Analysis of Methylation and mRNA Expression of Lin28B Gene Promoter Region in the Hypothalamus of Dolang Sheep During Pubertal Initiation.

Lin28B plays an important role in puberty initiation in sheep. This study aimed to discuss the correlation between different growth periods and the methylation status of cytosine-guanine dinucleotide (CpG) islands in the promoter region of the Lin28B gene in the Dolang sheep's hypothalamus. In this study, the sequence of the Lin28B gene promoter region in Dolang sheep was obtained by cloning and sequencing, and methyl groups of the CpG island of the Lin28B gene promoter in the hypothalamus were detected by bisulfite sequencing PCR during the three periods of prepuberty, adolescence, and postpuberty in Dolang sheep. Lin28B expression in the Dolang sheep's hypothalamus was detected by fluorescence quantitative PCR at three stages: prepuberty, puberty, and postpuberty. In this experiment, the 2993-bp Lin28B promoter region was obtained, and it was predicted that there was a CpG island containing 15 transcription factor binding sites and 12 CpG sites, which may play a role in gene expression regulation. Overall, methylation levels increased from prepuberty to postpuberty, while Lin28B expression levels decreased, indicating that Lin28B expression was negatively correlated with promoter methylation levels. Variance analysis showed significant differences in the methylation status of CpG5, CpG7, and CpG9 between pre- and postpuberty (p < 0.05). Our data show that Lin28B expression is increased by demethylation of promoter CpG islands, with CpG5, CpG7, and CpG9 implicated as critical regulatory sites.

Loss of histone methyltransferase SETD1B in oogenesis results in the redistribution of genomic histone 3 lysine 4 trimethylation.

Histone 3 lysine 4 trimethylation (H3K4me3) is an epigenetic mark found at gene promoters and CpG islands. H3K4me3 is essential for mammalian development, yet mechanisms underlying its genomic targeting are poorly understood. H3K4me3 methyltransferases SETD1B and MLL2 (KMT2B) are essential for oogenesis. We investigated changes in H3K4me3 in Setd1b conditional knockout (cKO) oocytes using ultra-low input ChIP-seq, with comparisons to DNA methylation and gene expression analyses. H3K4me3 was redistributed in Setd1b cKO oocytes showing losses at active gene promoters associated with downregulated gene expression. Remarkably, many regions also gained H3K4me3, in particular those that were DNA hypomethylated, transcriptionally inactive and CpG-rich, which are hallmarks of MLL2 targets. Consequently, loss of SETD1B disrupts the balance between MLL2 and de novo DNA methyltransferases in determining the epigenetic landscape during oogenesis. Our work reveals two distinct, complementary mechanisms of genomic targeting of H3K4me3 in oogenesis, with SETD1B linked to gene expression and MLL2 to CpG content.

Combination of pre-adapted bacteriophage therapy and antibiotics for treatment of fracture-related infection due to pandrug-resistant Klebsiella pneumoniae.

A 30-year-old bombing victim with a fracture-related pandrug-resistant Klebsiella pneumoniae infection after long-term (>700 days) antibiotic therapy is treated with a pre-adapted bacteriophage along with meropenem and colistin, followed by ceftazidime/avibactam. This salvage therapy results in objective clinical, microbiological and radiological improvement of the patient's wounds and overall condition. In support, the bacteriophage and antibiotic combination is highly effective against the patient's K. pneumoniae strain in vitro, in 7-day mature biofilms and in suspensions.

Salt stress and salt shock differently affect DNA methylation in salt-responsive genes in sugar beet and its wild, halophytic ancestor.

Here we determined the impact of salt shock and salt stress on the level of DNA methylation in selected CpG islands localized in promoters or first exons of sixteen salt-responsive genes in beets. Two subspecies differing in salt tolerance were subjected for analysis, a moderately salt-tolerant sugar beet Beta vulgaris ssp. vulgaris cv. Huzar and a halophytic beet, Beta vulgaris ssp. maritima. The CpG island methylation status was determined. All target sequences were hyper- or hypomethylated under salt shock and/or salt stress in one or both beet subspecies. It was revealed that the genomic regions analyzed were highly methylated in both, the salt treated plants and untreated controls. Methylation of the target sequences changed in a salt-dependent manner, being affected by either one or both treatments. Under both shock and stress, the hypomethylation was a predominant response in sugar beet. In Beta vulgaris ssp. maritima, the hypermethylation occurred with higher frequency than hypomethylation, especially under salt stress and in the promoter-located CpG sites. Conversely, the hypomethylation of the promoter-located CpG sites predominated in sugar beet plants subjected to salt stress. This findings suggest that DNA methylation may be involved in salt-tolerance and transcriptomic response to salinity in beets.

