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Viral and cellular gene expression are regulated by epigenetic alterations, including DNA methylation, histone modifications, nucleosome positioning, and chromatin looping. Human T-cell leukemia virus type 1 (HTLV-1) is a pathogenic retrovirus associated with inflammatory disorders and T-cell lymphoproliferative malignancy. The transforming activity of HTLV-1 is driven by the viral oncoprotein Tax, which acts as a transcriptional activator of the cAMP response element-binding protein (CREB) and nuclear factor kappa B (NFκB) pathways. The epigenetic effects of Tax and the induction of lymphoproliferative malignancy include alterations in DNA methylation and histone modifications. In addition, alterations in nucleosome positioning and DNA looping also occur in HTLV-1-induced malignant cells. A mechanistic definition of these effects will pave the way to new therapies for HTLV-1-associated disorders.

Tetralogy of Fallot (TOF) is the most common form of cyanotic congenital heart disease (CHD). Although a lower methylation level of whole genome has been demonstrated in TOF patients, little is known regarding the DNA methylation changes in specific gene and its associations with TOF development. NOTCH4 is a mediator of the Notch signalling pathway that plays an important role in normal cardiac development. However, the role of epigenetic regulation of the NOTCH4 gene in the pathogenesis of TOF remains unclear. Considering the NOTCH4 low mutation frequency and reduced expression in the TOF patients, we hypothesized that abnormal DNA methylation change of NOTCH4 gene may influence its expression and responsible for TOF development. In this study, we measured the promoter methylation status of NOTCH4 and was measured and its regulation mechanism was explored, which may be related to TOF disease. Additionally, the promoter methylation statuses of NOTCH4 was measured in order to further understand epigenetic mechanisms that may serve a role in the development of TOF. Immunohistochemical analysis was used to examine NOTCH4 expression in right ventricular outflow tract myocardial tissues in patients with TOF. Compared with healthy controls, patients with TOF displayed significantly reduced in NOTCH4 expression (P=0.0055). Moreover, bisulphite sequencing suggested that the methylation levels of CpG site 2 in the NOTCH4 promoter was significantly higher in the patients than in the controls (P=0.0459). NOTCH4 expression was negatively associated with CpG site 2 methylation levels (r=−0.51; P=0.01). ETS1 transcription factor can serve as transcriptional activators by binding to specific DNA sequences of target genes, such as DLL4 and NOTCH4, which serves an important role in normal heart development. Dual-luciferase reporter and electrophoretic mobility shift assays indicated that the ETS1 transcription factor could bind to the NOTCH4 promoter region. However, binding of ETS1 to the NOTCH4 promoter was abrogated by methylation at the putative ETS1 binding sites. These findings suggested that decreased NOTCH4 expression in patients with TOF may be associated with hypermethylation of CpG site 2 in the NOTCH4 promoter region, due to impaired binding of ETS1. [ABSTRACT FROM AUTHOR]

Cryptorchidism is the most common disorder of sex development (DSD) in dogs. This malformation is associated with reduced fertility and with a higher risk of gonadal cancer. Testicular descent is a complex process, and the functions of many environmental and genetic factors are crucial for the proper migration of fetal gonads into the scrotum. Among these, the hormone INSL3 (insulin-like peptide 3) and its receptor RXFP2 (relaxin family peptide receptor 2) play crucial roles in the transabdominal migration of the testes. The genetic background of canine cryptorchidism is poorly elucidated. The aim of this study was to compare the transcript and methylation levels of INSL3 and RXFP2 genes in undescended and descended testes of isolated unilateral cryptorchids, and in gonads of control male dogs with scrotal testes. Next, we searched for polymorphic variants in the 5′-regulatory regions of both genes associated with predispositions to cryptorchidism. The INSL3 transcript level was significantly higher in the undescended testes than in the descended testes of both the affected and control dogs. On the other hand, the mRNA level of RXFP2 was significantly lower in the retained gonads of cryptorchids than in the scrotal testes. The methylation level of a single CpG site located 15 bp upstream of the translation start codon in INSL3 was significantly higher in the testes of the control dogs than in both gonads of cryptorchids. The methylation level of 14 CpG sites in the coding region of INSL3 was significantly higher in undescended testes than in the scrotal testes, which may be associated with the higher mRNA levels of INSL3 observed in these samples. The methylation pattern of two CpG sites in the 5′-flanking region of RXFP2 was similar in both descended and undescended testes. We detected three and seven single nucleotide polymorphisms (SNPs) in the 5′-regulatory regions of INSL3 and RXFP2 , respectively. Among these, the frequency of A > C substitution (ss7093349755) located 495 bp upstream of the transcription start site of RXFP2 differed significantly between cryptorchids and control dogs. Our study showed two possible genetic biomarkers associated with canine cryptorchidism: a hypomethylation of a single CpG site in the 5′-flanking region of INSL3 , and the ss7093349755 SNP in the 5′-flanking region of RXFP2. • Genetic and epigenetic background of canine cryptorchidism is poorly elucidated. • mRNA profiles of INSL3 and RXFP2 are different in descended and undescended testes. • CpG site in 5′ region of INSL3 is hypomethylated in testes of unilateral cryptorchids. • Methylation of CpG sites in exon 1 of INSL3 is elevated in undescended testes. • SNP in 5′ region of RXFP2 potentially increases risk of cryptorchidism. [ABSTRACT FROM AUTHOR]

CD8 T cells play a crucial role in immune responses to virus infections and tumors. Naïve CD8 T lymphocytes after TCR stimulation undergo differentiation into CTLs and memory cells, which are essential sources of IFN‐γ. We investigated IFN‐γ production by CD8 T cell subsets found in nonimmune mice. A minor fraction of in vitro TCR‐stimulated CD8 T cells produce IFN‐γ, and it is regulated at the transcriptional level. Antigen inexperienced C57BL/6 mice present the coexistence of 2 populations. The main population exhibits a CD44lowCD122low profile, which is compatible with naïve lymphocytes. The minor expresses a phenotype of immunologic memory, CD44hiCD122hi. Both subsets are able to produce IL‐2 in response to TCR activation, but only the memory‐like population is responsible for IFN‐γ production. Similar to memory CD8 T cells, CD44hiCD8+ T cells also present a higher level of the transcriptional factor Eomes and a lower level of T‐bet (Tbx21) mRNA than CD44lowCD8+ T cells. The presence of the CD44hiCD8+ T cell population in nonimmune OT‐I transgenic mice reveals that the population is generated independently of antigenic stimulation. CpG methylation is an efficient epigenetic mechanism for gene silencing. DNA methylation at posttranscriptional CpG sites in the Ifng promoter is higher in CD44lowCD8+ T cells than in CD44hiCD8+ T cells. Thus, memory‐like CD8 T cells have a distinct epigenetic pattern in the Ifng promoter and can rapidly produce IFN‐γ in response to TCR stimulation. [ABSTRACT FROM AUTHOR]

The development of methyl-transverse relaxation-optimized spectroscopy (methyl-TROSY)-based NMR methods, in concert with robust strategies for incorporation of methyl-group probes of structure and dynamics into the protein of interest, has facilitated quantitative studies of high-molecular-weight protein complexes. Here we develop a one-pot in vitro reaction for producing NMR quantities of methyl-labeled DNA at the C5 and N6 positions of cytosine (5mC) and adenine (6mA) nucleobases, respectively, enabling the study of high-molecular-weight DNA molecules using TROSY approaches originally developed for protein applications. Our biosynthetic strategy exploits the large number of naturally available methyltransferases to specifically methylate DNA at a desired number of sites that serve as probes of structure and dynamics. We illustrate the methodology with studies of the 153-base pair Widom DNA molecule that is simultaneously methyl-labeled at five sites, showing that high-quality 13C-¹H spectra can be recorded on 100 µM samples in a few minutes. NMR spin relaxation studies of labeled methyl groups in both DNA and the H2B histone protein component of the 200-kDa nucleosome core particle (NCP) establish that methyl groups at 5mC and 6mA positions are, in general, more rigid than Ile, Leu, and Val methyl probes in protein side chains. Studies focusing on histone H2B of NCPs wrapped with either wild-type DNA or DNA methylated at all 26 CpG sites highlight the utility of NMR in investigating the structural dynamics of the NCP and how its histone core is affected through DNA methylation, an important regulator of transcription. [ABSTRACT FROM AUTHOR]

