Genetic and epigenetic basis of hepatoblastoma diversity.

Hepatoblastoma (HB) is the most common pediatric liver malignancy; however, hereditary predisposition and acquired molecular aberrations related to HB clinicopathological diversity are not well understood. Here, we perform an integrative genomic profiling of 163 pediatric liver tumors (154 HBs and nine hepatocellular carcinomas) based on the data acquired from a cohort study (JPLT-2). The total number of somatic mutations is precious low (0.52/Mb on exonic regions) but correlated with age at diagnosis. Telomerase reverse transcriptase (TERT) promoter mutations are prevalent in the tween HBs, selective in the transitional liver cell tumor (TLCT, > 8 years old). DNA methylation profiling reveals that classical HBs are characterized by the specific hypomethylated enhancers, which are enriched with binding sites for ASCL2, a regulatory transcription factor for definitive endoderm in Wnt-pathway. Prolonged upregulation of ASCL2, as well as fetal-liver-like methylation patterns of IGF2 promoters, suggests their "cell of origin" derived from the premature hepatoblast, similar to intestinal epithelial cells, which are highly proliferative. Systematic molecular profiling of HB is a promising approach for understanding the epigenetic drivers of hepatoblast carcinogenesis and deriving clues for risk stratification.

TET1 upregulation drives cancer cell growth through aberrant enhancer hydroxymethylation of HMGA2 in hepatocellular carcinoma.

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.

Accumulation of Molecular Aberrations Distinctive to Hepatocellular Carcinoma Progression.

Cancer develops through the accumulation of genetic and epigenetic aberrations. To identify sequential molecular alterations that occur during the development of hepatocellular carcinoma (HCC), we compared 52 early and 108 overt HCC samples by genome sequencing. Gene mutations in the p53/RB1 pathway, WNT pathway, MLL protein family, SWI/SNF complexes, and AKT/PI3K pathway were common in HCC. In the early phase of all entities, TERT was the most frequently upregulated gene owing to diverse mechanisms. Despite frequent somatic mutations in driver genes, including CTNNB1 and TP53, early HCC was a separate molecular entity from overt HCC, as each had a distinct expression profile. Notably, WNT target genes were not activated in early HCC regardless of CTNNB1 mutation status because ß-catenin did not translocate into the nucleus due to the E-cadherin/ß-catenin complex at the membrane. Conversely, WNT targets were definitively upregulated in overt HCC, with CTNNB1 mutation associated with downregulation of CDH1 and hypomethylation of CpG islands in target genes. Similarly, cell-cycle genes downstream of the p53/RB pathway were upregulated only in overt HCC, with TP53 or RB1 gene mutations associated with chromosomal deletion of 4q or 16q. HCC was epigenetically distinguished into four subclasses: normal-like methylation, global-hypomethylation (favorable prognosis), stem-like methylation (poor prognosis), and CpG island methylation. These methylation statuses were globally maintained through HCC progression. Collectively, these data show that as HCC progresses, additional molecular events exclusive of driver gene mutations cooperatively contribute to transcriptional activation of downstream targets according to methylation status. SIGNIFICANCE: In addition to driver gene mutations in the WNT and p53 pathways, further molecular events are required for aberrant transcriptional activation of these pathways as HCC progresses.

Cooperation of PU.1 With IRF8 and NFATc1 Defines Chromatin Landscapes During RANKL-Induced Osteoclastogenesis.