IGF1 gene is epigenetically activated in preterm infants with intrauterine growth restriction.

BACKGROUND: IGF1 is a key molecule in the regulation of growth and metabolism. Low IGF1 secretion is known to cause growth restriction in childhood, as well as deregulated lipid metabolism, cardiovascular disease, and diabetes in adulthood. The IGF1 gene P2 promoter is highly methylated, resulting in low secretion of IGF1 in small infants and children. However, it is unknown when this methylation occurs. The aim of study was to clarify the point when this epigenetic program occurs during intrauterine development. We analyzed 56 preterm infants born before 32 weeks of gestation, including 19 intrauterine growth restriction (IUGR) infants whose birth weights were lower than - 2SD calculated by the Japanese datasets. We extracted genomic DNA from whole blood at birth; methylation of the six CpG sites in the IGF1 P2 promoter was analyzed by the bisulfite amplicon method using the MiSeq platform. RESULTS: In contrast to term infants and children, the methylation of all six CpG sites positively correlated with body weight and body length at birth. IGF1 P2 promoter methylation levels were significantly reduced in all six CpG sites in infants with IUGR. CONCLUSIONS: These findings indicated that the IGF1 gene is epigenetically activated before 32 weeks of gestation in infants with IUGR and that the activated gene may become suppressed after this time point. This study may provide new insights to prevent the onset of adult diseases and to aid in nutritional management for preterm birth infants in neonatal intensive care units.

Dipeptidyl peptidase like 6 promoter methylation is a potential prognostic biomarker for pancreatic ductal adenocarcinoma.

BACKGROUND: Hypermethylation of gene promoters plays an important role in tumorigenesis. The present study aimed to identify and validate promoter methylation-driven genes (PMDGs) for pancreatic ductal adenocarcinoma (PDAC). METHODS: Based on GSE49149 and the PDAC cohort of The Cancer Genome Atlas (TCGA), differential analyses of promoter methylation, correlation analysis, and Cox regression analysis were performed to identify PMDGs. The promoter methylation level was assessed by bisulfite sequencing polymerase chain reaction (BSP) in paired tumor and normal tissues of 72 PDAC patients. Kaplan-Meier survival analyses were performed to evaluate the clinical value of PMDGs. RESULTS: In GSE49149, the ß-value of the dipeptidyl peptidase like 6 (DPP6) promoter was significantly higher in tumor compared with normal samples (0.50 vs. 0.24, P<0.001). In the PDAC cohort of TCGA, the methylation level of the DPP6 promoter was negatively correlated with mRNA expression (r = -0.54, P<0.001). In a multivariate Cox regression analysis, hypermethylation of the DPP6 promoter was an independent risk factor for PDAC (hazard ratio (HR) = 543.91, P=0.002). The results of BSP revealed that the number of methylated CG sites in the DPP6 promoter was greater in tumor samples than in normal samples (7.43 vs. 2.78, P<0.001). The methylation level of the DPP6 promoter was moderately effective at distinguishing tumor from normal samples (area under ROC curve (AUC) = 0.74, P<0.001). Hypermethylation of the DPP6 promoter was associated with poor overall (HR = 3.61, P<0.001) and disease-free (HR = 2.01, P=0.016) survivals for PDAC patients. CONCLUSION: These results indicate that DPP6 promoter methylation is a potential prognostic biomarker for PDAC.

Highly accurate skin-specific methylome analysis algorithm as a platform to screen and validate therapeutics for healthy aging.

