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Cryotherapy is one of the most common treatments for trauma or fatigue in the field of sports medicine. However, the molecular biological effects of acute cold exposure on skeletal muscle remain unclear. Therefore, we used zebrafish, which have recently been utilized as an animal model for skeletal muscle, to comprehensively investigate and selectively clarify the time-course changes induced by cryotherapy. Zebrafish were exposed intermittently to cold stimulation three times for 15 min each. Thereafter, skeletal muscle samples were collected after 15 min and 1, 2, 4, and 6 h. mRNA sequencing revealed the involvement of trim63a, fbxo32, fbxo30a, and klhl38b in "protein ubiquitination" from the top 10 most upregulated genes. Subsequently, we examined the time-course changes of the four genes by quantitative PCR, and their expression peaked 2 h after cryotherapy and returned to baseline after 6 h. Moreover, the proteins encoded by trim63a and fbxo32 (muscle-specific RING finger protein 1 [MuRF1] and muscle atrophy F-box, respectively), which are known to be major genes encoding E3 ubiquitin ligases, were examined by western blotting, and MuRF1 expression displayed similar temporal changes as trim63a expression. These findings suggest that acute cold exposure transiently upregulates E3 ubiquitin ligases, especially MuRF1; thus, cryotherapy may contribute to the treatment of trauma or fatigue by promoting protein processing.

Vascular endothelial growth factor (VEGF) signaling plays a central role in vascular development and maintenance of vascular homeostasis. In endothelial cells (ECs), VEGF activates the gene expression of angiogenic transcription factors (TFs), followed by induction of downstream angiogenic responsive genes. Recent findings support that histone modification dynamics contribute to the transcriptional control of genes that are important for EC functions. Lysine demethylase 2B (KDM2B) demethylates histone H3K4me3 and H3K36me2/3 and mediates the monoubiquitination of histone H2AK119. KDM2B functions as a transcriptional repressor in somatic cell reprogramming and tumor development. However, the role of KDM2B in VEGF signaling remains to be elucidated. Here, we show that KDM2B knockdown enhances VEGF-induced angiogenesis in cultured human ECs via increased migration and proliferation. In contrast, ectopic expression of KDM2B inhibits angiogenesis. The function of KDM2B may depend on its catalytic Jumonji C domain. Genome-wide analysis further reveals that KDM2B selectively controls the transcription of VEGF-induced angiogenic TFs that are associated with increased H3K4me3/H3K36me3 and decreased H2AK119ub. These findings suggest an essential role of KDM2B in VEGF signaling in ECs. As dysregulation of VEGF signaling in ECs is involved in various diseases, including cancer, KDM2B may be a potential therapeutic target in VEGF-mediated vasculopathic diseases.

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PDF file - 24K, List of genes upregulated in response to hypoxia and nutrient starvation double stress in human and mouse cancer cells.

PDF file - 37K, The sequence of specific primers for angiogenic factors and JMJD1A in real-time polymerase chain reaction analysis.

PDF file - 47K, List of 769 genes down-regulated by JMJD1A siRNA in vivo (c.f. Fig. S20).

PDF file - 38K, List of genes up- and down-regulated in response to hypoxia and nutrient starvation double stress (DDS) in human cancer cells.

Acute and chronic inflammation is a basic pathological event that contributes to atherosclerosis, cancer, infectious diseases, and immune disorders. Inflammation is an adaptive process to both external and internal stimuli experienced by the human body. Although the mechanism of gene transcription is highly complicated and orchestrated in a timely and spatial manner, recent developments in next-generation sequencing, genome-editing, cryo-electron microscopy, and single cell-based technologies could provide us with insights into the roles of super enhancers (SEs). Initially, SEs were implicated in determining cell fate; subsequent studies have clarified that SEs are associated with various pathological conditions, including cancer and inflammatory diseases. Recent technological advances have unveiled the molecular mechanisms of SEs, which involve epigenetic histone modifications, chromatin three-dimensional structures, and phase-separated condensates. In this review, we discuss the relationship between inflammation and SEs and the therapeutic potential of SEs for inflammatory diseases.

