Bisulfite Genomic DNA Sequencing Research Articles

Epigenetic Deregulation of Protein Tyrosine Kinase 6 Promotes Carcinogenesis of Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) accounts for over 90% of oral cancers and causes considerable morbidity and mortality. Epigenetic deregulation is a common mechanism underlying carcinogenesis. DNA methylation deregulation is the epigenetic change observed during the transformation of normal cells to precancerous and eventually cancer cells. This study investigated the DNA methylation patterns of PTK6 during the development of OSCC. Bisulfite genomic DNA sequencing was performed to determine the PTK6 methylation level. OSCC animal models were established to examine changes in PTK6 expression in the different stages of OSCC development. The DNA methylation of PTK6 was decreased during the development of OSCC. The mRNA and protein expression of PTK6 was increased in OSCC cell lines compared with human normal oral keratinocytes. In mice, the methylation level of PTK6 decreased after treatment with 4-nitroquinoline 1-oxide and arecoline, and the mRNA and protein expression of PTK6 was increased. PTK6 hypomethylation can be a diagnostic marker of OSCC. Upregulation of PTK6 promoted the proliferation, migration, and invasion of OSCC cells. PTK6 promoted carcinogenesis and metastasis by increasing STAT3 phosphorylation and ZEB1 expression. The epigenetic deregulation of PTK6 can serve as a biomarker for the early detection of OSCC and as a treatment target.

Methylation related genes affect sex differentiation in dioecious and gynodioecious papaya.

Morphological, genic and epigenetic differences often exist in separate sexes of dioecious and trioecious plants. However, the connections and relationships among them in different breeding systems are still unclear. Papaya has three sex types, which is genetically determined and epigenetically regulated, and was chosen as a model to study sex differentiation. Bisulfite sequencing of genomic DNA extracted from early-stage flowers revealed sex-specific genomic methylation landscapes and seasonally methylome reprogramming processes in dioecious and gynodioecious papaya grown in spring and summer. Extensive methylation of sex-determining region (SDR) was the distinguishing epigenetic characteristics of nascent XY sex chromosomes in papaya. Seasonal methylome reprogramming of early-stage flowers in both dioecy and gynodioecy systems were detected, resulting from transcriptional expression pattern alterations of methylation-modification-related and chromatin-remodeling-related genes, particularly from those genes involved in active demethylation. Genes involved in phytohormone signal transduction pathway in male flowers have played an important role in the formation of male-specific characteristics. These findings enhanced the understanding of the genetic and epigenetic contributions to sex differentiation and the complexity of sex chromosome evolution in trioecious plants.

A genomic atlas of systemic interindividual epigenetic variation in humans

BackgroundDNA methylation is thought to be an important determinant of human phenotypic variation, but its inherent cell type specificity has impeded progress on this question. At exceptional genomic regions, interindividual variation in DNA methylation occurs systemically. Like genetic variants, systemic interindividual epigenetic variants are stable, can influence phenotype, and can be assessed in any easily biopsiable DNA sample. We describe an unbiased screen for human genomic regions at which interindividual variation in DNA methylation is not tissue-specific.ResultsFor each of 10 donors from the NIH Genotype-Tissue Expression (GTEx) program, CpG methylation is measured by deep whole-genome bisulfite sequencing of genomic DNA from tissues representing the three germ layer lineages: thyroid (endoderm), heart (mesoderm), and brain (ectoderm). We develop a computational algorithm to identify genomic regions at which interindividual variation in DNA methylation is consistent across all three lineages. This approach identifies 9926 correlated regions of systemic interindividual variation (CoRSIVs). These regions, comprising just 0.1% of the human genome, are inter-correlated over long genomic distances, associated with transposable elements and subtelomeric regions, conserved across diverse human ethnic groups, sensitive to periconceptional environment, and associated with genes implicated in a broad range of human disorders and phenotypes. CoRSIV methylation in one tissue can predict expression of associated genes in other tissues.ConclusionsIn addition to charting a previously unexplored molecular level of human individuality, this atlas of human CoRSIVs provides a resource for future population-based investigations into how interindividual epigenetic variation modulates risk of disease.