Interleukin-1 beta (IL1B) is a key inducer of inflammation and an important factor in the regulation of hematopoietic stem cells and mesenchymal stromal progenitors. Irradiation of mice with ionizing radiation has been shown to induce a lasting increase in IL1B concentration in peripheral blood. One of the possible mechanisms may be demethylation of CpG cytosines in the Il1b promoter, which has not been characterized in detail for the mouse. In this study, the methylation level of CpGs located in a region between −3562 and −208 bp upstream of the start of transcription is studied in muscles, bones, liver, thymus, spleen, bone marrow, lymph nodes, lungs, and brain. The methylation level is compared to Il1b expression. Tissue-specific features of CpG methylation are established. It is demonstrated that the region between −2420 and −2406 bp is likely a part of the mouse Il1b promoter/enhancer and may determine the base level of Il1b expression in various tissues. Irradiation at a dose of 6 Gy does not change the methylation profile of most studied CpGs, and therefore, the cause of the stably increased IL1B level after irradiation is unlikely to be a change in the methylation of the studied CpGs in investigated tissues. [ABSTRACT FROM AUTHOR]

Study Question: Are there any differences between in vivo (IVV) and in vitro (IVT) matured metaphase II (MII) oocytes at the molecular level?Summary Answer: Between IVV and IVT oocytes, 507 differentially expressed genes (DEGs) were identified; the non-CpG methylomes were significantly different, but the CpG methylomes and genomic copy number variations (CNVs) were similar.What Is Known Already: A previous study using microarray and single-cell RNA-seq analysis revealed that numerous genes were differentially expressed between IVV and IVT oocytes. Independent studies of DNA methylation profiling in human oocytes have revealed negative correlations between gene transcription and the DNA methylation level at gene promoter regions. No study has compared global CpG or non-CpG methylation between these two groups of oocytes. Although a high level of aneuploidy has been reported in MII oocytes, no direct comparison of IVV and IVT oocytes based on single-cell sequencing data has been performed.Study Design, Size, Duration: We collected eight IVV oocytes from six patients and seven IVT oocytes from seven patients and then analysed each oocyte using the previously established single-cell triple omics sequencing (scTrioseq) analysis to determine associations among the transcriptome, DNA methylome and chromosome ploidy in the oocytes.Participants/materials, Setting, Methods: All IVV oocytes were donated by patients who received 150 IU gonadotropin per day from the third day of their menstrual cycle, followed by GnRH antagonist after 5 days of gonadotropin stimulation. All IVT oocytes were from immature oocytes which were donated by volunteers undergoing delivery by caesarean section then cultured in oocyte maturation medium containing 75 mIU/ml hMG for 24 to 48 h. Every single oocyte was analysed using the previously established single-cell multiomic sequencing analysis.Main Results and the Role Of Chance: There were 507 genes differentially expressed between the IVV (n = 8) and IVT (n = 7) oocytes, even though their global transcriptome profiles were similar. The enriched genes in IVV oocytes were related to the cell cycle process while those in IVT oocytes were related to mitochondrial respiration biogenesis. Although the global CpG methylation of the two groups of oocytes was similar, the non-CpG methylation level in IVV oocytes was higher than that in IVT oocytes. A high aneuploidy ratio was found in both groups, but the aneuploidy did not affect transcription according to the correlation analysis.Large-scale Data: N/A.Limitations and Reasons For Caution: Due to the difficulty in collecting MII oocytes, especially IVV matured oocytes, the sample size was limited.Wider Implications Of the Findings: Our findings indicate that single-cell multiomic sequencing can be utilised to examine the similarity and differences between IVV and IVT matured MII oocytes.Study Funding/competing Interest(s): This work was supported by the Ministry of Science and Technology of China, National Key R&D Program of China (No. 2017YFC1001601). The donated oocytes were collected by Shanghai Tenth People's Hospital. The authors declare no competing interests. [ABSTRACT FROM AUTHOR]

Loss of the tumor suppressor tuberous sclerosis complex 1 (Tsc1) in the liver promotes gluconeogenesis and glucose intolerance. We asked whether this could be attributed to aberrant expression of small RNAs. We performed small-RNA sequencing on liver of Tsc1-knockout mice, and found that miRNAs of the delta-like homolog 1 (Dlk1)-deiodinase iodothyronine type III (Dio3) locus are up-regulated in an mTORC1-dependent manner. Sustained mTORC1 signaling during development prevented CpG methylation and silencing of the Dlk1-Dio3 locus, thereby increasing miRNA transcription. Deletion of miRNAs encoded by the Dlk1-Dio3 locus reduced gluconeogenesis, glucose intolerance, and fasting blood glucose levels. Thus, miRNAs contribute to the metabolic effects observed upon loss of TSC1 and hyperactivation of mTORC1 in the liver. Furthermore, we show that miRNA is a downstream effector of hyperactive mTORC1 signaling. [ABSTRACT FROM AUTHOR]

Seasonal influenza is related to lifestyle-associated risk factors and it has been suggested that the epigenetic state of the individual plays an important role in the severity of infection. It is well known that epigenetics stringently regulate gene expression in each tissue and that aberrant epigenetic states can influence disease development. Despite some studies, limited information is available on changes in epigenetic states before and after influenza virus infection; in particular, it is unknown whether the epigenetic state at specific sites affects subsequent infection. Here, we analyzed CpG methylation states in clones derived from human primary small airway epithelial cells with the same genetic background but different viral replication rates. Our study revealed that demethylating CpGs downstream of the IFN-β transcription start site using a CRISPR/dCas9 system suppressed viral replication during subsequent influenza virus infection. Thus, our observations suggest that epigenome editing might provide adequate protection against the influenza virus. • Epigenetic state is related to influenza infection. • We analyzed CpG methylation state among human cells with the same genetic background. • Demethylation at CpGs downstream of IFN-β start codon suppressed viral replication. • Epigenome editing might be protective against influenza virus. [ABSTRACT FROM AUTHOR]

Lower-grade glioma (LGG) is a diffuse infiltrative tumor of the central nervous system, which lacks targeted therapy. We investigated the role of Podocan-like 1 (PODNL1) methylation in LGG clinical outcomes using the TCGA-LGG transcriptomics dataset. We identified four PODNL1 CpG sites, cg07425555, cg26969888, cg18547299, and cg24354933, which were associated with unfavorable overall survival (OS) and disease-free survival (DFS) in univariate and multivariate analysis after adjusting for age, gender, tumor-grade, and IDH1-mutation. In multivariate analysis, the OS and DFS hazard ratios ranged from 0.44 to 0.58 (p < 0.001) and 0.62 to 0.72 (p < 0.001), respectively, for the four PODNL1 CpGs. Enrichment analysis of differential gene and protein expression and analysis of 24 infiltrating immune cell types showed significantly increased infiltration in LGGs and its histological subtypes with low-methylation levels of the PODNL1 CpGs. High PODNL1 expression and low-methylation subgroups of the PODNL1 CpG sites were associated with significantly increased PD-L1, PD-1, and CTLA4 expressions. PODNL1 methylation may thus be a potential indicator of immune checkpoint blockade response, and serve as a biomarker for determining prognosis and immune subtypes in LGG.