Ten‐eleven translocation 1 (TET1) is an essential methylcytosine dioxygenase of the DNA demethylation pathway. Despite its dysregulation being known to occur in human cancer, the role of TET1 remains poorly understood. In this study, we report that TET1 promotes cell growth in human liver cancer. The transcriptome analysis of 68 clinical liver samples revealed a subgroup of TET1‐upregulated hepatocellular carcinoma (HCC), demonstrating hepatoblast‐like gene expression signatures. We performed comprehensive cytosine methylation and hydroxymethylation (5‐hmC) profiling and found that 5‐hmC was aberrantly deposited preferentially in active enhancers. TET1 knockdown in hepatoma cell lines decreased hmC deposition with cell growth suppression. HMGA2 was highly expressed in a TET1high subgroup of HCC, associated with the hyperhydroxymethylation of its intronic region, marked as histone H3K4–monomethylated, where the H3K27‐acetylated active enhancer chromatin state induced interactions with its promoter. Collectively, our findings point to a novel type of epigenetic dysregulation, methylcytosine dioxygenase TET1, which promotes cell proliferation via the ectopic enhancer of its oncogenic targets, HMGA2, in hepatoblast‐like HCC., Ten‐eleven translocation 1 (TET1), a methylcytosine dioxygenase of the DNA demethylation pathway, is overexpressed in the subgroup of hepatocellular carcinoma with the hepatoblast‐like expression pattern. Comprehensive epigenome profilings revealed the ectopic enhancer activation of HMGA2 through cytosine hydroxymethylation, H3K27 acetylation, H3K4 monomethylation, and promoter‐enhancer interaction in a TET1‐dependent manner.

The World Anti-Doping Agency (WADA) has prohibited the use of autologous blood transfusion (ABT) as a doping method by athletes. It is difficult to detect this doping method in laboratory tests, and a robust testing method has not yet been established. We conducted an animal experiment and used total RNA sequencing (RNA-Seq) to identify novel RNA markers to detect ABT doping within red blood cells (RBCs) as a pilot study before human trials. This study used whole blood samples from Wistar rats. The whole blood samples were mixed with a citrate–phosphate–dextrose solution with adenine (CPDA) and then stored in a refrigerator at 4 °C for 0 (control), 10, or 20 days. After each storage period, total RNA-Seq and bioinformatics were performed following RNA extraction and the purification of the RBCs. In the results, clear patterns of expression fluctuations were observed depending on the storage period, and it was found that there were large numbers of genes whose expression decreased in the 10- and 20-day periods compared to the control. Moreover, additional bioinformatic analysis identified three significant genes whose expression levels were drastically decreased according to the storage period. These results provide novel insights that may allow future studies to develop a testing method for ABT doping.

Recently, fasting has been spotlighted from a healthcare perspective. However, the de-tailed biological mechanisms and significance by which the effects of fasting confer health benefits are not yet clear. Due to certain advantages of the zebrafish as a vertebrate model, it is widely utilized in biological studies. However, the biological responses to nutrient metabolism within zebrafish skeletal muscles have not yet been amply reported. Therefore, we aimed to reveal a gene expression profile in zebrafish skeletal muscles in response to fasting-refeeding. Accordingly, mRNA-sequencing and bioinformatics analysis were performed to examine comprehensive gene expression changes in skeletal muscle tissues during fasting-refeeding. Our results produced a novel set of nutrition-related genes under a fasting-refeeding protocol. Moreover, we found that five genes were dramatically upregulated in each fasting (for 24 h) and refeeding (after 3 h), exhibiting a rapid response to the provided conditional changes. The assessment of the gene length revealed that the gene set whose expression was elevated only after 3 h of refeeding had a shorter length, suggesting that nutrition-related gene function is associated with gene length. Taken together, our results from the bioinformatics analyses provide new insights into biological mechanisms induced by fasting-refeeding conditions within zebrafish skeletal muscle.

In normal development, the rate of cell differentiation is tightly controlled and critical for normal development and stem cell differentiation. However, the underlying mechanisms regulating the rate of the differentiation are unknown, and manipulation of the rate of the stem cell differentiation is currently difficult. Here we show that activation of protein kinase A (PKA) accelerates the rate of mouse embryonic stem cell (ESC) differentiation through an early loss of ESC pluripotency markers and early appearance of mesodermal and other germ layer cells. The activation of PKA hastened differentiation by increasing the expression of a histone H3 lysine 9 (H3K9) dimethyltransferase, G9a protein, and the level of a negative epigenetic histone mark, H3K9 dimethylation (H3K9me2), in the promoter regions of the pluripotency markers Nanog and Oct4. These results elucidate a novel role of PKA on ESC differentiation and offer an experimental model for controlling the rate of ESC differentiation.

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by excessive pulmonary vascular remodeling. However, despite advances in therapeutic strategies, patients with PAH bearing mutations in the bone morphogenetic protein receptor type 2 (

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by excessive pulmonary vascular remodeling. However, despite advances in therapeutic strategies, patients with PAH bearing mutations in the bone morphogenetic protein receptor type 2 (BMPR2)-encoding gene present severe phenotypes and outcomes. We sought to investigate the effect of PER-like kinase (PERK), which participates in one of three major pathways associated with the unfolded protein response (UPR), on PAH pathophysiology in BMPR2 heterozygous mice. BMPR2 heterozygosity in pulmonary artery smooth muscle cells (PASMCs) decreased the abundance of the antiapoptotic microRNA miR124-3p through the arm of the UPR mediated by PERK. Hypoxia promoted the accumulation of unfolded proteins in BMPR2 heterozygous PASMCs, resulting in increased PERK signaling, cell viability, cellular proliferation, and glycolysis. Proteomic analyses revealed that PERK ablation suppressed PDGFRβ-STAT1 signaling and glycolysis in hypoxic BMPR2 heterozygous PASMCs. Furthermore, PERK ablation or PERK inhibition ameliorated pulmonary vascular remodeling in the Sugen/chronic hypoxia model of PAH, irrespective of BMPR2 status. Hence, these findings suggest that PERK inhibition is a promising therapeutic strategy for patients with PAH with or without BMPR2 mutation.