BACKGROUND: DNA methylation (DNAm) age constitutes a powerful tool to assess the molecular age and overall health status of biological samples. Recently, it has been shown that tissue-specific DNAm age predictors may present superior performance compared to the pan- or multi-tissue counterparts. The skin is the largest organ in the body and bears important roles, such as body temperature control, barrier function, and protection from external insults. As a consequence of the constant and intimate interaction between the skin and the environment, current DNAm estimators, routinely trained using internal tissues which are influenced by other stimuli, are mostly inadequate to accurately predict skin DNAm age. RESULTS: In the present study, we developed a highly accurate skin-specific DNAm age predictor, using DNAm data obtained from 508 human skin samples. Based on the analysis of 2,266 CpG sites, we accurately calculated the DNAm age of cultured skin cells and human skin biopsies. Age estimation was sensitive to the biological age of the donor, cell passage, skin disease status, as well as treatment with senotherapeutic drugs. CONCLUSIONS: This highly accurate skin-specific DNAm age predictor constitutes a holistic tool that will be of great use in the analysis of human skin health status/molecular aging, as well as in the analysis of the potential of established and novel compounds to alter DNAm age.

Complex context relationships between DNA methylation and accessibility, histone marks, and hTERT gene expression in acute promyelocytic leukemia cells: perspectives for all-trans retinoic acid in cancer therapy.

Telomerase (hTERT) reactivation and sustained expression is a key event in the process of cellular transformation. Therefore, the identification of the mechanisms regulating hTERT expression is of great interest for the development of new anticancer therapies. Although the epigenetic state of hTERT gene promoter is important, we still lack a clear understanding of the mechanisms by which epigenetic changes affect hTERT expression. Retinoids are well-known inducers of granulocytic maturation in acute promyelocytic leukemia (APL). We have previously shown that retinoids repressed hTERT expression in the absence of maturation leading to growth arrest and cell death. Exploring the mechanisms of this repression, we showed that transcription factor binding was dependent on the epigenetic status of hTERT promoter. In the present study, we used APL cells lines and publicly available datasets from APL patients to further investigate the integrated epigenetic events that promote hTERT promoter transition from its silent to its active state, and inversely. We showed, in APL patients, that the methylation of the distal domain of hTERT core promoter was altered and correlated with the outcome of the disease. Further studies combining complementary approaches carried out on APL cell lines highlighted the significance of a domain outside the minimal promoter, localized around 5 kb upstream from the transcription start site, in activating hTERT. This domain is characterized by DNA hypomethylation and H3K4Me3 deposition. Our findings suggest a cooperative interplay between hTERT promoter methylation, chromatin accessibility, and histone modifications that force the revisiting of previously proposed concepts regarding hTERT epigenetic regulation. They represent, therefore, a major advance in predicting sensitivity to retinoid-induced hTERT repression and, more generally, in the potential development of therapies targeting hTERT expression in cancers.

Inhibition of PHLDA2 transcription by DNA methylation and YY1 in goat placenta.

Pleckstrin homology-like domain family A member 2 (PHLDA2) is essential for placental development in mammals. This study was conducted to investigate transcriptional regulation of goat PHLDA2 in the placenta. Real-time PCR and Western blot analyses showed different expression of the PHLDA2 in goat placentas during gestation with highest expression at 30 and 45 days post coitus (P < 0.05). Luciferase reporter assays demonstrated the highest promoter activity in the region of -1023/+20 (P < 0.05). A CpG island was defined within -631/+379 region, where lower level of CpG-methylation was detected with bisulfite sequencing PCR in the placenta than that in the spleen and liver (P < 0.05). Meanwhile, in vitro experiments showed that 5-AzaC enhanced the gene expression in a dose-dependent manner. Site-directed mutation in vitro demonstrated that transcription factor Ying-yang 1 (YY1) had an inhibitory effect on the PHLDA2 expression, and the inhibition was further confirmed with overexpression and siRNA constructs of YY1. ChIP and RE-ChIP analyses further identified the binding of YY1 to the PHLDA2 promoter by interaction with histone deacetylase 1 (HDAC1) and HDAC3. This study uncovers the negative regulation of the CpG-methylation and YY1 on goat PHLDA2 expression. YY1 prefers binding to CpG-methylation sequences, and inhibits goat PHLDA2 expression via recruiting HDAC1 and 3.

Thyroid hormone influences brain gene expression programs and behaviors in later generations by altering germ line epigenetic information.