Methylation of cytosine in CpG dinucleotides is the major DNA modification in mammalian cells that is a key component of stable epigenetic marks. This modification, which on the one hand is reversible, while on the other hand, can be maintained through successive rounds of replication plays roles in gene regulation, genome maintenance, transgenerational epigenetic inheritance, and imprinting. Disturbed DNA methylation contributes to a wide array of human diseases from single-gene disorders to sporadic metabolic diseases or cancer. DNA methylation was also shown to affect several neurodegenerative disorders, including Huntington’s disease (HD), a fatal, monogenic inherited disease. HD is caused by a polyglutamine repeat expansion in the Huntingtin protein that brings about a multifaceted pathogenesis affecting several cellular processes. Research of the last decade found complex, genome-wide DNA methylation changes in HD pathogenesis that modulate transcriptional activity and genome stability. This article reviews current evidence that sheds light on the role of DNA methylation in HD.

DNA methylation is a major epigenetic factor that has been postulated to regulate cell lineage differentiation. We report here that conditional gene deletion of the maintenance DNA methyltransferase I (Dnmt1) in neural progenitor cells (NPCs) results in DNA hypomethylation and precocious astroglial differentiation. The developmentally regulated demethylation of astrocyte marker genes as well as genes encoding the crucial components of the gliogenic JAK-STAT pathway is accelerated in Dnmt1(-/-) NPCs. Through a chromatin remodeling process, demethylation of genes in the JAK-STAT pathway leads to an enhanced activation of STATs, which in turn triggers astrocyte differentiation. Our study suggests that during the neurogenic period, DNA methylation inhibits not only astroglial marker genes but also genes that are essential for JAK-STAT signaling. Thus, demethylation of these two groups of genes and subsequent elevation of STAT activity are key mechanisms that control the timing and magnitude of astroglial differentiation.

G-quadruplex (G4) sites in the human genome frequently colocalize with CCCTC-binding factor (CTCF)-bound sites in CpG islands (CGIs). We aimed to clarify the role of G4s in CTCF positioning. Molecular modeling data suggested direct interactions, so we performed in vitro binding assays with quadruplex-forming sequences from CGIs in the human genome. G4s bound CTCF with Kd values similar to that of the control duplex, while respective i-motifs exhibited no affinity for CTCF. Using ChIP-qPCR assays, we showed that G4-stabilizing ligands enhance CTCF occupancy at a G4-prone site in STAT3 gene. In view of the reportedly increased CTCF affinity for hypomethylated DNA, we next questioned whether G4s also facilitate CTCF recruitment to CGIs via protecting CpG sites from methylation. Bioinformatics analysis of previously published data argued against such a possibility. Finally, we questioned whether G4s facilitate CTCF recruitment by affecting chromatin structure. We showed that three architectural chromatin proteins of the high mobility group colocalize with G4s in the genome and recognize parallel-stranded or mixed-topology G4s in vitro. One of such proteins, HMGN3, contributes to the association between G4s and CTCF according to our bioinformatics analysis. These findings support both direct and indirect roles of G4s in CTCF recruitment.

Purpose: Molecular mechanisms of response to hypomethylating agents in patients with myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) still remain largely unknown. Therefore, the effects of 5-Azacytidine (Aza) on clonal architecture and DNA methylation were investigated in this study. Methods: Using next-generation sequencing (NGS), 30 myeloid leukemia-associated genes were analyzed in 15 MDS/CMML patients with excellent response to Aza. Effects on methylation levels were analyzed by quantitative methylation analysis using pyrosequencing for the global methylation marker LINE-1 in patients and myeloid cell lines. Various myeloid cell lines and a healthy cohort were screened for methylation levels in 23 genes. Selected targets were verified on the MDS/CMML cohort. Results: The study presented here showed a stable variant allele frequency and stable global methylation levels in responding patients. A significant demethylation of EZH2 and NOTCH1 was revealed in patients with Aza response. Conclusions: A response to Aza is not associated with eradication of malignant clones, but rather with a stabilization of the clonal architecture. We suggest changes in CpG methylation levels of EZH2 and NOTCH1 as potential targets of epigenetic response to Aza treatment which may also serve as useful biomarkers after clinical evaluation. [ABSTRACT FROM AUTHOR]

CpG methylation of DNA is an epigenetic marker that is highly related to the regulation of transcription initiation. For analysis of CpG methylation in genomic DNA sequences, bisulfite-induced modification in combination with polymerase chain reaction (PCR) is usually utilized, but it cannot be straightforwardly applied to methylated short- and middle-sized DNAs, such as < 500 base pairs (bp), which are often utilized in structural biology studies. In the present study, we applied nano-electrospray ionization mass spectrometry (nano-ESI-MS) for the characterization of methylated DNA with < 400 bp prepared in vitro. First, double-stranded DNA oligomers were methylated with recombinant M.SssI DNA methylase, which has been reported to modify completely and exclusively CpG sites in the sequence. The fragments generated by the digestion with methylation-insensitive restriction nuclease were then analyzed to identify the methylation levels by nano-ESI-MS, without liquid chromatography (LC) separation. By methylation-insensitive nuclease digestion, we divided the DNA strands into several fragments, and nano-ESI-MS enabled the accurate analysis of methylation levels in the DNA fragments with a relatively small amount of DNA sample prepared under optimized conditions. Furthermore, it was revealed that M.SssI methylase hardly modifies the CpG sites closely positioned at the ends of linear DNA. The present method is similar to the strategy for post-translational modification analysis of proteins and is promising for the rapid and definitive characterization of methylated DNA that may be used in structural biology studies. [ABSTRACT FROM AUTHOR]

The Developmental Origins of Health and Disease (DOHaD) hypothesis posits that in utero and early life conditions can disrupt normal fetal development and program susceptibility to later-life disease. Metastable epialleles are genomic loci in which CpG methylation patterning is responsive to maternal diet and conserved across time and tissues. Thus, these sites could serve as 'signatures' of gestational environment conditions. Here, we sought to determine if methylation of metastable epialleles was associated with changes in childhood body mass index (BMI) z-scores across ages one, two and ten in the Extremely Low Gestational Age Newborns (ELGAN) cohort. CpG methylation of 250 probes (corresponding to 111 genes) within metastable epiallele regions was measured in placental tissue. Linear mixed effects models were fit to evaluate the overall and sex-stratified associations between methylation and changes in BMI z-score over time. In total, 26 probes were associated (p < 0.05) with changes in BMI z-score overall, including probes within Mesoderm Specific Transcript (MEST) and Histone Deacetylase 4 (HDAC4), which have previously been associated with childhood obesity and adipogenesis. Sex-stratified analyses revealed a significant association, after adjusting for multiple comparisons (q < 0.05), within female placentas for one probe annotated to the imprinted gene PLAG1 Like Zinc Finger 1 (PLAGL1). These findings suggest epigenetic marks may be involved in programming susceptibility to obesity in utero and highlight the potential to use placental tissues in predicting growth rate trajectories among premature infants. [ABSTRACT FROM AUTHOR]

Ginsenoside Rh2, a major bioactive ingredient abundant in red ginseng, has an antiproliferative effect on various cancer cells. In this study, we report a novel long noncoding RNA, C3orf67-AS1, which was identified as being hypermethylated at a CpG site of the promoter by Rh2 in MCF-7 cancer cells. Rh2-induced hypermethylation was responsible for the lower gene expression; the expression was recovered following treatment with a methyltransferase inhibitor, 5-aza-2′-deoxycytidine. When C3orf67-AS1 was downregulated by a siRNA, the cell growth rate was decreased, demonstrating the RNA's oncogenic activity. Accordingly, breast cancer patients showed a lower methylation and higher expression level of C3orf67-AS1. Within 800 kb flanking C3orf67-AS1 on the chromosome, eight genes were found, and four genes including C3orf67 (the sense strand gene of C3orf67-AS1) were downregulated by Rh2. In particular, C3orf67 was downregulated when C3orf67-AS1 was suppressed by a siRNA; however, the expression of C3orf67-AS1 was not affected by C3orf67. Taken together, this study identifies a novel noncoding RNA, C3orf67-AS1, of which the expression could be suppressed by Rh2 via promoter methylation, thereby mediating the anti-proliferative effect of the ginsenoside. [ABSTRACT FROM AUTHOR]