Expression and function of Uc.160+, a transcribed ultraconserved region, in gastric cancer.

Transcribed ultraconserved regions (T-UCRs) are a novel class of noncoding RNAs that are highly conserved among the orthologous regions in most vertebrates. It has been reported that T-UCRs have distinct signatures in human cancers. We previously discovered the downregulation of T-UCR expression in gastric cancer (GC), indicating that T-UCRs could play an important role in GC biology. Uc.160+, a T-UCR reported to be downregulated in human cancer, has not been examined in GC. We analyzed the expression pattern of Uc.160+ in nonneoplastic and tumor tissues of the stomach by using uantitative reverse transcription polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH), specifically focusing on the mechanism of transcriptional regulation and target genes that are regulated by T-UCRs. We also attempted to determine the effect of Uc.160+ expression on biological features of GC cell lines by Western blotting. On the basis of the qRT-PCR and ISH results, Uc.160+ expression in adenoma and GC tissues was clearly downregulated compared with that in nonneoplastic mucosa tissues of the stomach. Cancer-specific DNA methylation in the promoter region of Uc.160 was observed by bisulfite genomic DNA sequencing analysis. The effect of DNA methylation on Uc.160+ expression was further confirmed by reporter gene assay. We also revealed that Uc.160+ inhibited the phosphorylation of Akt by regulating phosphatase and tensin homolog (PTEN) expression. These results indicate that Uc.160+ could possibly have a tumor suppressive role in GC.

High resolution methylation analysis of the HoxA5 regulatory region in different somatic tissues of laboratory mouse during development

Homeobox genes encode a group of DNA binding regulatory proteins whose key function occurs in the spatial-temporal organization of genome during embryonic development and differentiation. The role of these Hox genes during ontogenesis makes it an important model for research. HoxA5 is a member of Hox gene family playing a central role during axial body patterning and morphogenesis. DNA modification studies have shown that the function of Hox genes is partly governed by the methylation-mediated gene expression regulation. Therefore the study aimed to investigate the role of epigenetic events in regulation of tissue-specific expression pattern of HoxA5 gene during mammalian development. The methodology adopted were sodium bisulfite genomic DNA sequencing, quantitative real-time PCR and chromatin-immunoprecipitation (ChIP). Methylation profiling of HoxA5 gene promoter shows higher methylation in adult as compared to fetus in various somatic tissues of mouse being highest in adult spleen. However q-PCR results show higher expression during fetal stages being highest in fetal intestine followed by brain, liver and spleen. These results clearly indicate a strict correlation between DNA methylation and tissue-specific gene expression. The findings of chromatin-immunoprecipitation (ChIP) have also reinforced that epigenetic event like DNA methylation plays important role in the regulation of tissue specific expression of HoxA5.

Reelin expression is up-regulated in mice colon in response to acute colitis and provides resistance against colitis

Reelin is an extracellular matrix protein first known for its key role in neuronal migration. Studies in rodent small intestine suggested that reelin protects the organism from intestinal pathology. Here we determined in mice colon, by real time-PCR and immunological assays, the expression of the reelin signalling system; its response to dextran sulphate sodium (DSS) and the response of wild-type and reeler mice to DSS-treatment. DNA methylation was determined by bisulfite modification and sequencing of genomic DNA. In the colon mucosa reelin expression is restricted to the myofibroblasts, whereas both epithelial cells and myofibroblasts express reelin receptors (ApoER2 and VLDLR) and its effector protein Dab1. The muscle layer also expresses reelin. DSS-treatment reduces reelin expression in the muscle but it is activated in the mucosa. Activation of mucosal reelin is greater in magnitude and is delayed until after the activation of the myofibroblasts marker, α-SMA. This indicates that the DSS-induced reelin up-regulation results from changes in the reelin gene expression rather than from myofibroblasts proliferation. DSS-treatment does not modify Sp1 or Tbr1 mRNA abundance, but increases that of TGF-β1 and ApoER2, decreases that of CASK and DNMT1 and it also decreases the reelin promoter methylation. Finally, the reeler mice exhibit higher inflammatory scores than wild-type mice, indicating that the mutation increases the susceptibility to DSS-colitis. In summary, this data are the first to demonstrate that mouse distal colon increases reelin production in response to DSS-colitis via a DNMT1-dependent hypo-methylation of the gene promoter region and that reelin provides protection against colitis.

