N(1)-meA and N(3)-meC are cytotoxic DNA base methylation lesions that can accumulate in the genomes of various organisms in the presence of S(N)2 type methylating agents. We report here the structural characterization of these base lesions in duplex DNA using a cross-linked protein-DNA crystallization system. The crystal structure of N(1)-meA:T pair shows an unambiguous Hoogsteen base pair with a syn conformation adopted by N(1)-meA, which exhibits significant changes in the opening, roll and twist angles as compared to the normal A:T base pair. Unlike N(1)-meA, N(3)-meC does not establish any interaction with the opposite G, but remains partially intrahelical. Also, structurally characterized is the N(6)-meA base modification that forms a normal base pair with the opposite T in duplex DNA. Structural characterization of these base methylation modifications provides molecular level information on how they affect the overall structure of duplex DNA. In addition, the base pairs containing N(1)-meA or N(3)-meC do not share any specific characteristic properties except that both lesions create thermodynamically unstable regions in a duplex DNA, a property that may be explored by the repair proteins to locate these lesions.

Three new bianthraquinone derivatives, alterporriol K (1), L (2) and M (3), along with six known compounds were obtained from extracts of the endophytic fungus Alternaria sp. ZJ9-6B, isolated from the mangrove Aegiceras corniculatum collected in the South China Sea. Their structures were elucidated by one- and two-dimensional NMR spectroscopy, MS data analysis and circular dichroism measurements. Compounds 1, 2 and 3 were first isolated alterporriols with a C-2-C-2' linkage. The crystallographic data of tetrahydroaltersolanol B (7) was reported for the first time. In the primary bioassays, alterporriol K and L exhibited moderate cytotoxic activity towards MDA-MB-435 and MCF-7 cells with IC₅₀ values ranging from 13.1 to 29.1 μM.

Covalent modification of DNA distinguishes cellular identities and is crucial for regulating the pluripotency and differentiation of embryonic stem (ES) cells. The recent demonstration that 5-methylcytosine (5-mC) may be further modified to 5-hydroxymethylcytosine (5-hmC) in ES cells has revealed a novel regulatory paradigm to modulate the epigenetic landscape of pluripotency. To understand the role of 5-hmC in the epigenomic landscape of pluripotent cells, here we profile the genome-wide 5-hmC distribution and correlate it with the genomic profiles of 11 diverse histone modifications and six transcription factors in human ES cells. By integrating genomic 5-hmC signals with maps of histone enrichment, we link particular pluripotency-associated chromatin contexts with 5-hmC. Intriguingly, through additional correlations with defined chromatin signatures at promoter and enhancer subtypes, we show distinct enrichment of 5-hmC at enhancers marked with H3K4me1 and H3K27ac. These results suggest potential role(s) for 5-hmC in the regulation of specific promoters and enhancers. In addition, our results provide a detailed epigenomic map of 5-hmC from which to pursue future functional studies on the diverse regulatory roles associated with 5-hmC.

Eph receptor (Eph) and ephrin signaling can play central roles in prostate cancer and other cancer types. Exposed to ephrin-A1 PC3 prostate cancer cells alter adhesion to extracellular matrix (ECM) proteins. However, whether PC3 cells increase or reduce adhesion, and by which mechanisms they change adhesion to the ECM remains to be characterized. Here, we assay how ephrin-A1 stimulates PC3 cells to adhere to ECM proteins using single-cell force spectroscopy. We find that PC3 cells binding to immobilized ephrin-A1 but not to solubilized ephrin-A1 specifically strengthen adhesion to collagen I. This Eph-ephrin-A1 signaling, which we suppose is based on mechanotransduction, stimulates β1-subunit containing integrin adhesion via the protein kinase Akt and the guanine nucleotide-exchange factor cytohesin. Inhibiting the small GTPases, Rap1 or Rac1, generally lowered adhesion of PC3 prostate cancer cells. Our finding suggests a mechanism by which PC3 prostate cancer cells exposed to ephrins crosstalk to β1-integrins and preferably metastasize in bone, a collagen I rich tissue.