The biological function of the aromatase gene (cyp19a) is to transform testosterone into estradiol, which is involved in ovarian differentiation and development. In the present study, in order to explore the role of cyp19a in sex differentiation of Culter alburnus, the full-length cDNA sequence was obtained, and the relationship between promoter CpG methylation and gene expression level of cyp19a during gonad development was analyzed. Our results showed that the C. alburnus cyp19a was 2009 bp in length, including a 364 bp 5′-UTR, a 91 bp 3′-UTR, and a 1554 bp open reading frame encoding 517 amino acids. Multi-alignment of CYP19a amino acid sequences in different vertebrates revealed that C. alburnus had high identity with Cyprinus carpio and Danio rerio, followed by Oryzias laticeps, Monopterus albus, and Cynoglossus semilaevis, and had close to 50% homology with Xenopus laevis, Trachemys scripta, Homo sapiens, and Mus musculus. The results of this phylogenetic tree were consistent with those arrived at by traditional morphology and biochemistry, revealing a similar evolutionary status. qRT-PCR experiments showed that cyp19a was only highly expressed in the ovary, was less abundant in the brain, and almost undetectable in other tissues. Moreover, sodium bisulfite genome sequencing found that promoter CpGs of cyp19a was hypermethylated in the testis, while a few scattered CGs were methylated in the ovary, which showed an inverse relationship between methylation level and gene expression. These findings demonstrate that C. alburnus cyp19a is a sex-related gene with sexual dimorphic expression, and epigenetic modifications may play an important role during gonad differentiation. [ABSTRACT FROM AUTHOR]

Supplemental Digital Content is available in the text. Background: The adult mammalian cardiomyocytes lose their proliferative capacity, which is responsible for cardiac dysfunction and heart failure following injury. The molecular mechanisms underlying the attenuation of adult cardiomyocyte proliferation remain largely unknown. Because long noncoding RNAs (lncRNAs) have a critical role in the development of cardiovascular problems, we investigated whether lncRNAs have any role in the regulation of cardiomyocyte proliferation and cardiac repair. Methods: Using bioinformatics and initial analysis, we identified an lncRNA, named CPR (cardiomyocyte proliferation regulator), that has a potential regulatory role in cardiomyocyte proliferation. For in vivo experiments, we generated CPR knockout and cardiac-specific CPR-overexpressing mice. In isolated cardiomyocytes, we used adenovirus for silencing (CPR–small interfering RNA) or overexpressing CPR. To investigate the mechanisms of CPR function in cardiomyocyte proliferation, we performed various analyses including quantitative reverse transcription–polymerase chain reaction, Western blot, histology, cardiac function (by echocardiography), transcriptome analyses (microarray assay), RNA pull-down assay, and chromatin immunoprecipitation assay. Results: CPR level is comparatively higher in the adult heart than in the fetal stage. The silencing of CPR significantly increased cardiomyocyte proliferation in postnatal and adult hearts. Moreover, CPR deletion restored the heart function after myocardial injury, which was evident from increased cardiomyocyte proliferation, improvement of myocardial function, and reduced scar formation. In contrast, the neonatal cardiomyocyte proliferation and cardiac regeneration were remarkably suppressed in CPR-overexpressing mice or adeno-associated virus serotype 9–CPR—overexpressing heart. These results indicate that CPR acts as a negative regulator of cardiomyocyte proliferation and regeneration. Next, we found that CPR targets minichromosome maintenance 3, an initiator of DNA replication and cell cycle progression, to suppress cardiomyocyte proliferation. CPR silenced minichromosome maintenance 3 expression through directly interacting and recruiting DNMT3A to its promoter cysteine-phosphate-guanine sites, as evident from decreased minichromosome maintenance 3 promoter methylation and increased minichromosome maintenance 3 expression in CPR knocked-down cardiomyocytes and CPR knockout mouse heart. These results were confirmed in CPR-overexpressing cardiomyocytes and CPR-overexpressing mouse heart. Conclusions: Together, our findings identified that CPR is a suppressor of cardiomyocyte proliferation and indicated that lncRNAs take part in the regulation of cardiomyocyte proliferation and cardiac repair. Our study provides an lncRNA-based therapeutic strategy for effective cardiac repair and regeneration. [ABSTRACT FROM AUTHOR]

Abstract In eukaryotes, cytosine methylation of nuclear DNA at CpG sequences (5mCpG) regulates epigenetic inheritance through alterations in chromatin structure. However, mitochondria lack nucleosomal chromatin, therefore the molecular mechanisms by which 5mCpG influences mitochondria must be different and are as yet unknown. Mitochondrial Transcription Factor A (TFAM) is both the primary DNA-compacting protein in the mitochondrial DNA (mtDNA) nucleoid and a transcription-initiation factor. TFAM must encounter hundreds of CpGs in mtDNA, so the occurrence of 5mCpG has the potential to impact TFAM-DNA recognition. We used biophysical approaches to determine whether 5mCpG alters any TFAM-dependent activities. 5mCpG in the heavy strand promoter (HSP1) increased the binding affinity of TFAM and induced TFAM multimerization with increased cooperativity compared to nonmethylated DNA. However, 5mCpG had no apparent effect on TFAM-dependent DNA compaction. Additionally, 5mCpG had a clear and context-dependent effect on transcription initiating from the three mitochondrial promoters. Taken together, our findings demonstrate that 5mCpG in the mitochondrial promoter region does impact TFAM-dependent activities in vitro. Highlights • Methylation of specific mitochondrial DNA increases the binding affinity of TFAM. • Multimerization of TFAM on site-specific promoter DNA is enhanced by methylation. • Effect of DNA methylation on transcription initiation is promoter context dependent. [ABSTRACT FROM AUTHOR]

Cadmium, a ubiquitous heavy metal, is a toxic industrial and environmental pollutant. The initial biological response to cadmium exposure is induction of metallothioneins (MTs), a family of cysteine-rich, low-molecular-weight proteins that bind primarily zinc, cadmium, or both. This MT induction protects against cadmium toxicity by quenching cadmium. However, the effects of long-term cadmium exposure on MT1 gene expression are largely unknown. To investigate these effects, we used P1798 mouse lymphosarcoma cells, in which the MT1 gene is suppressed. As previously reported, MT1 expression remained unchanged after cadmium treatment. However, MT1 induction was observed in cells treated with 0.1 µM cadmium for 7 days, then exposed to 10 µM cadmium for 3 hr. In cells treated with 0.1 µM cadmium for 7 days, the transfected MT1 promoter reporter gene transcription and the cadmium incorporation in response to 10 µM cadmium induction were similar to those in untreated P1798 cells. Bisulfite genomic sequencing revealed that 7 day treatment with 0.1 µM cadmium slightly decreased CpG methylation in the 5' flanking region of the MT1 gene. Our results together show that cadmium treatment results in MT1 induction and epigenetic modification of the MT1 gene. [ABSTRACT FROM AUTHOR]