Methylation of the Sox9 and Oct4 promoters and its correlation with gene expression during testicular development in the laboratory mouse.

Sox9 and Oct4 are two important regulatory factors involved in mammalian development. Sox9, a member of the group E Sox transcription factor family, has a crucial role in the development of the genitourinary system, while Oct4, commonly known as octamer binding transcription factor 4, belongs to class V of the transcription family. The expression of these two proteins exhibits a dynamic pattern with regard to their expression sites and levels. The aim of this study was to investigate the role of de novo methylation in the regulation of the tissue- and site-specific expression of these proteins. The dynamics of the de novo methylation of 15 CpGs and six CpGs in Sox9 and Oct4 respectively, was studied with sodium bisulfite genomic DNA sequencing in mouse testis at different developmental stages. Consistent methylation of three CpGs was observed in adult ovary in which the expression of Sox9 was feeble, while the level of methylation in somatic tissue was greater in Oct4 compared to germinal tissue. The promoter-chromatin status of Sox9 was also studied with a chromatin immune-precipitation assay.

Abstract 1070: Epigenetic silencing of galectin-12 in colorectal cancer

Abstract Aim/Background: Galectins constitute a family of ß-galactoside binding proteins some of them known to be involved in colorectal tumorigenesis. Our previous work suggests that sodium butyrate can induce galectin-12 expression in CRC cell lines. How this might be exerted is largely unexplored. In the present study we examined the potential epigenetic regulation of galectin-12 expression in colorectal cancer cell lines and primary tumors. Material and Methods: For DNA-methylation analysis 4 microsatellite unstable (MSI) and 5 microsatellite stable (MSS) human CRC cell lines were treated with 5-Aza-2`deoxycytidine (5-Aza-dC) for 72h. Changes in galectin-12 expression were evaluated by RT-PCR analysis. Galectin-12 promoter methylation was determined by bisulfite treatment and genomic DNA sequencing of single clones in the CRC cell line SW707 as well as in FFPE specimens from 10 primary colorectal tumors (MSI: n = 5; MSS: n = 5). Results: Treatment with 5-Aza-dC led to de novo expression of galectin-12 transcript in 4/9 (44%) of colorectal cancer cell lines. We identified a CpG island in the galectin-12 promoter region that was found to be hypomethylated upon 5-Aza-dC treatment in SW707 cells and to be associated with concomitant de novo galectin-12 mRNA expression. When primary tissues were examined we observed galectin-12 promoter hypomethylation in healthy colon mucosa but significant hypermethylation of this promoter region in corresponding tumor tissue. Conclusions: This study for the first time shows that galectin-12 promoter methylation represses galectin-12 expression in colorectal tumor cells independent of their MSI status. In CRC cell lines this effect can be reversed by butyrate but in human colon primary tumors might be refractory to these effects of butyrate. Re-expression of galectin-12 may be a potential therapeutic strategy to constrain colorectal tumor growth. Citation Format: Eva-Maria Katzenmaier, Johannes Gebert, Matthias Kloor, Jürgen Kopitz. Epigenetic silencing of galectin-12 in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1070. doi:10.1158/1538-7445.AM2015-1070

Epigenetic DNA methylation of antioxidative stress regulator NRF2 in human prostate cancer.