Nrf2 is the key transcription factor regulating the antioxidant response which is crucial for cytoprotection against extracellular stresses. Numerous in vivo studies indicate that Nrf2 plays a protective role in anti-inflammatory response. 3-(3-Pyridylmethylidene)-2-indolinone (PMID) is a synthesized derivative of 2-indolinone compounds. Our previous study suggested that PMID induces the activation of Nrf2/ARE pathway, then protecting against oxidative stress-mediated cell death. However, little is known regarding the anti-inflammatory properties of PMID in severe inflammatory phenotypes. In the present study we determined if PMID treatment protects mice from dextran sodium sulphate- (DSS-) induced colitis. The result suggests that treatment with PMID prior to colitis induction significantly reduced body weight loss, shortened colon length, and decreased disease activity index compared to control mice. Histopathological analysis of the colon revealed attenuated inflammation in PMID pretreated animals. The levels of inflammatory markers in colon tissue and serum were reduced associated with inhibition of NF-κB activation. The expression levels of Nrf2-dependent genes such as HO-1, NQO1, and Nrf2 were increased in PMID pretreated mice. However, PMID pretreatment did not prevent DSS-induced colitis in Nrf2 knockout mice. These data indicate that PMID pretreatment in mice confers protection against DSS-induced colitis in Nrf2-dependent manner, suggesting a potential role of PMID in anti-inflammatory response.

Recent discoveries of reversible N(6)-methyladenosine (m(6)A) methylation on messenger RNA (mRNA) and mapping of m(6)A methylomes in mammals and yeast have revealed potential regulatory functions of this RNA modification. In plants, defects in m(6)A methyltransferase cause an embryo-lethal phenotype, suggesting a critical role of m(6)A in plant development. Here, we profile m(6)A transcriptome-wide in two accessions of Arabidopsis thaliana and reveal that m(6)A is a highly conserved modification of mRNA in plants. Distinct from mammals, m(6)A in A. thaliana is enriched not only around the stop codon and within 3'-untranslated regions, but also around the start codon. Gene ontology analysis indicates that the unique distribution pattern of m(6)A in A. thaliana is associated with plant-specific pathways involving the chloroplast. We also discover a positive correlation between m(6)A deposition and mRNA abundance, suggesting a regulatory role of m(6)A in plant gene expression.

Hypoxia induces the epithelial-mesenchymal transition, EMT, to promote cancer metastasis. In addition to transcriptional regulation mediated by hypoxia-inducible factors, HIFs, other epigenetic mechanisms of gene regulation, such as histone modifications and DNA methylation, are utilized under hypoxia. However, whether DNA demethylation mediated by TET1, a DNA dioxygenase converting 5-methylcytosine, 5mC, into 5-hydroxymethylcytosine, 5hmC, plays a role in hypoxia-induced EMT is largely unknown.

N(6)-methyladenosine (m(6)A) is the most abundant modification in mammalian mRNA and long noncoding RNA (lncRNA). Recent discoveries of two m(6)A demethylases and cell-type and cell-state-dependent m(6)A patterns indicate that m(6)A modifications are highly dynamic and likely play important biological roles for RNA akin to DNA methylation or histone modification. Proposed functions for m(6)A modification include mRNA splicing, export, stability, and immune tolerance; but m(6)A studies have been hindered by the lack of methods for its identification at single nucleotide resolution. Here, we develop a method that accurately determines m(6)A status at any site in mRNA/lncRNA, termed site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET). The method determines the precise location of the m(6)A residue and its modification fraction, which are crucial parameters in probing the cellular dynamics of m(6)A modification. We applied the method to determine the m(6)A status at several sites in two human lncRNAs and three human mRNAs and found that m(6)A fraction varies between 6% and 80% among these sites. We also found that many m(6)A candidate sites in these RNAs are however not modified. The precise determination of m(6)A status in a long noncoding RNA also enables the identification of an m(6)A-containing RNA structural motif.

Hepassocin (HPS) is a secreted protein with mitogenic activity on primary hepatocytes and protects hepatocytes from chemically-induced injury. Our previous studies showed that HPS stimulates proliferation of hepatocytes in an ERK pathway-dependent manner. However, the molecular mechanism of HPS-induced activation of the ERK pathway remains unclear. In this study, we found that HPS induced the phosphorylation of the epidermal growth factor receptor (EGFR) in the human L02 hepatocyte cell line, and this event was concomitant with the activation of the non-receptor tyrosine kinase Src. Specific inhibition of EGFR kinase activity by gefitinib or down-regulation of EGFR by specific EGFR siRNAs prevented HPS-induced activation of the ERK pathway and proliferation of L02 cells. Furthermore, inhibition of Src activity significantly blocked HPS-induced activation of the EGFR, which was suggestive of a ligand-independent transactivation mechanism of EGFR itself as well as ERK phosphorylation and proliferation of L02 cells. These results indicate that EGFR plays an important role in the mitogenic signaling induced by HPS in L02 cell lines and may further stimulate research on the role of HPS in hepatocytes within biological processes in human health and disease.

