by Ezh2 loss in JAK2V617F/Ezh2Δ/Δ HSPCs, suggesting that Hmga2 may facilitate upregulation of Ezh2 targets. Correspondingly, deletion of Hmga2 ameliorated anemia and splenomegaly in JAK2V617F/Ezh2Δ/wild-type mice, and MIRlet-7 suppression and PRC2 mutations correlated with the elevated HMGA2 mRNA levels in patients with MPNs, especially myelofibrosis. These findings suggest the crucial role of HMGA2 in MPN progression.

The “bivalent domain,” a distinctive histone modification signature, is characterized by repressive trimethylation of histone H3 at lysine 27 (H3K27me3) and active trimethylation of histone H3 at lysine 4 (H3K4me3) marks. Maintenance and dynamic resolution of these histone marks play important roles in regulating differentiation processes in various stem cell systems. However, little is known regarding their roles in hepatic stem/progenitor cells. In the present study, we conducted the chromatin immunoprecipitation (ChIP) assay followed by high-throughput DNA sequencing (ChIP-seq) analyses in purified delta-like 1 protein (Dlk+) hepatic stem/progenitor cells and successfully identified 562 genes exhibiting bivalent domains within 2 kb of the transcription start site. Gene ontology analysis revealed that these genes were enriched in developmental functions and differentiation processes. Microarray analyses indicated that many of these genes exhibited derepression after differentiation toward hepatocyte and cholangiocyte lineages. Among these, 72 genes, including Cdkn2a and Sox4, were significantly upregulated after differentiation toward hepatocyte or cholangiocyte lineages. Knockdown of Sox4 in Dlk+ cells suppressed colony propagation and resulted in increased numbers of albumin+/cytokeratin 7+ progenitor cells in colonies. These findings implicate that derepression of Sox4 expression is required to induce normal differentiation processes. In conclusion, combined ChIP-seq and microarray analyses successfully identified bivalent genes. Functional analyses of these genes will help elucidate the epigenetic machinery underlying the terminal differentiation of hepatic stem/progenitor cells.

TIF1β is a transcriptional corepressor that recruits repressive chromatin modifiers to target genes. Its biological function and physiological targets in somatic stem cells remain largely unknown. Here, we show that TIF1β is essential for the maintenance of hematopoietic stem cells (HSCs). Deletion of Tif1b in mice induced active cycling and apoptosis of HSCs and promoted egression of HSCs from the bone marrow, leading to rapid depletion of HSCs. Strikingly, Tif1b-deficient HSCs showed a strong trend of ectopic expression of nonhematopoietic genes. Levels of heterochromatin protein 1 (HP1α, β and γ) proteins, which form a complex with TIF1β, were significantly reduced in the absence of TIF1β and depletion of HP1 recapitulated a part of the phenotypes of Tif1b-deficient HSCs. These results demonstrate that the TIF1β-HP1 system functions as a critical repressive machinery that targets genes not normally activated in the hematopoietic compartment, thereby maintaining the transcriptional signature specific to HSCs.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Polycomb-group RING finger proteins (Pcgf1-Pcgf6) are components of Polycomb repressive complex 1 (PRC1)-related complexes that catalyze monoubiquitination of histone H2A at lysine 119 (H2AK119ub1), an epigenetic mark associated with repression of genes. Pcgf5 has been characterized as a component of PRC1.5, one of the non-canonical PRC1, consisting of Ring1a/b, Rybp/Yaf2 and Auts2. However, the biological functions of Pcgf5 have not yet been identified. Here we analyzed the impact of the deletion of Pcgf5 specifically in hematopoietic stem and progenitor cells (HSPCs). Pcgf5 is expressed preferentially in hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) compared with committed myeloid progenitors and differentiated cells. We transplanted bone marrow (BM) cells from Rosa::Cre-ERT control and Cre-ERT;Pcgf5fl/fl mice into lethally irradiated recipient mice. At 4 weeks post-transplantation, we deleted Pcgf5 by injecting tamoxifen, however, no obvious changes in hematopoiesis were detected including the number of HSPCs during a long-term observation period following the deletion. Competitive BM repopulating assays revealed normal repopulating capacity of Pcgf5-deficient HSCs. Nevertheless, Pcgf5-deficient HSPCs showed a significant reduction in H2AK119ub1 levels compared with the control. ChIP-sequence analysis confirmed the reduction in H2AK119ub1 levels, but revealed no significant association of changes in H2AK119ub1 levels with gene expression levels. Our findings demonstrate that Pcgf5-containing PRC1 functions as a histone modifier in vivo, but its role in HSPCs is limited and can be compensated by other PRC1-related complexes in HSPCs.