Poor homing of cells after transplantation is an unresolved common issue in cardiac cell therapies. To enhance stem cell homing, the ligand CXC motif chemokine 12 (CXCL12) and its specific receptor CXC receptor type 4 (CXCR4) have been employed as a system in this study to show that induced expression of the endogenous CXCR4 gene in mouse-induced pluripotent stem cells (iPSCs) improved the cell migration. Loci-specific epigenome editing in the form of CpG demethylation at CXCR4 promoter region of the mouse iPSCs was accomplished with CXCR4b-TAL-Tet1c, chimeric fusion proteins of the catalytic domain of ten-eleven translocation 1 (TET1) to the C-terminal end of the DNA binding domains of predesigned synthetic transcription activator-like effectors (TALEs) that recognize specific DNA sequences within the mouse CXCR4 promoter region. Infection of the mouse iPSCs with the engineered CXCR4b-TAL-Tet1c in the form of lentiviral particles induced the loci-specific CpG demethylation and subsequent activation of CXCR4 expression in mouse iPSCs. As expected, the CXCR4-overexpressing iPSCs exhibited 3.9-fold greater migration than the control iPSCs did without alteration of the stemness and activated phosphorylation of AKT significantly. These results set a sound foundation for subsequent in vivo iPSCs transplantation studies in rodent models of acute myocardial infarction and heart failure. We show that TALEs can enhance the expression of CXCR4 by CpG methylation, and may retain the stemness. Migration of iPSCs activated by CXCL12 is associated with significant phosphorylation of AKT, not ERK1/2. [ABSTRACT FROM AUTHOR]

Analysis of allele-specific expression may help to elucidate the genetic architecture of complex traits including fat deposition in pigs. Here, we used pyrosequencing to investigate the allele proportions of candidate genes (ACACA, ADIPOR1, FASN, LEP, ME1, SCD, and TNF) involved in regulation of lipid metabolism in two fat deposits (subcutaneous and visceral fat) and longissimus dorsi muscle of pigs representing Polish Large White, Polish Landrace, Duroc, and Pietrain breeds. We detected differential allelic expression of ACACA, LEP, SCD, and TNF in all tissues analyzed. To search for putative cis-regulatory elements involved in allele-specific expression, we quantified the methylation level within CpG islands located in 5′-flanking regions of ACACA and SCD. Comparison between samples showing markedly disproportionate allelic expression and control groups with similar levels of both alleles did not reveal significant differences. We also assessed the association of rs321308225 (c.*195C>A) an SNP located in the 3′UTR of ACACA with its allelic expression in Polish Landrace pigs, but it was not significant. We conclude that allelic imbalance occurs frequently in regard to genes involved in regulation of lipid deposition in pigs, and further studies are necessary to identify cis-regulatory elements affecting ACACA, LEP, SCD, and TNF expression in porcine fat tissues and skeletal muscle. [ABSTRACT FROM AUTHOR]

Dmrt1, a member of the Dmrt family, is an important transcription regulator of gender determination. To study the biological function of dmrt1 in sexual differentiation and its potential implication in breeding technology, we obtained the full-length cDNA and proximal promoter sequence of dmrt1 in Culter alburnus, and analyzed the impact of promoter CpG methylation on the gene expression pattern of dmrt1 during gonad development. Dmrt1 was 922 bp in length and consisted a 150 bp 5′-UTR, a 28 bp 3′-UTR, and a 744 bp open reading frame (ORF). Based on the coding sequence of the dmrt1 gene, the deduced amino acid sequence was detected, and the protein structure of this gene was predicted in C. alburnus. The results indicate that the structure and function of dmrt1 were highly conservative compared to other vertebrates. The expression level of dmrt1 mRNA in different tissues was explored by qRT-PCR, which was only highly expressed in the testes and almost undetectable in other tissues. The CpG methylation pattern of the dmrt1 promoter was studied using DNA sequencing of sodium bisulfite in adult testes and ovaries, and it was found that dmrt1 promoter CpGs were not methylated in the testes, whereas hypermethylated in the ovaries. These findings demonstrate that DNA methylation can regulate sexual dimorphic expression of dmrt1, and therefore epigenetic modifications may play a critical role in the gonad differentiation of C. alburnus. [ABSTRACT FROM AUTHOR]

Background/Aims: Interferon consensus sequence-binding protein 8 (IRF8) belongs to a family of interferon (IFN) regulatory factors that modulates various important physiological processes including carcinogenesis. As reported by others and our group, IRF8 expression is silenced by DNA methylation in both human solid tumors and hematological malignancies. However, the role of IRF8 in lung carcinoma remains elusive. In this study, we determined IRF8 epigenetic regulation, biological functions, and the signaling pathway involved in non-small cell lung cancer (NSCLC). Methods: IRF8 expression were determined by Q- PCR. MSP and A+T determined promotor methylation. MTS, clonogenic, Transwell assay, Flow cytometry, three-dimensional culture and AO/EB stain verified cell function. In vivo tumorigenesis examed the in vivo effects. By Chip-QPCR, RT-PCR, Western blot and Immunofluorescence staining, the mechanisms were studied. Results: IRF8 was significantly downregulated in lung tumor tissues compared with adjacent non-cancerous tissues. Furthermore, methylation-specific PCR analyses revealed that IRF8 methylation in NSCLC was a common event, and demethylation reagent treatment proved that downregulation of IRF8 was due to its promoter CpG hypermethylation. Clinical data showed that the IRF8 methylation was associated with tumor stage, lymph node metastasis status, patient outcome, and tumor histology. Exogenous expression of IRF8 in the silenced or downregulated lung cancer cell lines A549 and H1299 at least partially restored the sensitivity of lung cancer cells to apoptosis, and arrested cells at the G0/G1 phase. Cell viability, clonogenicity, and cell migration and invasive abilities were strongly inhibited by restored expression of IRF8. A three-dimensional culture system demonstrated that IRF8 changed the cells to a more spherical phenotype. Moreover, ectopic expression of IRF8 enhanced NSCLC chemosensitivity to cisplatin. Furthermore, as verified by Chip-qPCR, immunofluorescence staining, and western blotting, IRF8 bound to the T-cell factor/lymphoid enhancer factor (TCF /LEF) promoter, thus repressing β-catenin nuclear translocation and its activation. IRF8 significantly disrupted the effects of Wnt agonist, bml284, further suggesting its involvement in the Wnt/β-catenin pathway. Conclusion: IRF8 acted as a tumor suppressor gene through the transcriptional repression of β-catenin-TCF/LEF in NSCLC. IRF8 methylation may serve as a potential biomarker in NSCLC prognosis. [ABSTRACT FROM AUTHOR]

Ginsenoside Rg3 exerts antiproliferation activity on cancer cells by regulating diverse noncoding RNAs. However, little is known about the role of long noncoding RNAs (lncRNAs) or their relationship with competitive endogenous RNA (ceRNA) in Rg3-treated cancer cells. Here, a lncRNA (ATXN8OS) was found to be downregulated via Rg3-mediated promoter hypermethylation in MCF-7 breast cancer cells. SiRNA-induced downregulation of ATXN8OS decreased cell proliferation but increased apoptosis, suggesting that the noncoding RNA possessed proproliferation activity. An in silico search for potential ATXN8OS-targeting microRNAs (miRs) identified a promising candidate (miR-424-5p) based on its high binding score. As expected, miR-424-5p suppressed proliferation and stimulated apoptosis of the MCF-7 cells. The in silico miR-target-gene prediction identified 200 potential target genes of miR-424-5p, which were subsequently narrowed down to seven that underwent hypermethylation at their promoter by Rg3. Among them, three genes (EYA1, DACH1, and CHRM3) were previously known oncogenes and were proven to be oppositely regulated by ATXN8OS and miR-424-5p. When taken together, Rg3 downregulated ATXN8OS that inhibited the tumor-suppressive miR-424-5p, leading to the downregulation of the oncogenic target genes.

Two human endogenous retroviruses of the HERV-W family can act as cofactors triggering multiple sclerosis (MS): MS-associated retrovirus (MSRV) and ERVWE1. Endogenous retroviral elements are believed to have integrated in our ancestors’ DNA millions of years ago. Their involvement in the pathogenesis of various diseases, including neurodegenerative pathologies, has been demonstrated. Numerous studies have shown a correlation between the deterioration of patients’ health and increased expression of endogenous retroviruses. The exact causes and mechanisms of endogenous retroviruses activation remains unknown, which hampers development of therapeutics. In this review, we will summarize the main characteristics of human endogenous W retroviruses and describe the putative mechanisms of activation, including epigenetic mechanisms, humoral factors as well as the role of the exogenous viral infections.

