Nicotinamide mononucleotide (NMN) alleviates the poly(I:C)-induced inflammatory response in human primary cell cultures.

NMN is the direct precursor of nicotinamide adenine dinucleotide (NAD+) and is considered as a key factor for increasing NAD+ levels and mitochondrial activity in cells. In this study, based on transcriptome analysis, we showed that NMN alleviates the poly(I:C)-induced inflammatory response in cultures of two types of human primary cells, human pulmonary microvascular endothelial cells (HPMECs) and human coronary artery endothelial cells (HCAECs). Major inflammatory mediators, including IL6 and PARP family members, were grouped into coexpressed gene modules and significantly downregulated under NMN exposure in poly(I:C)-activated conditions in both cell types. The Bayesian network analysis of module hub genes predicted common genes, including eukaryotic translation initiation factor 4B (EIF4B), and distinct genes, such as platelet-derived growth factor binding molecules, in HCAECs, which potentially regulate the identified inflammation modules. These results suggest a robust regulatory mechanism by which NMN alleviates inflammatory pathway activation, which may open up the possibility of a new role for NMN replenishment in the treatment of chronic or acute inflammation.

[Klinefelter's syndrome diagnosed at the onset of acute myeloid leukemia with inv (16) following treatment for germ cell tumor].

A 22-year-old man with a history of mediastinal germ cell tumor, which was diagnosed at age 20 and remained disease-free after chemotherapy, was diagnosed with acute myeloid leukemia (AML) M2 in January 2020. Karyotype analysis of bone marrow (BM) specimen at diagnosis detected 47,XXY, inv (16) in all cells. Following induction treatment, he achieved complete remission with a remarkable decrease in the minimal residual disease marker. Although considered related to therapy, the AML had a prognostically favorable karyotype, and the initial treatment response was very good. He had no human leukocyte antigen-matched sibling donor candidate. Thus, allogeneic hematopoietic stem cell transplantation was not scheduled at the first complete remission. After three cycles of consolidation therapy, he remained disease-free for over one year. Karyotype analysis of BM during remission revealed that all analyzed cells harbored 47,XXY, and Klinefelter syndrome (KS) was diagnosed. Although the patient experienced an adjustment disorder on KS diagnosis, he had overcome the difficulty with the assistance of psycho-oncologists, clinical psychologists, and genetic counselors. Herein, we report this rare case of KS that manifested after AML diagnosis following mediastinal germ cell tumor treatment.

Gene Amplification and the Extrachromosomal Circular DNA.

Oncogene amplification is closely linked to the pathogenesis of a broad spectrum of human malignant tumors. The amplified genes localize either to the extrachromosomal circular DNA, which has been referred to as cytogenetically visible double minutes (DMs), or submicroscopic episome, or to the chromosomal homogeneously staining region (HSR). The extrachromosomal circle from a chromosome arm can initiate gene amplification, resulting in the formation of DMs or HSR, if it had a sequence element required for replication initiation (the replication initiation region/matrix attachment region; the IR/MAR), under a genetic background that permits gene amplification. In this article, the nature, intracellular behavior, generation, and contribution to cancer genome plasticity of such extrachromosomal circles are summarized and discussed by reviewing recent articles on these topics. Such studies are critical in the understanding and treating human cancer, and also for the production of recombinant proteins such as biopharmaceuticals by increasing the recombinant genes in the cells.

SIRT1 stabilizes extrachromosomal gene amplification and contributes to repeat-induced gene silencing.

Sirtuin 1 (SIRT1) is a protein deacetylase that maintains genome stability by preventing the activation of latent replication origins. Amplified genes in cancer cells localize on either extrachromosomal double minutes (DMs) or the chromosomal homogeneously staining region. Previously, we found that a plasmid with a mammalian replication initiation region and a matrix attachment region spontaneously mimics gene amplification in cultured animal cells and efficiently generates DMs and/or an homogeneously staining region. Here, we addressed the possibility that SIRT1 might be involved in initiation region/matrix attachment region-mediated gene amplification using SIRT1-knockout human COLO 320DM cells. Consequently, we found that extrachromosomal amplification was infrequent in SIRT1-deficient cells, suggesting that DNA breakage caused by latent origin activation prevented the formation of stable extrachromosomal amplicons. Moreover, we serendipitously found that reporter gene expression from the amplified repeats, which is commonly silenced by repeat-induced gene silencing (RIGS) in SIRT1-proficient cells, was strikingly higher in SIRT1-deficient cells, especially in the culture treated with the histone deacetylase inhibitor butyrate. Compared with the SIRT1-proficient cells, the gene expression per copy was up to thousand-fold higher in the sorter-isolated highest 10% cells among the SIRT1-deficient cells. These observations suggest that SIRT1 depletion alleviates RIGS. Thus, SIRT1 may stabilize extrachromosomal amplicons and facilitate RIGS. This result could have implications in cancer malignancy and protein expression.