Epigenetic control of NRF2, a master regulator of many critical antioxidative stress defense genes in human prostate cancer (CaP), is unknown. Our previous animal study found decreased Nrf2 expression through promoter CpG methylation/histone modifications during prostate cancer progression in TRAMP mice. In this study, we evaluated CpG methylation of human NRF2 promoter in 27 clinical prostate cancer samples and in LNCaP cells using MAQMA analysis and bisulfite genomic DNA sequencing. Prostate cancer tissue microarray (TMA) containing normal and prostate cancer tissues was studied by immunohistochemistry. Luciferase reporter assay using specific human NRF2 DNA promoter segments and chromatin immunoprecipitation (ChIP) assay against histone modifying proteins were performed in LNCaP cells. Three specific CpG sites in the NRF2 promoter were found to be hypermethylated in clinical prostate cancer samples (BPH<ADT-RCaP<AS-CaP). NRF2 staining in human prostate cancer TMA showed a decreasing trend for both intensity and percentage of positive cells from normal tissues to advanced-stage prostate cancer (Gleason score from 3-9). Reporter assays in the LNCaP cells containing these three CpG sites showed methylation-inhibited transcriptional activity of the NRF2 promoter. LNCaP cells treated with 5-aza/TSA restored the expression of NRF2 and NRF2 downstream target genes, decreased expression levels of DNMT and HDAC proteins, and ChIP assays showed increased RNA Pol II and H3Ac with a concomitant decrease in H3K9me3, MBD2, and MeCP2 at CpG sites of human NRF2 promoter. Taken together, these findings suggest that epigenetic modification may contribute to the regulation of transcription activity of NRF2, which could be used as prevention and treatment target of human prostate cancer.

Abstract 1377: Transcriptional silencing of MST1/Hippo by a coordinated MYC and EZH2 signaling in prostate cancer

Abstract Prostate cancer is a major cause of cancer-related death in western countries because of the poorly understood disease mechanisms. Hippo-like MST1 protein kinase regulates cell growth, stem cell self-renewal and carcinogenesis. The loss of Mst1 expression is implicated in the etiology several types of cancers with poor prognosis, including prostate cancer. However, the underlying mechanism of Mst1 transcriptional downregulation in prostate cancer is unknown. Here, we report that MYC and EZH2 function as epigenetic suppressors of MST1 in human prostate cancer cells. We showed that decline in MST1 expression correlated with EZH2 and MYC upregulation as demonstrated by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and western blot. Using methylation sensitive PCR and bisulfite genomic DNA sequencing, we showed that Mst1 promoter DNA was methylated at CpG sites that led to a reduction in MST1 expression. Our pharmacologic or RNAi experiments revealed that EZH2 contributed to MST1 silencing by inducing MST1 promoter DNA methylation and H3K27me3 modification that was accompanied by a reduced H3K4me3 mark and RNA polymerase II occupancy on MST1 promoter and thereby reducing its promoter activity and expression. On the other hand, overexpression of MYC stimulated EZH2 and conversely reduced MST1 promoter activation and its mRNA levels. Potent EZH2 inhibitors reversed the MYC effects on MST1, indicating that MYC attenuates MST1 through EZH2. Moreover, MST1 knockdown caused cell resistance to androgen deficiency and to EZH2, or MYC inhibitor-induced growth retardation. These findings indicate that MYC cooperates with EZH2 to epigenetically silence MST1 and suggest that the loss of MST1/Hippo is critical for MYC- and EZH2-driven cancer cell survival. Citation Format: Gamze Kuser Abali, Ahmet Alptekin, Bekir Cinar. Transcriptional silencing of MST1/Hippo by a coordinated MYC and EZH2 signaling in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1377. doi:10.1158/1538-7445.AM2014-1377

Study of promoter DNA methylation of Sox11 and its correlation with tissue-specific expression in the laboratory mouse