Weight control through either dietary calorie restriction (DCR) or exercise has been associated with cancer prevention in animal models. However, the underlying mechanisms are not fully defined. Bioinformatics using genomics, proteomics and lipidomics was employed to elucidate the molecular targets of weight control in a mouse skin cancer model. SENCAR mice were randomly assigned into four groups for 10 weeks: ad-libitum-fed sedentary control, ad-libitum-fed exercise (AE), exercise but pair-fed isocaloric amount of control (PE) and 20% DCR. Two hours after topical TPA treatment, skin epidermis was analyzed by Affymetrix for gene expression, DIGE for proteomics and lipidomics for phospholipids. Body weights were significantly reduced in both DCR and PE but not AE mice versus the control. Among 39,000 transcripts, 411, 67 and 110 genes were significantly changed in DCR, PE and AE, respectively. The expression of genes relevant to PI3K-Akt and Ras-MAPK signaling was effectively reduced by DCR and PE but not AE as measured through GenMAPP software. Proteomics analysis identified ~120 proteins, with 27 proteins significantly changed by DCR, including up-regulated apolipoprotein A-1, a key antioxidant protein that decreases Ras-MAPK activity. Of the total 338 phospholipids analyzed by lipidomics, 57 decreased by PE including 5 phophatidylinositol species that serve as PI3K substrates. Although a full impact has not been determined yet, it appears that the reduction of both Ras-MAPK and PI3K-Akt signaling pathways is a cancer preventive target that has been consistently demonstrated by three bioinformatics approaches.

Infection with the flavivirus Zika (ZIKV) causes neurological, immunological, and developmental defects through incompletely understood mechanisms. We report that ZIKV infection affects viral and human RNAs by altering the topology and function of N6-adenosine methylation (m6A), a modification affecting RNA structure and function. m6A nucleosides are abundant in ZIKV RNA, with twelve m6A peaks identified across full-length ZIKV RNA. m6A in ZIKV RNA is controlled by host methyltransferases METTL3 and METTL14 and demethylases ALKBH5 and FTO, and knockdown of methyltransferases increases, while silencing demethylases decreases, ZIKV production. YTHDF family proteins, which regulate the stability of m6A-modified RNA, bind to ZIKV RNA, and their silencing increases ZIKV replication. Profiling of the m6A methylome of host mRNAs reveals that ZIKV infection alters m6A location in mRNAs, methylation motifs, and target genes modified by methyltransferases. Our results identify a mechanism by which ZIKV interacts with and alters host cell functions.

Myocardial injuries in viral myocarditis (VMC) are caused by viral infection and related autoimmune disorders. Recent studies suggest that IL-9 mediated both antimicrobial immune and autoimmune responses in addition to allergic diseases. However, the role of IL-9 in viral infection and VMC remains controversial and uncertain. In this study, we infected Balb/c mice with Coxsackievirus B3 (CVB3), and found that IL-9 was enriched in the blood and hearts of VMC mice on days 5 and 7 after virus infection. Most of IL-9 was secreted by CD8+ T cells on day 5 and CD4+ T cells on day 7 in the myocardium. Further, IL-9 knockout exacerbated cardiac damage following CVB3 infection, along with a sharp increase in viral replication and IL-17a expression, as well as a decrease in TGF-β. In contrast, the repletion of IL-9 in Balb/c mice with CVB infection induced the opposite effect. Studies in vitro further revealed that IL-9 directly inhibited viral replication in cardiomyocytes by reducing coxsackie and adenovirus receptor expression, which might be associated with upregulation of TGF-β autocrine effect in these cells. However, IL-9 had no direct effect on apoptosis in cardiomyocytes. Our data indicated that IL-9 played a protective role in disease progression by inhibiting CVB3 replication in the early stages of VMC.