Genetic factors do not fully account for the relatively high heritability of neurodevelopmental conditions, suggesting that non-genetic heritable factors contribute to their etiology. To evaluate the potential contribution of aberrant thyroid hormone status to the epigenetic inheritance of neurological phenotypes, we examined genetically normal F2 generation descendants of mice that were developmentally overexposed to thyroid hormone due to a Dio3 mutation. Hypothalamic gene expression profiling in postnatal day 15 F2 descendants on the paternal lineage of ancestral male and female T3-overexposed mice revealed, respectively, 1089 and 1549 differentially expressed genes. A large number of them, 675 genes, were common to both sets, suggesting comparable epigenetic effects of thyroid hormone on both the male and female ancestral germ lines. Oligodendrocyte- and neuron-specific genes were strongly overrepresented among genes showing, respectively, increased and decreased expression. Altered gene expression extended to other brain regions and was associated in adulthood with decreased anxiety-like behavior, increased marble burying and reduced physical activity. The sperm of T3-overexposed male ancestors revealed significant hypomethylation of CpG islands associated with the promoters of genes involved in the early development of the central nervous system. Some of them were candidates for neurodevelopmental disorders in humans including Nrg3, Nrxn1, Gabrb3, Gabra5, Apba2, Grik3, Reln, Nsd1, Pcdh8, En1, and Elavl2. Thus, developmental levels of thyroid hormone influence the epigenetic information of the germ line, disproportionately affecting genes with critical roles in early brain development, and leading in future generations to disease-relevant alterations in postnatal brain gene expression and adult behavior.

Effects of Dietary Lipid Composition and Fatty Acid Desaturase 2 Expression in Broodstock Gilthead Sea Bream on Lipid Metabolism-Related Genes and Methylation of the fads2 Gene Promoter in Their Offspring.

Polyunsaturated fatty acids (PUFA) in parental diets play a key role in regulating n-3 LC-PUFA metabolism of the offspring. However, it is not clear whether this metabolic regulation is driven by the precursors presented in the diet or by the parental ability to synthesize them. To elucidate this, broodstocks of gilthead sea bream with different blood expression levels of fads2, which encodes for the rate-limiting enzyme in the n-3 LC-PUFA synthesis pathway, were fed either a diet supplemented with alpha-linolenic acid (ALA, 18:3n-3) or a control diet. The progenies obtained from these four experimental groups were then challenged with a low LC-PUFA diet at the juvenile stage. Results showed that the offspring from parents with high fads2 expression presented higher growth and improved utilization of low n-3 LC-PUFA diets compared to the offspring from parents with low fads2 expression. Besides, an ALA-rich diet during the gametogenesis caused negative effects on the growth of the offspring. The epigenetic analysis demonstrated that methylation in the promoter of fads2 of the offspring was correlated with the parental fads2 expression levels and type of the broodstock diet.

Hypothalamic DNA methylation in rats with dihydrotestosterone-induced polycystic ovary syndrome: effects of low-frequency electro-acupuncture.

NEW FINDINGS: What is the central question of this study? What is the role of hypothalamic DNA methylation in the development of polycystic ovary syndrome (PCOS) and the response to electro-acupuncture treatment. What is the main finding and its importance? Global DNA methylation and expression of DNA methyltransferases (DNMTs) were increased in PCOS-like rats, and electro-acupuncture (EA) decreased global DNA methylation and DNMT3b expression. Pyrosequencing showed that the DNA methylation of some PCOS candidate genes was changed in the PCOS and PCOS+EA groups, suggesting that hypothalamic DNA methylation plays an important role in the development of PCOS and in mediating the effects of electro-acupuncture treatment. ABSTRACT: Polycystic ovary syndrome (PCOS) is a common reproductive and endocrine disease of unknown aetiology. Recently, epigenetic studies focusing on DNA methylation in PCOS have received much attention, but the mechanisms are still unclear. In the present study, we used the 5α-dihydrotestosterone-induced PCOS-like rat model and treated the rats with electro-acupuncture (EA). Rats were randomly divided into four groups - controls, diet-induced obesity, PCOS and PCOS+EA. We examined the reproductive, metabolic and behavioural phenotypes, validated the effect of EA, and explored the role of hypothalamic DNA methylation by analysing the methylation of global DNA and selected candidate genes. The PCOS rats presented with reproductive dysfunctions such as lack of regular oestrous cyclicity, metabolic disorders such as increased body weight and insulin resistance, and depression and anxiety-like behaviours. EA improved the reproductive functions, decreased body weight and improved experimental depressive behaviour. Furthermore, global DNA methylation and the expression of DNA methyltransferases (DNMTs) were increased in PCOS rats compared to the control group, and EA decreased the global DNA methylation and the expression of DNMT3b. In addition, pyrosequencing showed that the DNA methylation of certain CpG sites in targeted genes (Plcg1, Camk2b, Esr2 and Pgr) was increased in the PCOS group, but the DNA methylation of Camk2b and Ar was decreased after EA treatment. These results indicate that hypothalamic DNA methylation might be correlated with the development of PCOS and that EA has an effect on hypothalamic DNA methylation in PCOS rats.