Peripartum cardiomyopathy (PPCM) is a life-threatening heart disease arising in the peripartum period. 16 kDa-prolactin (16K PRL) and its subproduct microRNA (miR)-146a-5p are candidate biomarkers for PPCM. However, we showed that miR-146a-5p is not well upregulated in the sera of PPCM patients. Here, we found that miR-320a/b were the most specific miRNAs that suppressed the expression of protein kinase RNA-like endoplasmic reticulum kinase (PERK). We transfected neonatal rat cardiomyocytes with plasmids containing full-length prolactin (23K PRL) or 16K PRL in vitro. We found that 23K PRL, but not 16K PRL, upregulated PERK signaling, and hypoxia promoted this effect. During the perinatal period, cardiomyocyte-specific PERK homogenous knockout (CM-KO) mice showed PPCM phenotypes after consecutive deliveries. Downregulation of PERK or JAK/STAT signaling and upregulation of apoptosis were observed in CM-KO mouse hearts. These results demonstrated that interruption of 23K PRL-PERK interaction by miR-320a/b could be a therapeutic strategy for PPCM. Funding Statement: This study was supported by the Japan Society for the Promotion of Science KAKENHI grant number JP18K16756 and JP20K17210 (T.S.) and by the research grant from Public Trust Cardiovascular Research Fund (T.S.). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The institutional review board of National Cerebral and Cardiovascular Center in Osaka approved this study. Eligible women were enrolled after providing written informed consent. Animal Ethics Approval Statement: All experiments were approved by the University of Tokyo Ethics Committee for Animal Experiments and strictly adhered to the guidelines for animal experiments of the University of Tokyo.

Peripartum cardiomyopathy (PPCM) is a life-threatening heart failure occurring in the peripartum period. Although mal-angiogenesis, induced by the 16-kDa N-terminal prolactin fragment (16 K PRL), is involved in the pathogenesis, the effect of full-length prolactin (23 K PRL) is poorly understood. We transfected neonate rat cardiomyocytes with plasmids containing 23 K PRL or 16 K PRL in vitro and found that 23 K PRL, but not 16 K PRL, upregulated protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling, and hypoxia promoted this effect. During the perinatal period, cardiomyocyte-specific PERK homogenous knockout (CM-KO) mice showed PPCM phenotypes after consecutive deliveries. Downregulation of PERK or JAK/STAT signaling and upregulation of apoptosis were observed in CM-KO mouse hearts. Moreover, in bromocriptine-treated CM-KO mice, cardiac function did not improve and cardiomyocyte apoptosis was not suppressed during the peripartum period. These results demonstrate that interaction between 23 K PRL and PERK signaling is cardioprotective during the peripartum term.

Temporal and spatial colinear expression of the Hox genes determines the specification of positional identities during vertebrate development. Post-translational modifications of histones contribute to transcriptional regulation. Lysine demethylase 7A (Kdm7a) demethylates lysine 9 or 27 di-methylation of histone H3 (H3K9me2, H3K27me2) and participates in the transcriptional activation of developmental genes. However, the role of Kdm7a during mouse embryonic development remains to be elucidated. Herein, we show that Kdm7a−/− mouse exhibits an anterior homeotic transformation of the axial skeleton, including an increased number of presacral elements. Importantly, posterior Hox genes (caudally from Hox9) are specifically downregulated in the Kdm7a−/− embryo, which correlates with increased levels of H3K9me2, not H3K27me2. These observations suggest that Kdm7a controls the transcription of posterior Hox genes, likely via its demethylating activity, and thereby regulating the murine anterior-posterior development. Such epigenetic regulatory mechanisms may be harnessed for proper control of coordinate body patterning in vertebrates., Higashijima et al show that mice lacking the Kdm7a demethylase exhibits anterior homeotic transformation of the axial skeleton and downregulation of posterior Hox gene transcription and these changes are associated with increased H3K9me2 at posterior Hox loci. These findings provide insights into the epigenetic control of Hox-mediated patterning in embryogenesis.