Receptor activator of nuclear factor κB ligand (RANKL) induces osteoclast (OC) differentiation from bone marrow-derived macrophages (BMMs). The transcription factors nuclear factor of activated T cells 1 (NFATc1) and interferon regulatory factor (IRF) 8 play positive and negative roles, respectively, in this process. However, genomewide mapping of the active cis-regulatory elements regulating OC differentiation has not been performed, and little is known about the global landscape of OC-specific gene regulation. We used chromatin immunoprecipitation and formaldehyde-assisted isolation of regulatory elements followed by sequencing to show that PU.1 transcription factor binding motifs were overrepresented at active cis-regulatory regions in both murine BMMs and OCs, while IRF and NFAT binding motifs were selectively enriched at these regions in BMMs and OCs, respectively. We also found that RANKL induced the downregulation of Irf8 and upregulation of Nfatc1 expression, which was associated with dramatic alterations in histone modification. BMM-specific PU.1 binding sites were observed to overlap with IRF8 binding sites in BMMs, and this also occurred for OC-specific PU.1 binding sites and NFATc1 binding sites in OCs. The expression of genes with IRF8 peaks within BMM-specific PU.1 binding sites was significantly higher in BMMs than in OCs, while that of genes with NFATc1 peaks within OC-specific PU.1 binding sites was significantly higher in OCs than in BMMs. Our results suggest that PU.1 switches its transcription partner from IRF8 to NFATc1 and alters the binding regions during RANKL-induced osteoclastogenesis, which is associated with changes in epigenetic profiles and the control of cell type-specific gene expression. © 2019 American Society for Bone and Mineral Research.

Cardiomyocyte gene programs encoding morphological and functional signatures in cardiac hypertrophy and failure.

Pressure overload induces a transition from cardiac hypertrophy to heart failure, but its underlying mechanisms remain elusive. Here we reconstruct a trajectory of cardiomyocyte remodeling and clarify distinct cardiomyocyte gene programs encoding morphological and functional signatures in cardiac hypertrophy and failure, by integrating single-cardiomyocyte transcriptome with cell morphology, epigenomic state and heart function. During early hypertrophy, cardiomyocytes activate mitochondrial translation/metabolism genes, whose expression is correlated with cell size and linked to ERK1/2 and NRF1/2 transcriptional networks. Persistent overload leads to a bifurcation into adaptive and failing cardiomyocytes, and p53 signaling is specifically activated in late hypertrophy. Cardiomyocyte-specific p53 deletion shows that cardiomyocyte remodeling is initiated by p53-independent mitochondrial activation and morphological hypertrophy, followed by p53-dependent mitochondrial inhibition, morphological elongation, and heart failure gene program activation. Human single-cardiomyocyte analysis validates the conservation of the pathogenic transcriptional signatures. Collectively, cardiomyocyte identity is encoded in transcriptional programs that orchestrate morphological and functional phenotypes.

TRPV4-dependent induction of a novel mammalian cold-inducible protein SRSF5 as well as CIRP and RBM3.

Cold-inducible RNA-binding protein (CIRP) and RNA-binding motif protein 3 (RBM3) are two evolutionarily conserved RNA-binding proteins that are structurally related to hnRNPs and upregulated in response to moderately low temperatures in mammalian cells. Although contributions of splicing efficiency, the gene promoters activated upon mild hypothermia and the transcription factor Sp1 to induction of CIRP have been reported, precise mechanisms by which hypothermia and other stresses induce the expression of mammalian cold-inducible proteins (CIPs) are poorly understood. By screening the serine/arginine-rich splicing factors (SRSFs), we report that the transcript and protein levels of SRSF5 were increased in mammalian cells cultured at 32 °C. Expression of SRSF5 as well as CIRP and RBM3 were also induced by DNA damage, hypoxia, cycloheximide and hypotonicity. Immunohistochemical studies demonstrated that SRSF5 was constitutively expressed in male germ cells and the level was decreased in human testicular germ cell tumors. SRSF5 facilitated production of p19 H-RAS, and increased sensitivity to doxorubicin in human U-2 OS cells. Induction of CIPs was dependent on transient receptor potential vanilloid 4 (TRPV4) channel protein, but seemed independent of its ion channel activity. These findings indicate a previously unappreciated role for the TRP protein in linking environmental stress to splicing.

Negative feedback loop of bone resorption by NFATc1-dependent induction of Cadm1.