Sox11, a member of the group C Sox transcription factor family, is predominantly expressed in the neurogenic areas of the adult brain. In the embryo, Sox11 is expressed in the developing nervous system in both glial and neuronal lineages. Its expression exhibits a dynamic pattern with respect to both expression sites and expression levels. The aim of the present study is to investigate the role of de novo methylation in regulation of its tissue specific expression. The dynamics of de novo methylation of 24 CpGs is studied with the help of sodium bisulfite genomic DNA sequencing in the mouse brain, kidney and testis at different developmental stages i.e. 12.5 dpc, 18.5 dpc, and 5 dpp and adult stage. More sites were methylated in adult stages of tissues (brain, kidney and testis) in comparison to fetal as well as neonatal tissues. On the contrary Sox11 expression was high in fetal and neonatal stages in these tissues. Adult tissues show low expression of Sox11 gene. Thus Sox11, a developmentally important gene, sets a beautiful inverse correlation between methylation and expression.

Apigenin reactivates Nrf2 anti-oxidative stress signaling in mouse skin epidermal JB6 P + cells through epigenetics modifications.

Nrf2 is a crucial transcription factor that controls a critical anti-oxidative stress defense system and is implicated in skin homeostasis. Apigenin (API), a potent cancer chemopreventive agent, protects against skin carcinogenesis and elicits multiple molecular signaling pathways. However, the potential epigenetic effect of API in skin cancer chemoprotection is not known. In this study, bisulfite genomic DNA sequencing and methylated DNA immunoprecipitation were utilized to investigate the demethylation effect of API at 15 CpG sites in the Nrf2 promoter in mouse skin epidermal JB6 P + cells. In addition, qPCR and Western blot analyses were performed to evaluate the mRNA and protein expression of Nrf2 and the Nrf2 ARE downstream gene, NQO1. Finally, the protein expression levels of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) were evaluated using API and the DNMT/HDAC inhibitor 5-aza/ trichostatin A. Our results showed that API effectively reversed the hypermethylated status of the 15 CpG sites in the Nrf2 promoter in a dose-dependent manner. API enhanced the nuclear translocation of Nrf2 and increased the mRNA and protein expression of Nrf2 and the Nrf2 downstream target gene, NQO1. Furthermore, API reduced the expression of the DNMT1, DNMT3a, and DNMT3b epigenetic proteins as well as the expression of some HDACs (1-8). Taken together, our results showed that API can restore the silenced status of Nrf2 in skin epidermal JB6 P + cells by CpG demethylation coupled with attenuated DNMT and HDAC activity. These results may provide new therapeutic insights into the prevention of skin cancer by dietary phytochemicals.

Methylglyoxal induces endoplasmic reticulum stress and DNA demethylation in the Keap1 promoter of human lens epithelial cells and age-related cataracts

Age-related cataracts are a leading cause of blindness. Previously, we have demonstrated the association of the unfolded protein response with various cataractogenic stressors. However, DNA methylation alterations leading to suppression of lenticular antioxidant protection remains unclear. Here, we report the methylglyoxal-mediated sequential events responsible for Keap1 promoter DNA demethylation in human lens epithelial cells, because Keap1 is a negative regulatory protein that regulates the Nrf2 antioxidant protein. Methylglyoxal induces endoplasmic reticulum stress and activates the unfolded protein response leading to overproduction of reactive oxygen species before human lens epithelial cell death. Methylglyoxal also suppresses Nrf2 and DNA methyltransferases but activates the DNA demethylation pathway enzyme TET1. Bisulfite genomic DNA sequencing confirms the methylglyoxal-mediated Keap1 promoter DNA demethylation leading to overexpression of Keap1 mRNA and protein. Similarly, bisulfite genomic DNA sequencing shows that human clear lenses (n = 15) slowly lose 5-methylcytosine in the Keap1 promoter throughout life, at a rate of 1% per year. By contrast, diabetic cataractous lenses (n = 21) lose an average of 90% of the 5-methylcytosine regardless of age. Overexpressed Keap1 protein is responsible for decreasing Nrf2 by proteasomal degradation, thereby suppressing Nrf2-dependent stress protection. This study demonstrates for the first time the associations of unfolded protein response activation, Nrf2-dependent antioxidant system failure, and loss of Keap1 promoter methylation because of altered active and passive DNA demethylation pathway enzymes in human lens epithelial cells by methylglyoxal. As an outcome, the cellular redox balance is altered toward lens oxidation and cataract formation.