Tubulointerstitial fibrosis has been recently reported to be caused by the collapse of the epigenetic regulation of kidney diseases. We examined whether pharmacological inhibition of histone modification is effective against renal fibrosis. DZNep (3-deazaneplanocin A) was originally developed as an anti-cancer drug to inhibit the repressive histone mark, H3K27me3. We used a model of chronic tubulointerstitial fibrosis induced by unilateral ischaemia/reperfusion and administered DZNep intravenously to the mice for 8 weeks. We found DZNep contributes to the reduction of tubulointerstitial fibrosis. We selected only tubular cells from in vivo samples using laser-capture microdissection because epigenetic regulation is specific to the cell types, and we focused on the changes in the tubular cells. We performed a genome-wide analysis of tubular cells using high-throughput sequencing (RNA-seq) to identify novel epigenetic factors associated with renal fibrosis. We found that pro-fibrotic genes such as COL3A1 (collagen type 3a1) and TIMP2 (tissue inhibitor of metalloproteinase 2) were suppressed by DZNep in vivo. In addition, pro-fibrotic genes such as COL4A1 (collagen type 4a1), TIMP2 and MMP14 were down-regulated by DZNep in vitro. In conclusion, we found that pharmacological epigenetic modification by DZNep decreased the expression levels of fibrogenic genes in tubular cells and inhibited tubulointerstitial fibrosis.

Summary: Here, we find that human-induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM)-fated progenitors (CFPs) that express a tetraspanin family glycoprotein, CD82, almost exclusively differentiate into CMs both in vitro and in vivo. CD82 is transiently expressed in late-stage mesoderm cells during hiPSC differentiation. Purified CD82+ cells gave rise to CMs under nonspecific in vitro culture conditions with serum, as well as in vivo after transplantation to the subrenal space or injured hearts in mice, indicating that CD82 successfully marks CFPs. CD82 overexpression in mesoderm cells as well as in undifferentiated hiPSCs increased the secretion of exosomes containing β-catenin and reduced nuclear β-catenin protein, suggesting that CD82 is involved in fated restriction to CMs through Wnt signaling inhibition. This study may contribute to the understanding of CM differentiation mechanisms and to cardiac regeneration strategies. : Takeda et al. find that CD82+ is a cell-surface marker on cardiomyocyte-fated progenitors made from human iPSCs. Keywords: iPSCs, CD82, cardiomyocytes, progenitors, exosome, Wnt inhibition

Despite the World Anti-Doping Agency (WADA) ban on gene doping in the context of advancements in gene therapy, the risk of EPO gene-based doping among athletes is still present. To address this and similar risks, gene-doping tests are being developed in doping control laboratories worldwide. In this regard, the present study was performed with two objectives: to develop a robust gene-doping mouse model with the human EPO gene (hEPO) transferred using recombinant adenovirus (rAdV) as a vector and to develop a detection method to identify gene doping by using this model. The rAdV including the hEPO gene was injected intravenously to transfer the gene to the liver. After injection, the mice showed significantly increased whole-blood red blood cell counts and increased expression of hematopoietic marker genes in the spleen, indicating successful development of the gene-doping model. Next, direct and potentially indirect proof of gene doping were evaluated in whole-blood DNA and RNA by using a quantitative PCR assay and RNA sequencing. Proof of doping could be detected in DNA and RNA samples from one drop of whole blood for approximately a month, furthermore, the overall RNA expression profiles showed significant changes, allowing advanced detection of hEPO gene doping.

Although studies of the differentiation from mouse embryonic stem (ES) cells to vascular endothelial cells (ECs) provide an excellent model for investigating the molecular mechanisms underlying vascular development, temporal dynamics of gene expression and chromatin modifications have not been well studied. Herein, using transcriptomic and epigenomic analyses based on H3K4me3 and H3K27me3 modifications at a genome-wide scale, we analysed the EC differentiation steps from ES cells and crucial epigenetic modifications unique to ECs. We determined that Gata2, Fli1, Sox7 and Sox18 are master regulators of EC that are induced following expression of the haemangioblast commitment pioneer factor, Etv2. These master regulator gene loci were repressed by H3K27me3 throughout the mesoderm period but rapidly transitioned to histone modification switching from H3K27me3 to H3K4me3 after treatment with vascular endothelial growth factor. SiRNA knockdown experiments indicated that these regulators are indispensable not only for proper EC differentiation but also for blocking the commitment to other closely aligned lineages. Collectively, our detailed epigenetic analysis may provide an advanced model for understanding temporal regulation of chromatin signatures and resulting gene expression profiles during EC commitment. These studies may inform the future development of methods to stimulate the vascular endothelium for regenerative medicine.