Th17 cells play a key role in the progression of coxsackievirus B3 (CVB3)-induced acute viral myocarditis (AVMC). However, the direct effect of virus on Th17 cell differentiation is still unknown. Recently, nucleoporin (Nup) 98 has been proved to be associated with lymphocyte differentiation. Therefore, we investigated whether Nup98 mediated Th17 cell differentiation in AVMC. In our study, patients with AVMC and healthy controls were recruited. The results showed that CVB3 could enter into the CD4+ T cells in AVMC patients and healthy controls. After transfecting purified CD4+ T cells with CVB3 in vitro, the Th17 cell frequency, IL-17 secretion, and RORγT synthesis were increased while the Nup98 levels were decreased. Furthermore, down-regulating Nup98 expression by siRNA-Nup98 in CD4+ T cells resulted in the elevated Th17 cell frequency and IL-17 secretion, along with enhanced levels of RORγT, dissociative p300/CBP, and acetylated Stat3. Up-regulation of Nup98 expression by pcDNA3.1-Nup98 showed the opposite effects. Our results suggested that CVB3 directly induced CD4+ T cell differentiation into Th17 cells by inhibiting Nup98 expression, representing a therapeutic target in AVMC.

The epithelial-mesenchymal transition (EMT) is one of the crucial procedures for cancer invasion and distal metastasis. Despite undergoing intensive studies, the mechanisms underlying EMT remain to be completely elucidated. Here, we identified that apoptosis-stimulating protein of p53-2 (ASPP2) is a novel target of MiR-205 in various cancers. Interestingly, the binding site of MiR-205 at the 3'-untranslated region of ASPP2 was highly conserved among different species. An inverse correlation between MiR-205 and ASPP2 was further observed in vivo in cervical cancers, suggesting MiR-205 may be an important physiological inhibitor of ASPP2. Hypoxia is a hallmark of solid tumor microenvironment and one of such conditions to induce EMT. Notably, MiR-205 was remarkably induced by hypoxia in cervical and lung cancer cells. A marked suppression of ASPP2 was observed simultaneously. Further studies confirmed that hypoxia-induced ASPP2 suppression was mainly attributed to the elevated MiR-205. Interestingly, the alteration of MiR-205/ASPP2 under hypoxia was accompanied with the decreased epithelial marker E-cadherin and increased mesenchymal marker Vimentin, as well as a morphological transition from the typical cobblestone-like appearance to the mesenchymal-like structure. More importantly, MiR-205 mimics or ASPP2 silencing similarly promoted EMT process. By contrast, ASPP2 recovery or MiR-205 inhibitor reversed MiR-205-dependent EMT. Further studies demonstrated that the newly revealed MiR-205/ASPP2 axis promoted cell migration and also increased cell proliferation both in vivo and in vitro. These data together implicated a critical impact of MiR-205/ASPP2 on promoting EMT. MiR-205/ASPP2 may be potential diagnostic and therapeutic biomarkers in cervical and lung cancers.

The purpose of this study was to detect the differentially expressed genes between ossified herniated discs and herniated discs without ossification. In addition, we sought to identify a few candidate genes and pathways by using bioinformatics analysis. We analyzed 6 samples each of ossified herniated discs (experimental group) and herniated discs without ossification (control group). Purified mRNA and cDNA extracted from the samples were subjected to sequencing. The NOISeq method was used to statistically identify the differentially expressed genes (DEGs) between the 2 groups. An in-depth analysis using bioinformatics tools based on the DEGs was performed using Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and protein-protein interaction network analysis. The top 6 DEGs were verified using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). A total of 132 DEGs was detected. A total of 129 genes in the ossified group were upregulated and 3 genes were found to be downregulated as compared to the control group. The top 3 cellular components in GO ontologies analysis were extracellular matrix components. GO functions were mainly related to the glycoprotein in the cell membrane and extracellular matrix. The GO process was related to completing response to stimulus, immune reflex and defense. The top 5 KEGG enrichment pathways were associated with infection and inflammation. Three of the top 20 DEGs [sclerostin (SOST), WNT inhibitory factor 1 (WIF1) and secreted frizzled related protein 4 (SFRP4)] were related to the inhibition of the Wnt pathway. The ossified discs exhibited a higher expression of the top 6 DEGs [SOST, joining chain of multimeric IgA and IgM (IGJ; also known as JCHAIN), defensin alpha 4 (DEFA4), SFRP4, proteinase 3 (PRTN3) and cathepsin G (CTSG)], with the associated P-values of 0.045, 0.000, 0.008, 0.010, 0.015 and 0.002, respectively, as calculated by the independent sample t-test. The gene expression profiling of the 2 groups revealed differential gene expression. Thus, our data suggest that Wnt pathway abnormality and local inflammation may be related to disc ossification.