Overexpression of MYC and EZH2 cooperates to epigenetically silence MST1 expression

Hippo-like MST1 protein kinase regulates cell growth, organ size, and carcinogenesis. Reduction or loss of MST1 expression is implicated in poor cancer prognosis. However, the mechanism leading to MST1 silencing remains elusive. Here, we report that both MYC and EZH2 function as potent suppressors of MST1 expression in human prostate cancer cells. We demonstrated that concurrent overexpression of MYC and EZH2 correlated with the reduction or loss of MST1 expression, as shown by RT-qPCR and immunoblotting. Methylation sensitive PCR and bisulfite genomic DNA sequencing showed that DNA methylation caused MST1 silencing. Pharmacologic and RNAi experiments revealed that MYC and EZH2 silenced MST1 expression by inhibiting its promoter activity, and that EZH2 was a mediator of the MYC-induced silencing of MST1. In addition, MYC contributed to MST1 silencing by partly inhibiting the expression of microRNA-26a/b, a negative regulator of EZH2. As shown by ChIP assays, EZH2-induced DNA methylation and H3K27me3 modification, which was accompanied by a reduced H3K4me3 mark and RNA polymerase II occupancy on the MST1 promoter CpG region, were the underlying cause of MST1 silencing. Moreover, potent pharmacologic inhibitors of MYC or EZH2 suppressed prostate cancer cell growth in vitro, and the knockdown of MST1 caused cells’ resistance to MYC and EZH2 inhibitor-induced growth retardation. These findings indicate that MYC, in concert with EZH2, epigenetically attenuates MST1 expression and suggest that the loss of MST1/Hippo functions is critical for the MYC or EZH2 mediation of cancer cell survival.

Malondialdehyde mediates oxidized LDL-induced coronary toxicity through the Akt-FGF2 pathway via DNA methylation

BackgroundOxidized LDL (oxLDL) is involved in the development of atherosclerotic heart disease through a mechanism that is not fully understood. In this study, we examined the role of malondialdehyde (MDA), an important oxidative stress epitope of oxLDL, in mediating coronary endothelial cytotoxicity.ResultsHuman coronary artery endothelial cells (HCAECs) were treated with oxLDL in the presence or absence of antibody against MDA (anti-MDA) or apoB100 (anti-apoB100). In HCAECs treated with oxLDL (100 μg/ml) alone, DNA synthesis, cell viability, and expression of prosurvival fibroblast growth factor 2 (FGF2) were significantly reduced (P < 0.01 vs phosphate buffered saline–treated cells). These inhibitory effects of oxLDL were significantly attenuated in HCAECs cotreated with anti-MDA (0.15 μg/ml; P < 0.05 vs oxLDL-treated cells), but not in those cotreated with anti-apoB100. When we tested the effects of a panel of signal transduction modifiers on the signal transduction pathways of MDA in oxLDL-treated HCAECs, we found that MDA-induced cytotoxicity was mediated partly through the Akt pathway. Using a reporter gene assay, we identified an oxLDL-response element in the FGF2 promoter that was responsible for the transcriptional repression of FGF2 by oxLDL. The results of bisulfite genomic DNA sequencing showed that in HCAECs treated with oxLDL, the GC-rich promoter of FGF2 was heavily methylated at cytosine residues, whereas cotreatment with anti-MDA markedly reduced oxLDL-induced FGF2 promoter methylation.ConclusionOxLDL disrupts the growth and survival of HCAECs through an MDA-dependent pathway involving methylation of the FGF2 promoter and repression of FGF2 transcription. This novel epigenetic mechanism of oxLDL may underlie its atherogenicity in patients with atherosclerotic cardiovascular disease.