SUMMARYTemporal and spatial colinear expression of theHoxgenes determines the specification of positional identities during vertebrate development. Post-translational modifications of histones contribute to transcriptional regulation. Lysine demethylase 7A (Kdm7a) demethylates lysine 9 di-methylation of histone H3 (H3K9me2) and participates in the transcriptional activation of developmental genes. However, the role of Kdm7a during mouse embryonic development remains to be elucidated. Here, we show thatKdm7a−/−mouse exhibits an anterior homeotic transformation of the axial skeleton, including an increased number of presacral elements. Importantly, posteriorHoxgenes (caudally fromHox9) are specifically downregulated in theKdm7a−/−embryo, which correlates with increased levels of H3K9me2. These observations suggest that Kdm7a controls the transcription of posteriorHoxgenes, likely via its demethylating activity, and thereby regulating the murine anterior-posterior development. Such epigenetic regulatory mechanisms may be harnessed for the proper control of coordinate body patterning in vertebrates.

Summary: Tolerance to severe tumor microenvironments, including hypoxia and nutrient starvation, is a common feature of aggressive cancer cells and can be targeted. However, metabolic alterations that support cancer cells upon nutrient starvation are not well understood. Here, by comprehensive metabolome analyses, we show that glutamine deprivation leads to phosphoethanolamine (PEtn) accumulation in cancer cells via the downregulation of PEtn cytidylyltransferase (PCYT2), a rate-limiting enzyme of phosphatidylethanolamine biosynthesis. PEtn accumulation correlated with tumor growth under nutrient starvation. PCYT2 suppression was partially mediated by downregulation of the transcription factor ELF3. Furthermore, PCYT2 overexpression reduced PEtn levels and tumor growth. In addition, PEtn accumulation and PCYT2 downregulation in human breast tumors correlated with poor prognosis. Thus, we show that glutamine deprivation leads to tumor progression by regulating PE biosynthesis via the ELF3-PCYT2 axis. Furthermore, manipulating glutamine-responsive genes could be a therapeutic approach to limit cancer progression. : Osawa et al. find that accumulation of phosphoethanolamine (PEtn) protects cancer cells under glutamine starvation through the downregulation of PCYT2. Glutamine regulates PE biosynthesis through PCYT2, resulting in pro-tumorigenic metabolite PEtn accumulation. PEtn stimulates the tolerance of cancer cells to starvation, and lowered PCYT2 expression correlates with decreased survival in patients. Keywords: cancer metabolism, tumor microenvironments, hypoxia, nutrient starvation, glutamine deprivation, amino acids, phosphoethanolamine, PCYT2, PE biosynthesis

Summary Oxidative/nitrosative stress is a major trigger of cardiac dysfunction, involving the unfolded protein response and mitochondrial dysfunction. Activation of nitric oxide-cyclic guanosine monophosphate-protein kinase G signaling by sildenafil improves cardiac mal-remodeling during pressure-overload-induced heart failure. Transcriptome analysis was conducted in failing hearts with or without sildenafil treatment. Protein kinase R–like endoplasmic reticulum (ER) kinase (PERK) downstream signaling pathways, EIF2 and NRF2, were significantly altered. Although EIF2 signaling was suppressed, NRF2 signaling was upregulated, inhibiting the maturation of miR 24-3p through EGFR-mediated Ago2 phosphorylation. To study the effect of sildenafil on these pathways, we generated cardiac-specific PERK knockout mice. In these mice, sildenafil could not inhibit the maturations, the nuclear translocation of NRF2 was suppressed, and mitochondrial dysfunction advanced. Altogether, these results show that PERK-mediated suppression of miRNAs by sildenafil is vital for maintaining mitochondrial homeostasis through NRF2-mediated oxidative stress response., Graphical Abstract, Highlights • RBM3 inhibited the PERK arm of UPR in chronic heart failure (HF) • Sildenafil significantly altered PERK downstream signaling pathways, EIF2 and NRF2 • PERK-mediated miRNA suppression by sildenafil was vital for mitochondrial homeostasis • Ago2 phosphorylation by EGFR was vital for inhibiting HF-induced miRNA maturation, Biological Sciences; Molecular Biology; Cell Biology

SUMMARYLysine 9 di-methylation and lysine 27 tri-methylation of histone H3 (H3K9me2 and H3K27me3) are generally linked to gene repression. However, the functions of repressive histone methylation dynamics during inflammatory responses remain enigmatic. We found that tumor necrosis factor (TNF)-α rapidly induces the co-occupancy of lysine demethylases 7A (KDM7A) and 6A (UTX) with nuclear factor kappa-B (NF-κB) recruited elements in human endothelial cells. KDM7A and UTX demethylate H3K9me2 and H3K27me3, respectively, and both are required for activation of NF-κB-dependent inflammatory genes. Chromosome conformation capture-based methods demonstrated increased interactions between TNF-α-induced super enhancers at NF-κB-relevant loci, coinciding with KDM7A- and UTX-recruitment. Simultaneous inhibition of KDM7A and UTX significantly reduced leukocyte adhesion in mice, establishing the biological and potential translational relevance of this mechanism. Collectively, these findings suggest that rapid erasure of repressive histone marks by KDM7A and UTX is essential for NF-κB-dependent regulation of genes that control inflammatory responses of endothelial cells.HIGHLIGHTSKDM7A and UTX cooperatively control NF-κB-dependent transcription in vascular endothelial cells.Demethylation of repressive histone marks by KDM7A and UTX is critical for early inflammatory responses.KDM7A and UTX are associated with TNF-α-induced looping of super enhancers.Pharmacological inhibition of KDM7A and UTX reduces leukocyte adhesive interactions with endothelial cells in mice.

