Plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) are spontaneously amplified in transfected mammalian cells, and such amplification generates chromosomal homogeneously staining regions (HSRs) or extrachromosomal double minutes (DMs). This method provides a novel, efficient, and rapid way to establish cells that stably produce high levels of recombinant proteins. However, because IR/MAR plasmids are amplified as repeats, they are frequently targeted by repeat-induced gene silencing (RIGS), which silences a variety of repeated sequences in transgenes and the genome. To address this problem, we developed a novel screening system using the IR/MAR plasmid to isolate human genome sequences that alleviate RIGS. The screen identified a 3,271 bp sequence (B-3-31) that elevated transgene expression without affecting the amplification process. Neither non-B structure (i.e., the inverted repeats or bending) nor known epigenetic modifier elements such as MARs, insulators, UCOEs, or STARs could explain the anti-silencing activity of B-3-31. Instead, the activity was distributed throughout the entire B-3-31 sequence, which was extremely A/T-rich and CpG-poor. Because B-3-31 effectively and reproducibly alleviated RIGS of repeated genes, it could be used to increase recombinant protein production.

We previously found that plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) efficiently initiate gene amplification and spontaneously increase their copy numbers in animal cells. In this study, this novel method was applied to the establishment of cells with high recombinant antibody production. The level of recombinant antibody expression was tightly correlated with the efficiency of plasmid amplification and the cytogenetic appearance of the amplified genes, and was strongly dependent on cell type. By using a widely used cell line for industrial protein production, CHO DG44, clones expressing very high levels of antibody were easily obtained. High-producer clones stably expressed the antibody over several months without eliciting changes in both the protein expression level and the cytogenetic appearance of the amplified genes. The integrity and reactivity of the protein produced by this method was fine. In serum-free suspension culture, the specific protein production rate in high-density cultures was 29.4 pg/cell/day. In conclusion, the IR/MAR gene amplification method is a novel and efficient platform for recombinant antibody production in mammalian cells, which rapidly and easily enables the establishment of stable high-producer cell clone.

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.

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.

The presence of micronuclei in mammalian cells is related to several mutagenetic stresses. In order to understand how micronuclei emerge, behave in cells, and affect cell fate, we performed extensive time-lapse microscopy of HeLa H2B-GFP cells in the presence of hydroxyurea at low concentration. Micronuclei formed after mitosis from lagging chromatids or chromatin bridges between anaphase chromosomes and were stably maintained in the cells for up to one cell cycle. Nuclear buds also formed from chromatin bridges or during interphase. If the micronuclei-bearing cells entered mitosis, they either produced daughter cells without micronuclei or, more frequently, produced cells with additional micronuclei. Low concentrations of hydroxyurea efficiently induced multipolar mitosis, which generated lagging chromatids or chromatin bridges, and also generated multinuclear cells that were tightly linked to apoptosis. We found that the presence of micronuclei is related to apoptosis but not to multipolar mitosis. Furthermore, the structural heterogeneity among micronuclei, with respect to chromatin condensation or the presence of lamin B, derived from the mechanism of micronuclei formation. Our study reinforces the notion that micronucleation has important implications in the genomic plasticity of tumor 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.

Gene amplification plays a pivotal role in malignant transformation of human cells. A plasmid with both a mammalian replication-initiation region (IR)/origin/replicator and a nuclear matrix-attachment region (MAR) is spontaneously amplified in transfected cells by a mechanism that involves amplification at the extrachromosomal site, followed by amplification at the chromosomal arm, ultimately generating a long homogeneously staining region (HSR). Several observations suggest that replication initiation from IR sequences might mediate amplification. To test this idea, we previously dissected c-myc and DHFR IRs to identify the minimum sequence required to support amplification. In this study, we applied an improved analysis that discriminates between two amplification steps to the ß-globin RepP IR, which contains separate elements already known to be essential for initiation on the chromosome arm. The IR sequence was required at least for the extrachromosomal amplification step. In addition to the vector-encoded MAR, amplification also required an AT-rich region and a MAR-like element, consistent with the results regarding replicator activity on the chromosome. However, amplification did not require the AG-rich tract necessary for replicator activity, but instead required a novel sequence containing another AG-rich tract. The differential sequence requirement might be a consequence of extrachromosomal replication.