Repeat induces not only gene silencing, but also gene activation in mammalian cells.

Repeat-induced gene silencing (RIGS) establishes the centromere structure, prevents the spread of transposons and silences transgenes, thereby limiting recombinant protein production. We previously isolated a sequence (B-3-31) that alleviates RIGS from the human genome. Here, we developed an assay system for evaluating the influence of repeat sequences on gene expression, based on in vitro ligation followed by our original gene amplification technology in animal cells. Using this assay, we found that the repeat of B-3-31, three core sequences of replication initiation regions (G5, C12, and D8) and two matrix attachment regions (AR1 and 32-3), activated the co-amplified plasmid-encoded d2EGFP gene in both human and hamster cell lines. This upregulation effect persisted for up to 82 days, which was confirmed to be repeat-induced, and was thus designated as a repeat-induced gene activation (RIGA). In clear contrast, the repeat of three bacterial sequences (lambda-phage, Amp, and ColE1) and three human retroposon sequences (Alu, 5'-untranslated region, and ORF1 of a long interspersed nuclear element) suppressed gene expression, thus reflecting RIGS. RIGS was CpG-independent. We suggest that RIGA might be associated with replication initiation. The discovery of RIGS and RIGA has implications for the repeat in mammalian genome, as well as practical value in recombinant production.

Double-strand breakage in the extrachromosomal double minutes triggers their aggregation in the nucleus, micronucleation, and morphological transformation.

Gene amplification plays a pivotal role in malignant transformation. Amplified genes often reside on extrachromosomal double minutes (DMs). Low-dose hydroxyurea induces DM aggregation in the nucleus which, in turn, generates micronuclei composed of DMs. Low-dose hydroxyurea also induces random double-strand breakage throughout the nucleus. In the present study, we found that double-strand breakage in DMs is sufficient for induction of DM aggregation. Here, we used CRISPR/Cas9 to introduce specific breakages in both natural and artificially tagged DMs of human colorectal carcinoma COLO 320DM cells. Aggregation occurred in the S phase but not in the G1 phase within 4 hours after breakage, which suggested the possible involvement of homologous recombination in the aggregation of numerous DMs. Simultaneous detection of DMs and the phosphorylated histone H2AX revealed that the aggregation persisted after breakage repair. Thus, the aggregate generated cytoplasmic micronuclei at the next interphase. Our data also suggested that micronuclear entrapment eliminated the DMs or morphologically transformed them into giant DMs or homogeneously staining regions (HSRs). In this study, we obtained a model explaining the consequences of DMs after double-strand breakage in cancer cells. Because double-strand breakage is frequently involved in cancer therapy, the model suggests how it affects gene amplification.

An efficient protein production system via gene amplification on a human artificial chromosome and the chromosome transfer to CHO cells.

Gene amplification methods play a crucial role in establishment of cells that produce high levels of recombinant protein. However, the stability of such cell lines and the level of recombinant protein produced continue to be suboptimal. Here, we used a combination of a human artificial chromosome (HAC) vector and initiation region (IR)/matrix attachment region (MAR) gene amplification method to establish stable cells that produce high levels of recombinant protein. Amplification of Enhanced green fluorescent protein (EGFP) was induced on a HAC carrying EGFP gene and IR/MAR sequences (EGFP MAR-HAC) in CHO DG44 cells. The expression level of EGFP increased approximately 6-fold compared to the original HAC without IR/MAR sequences. Additionally, anti-vascular endothelial growth factor (VEGF) antibody on a HAC (VEGF MAR-HAC) was also amplified by utilization of this IR/MAR-HAC system, and anti-VEGF antibody levels were approximately 2-fold higher compared with levels in control cells without IR/MAR. Furthermore, the expression of anti-VEGF antibody with VEGF MAR-HAC in CHO-K1 cells increased 2.3-fold compared with that of CHO DG44 cells. Taken together, the IR/MAR-HAC system facilitated amplification of a gene of interest on the HAC vector, and could be used to establish a novel cell line that stably produced protein from mammalian cells.