Chronic tubulointerstitial hypoxia plays an important role as the final common pathway to end‐stage renal disease. HIF‐1 (hypoxia‐inducible factor‐1) is a master transcriptional factor under hypoxia, regulating downstream target genes. Genome‐wide analysis of HIF‐1 binding sites using high‐throughput sequencers has clarified various kinds of downstream targets and made it possible to demonstrate the novel roles of HIF‐1. Our aim of this study is to identify novel HIF‐1 downstream epigenetic targets which may play important roles in the kidney. Immortalized tubular cell lines (HK2; human kidney‐2) and primary cultured cells (RPTEC; renal proximal tubular cell lines) were exposed to 1% hypoxia for 24–72 h. We performed RNA‐seq to clarify the expression of mRNA and long non‐coding RNA (lncRNA). We also examined ChIP‐seq to identify HIF‐1 binding sites under hypoxia. RNA‐seq identified 44 lncRNAs which are up‐regulated under hypoxic condition in both cells. ChIP‐seq analysis demonstrated that HIF‐1 also binds to the lncRNAs under hypoxia. The expression of novel lncRNA, DARS‐AS1 (aspartyl‐tRNA synthetase anti‐sense 1), is up‐regulated only under hypoxia and HIF‐1 binds to its promoter region, which includes two hypoxia‐responsive elements. Its expression is also up‐regulated with cobalt chloride exposure, while it is not under hypoxia when HIF‐1 is knocked down by siRNA. To clarify the biological roles of DARS‐AS1, we measured the activity of caspase 3/7 using anti‐sense oligo of DARS‐AS1. Knockdown of DARS‐AS1 deteriorated apoptotic cell death. In conclusion, we identified the novel lncRNAs regulated by HIF‐1 under hypoxia and clarified that DARS‐AS1 plays an important role in inhibiting apoptotic cell death in renal tubular cells.

Antiangiogenic strategies can be effective for cancer therapy, but like all therapies resistance poses a major clinical challenge. Hypoxia and nutrient starvation select for aggressive qualities that may render tumors resistant to antiangiogenic attack. Here, we show that hypoxia and nutrient starvation cooperate to drive tumor aggressiveness through epigenetic regulation of the histone demethylase JMJD1A (JHDM2A; KDM3A). In cancer cells rendered resistant to long-term hypoxia and nutrient starvation, we documented a stimulation of AKT phosphorylation, cell morphologic changes, cell migration, invasion, and anchorage-independent growth in culture. These qualities associated in vivo with increased angiogenesis and infiltration of macrophages into tumor tissues. Through expression microarray analysis, we identified a cluster of functional drivers such as VEGFA, FGF18, and JMJD1A, the latter which was upregulated in vitro under conditions of hypoxia and nutrient starvation and in vivo before activation of the angiogenic switch or the prerefractory phase of antiangiogenic therapy. JMJD1A inhibition suppressed tumor growth by downregulating angiogenesis and macrophage infiltration, by suppressing expression of FGF2, HGF, and ANG2. Notably, JMJD1A inhibition enhanced the antitumor effects of the anti-VEGF compound bevacizumab and the VEGFR/KDR inhibitor sunitinib. Our results form the foundation of a strategy to attack hypoxia- and nutrient starvation–resistant cancer cells as an approach to leverage antiangiogenic treatments and limit resistance to them. Cancer Res; 73(10); 3019–28. ©2013 AACR.

The effects of chronic low-dose radiation on human health have not been well established. Recent studies have revealed that neural progenitor cells are present not only in the fetal brain but also in the adult brain. Since immature cells are generally more radiosensitive, here we investigated the effects of chronic low-dose radiation on cultured human neural progenitor cells (hNPCs) derived from embryonic stem cells. Radiation at low doses of 31, 124 and 496 mGy per 72 h was administered to hNPCs. The effects were estimated by gene expression profiling with microarray analysis as well as morphological analysis. Gene expression was dose-dependently changed by radiation. By thirty-one mGy of radiation, inflammatory pathways involving interferon signaling and cell junctions were altered. DNA repair and cell adhesion molecules were affected by 124 mGy of radiation while DNA synthesis, apoptosis, metabolism and neural differentiation were all affected by 496 mGy of radiation. These in vitro results suggest that 496 mGy radiation affects the development of neuronal progenitor cells while altered gene expression was observed at a radiation dose lower than 100 mGy. This study would contribute to the elucidation of the clinical and subclinical phenotypes of impaired neuronal development induced by chronic low-dose radiation.