Cytosine-C5 methyltransferases (MTases) maintain methylation status in CpG islands (CpGIs), and their abnormal activities are implicated in various diseases and cancers. Simultaneous monitoring of multiple cytosine-C5 MTases has remained challenging due to diversity of DNA substrates and scarcity of methylation-independent restriction endonucleases (MIREs). Herein, we construct a CpG methylation-powered dynamic three-dimensional-DNAzyme walker for single-molecule monitoring of multiple cytosine-C5 MTases. The presence of M.SssI and M.CviPI catalyzes cytosines-C5 methylation in hairpin probes to form catalytic substrates of GlaI. Then GlaI specifically cleaves 5-mCs to yield two metal-specific DNAzymes. Upon the addition of substrate probes (SP1/SP2)-functionalized AuNPs, two DNAzymes selectively bind with SP1 and SP2, and autonomously complete 3D-DNAzymes walking with assistance of cofactors Mg2+ and Zn2+, inducing cyclic cleavage of substrate probes and dissociation of abundant Cy5 and Cy3 from AuNPs. Cy5 and Cy3 are simply measured through single-molecule imaging, with Cy5 indicating M.SssI and Cy3 indicating M.CviPI. This nanosensor achieves a limit of detection (LOD) of 3.944 × 10-9 U/μL for M.SssI and 5.046 × 10-9 U/μL for M.CviPI. It can discriminate cytosine-C5 MTases members, screen potential inhibitors, and quantify multiple cytosine-C5 MTases in human serum and diverse cell lysates, providing a powerful platform for biomedical research and clinical diagnostics. • We develop a CpG methylation-powered dynamic 3D-DNAzyme walker. • This nanosensor enables single-molecule monitoring of multiple cytosine-C5 MTases. • This nanosensor exhibits good specificity and high sensitivity. • This nanosensor can detect cytosine-C5 MTases in human serum and cell lysates. • This nanosensor can screen inhibitors. [ABSTRACT FROM AUTHOR]

Background: Prenatal exposure to inorganic arsenic (iAs) is associated with dysregulated fetal gene and protein expression. Potential biological mechanisms that underlie these changes include, but are not limited to, changes to the epigenome. Objective: The aim of the present study was to identify whether the expression of key genes, proteins, or both and their associated biological pathways are perturbed by compiling datasets from studies on prenatal arsenic exposure. Methods: We compiled datasets from 12 studies that analyzed the relationship between prenatal iAs exposure and changes to the fetal epigenome (5-methyl cytosine), transcriptome (mRNA expression), and/or proteome (protein expression). Findings: Across the 12 studies, a set of 845 unique genes was identified and found to enrich for their role in biological pathways, including the peroxisome proliferator-activated receptor, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, and the glucocorticoid receptor. Tumor necrosis factor was identified as a putative cellular regulator underlying most (n = 277) of the identified iAs-associated gene or protein expression changes. Conclusions: The identification of the common set of genes across numerous human cohorts suggests a conserved biological response to prenatal arsenic exposure. The genes/proteins and their associated pathways may be useful in future mechanistic investigations of iAs associated diseases.

Oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) is a method in which PCR amplification of a target sequence is inhibited in a sequence-specific manner by the hybridization of an ORN with the target sequence. Previously, we reported that ORNi-PCR could detect nucleotide mutations in DNA purified from cultured cancer cell lines or genome-edited cells. In this study, we investigated whether ORNi-PCR can discriminate nucleotide differences and CpG methylation status in damaged DNA, such as tissue specimen DNA and bisulfite-treated DNA. First, we showed that ORNi-PCR could discriminate nucleotide differences in DNA extracted from acetone-fixed paraffin-embedded rat liver specimens or formalin-fixed paraffin-embedded human specimens. Rat whole blood specimens were compatible with ORNi-PCR for the same purpose. Next, we showed that ORNi-PCR could discriminate CpG methylation status in bisulfite-treated DNA. These results demonstrate that ORNi-PCR can discriminate nucleotide differences and CpG methylation status in multiple types of DNA samples. Thus, ORNi-PCR is potentially useful in a wide range of fields, including molecular biology and medical diagnosis.

miR-218, consisting of miR-218-1 at 4p15.31 and miR-218-2 at 5q35.1, was significantly decreased in esophageal squamous cell carcinoma (ESCC) in our previous study. The aim of this study was to determine whether aberrant methylation is associated with miR-218 repression. Bisulfite sequencing analysis (BSP), methylation specific PCR (MSP), and 5-aza-2′-deoxycytidine treatment assay were applied to determine the methyaltion status of miR-218 in cells and clinical samples. In vitro assays were performed to explore the role of miR-218. Results showed that miR-218-1 was significantly CpG hypermethylated in tumor tissues (81%, 34/42) compared with paired non-tumor tissues (33%, 14/42) (p < 0.05). However, no statistical difference was found in miR-218-2. Accordingly, expression of miR-218 was negatively correlated with miR-218-1 methylation status (p < 0.05). After demethylation treatment by 5-aza-2′-deoxycytidine, there was a 2.53- and 2.40-fold increase of miR-218 expression in EC109 and EC9706, respectively. miR-218 suppressed cell proliferation and arrested cells at G1 phase by targeting 3′ untranslated region (3′UTR) of roundabout guidance receptor 1 (ROBO1). A negative correlation was found between miR-218 and ROBO1 mRNA expression in clinical samples. In conclusion, our results support that aberrant CpG hypermethylation at least partly accounts for miR-218 silencing in ESCC, which impairs its tumor-suppressive function.

DNA methyltransferases (MTases) catalyze the transfer of the activated methyl group of the cofactor S-adenosyl-l-methionine (AdoMet or SAM) to the exocyclic amino groups of adenine or cytosine or the C5 ring atom of cytosine within specific DNA sequences. The DNA adenine-N6 MTase from Thermus aquaticus (M.TaqI) is also capable of coupling synthetic N-adenosylaziridine cofactor analogues to its target adenine within the double-stranded 5′-TCGA-3′ sequence. This M.TaqI-mediated coupling reaction was exploited to sequence-specifically deliver fluorophores and biotin to DNA using N-adenosylaziridine derivatives carrying reporter groups at the 8-position of the adenine ring. However, these 8-modified aziridine cofactors were poor substrates for the DNA cytosine-C5 MTase from Haemophilus haemolyticus (M.HhaI). Based on the crystal structure of M.HhaI in complex with a duplex oligodeoxynucleotide and the cofactor product, we synthesized a stable 7-deazaadenosylaziridine derivative with a biotin group attached to the 7-position via a flexible linker. This 7-modified aziridine cofactor can be efficiently used by M.HhaI for the direct, quantitative and sequence-specific delivery of biotin to the second cytosine within 5′-GCGC-3′ sequences in short duplex oligodeoxynucleotides and plasmid DNA. In addition, we demonstrate that biotinylation by M.HhaI depends on the methylation status of the target cytosine and, thus, could provide a method for cytosine-C5 DNA methylation detection in mammalian DNA.