Experience-dependent neuroplasticity of the developing hypothalamus: integrative epigenomic approaches.

Augmented maternal care during the first postnatal week promotes life-long stress resilience and improved memory compared with the outcome of routine rearing conditions. Recent evidence suggests that this programming commences with altered synaptic connectivity of stress sensitive hypothalamic neurons. However, the epigenomic basis of the long-lived consequences is not well understood. Here, we employed whole-genome bisulfite sequencing (WGBS), RNA-sequencing (RNA-seq), and a multiplex microRNA (miRNA) assay to examine the effects of augmented maternal care on DNA cytosine methylation, gene expression, and miRNA expression. A total of 9,439 differentially methylated regions (DMRs) associated with augmented maternal care were identified in male offspring hypothalamus, as well as a modest but significant decrease in global DNA methylation. Differentially methylated and expressed genes were enriched for functions in neurotransmission, neurodevelopment, protein synthesis, and oxidative phosphorylation, as well as known stress response genes. Twenty prioritized genes were identified as highly relevant to the stress resiliency phenotype. This combined unbiased approach enabled the discovery of novel genes and gene pathways that advance our understanding of the epigenomic mechanisms underlying the effects of maternal care on the developing brain.

Genome-wide survey reveals dynamic effects of folate supplement on DNA methylation and gene expression during C2C12 differentiation.

Folic acid supplements taken during pregnancy can prevent neural tube defects and other developmental abnormalities. Here, we explored the effects of folate supplementation on gene expression and DNA methylation during C2C12 differentiation. Based on the folic acid concentration, this study comprised three groups: low folate (L), normal folate (N), and high-folate supplement (H). Our analyses revealed that differentiation and the mRNA expression of the gene myogenin in C2C12 cell were enhanced by folic acid; however, the overall methylation percentage in myogenin promoter between different treatment groups was not significantly different ( P > 0.05). The results of MeDIP-chip showed that hundreds of differentially methylated regions (DMRs) were identified between every two groups in both promoter and CpG islands, respectively. Genes with DMRs between N and L groups were mainly enriched in the processes of cell differentiation and cell development, whereas those with DMRs between H and N groups were frequently enriched in cellular process/cycle and cell metabolic processes. In addition, correlation analysis between methylation profile and expression profile revealed that some genes were regulated by methylation status directly. Together, these analyses suggest that folate deficiency and supplementation can influence the differentiation, genome-wide DNA methylation level and the expression of myogenesis-related genes including myogenin in the C2C12 cell line.

Asymmetric DNA methylation of CpG dyads is a feature of secondary DMRs associated with the Dlk1/Gtl2 imprinting cluster in mouse.