Tumour necrosis factor alpha (TNFα) is a potent cytokine that signals through nuclear factor kappa B (NFκB) to activate a subset of human genes. It is usually assumed that this involves RNA polymerases transcribing responsive genes wherever they might be in the nucleus. Using primary human endothelial cells, variants of chromosome conformation capture (including 4C and chromatin interaction analysis with paired-end tag sequencing), and fluorescence in situ hybridization to detect single nascent transcripts, we show that TNFα induces responsive genes to congregate in discrete 'NFκB factories'. Some factories further specialize in transcribing responsive genes encoding micro-RNAs that target downregulated mRNAs. We expect all signalling pathways to contain this extra leg, where responding genes are transcribed in analogous specialized factories.

Endothelial cell activation and dysfunction underlie many vascular disorders, including atherosclerosis and inflammation. Here, we show that interleukin-4 (IL-4) markedly induced vascular cell adhesion molecule 1 (VCAM-1), both in cultured endothelial cells and in the intact endothelium in mice. Combined treatment with IL-4 and tumor necrosis factor alpha (TNF-α) resulted in further, sustained induction of VCAM-1 expression. IL-4-mediated induction of VCAM-1 and secondary monocyte adhesion was predominantly regulated by the transcription factor STAT6. Genome-wide survey of IL-4-mediated STAT6 binding from sequential chromatin-immunoprecipitation with deep sequencing (chromatin immunoprecipitation sequencing [ChIP-seq]) in endothelial cells revealed regions of transient and sustained transcription factor binding. Through the combination of DNA microarrays and ChIP-seq at the same time points, the majority of IL-4-responsive genes were shown to be STAT6 dependent and associated with direct STAT6 binding to their promoter. IL-4-mediated stable binding of STAT6 led to sustained target gene expression. Moreover, our strategy led to the identification of a novel functionally important STAT6 binding site within 16 kb upstream of the VCAM-1 gene. Taken together, these findings support a critical role for STAT6 in mediating IL-4 signal transduction in endothelial cells. Identification of a novel IL-4-mediated VCAM-1 enhancer may provide a foundation for targeted therapy in vascular disease.

Endothelial cell (EC) plasticity in pathological settings has recently been recognized as a driver of disease progression. Endothelial-to-mesenchymal transition (EndMT), in which ECs acquire mesenchymal properties, has been described for a wide range of pathologies, including cancer. However, the mechanism regulating EndMT in the tumor microenvironment and the contribution of EndMT in tumor progression are not fully understood. Here, we found that combined knockdown of two ETS family transcription factors, ERG and FLI1, induces EndMT coupled with dynamic epigenetic changes in ECs. Genome-wide analyses revealed that ERG and FLI1 are critical transcriptional activators for EC-specific genes, among which microRNA-126 partially contributes to blocking the induction of EndMT. Moreover, we demonstrated that ERG and FLI1 expression is downregulated in ECs within tumors by soluble factors enriched in the tumor microenvironment. These data provide new insight into the mechanism of EndMT, functions of ERG and FLI1 in ECs, and EC behavior in pathological conditions., Author summary Differentiated cells possess unique characteristics to maintain vital activities. However, cells occasionally show abnormal behavior in pathological settings due to dysregulated gene expression. Endothelial-to-mesenchymal transition (EndMT) is a phenomenon in which endothelial cells lose their characteristics and acquire mesenchymal-like properties. Although EndMT is observed in various diseases including cancer, and augments fibrosis and vascular defects, the mechanism of EndMT induction is not fully understood. Here, we show that EndMT is triggered via reduced expression of ERG and FLI1, which have recently been recognized as pivotal transcription factors in endothelial cells (ECs). Mechanistically, ERG and FLI1 activate EC-specific genes and repress mesenchymal-like genes via epigenetic regulation to prevent EndMT. Furthermore, we demonstrate that microRNA-126, which is specifically expressed in ECs, is the key downstream target of ERG/FLI1 for regulating EndMT. Finally, we show that ERG and FLI1 expression is decreased in ECs within tumors, suggesting that EndMT is induced in the tumor microenvironment. Collectively, these findings indicate that loss of ERG and FLI1 leads to the aberrant behavior of ECs in pathological conditions.