Trimethylation of histone H3 lysine 4 and lysine 27 (H3K4me3 and H3K27me3) at gene promoter regions critically regulates gene expression. Key developmental genes tend to exhibit changes in histone modification patterns from the H3K4me3/H3K27me3 bivalent pattern to the H3K4me3 monovalent pattern. Using comprehensive chromatin immunoprecipitation followed by sequencing in bone marrow-derived macrophages (BMMs) and mature osteoclasts, we found that cell surface adhesion molecule 1 (Cadm1) is a direct target of nuclear factor of activated T cells 1 (NFATc1) and exhibits a bivalent histone pattern in BMMs and a monovalent pattern in osteoclasts. Cadm1 expression was upregulated in BMMs by receptor activator of nuclear factor kappa B ligand (RANKL), and blocked by a calcineurin/NFATc1 inhibitor, FK506. Cadm1-deficient mice exhibited significantly reduced bone mass compared with wild-type mice, which was due to the increased osteoclast differentiation, survival and bone-resorbing activity in Cadm1-deficient osteoclasts. These results suggest that Cadm1 is a direct target of NFATc1, which is induced by RANKL through epigenetic modification, and regulates osteoclastic bone resorption in a negative feedback manner.

Hypoxia-Inducible Factor-1α Activates the Transforming Growth Factor-ß/SMAD3 Pathway in Kidney Tubular Epithelial Cells.

BACKGROUND: Kidney injury, including chronic kidney disease and acute kidney injury, is a worldwide health problem. Hypoxia and transforming growth factor-ß (TGF-ß) are well-known factors that promote kidney injury. Hypoxia-inducible factor (HIF) and SMAD3 are their main downstream transcriptional factors. Hypoxia-HIF pathway and TGF-ß/SMAD3 pathway play a crucial role in the progression of kidney injury. However, reports on their interactions are limited, and the global transcriptional regulation under their control is almost unknown. METHODS: Kidney tubular epithelial cells were cultured and stimulated by hypoxia and TGF-ß. We detected global binding sites of HIF-1α and SMAD3 in cells using chromatin immunoprecipitation-sequencing (ChIP-Seq), and measured the gene expression using RNA-sequencing (RNA-Seq). ChIP-quantitative PCR (qPCR) was used to quantitatively evaluate bindings of SMAD3. RESULTS: ChIP-Seq revealed that 2,065 and 5,003 sites were bound by HIF-1α and SMAD3, respectively, with 614 sites co-occupied by both factors. RNA-Seq showed that hypoxia and TGF-ß stimulation causes synergistic upregulation of 249 genes, including collagen type I alpha 1 (COL1A1) and serpin peptidase inhibitor, clade E, member 1, which are well-known to be involved in fibrosis. Ontology of the 249 genes implied that the interaction of HIF-1α and SMAD3 is related to biological processes such as fibrosis. ChIP-qPCR of SMAD3 at HIF-1α binding sites near COL1A1 and SERPINE1 indicated that HIF-1α promotes the bindings of SMAD3, which is induced by TGF-ß. CONCLUSIONS: These findings suggest that HIF-1α induced by hypoxia activates the TGF-ß/SMAD3 pathway. This mechanism may promote kidney injury, especially by upregulating genes related to fibrosis.

Overeating at dinner time among Japanese workers: Is overeating related to stress response and late dinner times?

There are several known risk factors for overeating, including negative feelings and hunger. It was hypothesized that overtime work is associated with stress responses and later dinner times, leading to longer periods of time without eating, and that this, in turn, leads to a strong experience of hunger and consequent overeating at dinner. The aim of this study was to examine relationships among overeating at dinner, stress responses (e.g., fatigue, anxiety, and depression), and dinner times in Japanese male workers. In December 2012, 255 Japanese male workers at a leasing company completed a self-report questionnaire about overeating at dinner, psychological stress responses, physical stress responses, and dinner times. Each worker was sent an email with a link to the questionnaire website, where his answers were collected. Relationships between overeating at dinner and lifestyle issues were investigated using multiple linear regression analysis treating overeating as a dependent variable. Factors related to overeating at dinner included psychological stress response (ß = 0.251 p < 0.001) and dinner time (ß = 0.220, p = 0.004). These cross-sectional data suggest that overeating at dinner is related to dinner time in men and to stress responses.