BACKGROUND: Differential DNA methylation plays a critical role in the regulation of imprinted genes. The differentially methylated state of the imprinting control region is inherited via the gametes at fertilization, and is stably maintained in somatic cells throughout development, influencing the expression of genes across the imprinting cluster. In contrast, DNA methylation patterns are more labile at secondary differentially methylated regions which are established at imprinted loci during post-implantation development. To investigate the nature of these more variably methylated secondary differentially methylated regions, we adopted a hairpin linker bisulfite mutagenesis approach to examine CpG dyad methylation at differentially methylated regions associated with the murine Dlk1/Gtl2 imprinting cluster on both complementary strands. RESULTS: We observed homomethylation at greater than 90% of the methylated CpG dyads at the IG-DMR, which serves as the imprinting control element. In contrast, homomethylation was only observed at 67-78% of the methylated CpG dyads at the secondary differentially methylated regions; the remaining 22-33% of methylated CpG dyads exhibited hemimethylation. CONCLUSIONS: We propose that this high degree of hemimethylation could explain the variability in DNA methylation patterns at secondary differentially methylated regions associated with imprinted loci. We further suggest that the presence of 5-hydroxymethylation at secondary differentially methylated regions may result in hemimethylation and methylation variability as a result of passive and/or active demethylation mechanisms.

Genome-wide analysis of DNA methylation in hypothalamus and ovary of Capra hircus.

BACKGROUND: DNA methylation is a frequently studied epigenetic modification due to its role in regulating gene expression and hence in biological processes and in determining phenotypic plasticity in organisms. Rudimentary DNA methylation patterns for some livestock species are publically available: among these, goat methylome deserves to be further explored. RESULTS: Genome-wide DNA methylation maps of the hypothalamus and ovary from Saanen goats were generated using Methyl-CpG binding domain protein sequencing (MBD-seq). Analysis of DNA methylation patterns indicate that the majority of methylation peaks found within genes are located gene body regions, for both organs. Analysis of the distribution of methylated sites per chromosome showed that chromosome X had the lowest number of methylation peaks. The X chromosome has one of the highest percentages of methylated CpG islands in both organs, and approximately 50% of the CpG islands in the goat epigenome are methylated in hypothalamus and ovary. Organ-specific Differentially Methylated Genes (DMGs) were correlated with the expression levels. CONCLUSIONS: The comparison between transcriptome and methylome in hypothalamus and ovary showed that a higher level of methylation is not accompanied by a higher gene suppression. The genome-wide DNA methylation map for two goat organs produced here is a valuable starting point for studying the involvement of epigenetic modifications in regulating goat reproduction performance.

DNA Methylation Patterns in the Hypothalamus of Female Pubertal Goats.

Female pubertal development is tightly controlled by complex mechanisms, including neuroendocrine and epigenetic regulatory pathways. Specific gene expression patterns can be influenced by DNA methylation changes in the hypothalamus, which can in turn regulate timing of puberty onset. In order to understand the relationship between DNA methylation changes and gene expression patterns in the hypothalamus of pubertal goats, whole-genome bisulfite sequencing and RNA-sequencing analyses were carried out. There was a decline in DNA methylation levels in the hypothalamus during puberty and 268 differentially methylated regions (DMR) in the genome, with differential patterns in different gene regions. There were 1049 genes identified with distinct expression patterns. High levels of DNA methylation were detected in promoters, introns and 3'-untranslated regions (UTRs). Levels of methylation decreased gradually from promoters to 5'-UTRs and increased from 5'-UTRs to introns. Methylation density analysis demonstrated that methylation level variation was consistent with the density in the promoter, exon, intron, 5'-UTRs and 3'-UTRs. Analyses of CpG island (CGI) sites showed that the enriched gene contents were gene bodies, intergenic regions and introns, and these CGI sites were hypermethylated. Our study demonstrated that DNA methylation changes may influence gene expression profiles in the hypothalamus of goats during the onset of puberty, which may provide new insights into the mechanisms involved in pubertal onset.

Exosome-based tumor antigens-adjuvant co-delivery utilizing genetically engineered tumor cell-derived exosomes with immunostimulatory CpG DNA.