DNA methylation is a critical epigenetic regulator in the occurrence and development of diseases and is closely related to various functional responses in relation to spinal cord injury. To investigate the role of DNA methylation in spinal cord injury, we constructed a library with reduced-representation bisulfite sequencing data obtained at various time points (day 0-42) after spinal cord injury in mice. Global DNA methylation levels, specifically non-CpG (CHG and CHH) methylation levels, decreased modestly following spinal cord injury. Stages post-spinal cord injury were classified as early (day 0-3), intermediate (day 7-14), and late (day 28-42) based on similarity and hierarchical clustering of global DNA methylation patterns. The non-CpG methylation level, which included CHG and CHH methylation levels, was markedly reduced despite accounting for a minor proportion of total methylation abundance. At multiple genomic sites, including the 5' untranslated regions, promoter, exon, intron, and 3' untranslated regions, the non-CpG methylation level was markedly decreased following spinal cord injury, whereas the CpG methylation level remained unchanged at these locations. Approximately one-half of the differentially methylated regions were located in intergenic areas; the other differentially methylated regions in both CpG and non-CpG regions were clustered in intron regions, where the DNA methylation level was highest. The function of genes associated with differentially methylated regions in promoter regions was also investigated. From Gene Ontology analysis results, DNA methylation was implicated in a number of essential functional responses to spinal cord injury, including neuronal synaptic connection creation and axon regeneration. Notably, neither CpG methylation nor non-CpG methylation was implicated in the functional response of glial or inflammatory cells. In summary, our work elucidated the dynamic pattern of DNA methylation in the spinal cord following injury and identified reduced non-CpG methylation as an epigenetic target after spinal cord injury in mice., Competing Interests: None

Melanoma is among the most aggressive and treatment-resistant human cancers. Aberrant histone H3 methylation at Lys 9 (H3K9) correlates with carcinogenic gene silencing, but the significance of suppressor of variegation 3-9 homolog 1 (SUV39H1), an H3K9-specific methyltransferase, in melanoma initiation and progression remains unclear. Here, we show that SUV39H1-mediated H3K9 trimethylation facilitates retinoblastoma (RB)1 promoter CpG island methylation by interacting with DNA methyltransferase 3A and decreasing RB mRNA and protein in melanoma cells. Reduced RB abundance, in turn, impairs E2F1 transcriptional inhibition, leading to increased peptidyl-prolyl cis-trans isomerase never-in-mitosis A (NIMA)-interacting 1 (PIN1) levels, human keratinocyte neoplastic cell transformation, and melanoma tumorigenesis via enhanced rapidly accelerated fibrosarcoma 1(RAF1)-MEK-ERK signaling pathway activation. In a synergistic model with B16-F1 murine melanoma cells, SUV39H1 and PIN1 overexpression increased melanoma growth, which was abrogated by their inhibition in SUV39H1-overexpressing B16-F1 mice. SUV39H1 also positively correlated with PIN1 expression in human melanoma. Our studies establish SUV39H1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis. [ABSTRACT FROM AUTHOR]

Summary Genes in higher eukaryotes are regulated by long-range interactions, which can determine what combination of genes is expressed in a chromosomal segment. The choice of the genes can display exclusivity, independence, or co-occurrence. We introduced a simple measure to quantify this interdependence in gene expression and differentiated mouse embryonic stem cells to neurons to measure the single-cell expression of the gene isoforms in the protocadherin (Pcdh) cluster, a key component of neuronal diversity. As the neuronal progenitors mature into neurons, expression of the gene isoforms in the Pcdh array is initially concurrent. Even though the number of the expressed genes is increasing during differentiation, the expression shifts toward exclusivity. The expression frequency correlates highly with CTCF binding to the promoters and follows dynamically the changes in the binding during the differentiation. These findings aid in understanding the interplay between cellular differentiation and stochastic gene choice. Graphical Abstract Highlights • The protocadherin array was analyzed in single cells during neural differentiation • The expression of genes in an array can be exclusive, independent, or concurrent • Gene choice pattern shifts from co-occurrence toward exclusivity as neurons mature • The shift of gene choice pattern coincides with the rearrangement of CTCF binding Wada et al. devised an approach to quantitatively characterize possible gene choice patterns in gene arrays: exclusivity, independence, or co-occurrence. Single-cell analysis of the protocadherin gene isoforms revealed that the isoforms are expressed concurrently early during the differentiation of neuronal progenitor cells to neurons, which shifts to exclusivity later. [ABSTRACT FROM AUTHOR]

Background DNA methylation at the 5-position of cytosine is an epigenetic modification of CpG dinucleotides. In addition to CpG methylation, the G-quadruplex (G4) structure has been reported as a regulator of gene expression. The identification of G4 forming sequences in CpG islands suggests an involvement of CpG-methylated G4 structures in biological processes; however, few reports have addressed the effects of CpG methylation on G4 structure. Methods The thermostability of a methylated, 21-mer G4 structure located on the vascular endothelial growth factor ( VEGF ) gene promoter containing four CpG sites (C1, C6, C11, and C17) were investigated using circular dichroism (CD) spectral analysis. Results CD melting analysis revealed that VEGF G4 was stabilized by a single CpG methylation on C11 in the presence of Na + and Mg 2+ . However, either C1 or C11 methylation enhanced VEGF G4 thermal stability in the presence of K + . Conclusions Single CpG methylation appears to enhance VEGF G4 thermostability in a manner dependent on both the CpG methylation site and cation type. General significance These results are expected to contribute to the elucidation of the roles of CpG methylation-stabilized G4 structures in biological processes. [ABSTRACT FROM AUTHOR]

Aim: Klotho expression significantly declines in alveolar macrophages and airway epithelial cells in chronic obstructive pulmonary disease (COPD) patients, and cigarette smoke extract dramatically inhibits the expression and secretion of α-Klotho. This suggests that the silencing of Klotho is the major factor promoting COPD related inflammatory responses. This study aims to investigate the mechanism of Klotho downregulation and its effect on the inflammatory cytokines secretion and cell apoptosis. Methods: Expression of DNA methyltransferases (DNMTs) and Notch signaling activation were quantified in MH-S and 16HBE cells stimulated with cigarette smoke extract (CSE) solution. Specific inhibitors of DNMTs or Notch pathway were added together with CSE into treated and control cells. Inflammatory cytokines, cell viability and cell death were determined to explore the effect of Klotho on COPD related inflammation. Results: CSE treatment statistically increased the level of DNMTs expression, Klotho promoter methylation, and activated the Notch signaling pathway. Notch signal activation played a critical role in the process of modification of Klotho promoter methylation. The inhibition of DNMTs and Notch pathway rescued Klotho levels and inhibited inflammation and cell apoptosis after CSE treatment. Conclusion: Notch-mediated Klotho hypermethylation inhibited Klotho expression, which promoted inflammatory response and cell apoptosis that were associated with the development of COPD. [ABSTRACT FROM AUTHOR]

Arabidopsis ASYMMETRIC LEAVES2 (AS2) plays a critical role in leaf adaxial-abaxial partitioning by repressing expression of the abaxial-determining gene ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3). We previously reported that six CpG dinucleotides in its exon 6 are thoroughly methylated by METHYLTRASFERASE1, that CpG methylation levels are inversely correlated with ETT/ARF3 transcript levels and that methylation levels at three out of the six CpG dinucleotides are decreased in as2-1. All these imply that AS2 is involved in epigenetic repression of ETT/ARF3 by gene body DNA methylation. The mechanism of the epigenetic repression by AS2, however, is unknown. Here, we tested mutations of NUCLEOLIN1 (NUC1) and RNA HELICASE10 (RH10) encoding nucleolus-localized proteins for the methylation in exon 6 as these mutations enhance the level of ETT/ARF3 transcripts in as2-1. Methylation levels at three specific CpGs were decreased in rh10-1, and two of those three overlapped with those in as2-1. Methylation levels at two specific CpGs were decreased in nuc1-1, and one of those three overlapped with that in as2-1. No site was affected by both rh10-1 and nuc1-1. One specific CpG was unaffected by these mutations. These results imply that the way in which RH10, NUC1 and AS2 are involved in maintaining methylation at five CpGs in exon 6 might be through at least several independent pathways, which might interact with each other. Furthermore, we found that AS2 binds specifically the sequence containing CpGs in exon 1 of ETT/ARF3, and that the binding requires the zinc-finger-like motif in AS2 that is structurally similar to the zinc finger-CxxC domain in vertebrate DNA methyltransferase1. [ABSTRACT FROM AUTHOR]