Epigenomic Regulation of Smad1 Signaling During Cellular Senescence Induced by Ras Activation.

Epigenomic modification plays important roles in regulating gene expression during development, differentiation, and cellular senescence. When oncogenes are activated, cells fall into stable growth arrest to block cellular proliferation, which is called oncogene-induced senescence. We recently identified through genome-wide analyses that Bmp2-Smad1 signal and its regulation by harmonized epigenomic alteration play an important role in Ras-induced senescence of mouse embryonic fibroblasts. We describe in this chapter the methods for analyses of epigenomic alteration and Smad1 targets on genome-wide scale.

Overeating, late dinner, and perceived stress in Japanese workers.

OBJECTIVES: This study examined relationships among overeating at dinner, dinner time, perceived stress, and strategies for coping with stress among Japanese workers. METHODS: In December 2012, 255 male Japanese workers at a leasing company completed a questionnaire about overeating (score range: 5-20), dinner time, perceived stress, and strategies for coping with stress. Each worker was sent an email with a link to a website, where their answers were collected. Relationships among overeating, dinner time, perceived stress, and stress-related coping strategies were investigated using two-way analysis of variance. RESULTS: The analyses of cross-sectional data revealed no differences in the overeating scores among those who ate dinner before 21:00 according to their level of perceived stress. However, those who ate dinner after 21:00 and reported feeling stressed tended to overeat at dinner (F(1, 237)=5.62, p=0.019). Additionally, those with perceived stress engaged in emotional expression involving others, and those without perceived stress tended to seek help to solve their problems and change their mood. We found no significant interactions involving the items related to strategies for coping with stress. CONCLUSION: This study found that overeating at dinner was related to dinner time and perceived stress. Additionally, the combination of a late dinner time and perceived stress reinforced overeating at dinner. The results of this study do not identify a coping strategy to prevent overeating.

PRC2 overexpression and PRC2-target gene repression relating to poorer prognosis in small cell lung cancer.

Small cell lung cancer (SCLC) is a subtype of lung cancer with poor prognosis. Expression array analysis of 23 SCLC cases and 42 normal tissues revealed that EZH2 and other PRC2 members were highly expressed in SCLC. ChIP-seq for H3K27me3 suggested that genes with H3K27me3(+) in SCLC were extended not only to PRC2-target genes in ES cells but also to other target genes such as cellular adhesion-related genes. These H3K27me3(+) genes in SCLC were repressed significantly, and introduction of the most repressed gene JUB into SCLC cell line lead to growth inhibition. Shorter overall survival of clinical SCLC cases correlated to repression of JUB alone, or a set of four genes including H3K27me3(+) genes. Treatment with EZH2 inhibitors, DZNep and GSK126, resulted in growth repression of SCLC cell lines. High PRC2 expression was suggested to contribute to gene repression in SCLC, and may play a role in genesis of SCLC.

Cold-inducible RNA-binding protein (Cirp) interacts with Dyrk1b/Mirk and promotes proliferation of immature male germ cells in mice.