Targeted amplification of a sequence of interest in artificial chromosome in mammalian cells.

A plasmid with a replication initiation region (IR) and a matrix attachment region (MAR) initiates gene amplification in mammalian cells at a random chromosomal location. A mouse artificial chromosome (MAC) vector can stably carry a large genomic region. In this study we combined these two technologies with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas)9 strategy to achieve targeted amplification of a sequence of interest. We previously showed that the IR/MAR plasmid was amplified up to the extrachromosomal tandem repeat; here we demonstrate that cleavage of these tandem plasmids and MAC by Cas9 facilitates homologous recombination between them. The plasmid array on the MAC could be further extended to form a ladder structure with high gene expression by a breakage-fusion-bridge cycle involving breakage at mouse major satellites. Amplification of genes on the MAC has the advantage that the MAC can be transferred between cells. We visualized the MAC in live cells by amplifying the lactose operator array on the MAC in cells expressing lactose repressor-green fluorescent protein fusion protein. This targeted amplification strategy is in theory be applicable to any sequence at any chromosomal site, and provides a novel tool for animal cell technology.

Generation and maintenance of acentric stable double minutes from chromosome arms in inter-species hybrid cells.

BACKGROUND: Extrachromosomal acentric double minutes (DMs) contribute to human malignancy by carrying amplified oncogenes. Recent cancer genomics revealed that the pulverization of defined chromosome arms (chromothripsis) may generate DMs, however, nobody had actually generated DMs from chromosome arm in culture. Human chromosomes are lost in human-rodent hybrid cells. RESULTS: We found that human acentric DMs with amplified c-myc were stable in human-rodent hybrid cells, although the degree of stability depended on the specific rodent cell type. Based on this finding, stable human-rodent hybrids were efficiently generated by tagging human DMs with a plasmid with drug-resistance gene. After cell fusion, human chromosomes were specifically pulverised and lost. Consistent with chromothripsis, pulverization of human chromosome arms was accompanied by the incorporation into micronuclei. Such micronucleus showed different replication timing from the main nucleus. Surprisingly, we found that the hybrid cells retained not only the original DMs, but also new DMs without plasmid-tag and c-myc, but with human Alu. These DMs were devoid of telomeres and centromeres, and were stable in culture for more than 3 months. Microarray analysis showed that the new DMs were generated from several human chromosomal regions containing genes advantageous for cellular growth. Such regions were completely different from the original DMs. CONCLUSIONS: The inter-species hybrid mimics the chromothripsis in culture. This is the first report that experimentally demonstrates the generation of multiple stable acentric DMs from the chromosome arm.

Pressure overload inhibits glucocorticoid receptor transcriptional activity in cardiomyocytes and promotes pathological cardiac hypertrophy.

Glucocorticoid receptor (GR) is abundantly expressed in cardiomyocytes. However, the role of GR in regulating cardiac hypertrophy and heart failure in response to pressure overload remains unclear. Cardiomyocyte-specific GR knockout (GRcKO) mice, mineralocorticoid receptor (MR) knockout (MRcKO), and GR and MR double KO (GRMRdcKO) mice were generated using the Cre-lox system. In response to pressure overload, GRcKO mice displayed worse cardiac remodeling compared to control (GRf/f) mice, including a greater increase in heart weight to body weight ratio with a greater increase in cardiomyocytes size, a greater decline in left ventricular contractility, and higher reactivation of fetal genes. MRcKO mice showed a comparable degree of cardiac remodeling compared to control (MRf/f) mice. The worse cardiac remodeling in pressure overloaded GRcKO mice is not due to compensatory activation of cardiomyocyte MR, since pressure overloaded GRMRdcKO mice displayed cardiac remodeling to the same extent as GRcKO mice. Pressure overload suppressed GR-target gene expression in the heart. Although plasma corticosterone levels and subcellular localization of GR (nuclear/cytoplasmic GR) were not changed, a chromatin immunoprecipitation assay revealed that GR recruitment onto the promoter of GR-target genes was significantly suppressed in response to pressure overload. Rescue of the expression of GR-target genes to the same extent as sham-operated hearts attenuated adverse cardiac remodeling in pressure-overloaded hearts. Thus, GR works as a repressor of adverse cardiac remodeling in response to pressure overload, but GR-mediated transcription is suppressed under pressure overload. Therapies that maintain GR-mediated transcription in cardiomyocytes under pressure overload can be a promising therapeutic strategy for heart failure.