N(6)-methyladenosine (m(6)A) is the most prevalent internal (non-cap) modification present in the messenger RNA of all higher eukaryotes. Although essential to cell viability and development, the exact role of m(6)A modification remains to be determined. The recent discovery of two m(6)A demethylases in mammalian cells highlighted the importance of m(6)A in basic biological functions and disease. Here we show that m(6)A is selectively recognized by the human YTH domain family 2 (YTHDF2) 'reader' protein to regulate mRNA degradation. We identified over 3,000 cellular RNA targets of YTHDF2, most of which are mRNAs, but which also include non-coding RNAs, with a conserved core motif of G(m(6)A)C. We further establish the role of YTHDF2 in RNA metabolism, showing that binding of YTHDF2 results in the localization of bound mRNA from the translatable pool to mRNA decay sites, such as processing bodies. The carboxy-terminal domain of YTHDF2 selectively binds to m(6)A-containing mRNA, whereas the amino-terminal domain is responsible for the localization of the YTHDF2-mRNA complex to cellular RNA decay sites. Our results indicate that the dynamic m(6)A modification is recognized by selectively binding proteins to affect the translation status and lifetime of mRNA.

14-3-3 proteins regulate numerous cellular processes through interaction with a variety of proteins, and have been identified as HNF1α binding partner by mass spectrometry analysis in our previous study. In the present study, the interaction between 14-3-3ζ and HNF1α has been further validated by in vivo and in vitro assays. Moreover, we have found that overexpression of 14-3-3ζ potentiated the transcriptional activity of HNF1α in cultured cells, and silencing of 14-3-3ζ by RNA interference in HepG2 cells specifically affected the HNF1α-dependent gene expression. Furthermore, we have demonstrated that 14-3-3ζ is recruited to endogenous HNF1α responsive promoters and enhances HNF1α binding to its cognate DNA sequences. In addition, we have also provided evidence that the association between HNF1α and 14-3-3ζ is phosphorylation-dependent. Taken together, these results suggest that 14-3-3ζ may be an endogenous physiologic regulator of HNF1α.

Despite its biological importance, tRNA has not been adequately sequenced by standard methods because of its abundant post-transcriptional modifications and stable structure, which interfere with cDNA synthesis. We achieved efficient and quantitative tRNA sequencing in HEK293T cells by using engineered demethylases to remove base methylations and a highly processive thermostable group II intron reverse transcriptase to overcome these obstacles. Our method, DM-tRNA-seq, should be applicable to investigations of tRNA in all organisms.

We present a highly sensitive and selective chemical labeling and capture approach for genome-wide profiling of 5-hydroxylmethylcytosine (5hmC) using DNA isolated from ∼1,000 cells (nano-hmC-Seal). Using this technology, we assessed 5hmC occupancy and dynamics across different stages of hematopoietic differentiation. Nano-hmC-Seal profiling of purified Tet2-mutant acute myeloid leukemia (AML) murine stem cells allowed us to identify leukemia-specific, differentially hydroxymethylated regions that harbor known and candidate disease-specific target genes with differential 5hmC peaks compared to normal stem cells. The change of 5hmC patterns in AML strongly correlates with differential gene expression, demonstrating the importance of dynamic alterations of 5hmC in regulating transcription in AML. Together, covalent 5hmC labeling offers an effective approach to study and detect DNA methylation dynamics in in vivo disease models and in limited clinical samples.

MicroRNAs are subject to precise regulation and have key roles in tumorigenesis. In contrast to the oncogenic role of miR-22 reported in myelodysplastic syndrome (MDS) and breast cancer, here we show that miR-22 is an essential anti-tumour gatekeeper in de novo acute myeloid leukaemia (AML) where it is significantly downregulated. Forced expression of miR-22 significantly suppresses leukaemic cell viability and growth in vitro, and substantially inhibits leukaemia development and maintenance in vivo. Mechanistically, miR-22 targets multiple oncogenes, including CRTC1, FLT3 and MYCBP, and thus represses the CREB and MYC pathways. The downregulation of miR-22 in AML is caused by TET1/GFI1/EZH2/SIN3A-mediated epigenetic repression and/or DNA copy-number loss. Furthermore, nanoparticles carrying miR-22 oligos significantly inhibit leukaemia progression in vivo. Together, our study uncovers a TET1/GFI1/EZH2/SIN3A/miR-22/CREB-MYC signalling circuit and thereby provides insights into epigenetic/genetic mechanisms underlying the pathogenesis of AML, and also highlights the clinical potential of miR-22-based AML therapy.