Epigenetics confers adaptability and survival advantage to an organism. Most epigenetic processes demonstrate memory and heritability. DNA methylation is an epigenetic process that adds imprints which can be inherited during cell division and across generations. DNA methylation adds an additional level of information to the basic DNA sequence and can influence chromatin organization and the function of the DNA sequence. In bacteria, it works as a defence strategy and preserves genome integrity. DNA methylation in eukaryotes has been implicated in a large number of cellular regulatory processes and is implied in development, differentiation, life style diseases and cancer. Mammals have an intricate DNA methylation machinery with dNMT1, 3A and 3B enzymes. The human X chromosome inactivation, an example of differential regulation of homologous chromosomes, is known to involve many epigenetic processes with intricate interactions of lnc RNAs, miRNAs and DNA methylation. Drosophila possesses very low levels of DNA methylation with only dNMT2 gene. Since Drosophila is an important model organism for study of development and differentiation, the implications of this sparse DNA methylation and the lack of DNA methylation machinery in Drosophila is discussed. [ABSTRACT FROM AUTHOR]

IL2RA, a subunit of the high affinity receptor for interleukin-2 (IL2), plays a crucial role in immune homeostasis. Notably, IL2RA expression is induced in CD4+ T cells in response to various stimuli and is constitutive in regulatory T cells (Tregs). We selected for our study 18 CpGs located within cognate regulatory regions of the IL2RA locus and characterized their methylation in naive, regulatory, and memory CD4+ T cells. We found that 5/18 CpGs (notably CpG + 3502) show dynamic, active demethylation during the in vitro activation of naive CD4+ T cells. Demethylation of these CpGs correlates with appearance of IL2RA protein at the cell surface. We found no influence of cis located SNP alleles upon CpG methylation. Treg cells show constitutive demethylation at all studied CpGs. Methylation of 9/18 CpGs, including CpG +3502, decreases with age. Our data thus identify CpG +3502 and a few other CpGs at the IL2RA locus as coordinated epigenetic regulators of IL2RA expression in CD4+ T cells. This may contribute to unravel how the IL2RA locus can be involved in immune physiology and pathology. [ABSTRACT FROM AUTHOR]

Transposons are major drivers of mammalian genome evolution. To obtain new insights into the contribution of transposons to the regulation of protein translation, we here examined how transposons affected the genesis and function of upstream open reading frames (uORFs), which serve as cis‐acting elements to regulate translation from annotated ORFs (anORFs) located downstream of the uORFs in eukaryotic mRNAs. Among 39,786 human uORFs, 3,992 had ATG trinucleotides of a transposon origin, termed “transposon‐derived upstream ATGs” or TuATGs. Luciferase reporter assays suggested that many TuATGs modulate translation from anORFs. Comparisons with transposon consensus sequences revealed that most TuATGs were generated by nucleotide substitutions in non‐ATG trinucleotides of integrated transposons. Among these non‐ATG trinucleotides, GTG and ACG were converted into TuATGs more frequently, indicating a CpG methylation‐mediated process of TuATG formation. Interestingly, it is likely that this process accelerated human‐specific upstream ATG formation within transposon sequences in 5′ untranslated regions after divergence between human and nonhuman primates. Methylation‐mediated TuATG formation seems to be ongoing in the modern human population and could alter the expression of disease‐related proteins. This study shows that transposons have potentially been shaping the human proteome landscape via cis‐acting uORF creation. [ABSTRACT FROM AUTHOR]

Interferon (IFN)-γ-inducing interleukin (IL)-18 is a crucial inflammatory cytokine systemically provided by monocytes. It is counteracted by IL-18 binding protein (IL-18BP), a decoy receptor that displays IFNγ-inducibility thus curbing inflammation by negative feedback. Since IL18BP inducibility is pronounced in human epithelial cells but diminished in monocytes, differential IL18BP regulation was investigated herein in both types of cells. Interestingly, DNA-demethylating 5-aza-2′-deoxycytidine enhanced IFNγ-induced IL-18BP only in monocytic but not in epithelial cells. Subsequent promoter analysis brought into focus a specific CpG (coined CpG2) neighboring a γ-activated site responsible for IL18BP induction. CpG2 was consistently methylated in monocytic but unmethylated in epithelial cells. Notably, demethylation by 5-aza-2′-deoxycytidine treatment of monocytic cells impeded methyl-CpG-binding protein-2 (MeCP2) interaction with CpG2, increased adjacent histone H3K9-acetylation, and enhanced RNA-polymerase-II recruitment to the nearby IL18BP transcriptional start. Both latter observations are indicative of a gene locus displaying augmented transcriptional activity. Data suggest that epigenetic silencing by single CpG methylation determines differential IL18BP inducibility in monocytic versus epithelial cells. This regulatory principle should serve and control pivotal IL-18-related cell type-specific (patho)-physiological functions. Whereas epithelial IL-18BP evidently counteracts pathological inflammation at biological barriers, retarded IL18BP inducibility in monocytes may be key to combat blood-borne infections in IL-18-dependent manner. [ABSTRACT FROM AUTHOR]

(1) Background: TP53 deficiency remains a major adverse event in Multiple Myeloma (MM) despite therapeutic progresses. As it is not possible to target TP53 deficiency with pharmacological agents, we explored the possibility of activating another p53 family member, p73, which has not been well studied in myeloma. (2) Methods: Using human myeloma cell lines (HMCLs) with normal or abnormal TP53 status, we assessed TP73 methylation and expression. (3) Results: Using microarray data, we reported that TP73 is weakly expressed in 47 HMCLs and mostly in TP53 wild type (TP53wt) HMCLs (p = 0.0029). Q-RT-PCR assays showed that TP73 was expressed in 57% of TP53wt HMCLs (4 out of 7) and 11% of TP53 abnormal (TP53abn) HMCLs (2 out of 18) (p = 0.0463). We showed that TP73 is silenced by methylation in TP53abn HMCLs and that decitabine increased its expression, which, however, remained insufficient for significant protein expression. Alkylating drugs increased expression of TP73 only in TP53wt HMCLs but failed to synergize with decitabine in TP53abn HMCLs. (4) Conclusions: Decitabine and melphalan does not appear as a promising combination for inducing p73 and bypassing p53 deficiency in myeloma cells. [ABSTRACT FROM AUTHOR]

Hypoxia-dependent accumulation of vascular endothelial growth factor (VEGF) plays a major role in retinal diseases characterized by neovessel formation. In this study, we investigated whether the glial water channel Aquaporin-4 (AQP4) is involved in the hypoxia-dependent VEGF upregulation in the retina of a mouse model of oxygen-induced retinopathy (OIR). The expression levels of VEGF, the hypoxia-inducible factor-1α (HIF-1α) and the inducible form of nitric oxide synthase (iNOS), the production of nitric oxide (NO), the methylation status of the HIF-1 binding site (HBS) in the VEGF gene promoter, the binding of HIF-1α to the HBS, the retinal vascularization and function have been determined in the retina of wild-type (WT) and AQP4 knock out (KO) mice under hypoxic (OIR) or normoxic conditions. In response to 5 days of hypoxia, WT mice were characterized by ( i) AQP4 upregulation, ( ii) increased levels of VEGF, HIF-1α, iNOS and NO, ( iii) pathological angiogenesis as determined by engorged retinal tufts and ( iv) dysfunctional electroretinogram (ERG). AQP4 deletion prevents VEGF, iNOS and NO upregulation in response to hypoxia thus leading to reduced retinal damage although in the presence of high levels of HIF-1α. In AQP4 KO mice, HBS demethylation in response to the beginning of hypoxia is lower than in WT mice reducing the binding of HIF-1α to the VEGF gene promoter. We conclude that in the absence of AQP4, an impaired HBS demethylation prevents HIF-1 binding to the VEGF gene promoter and the relative VEGF transactivation, reducing the VEGF-induced retinal damage in response to hypoxia. [ABSTRACT FROM AUTHOR]