Publisher Correction: Direct reprogramming of fibroblasts into skeletal muscle progenitor cells by transcription factors enriched in undifferentiated subpopulation of satellite cells.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

LSD1 mediates metabolic reprogramming by glucocorticoids during myogenic differentiation.

The metabolic properties of cells are formed under the influence of environmental factors such as nutrients and hormones. Although such a metabolic program is likely initiated through epigenetic mechanisms, the direct links between metabolic cues and activities of chromatin modifiers remain largely unknown. In this study, we show that lysine-specific demethylase-1 (LSD1) controls the metabolic program in myogenic differentiation, under the action of catabolic hormone, glucocorticoids. By using transcriptomic and epigenomic approaches, we revealed that LSD1 bound to oxidative metabolism and slow-twitch myosin genes, and repressed their expression. Consistent with this, loss of LSD1 activity during differentiation enhanced the oxidative capacity of myotubes. By testing the effects of various hormones, we found that LSD1 levels were decreased by treatment with the glucocorticoid dexamethasone (Dex) in cultured myoblasts and in skeletal muscle from mice. Mechanistically, glucocorticoid signaling induced expression of a ubiquitin E3 ligase, JADE-2, which was responsible for proteasomal degradation of LSD1. Consequently, in differentiating myoblasts, chemical inhibition of LSD1, in combination with Dex treatment, synergistically de-repressed oxidative metabolism genes, concomitant with increased histone H3 lysine 4 methylation at these loci. These findings demonstrated that LSD1 serves as an epigenetic regulator linking glucocorticoid action to metabolic programming during myogenic differentiation.

Author Correction: Direct reprogramming of fibroblasts into skeletal muscle progenitor cells by transcription factors enriched in undifferentiated subpopulation of satellite cells.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Direct reprogramming of fibroblasts into skeletal muscle progenitor cells by transcription factors enriched in undifferentiated subpopulation of satellite cells.

Satellite cells comprise a functionally heterogeneous population of stem cells in skeletal muscle. Separation of an undifferentiated subpopulation and elucidation of its molecular background are necessary to identify the reprogramming factors to induce skeletal muscle progenitor cells. In this study, we found that intracellular esterase activity distinguishes a subpopulation of cultured satellite cells with high stemness using esterase-sensitive cell staining reagent, calcein-AM. Gene expression analysis of this subpopulation revealed that defined combinations of transcription factors (Pax3, Mef2b, and Pitx1 or Pax7, Mef2b, and Pitx1 in embryonic fibroblasts, and Pax7, Mef2b and MyoD in adult fibroblasts) reprogrammed fibroblasts into skeletal muscle progenitor cells. These reprogrammed cells formed Dystrophin-positive mature muscle fibers when transplanted into a mouse model of Duchenne muscular dystrophy. These results highlight the new marker for heterogenous population of cultured satellite cells, potential therapeutic approaches and cell sources for degenerative muscle diseases.

Phosphorylated SIRT1 associates with replication origins to prevent excess replication initiation and preserve genomic stability.

Chromatin structure affects DNA replication patterns, but the role of specific chromatin modifiers in regulating the replication process is yet unclear. We report that phosphorylation of the human SIRT1 deacetylase on Threonine 530 (T530-pSIRT1) modulates DNA synthesis. T530-pSIRT1 associates with replication origins and inhibits replication from a group of 'dormant' potential replication origins, which initiate replication only when cells are subject to replication stress. Although both active and dormant origins bind T530-pSIRT1, active origins are distinguished from dormant origins by their unique association with an open chromatin mark, histone H3 methylated on lysine 4. SIRT1 phosphorylation also facilitates replication fork elongation. SIRT1 T530 phosphorylation is essential to prevent DNA breakage upon replication stress and cells harboring SIRT1 that cannot be phosphorylated exhibit a high prevalence of extrachromosomal elements, hallmarks of perturbed replication. These observations suggest that SIRT1 phosphorylation modulates the distribution of replication initiation events to insure genomic stability.

Amplification of a transgene within a long array of replication origins favors higher gene expression in animal cells.