The gene amplification plays a critical role in the malignant transformation of mammalian cells. The most widespread method for amplifying a target gene in cell culture is the use of methotrexate (Mtx) treatment to amplify dihydrofolate reductase (Dhfr). Whereas, we found that a plasmid bearing both a mammalian origin of replication (initiation region; IR) and a matrix attachment region (MAR) was spontaneously amplified in mammalian cells. In this study, we attempted to uncover the underlying mechanism by which the IR/MAR sequence might accelerate Mtx induced Dhfr amplification. The plasmid containing the IR/MAR was extrachromosomally amplified, and then integrated at multiple chromosomal locations within individual cells, increasing the likelihood that the plasmid might be inserted into a chromosomal environment that permits high expression and further amplification. Efficient amplification of this plasmid alleviated the genotoxicity of Mtx. Clone-based cytogenetic and sequence analysis revealed that the plasmid was amplified in a chromosomal context by breakage-fusion-bridge cycles operating either at the plasmid repeat or at the flanking fragile site activated by Mtx. This mechanism explains how a circular molecule bearing IR/MAR sequences of chromosomal origin might be amplified under replication stress, and also provides insight into gene amplification in human cancer.

We here addressed the basic question, how does extrachromosomal DNA behave when it is placed in the nuclear or the cytoplasmic environment and how is it eliminated? To do this, we tracked microinjected DNA molecules in live cells. In the cytoplasm, the diffusion of microinjected DNA was inhibited in a size- and linearity-dependent manner, probably by the intermediate filament. This was followed by the rapid disappearance of the DNA fluorescent signal. In the nucleus, the diffusion was also dependent on the size of the molecule and was accompanied by the aggregation of the DNA. The aggregation may be due to a putative DNA-binding molecule, whose level is high during the G1 phase. Surprisingly, the injected DNA could move across the nuclear membrane and appeared in the cytoplasm, which suggests the presence of a transport system. The intracytoplasmic behavior and the elimination of such DNA was obviously different from the DNA that was directly injected at the cytoplasm. The DNA remaining in the nucleus appeared to be stable and persisted in the nucleus or, after cell division, in the cytoplasm, for more than one cell cycle. These findings provide a novel and basic understanding of the behavior and elimination of a wide variety of extrachromosomal genetic material.

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.

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.

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.

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.

Heterochromatin is critical for genome integrity, and recent studies have suggested the importance of transcription in heterochromatin for maintaining its silent state. We previously developed a method to generate a large homogeneously staining region (HSR) composed of tandem plasmid sequences in human cells that showed typical heterochromatin characteristics. In this study, we examined transcription in the HSR. We found that transcription of genes downstream to no-inducible SRalpha promoter was restricted to a few specific points inside the large HSR domain. Furthermore, the HSR localized to either to the surface or to the interior of the nucleolus, where it was more actively transcribed. The perinucleolar or intranucleolar locations were biased to late or early S-phase, and the location depended on either RNA polymerase II/III or I transcription, respectively. Strong activation of the inducible TRE promoter resulted in the reversible loosening of the HSR domain and the appearance of transcripts downstream of not only the TRE promoters, but also the SRalpha promoters. During this process, detection of HP1alpha or H3K9Me3 suggested that transcription was activated at many specific points dispersed inside large heterochromatin. The transcriptional rules obtained from studying artificial heterochromatin should be useful for understanding natural heterochromatin.

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.

Right ventricular hypertrophy (RVH) and right ventricular (RV) contractile dysfunction are major determinants of prognosis in pulmonary arterial hypertension (PAH) and PAH remains a severe disease. Recently, direct interruption of left ventricular hypertrophy has been suggested to decrease the risk of left-sided heart failure. Hexamethylene bis-acetamide inducible protein 1 (HEXIM1) is a negative regulator of positive transcription elongation factor b (P-TEFb), which activates RNA polymerase II (RNAPII)-dependent transcription and whose activation is strongly associated with left ventricular hypertrophy. We hypothesized that during the progression of PAH, increased P-TEFb activity might also play a role in RVH, and that HEXIM1 might have a preventive role against such process. We revealed that, in the mouse heart, HEXIM1 is highly expressed in the early postnatal period and its expression is gradually decreased, and that prostaglandin I(2), a therapeutic drug for PAH, increases HEXIM1 levels in cardiomyocytes. These results suggest that HEXIM1 might possess negative effect on cardiomyocyte growth and take part in cardiomyocyte regulation in RV. Using adenovirus-mediated gene delivery to cultured rat cardiomyocytes, we revealed that overexpression of HEXIM1 prevents endothelin-1-induced phosphorylation of RNAPII, cardiomyocyte hypertrophy, and mRNA expression of hypertrophic genes, whereas a HEXIM1 mutant lacking central basic region, which diminishes P-TEFb-suppressing activity, could not. Moreover, we created cardiomyocyte-specific HEXIM1 transgenic mice and revealed that HEXIM1 ameliorates RVH and prevents RV dilatation in hypoxia-induced PAH model. Taken together, these findings indicate that cardiomyocyte-specific overexpression of HEXIM1 inhibits progression to RVH under chronic hypoxia, most possibly via inhibition of P-TEFb-mediated enlargement of cardiomyocytes. We conclude that P-TEFb/HEXIM1-dependent transcriptional regulation may play a pathophysiological role in RVH and be a novel therapeutic target for mitigating RVH in PAH.

Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone.

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.

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.

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.