Precise understanding of radiation effects is critical to develop new modalities for the prevention and treatment of radiation-induced damage. We previously reported that non-lethal doses of X-ray irradiation induce DNA damage in human hematopoietic stem and progenitor cells (HSPCs) reconstituted in NOD/Shi-scid IL2rγnull (NOG) immunodeficient mice and severely compromise their repopulating capacity. In this study, we analyzed in detail the functional changes in human HSPCs in NOG mice following non-lethal radiation. We transplanted cord blood CD34+ HSPCs into NOG mice. At 12 weeks post-transplantation, the recipients were irradiated with 0, 0.5, or 1.0 Gy. At 2 weeks post-irradiation, human CD34+ HSPCs recovered from the primary recipient mice were transplanted into secondary recipients. CD34+ HSPCs from irradiated mice showed severely impaired reconstitution capacity in the secondary recipient mice. Of interest, non-lethal radiation compromised contribution of HSPCs to the peripheral blood cells, particularly to CD19+ B lymphocytes, which resulted in myeloid-biased repopulation. Co-culture of limiting numbers of CD34+ HSPCs with stromal cells revealed that the frequency of B cell-producing CD34+ HSPCs at 2 weeks post-irradiation was reduced more than 10-fold. Furthermore, the key B-cell regulator genes such as IL-7R and EBF1 were downregulated in HSPCs upon 0.5 Gy irradiation. Given that compromised repopulating capacity and myeloid-biased differentiation are representative phenotypes of aged HSCs, our findings indicate that non-lethal ionizing radiation is one of the critical external stresses that promote aging of human HSPCs in the bone marrow niche.

Tumor-initiating cells (TICs) play a central role in tumor development, metastasis, and recurrence. In the present study, we investigated the effect of disulfiram (DSF), an inhibitor of aldehyde dehydrogenase, toward tumor-initiating hepatocellular carcinoma (HCC) cells. DSF treatment suppressed the anchorage-independent sphere formation of both HCC cells. Flow cytometric analyses showed that DSF but not 5-fluorouracil (5-FU) drastically reduces the number of tumor-initiating HCC cells. The sphere formation assays of epithelial cell adhesion molecule (EpCAM)(+) HCC cells co-treated with p38-specific inhibitor revealed that DSF suppresses self-renewal capability mainly through the activation of reactive oxygen species (ROS)-p38 MAPK pathway. Microarray experiments also revealed the enrichment of the gene set involved in p38 MAPK signaling in EpCAM(+) cells treated with DSF but not 5-FU. In addition, DSF appeared to downregulate Glypican 3 (GPC3) in a manner independent of ROS-p38 MAPK pathway. GPC3 was co-expressed with EpCAM in HCC cell lines and primary HCC cells and GPC3-knockdown reduced the number of EpCAM(+) cells by compromising their self-renewal capability and inducing the apoptosis. These results indicate that DSF impaired the tumorigenicity of tumor-initiating HCC cells through activation of ROS-p38 pathway and in part through the downregulation of GPC3. DSF might be a promising therapeutic agent for the eradication of tumor-initiating HCC cells.

Metformin has been widely used as an oral drug for diabetes mellitus for approximately 60 years. Interestingly, recent reports showed that metformin exhibited an anti-tumor action in a wide range of malignancies including hepatocellular carcinoma (HCC). In the present study, we investigated its impact on tumor-initiating HCC cells. Metformin suppressed cell growth and induced apoptosis in a dose-dependent manner. Flow cytometric analysis showed that metformin treatment markedly reduced the number of tumor-initiating epithelial cell adhesion molecule (EpCAM)(+) HCC cells. Non-adherent sphere formation assays of EpCAM(+) cells showed that metformin impaired not only their sphere-forming ability, but also their self-renewal capability. Consistent with this, immunostaining of spheres revealed that metformin significantly decreased the number of component cells positive for hepatic stem cell markers such as EpCAM and α-fetoprotein. In a xenograft transplantation model using non-obese diabetic/severe combined immunodeficient mice, metformin and/or sorafenib treatment suppressed the growth of tumors derived from transplanted HCC cells. Notably, the administration of metformin but not sorafenib decreased the number of EpCAM(+) cells and impaired their self-renewal capability. As reported, metformin activated AMP-activated protein kinase (AMPK) through phosphorylation; however its inhibitory effect on the mammalian target of rapamycin (mTOR) pathway did not necessarily correlate with its anti-tumor activity toward EpCAM(+) tumor-initiating HCC cells. These results indicate that metformin is a promising therapeutic agent for the elimination of tumor-initiating HCC cells and suggest as-yet-unknown functions other than its inhibitory effect on the AMPK/mTOR pathway.

The polycomb-group (PcG) proteins function as general regulators of stem cells. We previously reported that retrovirus-mediated overexpression of Bmi1, a gene encoding a core component of polycomb repressive complex (PRC) 1, maintained self-renewing hematopoietic stem cells (HSCs) during long-term culture. However, the effects of overexpression of Bmi1 on HSCs in vivo remained to be precisely addressed.In this study, we generated a mouse line where Bmi1 can be conditionally overexpressed under the control of the endogenous Rosa26 promoter in a hematopoietic cell-specific fashion (Tie2-Cre;R26Stop(FL)Bmi1). Although overexpression of Bmi1 did not significantly affect steady state hematopoiesis, it promoted expansion of functional HSCs during ex vivo culture and efficiently protected HSCs against loss of self-renewal capacity during serial transplantation. Overexpression of Bmi1 had no effect on DNA damage response triggered by ionizing radiation. In contrast, Tie2-Cre;R26Stop(FL)Bmi1 HSCs under oxidative stress maintained a multipotent state and generally tolerated oxidative stress better than the control. Unexpectedly, overexpression of Bmi1 had no impact on the level of intracellular reactive oxygen species (ROS).Our findings demonstrate that overexpression of Bmi1 confers resistance to stresses, particularly oxidative stress, onto HSCs. This thereby enhances their regenerative capacity and suggests that Bmi1 is located downstream of ROS signaling and negatively regulated by it.