Cold-inducible RNA-binding protein (Cirp) was the first cold-shock protein identified in mammals. It is structurally quite different from bacterial cold-shock proteins and is induced in response to mild, but not severe, hypothermia. To clarify the physiological function of Cirp in vivo, we produced cirp-knockout mice. They showed neither gross abnormality nor defect in fertility, but the number of undifferentiated spermatogonia was significantly reduced and the recovery of spermatogenesis was delayed after treatment with a cytotoxic agent, busulfan. Cirp accelerated cell-cycle progression from G0 to G1 as well as from G1 to S phase in cultured mouse embryonic fibroblasts. Cirp directly bound to dual-specificity tyrosine-phosphorylation-regulated kinase 1B (Dyrk1b, also called Mirk) and inhibited its binding to p27, resulting in decreased phosphorylation and destabilization of p27. Cirp did not affect binding of Dyrk1b to cyclin D1 but inhibited phosphorylation of cyclin D1 by Dyrk1b, resulting in cyclin D1 stabilization. In the spermatogonial cell line GC-1spg, suppression of Cirp expression increased the protein level of p27, decreased that of cyclin D1, and decreased the growth rate, which depended on Dyrk1b. Consistent changes in the protein levels of p27 and cyclin D1 as well as the percentage of cells in G0 phase were observed in undifferentiated spermatogonia of cirp-knockout mice. In undifferentiated spermatogonia of wild-type mice, Cirp and Dyrk1b colocalized in the nucleus. Thus, our study demonstrates that Cirp functions to fine-tune the proliferation of undifferentiated spermatogonia by interacting with Dyrk1b.

Activation of Bmp2-Smad1 signal and its regulation by coordinated alteration of H3K27 trimethylation in Ras-induced senescence.

Cellular senescence involves epigenetic alteration, e.g. loss of H3K27me3 in Ink4a-Arf locus. Using mouse embryonic fibroblast (MEF), we here analyzed transcription and epigenetic alteration during Ras-induced senescence on genome-wide scale by chromatin immunoprecipitation (ChIP)-sequencing and microarray. Bmp2 was the most activated secreted factor with H3K4me3 gain and H3K27me3 loss, whereas H3K4me3 loss and de novo formation of H3K27me3 occurred inversely in repression of nine genes, including two BMP-SMAD inhibitors Smad6 and Noggin. DNA methylation alteration unlikely occurred. Ras-activated cells senesced with nuclear accumulation of phosphorylated SMAD1/5/8. Senescence was bypassed in Ras-activated cells when Bmp2/Smad1 signal was blocked by Bmp2 knockdown, Smad6 induction, or Noggin induction. Senescence was induced when recombinant BMP2 protein was added to Bmp2-knocked-down Ras-activated cells. Downstream Bmp2-Smad1 target genes were then analyzed genome-wide by ChIP-sequencing using anti-Smad1 antibody in MEF that was exposed to BMP2. Smad1 target sites were enriched nearby transcription start sites of genes, which significantly correlated to upregulation by BMP2 stimulation. While Smad6 was one of Smad1 target genes to be upregulated by BMP2 exposure, Smad6 repression in Ras-activated cells with increased enrichment of Ezh2 and gain of H3K27me3 suggested epigenetic disruption of negative feedback by Polycomb. Among Smad1 target genes that were upregulated in Ras-activated cells without increased repressive mark, Parvb was found to contribute to growth inhibition as Parvb knockdown lead to escape from senescence. It was revealed through genome-wide analyses in this study that Bmp2-Smad1 signal and its regulation by harmonized epigenomic alteration play an important role in Ras-induced senescence.

The oncoprotein gankyrin interacts with RelA and suppresses NF-kappaB activity.

Gankyrin is an oncoprotein commonly overexpressed in hepatocellular carcinomas. It interacts with multiple proteins and accelerates degradation of tumor suppressors Rb and p53. Since gankyrin consists of 7 ankyrin repeats and is structurally similar to IkappaBs, we investigated its interaction with NF-kappaB. We found that gankyrin directly binds to RelA. In HeLa and 293 cells, overexpression of gankyrin suppressed the basal as well as TNFalpha-induced transcriptional activity of NF-kappaB, whereas down-regulation of gankyrin increased it. Gankyrin did not affect the NF-kappaB DNA-binding activity or nuclear translocation of RelA induced by TNFalpha in these cells. Leptomycin B that inhibits nuclear export of RelA suppressed the NF-kappaB activity, which was further suppressed by gankyrin. The inhibitory effect of gankyrin was abrogated by nicotinamide as well as down-regulation of SIRT1, a class III histone deacetylase. Thus, gankyrin binds to NF-kappaB and suppresses its activity at the transcription level by modulating acetylation via SIRT1.