For cancer immunotherapy via tumor antigen vaccination in combination with an adjuvant, major challenges include the identification of a particular tumor antigen and efficient delivery of the antigen as well as adjuvant to antigen-presenting cells. In this study, we proposed an efficient exosome-based tumor antigens-adjuvant co-delivery system using genetically engineered tumor cell-derived exosomes containing endogenous tumor antigens and immunostimulatory CpG DNA. Murine melanoma B16BL6 cells were transfected with a plasmid vector encoding a fusion streptavidin (SAV; a protein that binds to biotin with high affinity)-lactadherin (LA; an exosome-tropic protein) protein, yielding genetically engineered SAV-LA-expressing exosomes (SAV-exo). SAV-exo were combined with biotinylated CpG DNA to prepare CpG DNA-modified exosomes (CpG-SAV-exo). Fluorescent microscopic observation revealed the successful modification of exosomes with CpG DNA by SAV-biotin interaction. CpG-SAV-exo showed efficient and simultaneous delivery of exosomes with CpG DNA to murine dendritic DC2.4 cells in culture. Treatment with CpG-SAV-exo effectively activated DC2.4 cells and enhanced tumor antigen presentation capacity. Immunization with CpG-SAV-exo exhibited stronger in vivo antitumor effects in B16BL6 tumor-bearing mice than simple co-administration of exosomes and CpG DNA. Thus, genetically engineered CpG-SAV-exo is an effective exosome-based tumor antigens-adjuvant co-delivery system that will be useful for cancer immunotherapy.

Folate deprivation induces cell cycle arrest at G0/G1 phase and apoptosis in hippocampal neuron cells through down-regulation of IGF-1 signaling pathway.

Folate deficiency contributes to impaired adult hippocampal neurogenesis, yet the mechanisms remain unclear. Here we use HT-22 hippocampal neuron cells as model to investigate the effect of folate deprivation (FD) on cell proliferation and apoptosis, and to elucidate the underlying mechanism. FD caused cell cycle arrest at G0/G1 phase and increased the rate of apoptosis, which was associated with disrupted expression of folate transport and methyl transfer genes. FOLR1 and SLC46A1 were (P<0.01) down-regulated, while SLC19A1 was up-regulated (P<0.01) in FD group. FD cells exhibited significantly (P<0.05) higher protein content of BHMT, MAT2b and DNMT3a, as well as increased SAM/SAH concentrations and global DNA hypermethylation. The expression of the total and all the 3 classes of IGF-1 mRNA variants was significantly (P<0.01) down-regulated and IGF-1 concentration was decreased (P<0.05) in the culture media. IGF-1 signaling pathway was also compromised with diminished activation (P<0.05) of STAT3, AKT and mTOR. CpG hypermethylation was detected in the promoter regions of IGF-1 and FOLR1 genes, while higher SLC19A1 mRNA corresponded to hypomethylation of its promoter. IGF-1 supplementation in FD media significantly abolished FD-induced decrease in cell viability. However, IGF-1 had limited effect in rescuing the cell phenotype when added 24h after FD. Taken together, down-regulation of IGF-1 expression and signaling is involved in FD-induced cell cycle arrest and apoptosis in HT-22 hippocampal neuron cells, which is associated with an abnormal activation of methyl transfer pathway and hypermethylation of IGF-1 gene promoter.

Expression of metastasis suppressor gene AES driven by a Yin Yang (YY) element in a CpG island promoter and transcription factor YY2.

We recently found that the product of the AES gene functions as a metastasis suppressor of colorectal cancer (CRC) in both humans and mice. Expression of amino-terminal enhancer of split (AES) protein is significantly decreased in liver metastatic lesions compared with primary colon tumors. To investigate its downregulation mechanism in metastases, we searched for transcriptional regulators of AES in human CRC and found that its expression is reduced mainly by transcriptional dysregulation and, in some cases, by additional haploidization of its coding gene. The AES promoter-enhancer is in a typical CpG island, and contains a Yin-Yang transcription factor recognition sequence (YY element). In human epithelial cells of normal colon and primary tumors, transcription factor YY2, a member of the YY family, binds directly to the YY element, and stimulates expression of AES. In a transplantation mouse model of liver metastases, however, expression of Yy2 (and therefore of Aes) is downregulated. In human CRC metastases to the liver, the levels of AES protein are correlated with those of YY2. In addition, we noticed copy-number reduction for the AES coding gene in chromosome 19p13.3 in 12% (5/42) of human CRC cell lines. We excluded other mechanisms such as point or indel mutations in the coding or regulatory regions of the AES gene, CpG methylation in the AES promoter enhancer, expression of microRNAs, and chromatin histone modifications. These results indicate that Aes may belong to a novel family of metastasis suppressors with a CpG-island promoter enhancer, and it is regulated transcriptionally.