Plasmids with both a mammalian replication initiation region (IR) and a matrix attachment region (MAR) are spontaneously amplified in transfected cells, and generate extrachromosomal double minute (DM) or chromosomal homogeneously staining region (HSR). We previously isolated the shortest core IR (G5) required for gene amplification. In this study, we ligated the G5 DNA to create direct or inverted repeats, mixed the repeats with an expression plasmid, and transfected the mixture into human COLO 320DM or hamster CHO DG44 cells. Consequently, we found that the transfected sequence generated DMs or HSR where, surprisingly, the plasmid sequence was embedded within a long stretch of G5 sequences. The amplified structure from the direct G5 repeats was stable, whereas that from the inverted repeats was not. The amplification might be explained by the efficient replication/multimerization of the G5 repeat and recombination with the co-transfected plasmid in an extrachromosomal context. The product might then be integrated into a chromosome arm to generate a HSR. The expression from the plasmid within the long G5 array was much higher than that from a simple plasmid repeat. Because G5 is a core IR that favors gene expression, a long array of G5 provides an excellent environment for gene expression from the embedded plasmid.

Role of skeletal muscle glucocorticoid receptor in systemic energy homeostasis.

Glucocorticoids (GCs) affect nearly every organ and tissue in the body, regulating diverse physiologic processes including energy homeostasis. The metabolic mission of GCs is to supply enough glucose into the circulation to fuel the brain and ensure survival of the organism under conditions of acute stress or starvation. Recent studies have revealed that GCs, via orchestration between multiple organs, physiologically elicit fine tuning of systemic energy metabolism.

[Novel role of skeletal muscle in systemic energy homeostasis -mechano-metabo coupling via muscle-liver-fat signaling axis.]

Skeletal muscle has a pleiotropic role in organismal energy metabolism, for example, by storing protein as an energy source, or by excreting endocrine hormones. Muscle proteolysis is an active process tightly controlled by specific signaling pathways and transcriptional programs, involving ubiquitin-proteasome and autophagy machineries. Glucocorticoid receptor(GR)transcriptionally controls expression levels of components in the both machineries, and modulates muscle-liver-fat signaling axis. This skeletal muscle-liver-fat signaling axis controls organismal energy distribution and may serve as a target for the development of therapies against various metabolic diseases, including obesity.

The histone 3 lysine 9 methyltransferase inhibitor chaetocin improves prognosis in a rat model of high salt diet-induced heart failure.

Histone acetylation has been linked to cardiac hypertrophy and heart failure. However, the pathological implications of changes in histone methylation and the effects of interventions with histone methyltransferase inhibitors for heart failure have not been fully clarified. Here, we focused on H3K9me3 status in the heart and investigated the effects of the histone H3K9 methyltransferase inhibitor chaetocin on prognoses in Dahl salt-sensitive rats, an animal model of chronic heart failure. Chaetocin prolonged survival and restored mitochondrial dysfunction. ChIP-seq analysis demonstrated that chronic stress to the heart induced H3K9me3 elevation in thousands of repetitive elements, including intronic regions of mitochondria-related genes, such as the gene encoding peroxisome proliferator-activated receptor-gamma coactivator 1 alpha. Furthermore, chaetocin reversed this effect on these repetitive loci. These data suggested that excessive heterochromatinization of repetitive elements of mitochondrial genes in the failing heart may lead to the silencing of genes and impair heart function. Thus, chaetocin may be a potential therapeutic agent for chronic heart failure.

The effects of bolus supplementation of branched-chain amino acids on skeletal muscle mass, strength, and function in patients with rheumatic disorders during glucocorticoid treatment.

OBJECTIVES: To test the effects of bolus supplementation of branched-chain amino acids (BCAA) on skeletal muscle mass, strength, and function in patients with rheumatic disorders taking glucocorticoid (GC). METHODS: Patients with rheumatic disorders treated with prednisolone (≥10 mg/day) were randomized to ingest additional daily 12 g of BCAA (n = 9) or not (n = 9) for 12 weeks. At baseline, and 4, 8, and 12 weeks, they underwent bioelectrical impedance analysis, muscle strength and functional tests, and computed tomography analysis for cross-sectional area of mid-thigh muscle. RESULTS: Disease activities of the patients were well controlled and daily GC dose was similarly reduced in both groups. Limb muscle mass was recovered in both groups. Whole-body muscle mass and muscle strength and functional mobility were increased only in BCAA (+) group. The effects of BCAA supplementation on recovering skeletal muscle mass were prominent in particular muscles including biceps femoris muscle. CONCLUSIONS: This trial is the first-in-man clinical trial to demonstrate that BCAA supplementation might be safe and, at least in part, improve skeletal muscle mass, strength, and function in patients with rheumatic disorders treated with GC.