VEGF is a key regulator of endothelial cell migration, proliferation, and inflammation, which leads to activation of several signaling cascades, including the calcineurin-nuclear factor of activated T cells (NFAT) pathway. NFAT is not only important for immune responses but also for cardiovascular development and the pathogenesis of Down syndrome. By using Down syndrome model mice and clinical patient samples, we showed recently that the VEGF-calcineurin-NFAT signaling axis regulates tumor angiogenesis and tumor metastasis. However, the connection between genome-wide views of NFAT-mediated gene regulation and downstream gene function in the endothelium has not been studied extensively. Here we performed comprehensive mapping of genome-wide NFATc1 binding in VEGF-stimulated primary cultured endothelial cells and elucidated the functional consequences of VEGF-NFATc1-mediated phenotypic changes. A comparison of the NFATc1 ChIP sequence profile and epigenetic histone marks revealed that predominant NFATc1-occupied peaks overlapped with promoter-associated histone marks. Moreover, we identified two novel NFATc1 regulated genes, CXCR7 and RND1. CXCR7 knockdown abrogated SDF-1- and VEGF-mediated cell migration and tube formation. siRNA treatment of RND1 impaired vascular barrier function, caused RhoA hyperactivation, and further stimulated VEGF-mediated vascular outgrowth from aortic rings. Taken together, these findings suggest that dynamic NFATc1 binding to target genes is critical for VEGF-mediated endothelial cell activation. CXCR7 and RND1 are NFATc1 target genes with multiple functions, including regulation of cell migration, tube formation, and barrier formation in endothelial cells.

Transcription factor GATA2 is highly expressed in hematopoietic stem cells and progenitors, whereas its expression declines after erythroid commitment of progenitors. In contrast, the start of GATA1 expression coincides with the erythroid commitment and increases along with the erythroid differentiation. We refer this dynamic transition of GATA factor expression to as the 'GATA factor switching'. Here, we examined contribution of the GATA factor switching to the erythroid differentiation. In Gata1-knockdown embryos that concomitantly express Gata2-GFP reporter, high-level expression of GFP reporter was detected in accumulated immature hematopoietic cells with impaired differentiation, demonstrating that GATA1 represses Gata2 gene expression in hematopoietic progenitors in vivo. We have conducted chromatin immunoprecipitation (ChIP) on microarray analyses of GATA2 and GATA1, and results indicate that the GATA1-binding sites widely overlap with the sites pre-occupied by GATA2 before the GATA1 expression. Importantly, erythroid genes harboring GATA boxes bound by both GATA1 and GATA2 tend to be expressed in immature erythroid cells, whereas those harboring GATA boxes to which GATA1 binds highly but GATA2 binds only weakly are important for the mature erythroid cell function. Our results thus support the contention that preceding binding of GATA2 helps the following binding of GATA1 and thereby secures smooth expression of the transient-phase genes.

The recent and rapid advent of next-generation sequencing (NGS) has made this technology broadly available not only to researchers in various molecular and cellular biology fields but also to those in kidney disease. In this paper, we describe the usage of ChIP-seq (chromatin immunoprecipitation with sequencing) and RNA-seq for sample preparation and interpretation of raw data in the investigation of biological phenomenon in renal diseases. ChIP-seq identifies genome-wide transcriptional DNA-binding sites as well as histone modifications, which are known to regulate gene expression, in the intragenic as well as in the intergenic regions. With regard to RNA-seq, this process analyzes not only the expression level of mRNA but also splicing variants, non-coding RNA, and microRNA on a genome-wide scale. The combination of ChIP-seq and RNA-seq allows the clarification of novel transcriptional mechanisms, which have important roles in various kinds of diseases, including chronic kidney disease. The rapid development of these techniques requires an update on the latest information and methods of NGS. In this review, we highlight the merits and characteristics of ChIP-seq and RNA-seq and discuss the use of the genome-wide analysis in kidney disease.

Hypoxia-inducible factor 1 (HIF1) is a master regulator of adaptive gene expression under hypoxia. However, a role for HIF1 in the epigenetic regulation remains unknown. Genome-wide analysis of HIF1 binding sites (chromatin immunoprecipitation [ChIP] with deep sequencing) of endothelial cells clarified that HIF1 mainly binds to the intergenic regions distal from transcriptional starting sites under both normoxia and hypoxia. Next, we examined the temporal profile of gene expression under hypoxic conditions by using DNA microarrays. We clarified that early hypoxia-responsive genes are functionally associated with glycolysis, including GLUT3 (SLC2A3). Acetylated lysine 27 of histone 3 covered the HIF1 binding sites, and HIF1 functioned as an enhancer of SLC2A3 by interaction with lysine (K)-specific demethylase 3A (KDM3A). Knockdown of HIF1α and KDM3A showed that glycolytic genes are regulated by both HIF1 and KDM3A and respond to hypoxia in a manner independent of cell type specificity. We elucidated that both the chromatin conformational structure and histone modification change under hypoxic conditions and enhance the expression of SLC2A3 based on the combined results of chromatin conformation capture (3C) and ChIP assays. KDM3A is recruited to the SLC2A3 locus in an HIF1-dependent manner and demethylates H3K9me2 so as to upregulate its expression. These findings provide novel insights into the interaction between HIF1 and KDM3A and also the epigenetic regulation of HIF1.