Unmethylated state of 5′ upstream CpG islands may be necessary but not sufficient for the testis-enriched expression of ZNF230/Znf230

The testis-enriched genes ZNF230/Znf230 are located on human chromosome 11p15/mouse chromosome 7 near conserved imprinting control regions. Typical CpG islands (CGIs) extend from the promoter to the first exon in each of these genes. To investigate the correlation between the methylation status of the above CGIs and the expression patterns of the two genes, we performed bisulfite genomic sequencing of genomic DNA from human and mouse tissues and cells. The results showed that the CGIs of ZNF230/Znf230 were completely unmethylated in all selected tissues and cells, regardless of the expression levels of the two genes. Further experiments using Znf230-second-exon-knockout mice to investigate the imprinting status of Znf230 showed that its expression was not affected by genomic imprinting. However, an in vitro methylation assay illustrated that the methylation of these CpG sites could repress the expression of the luciferase reporter gene. Furthermore, chromatin immunoprecipitation with anti-Specificity protein 1 (Sp1) antibody showed that Sp1 could bind to the CGIs in the ZNF230/Znf230 gene promoter. Thus, we propose that the unmethylated state of ZNF230/Znf230 CGIs may be a prerequisite for their expression but not sufficient for their abundant expression in the testis, and that Sp1 binding may be one factor involved in preserving the methylation-free state of ZNF230/Znf230 CGIs.

In-solution hybrid capture of bisulfite-converted DNA for targeted bisulfite sequencing of 174 ADME genes

DNA methylation is one of the most important epigenetic alterations involved in the control of gene expression. Bisulfite sequencing of genomic DNA is currently the only method to study DNA methylation patterns at single-nucleotide resolution. Hence, next-generation sequencing of bisulfite-converted DNA is the method of choice to investigate DNA methylation profiles at the genome-wide scale. Nevertheless, whole genome sequencing for analysis of human methylomes is expensive, and a method for targeted gene analysis would provide a good alternative in many cases where the primary interest is restricted to a set of genes.Here, we report the successful use of a custom Agilent SureSelect Target Enrichment system for the hybrid capture of bisulfite-converted DNA. We prepared bisulfite-converted next-generation sequencing libraries, which are enriched for the coding and regulatory regions of 174 ADME genes (i.e. genes involved in the metabolism and distribution of drugs). Sequencing of these libraries on Illumina’s HiSeq2000 revealed that the method allows a reliable quantification of methylation levels of CpG sites in the selected genes, and validation of the method using pyrosequencing and the Illumina 450K methylation BeadChips revealed good concordance.

Promoter DNA Methylation Regulates Murine SUR1 (Abcc8) and SUR2 (Abcc9) Expression in HL-1 Cardiomyocytes

Two mammalian genes encode the SURx (SUR1, Abcc8 and SUR2, Abcc9) subunits that combine with Kir6.2 (Kcnj11) subunits to form the ATP-sensitive potassium (KATP) channel in cardiac myocytes. Different isoform combinations endow the channel with distinct physiological and pharmacological properties, and we have recently reported that the molecular composition of sarcolemmal KATP channels is chamber specific in the mouse heart. KATP channel composition is determined by what subunits are expressed in a cell or tissue. In the present study, we explore the role of CpG methylation in regulating SUR1 and SUR2 expression. In HL-1 cardiomyocytes, as in atrial myocytes, SUR1 expression is markedly greater than SUR2. Consistent with CpG methylation-dependent silencing of SUR2 expression, bisulfite sequencing of genomic DNA isolated from HL-1 cells demonstrates that 57.6% of the CpGs in the promoter region of the SUR2 gene are methylated, compared with 0.14% of the the CpG residues in the SUR1 sequence. Moreover, treatment with 10 µM 5-aza-2′-deoxycytidine (Aza-dC) significantly increased both the unmethylated fraction of the SUR2 CpG island and mRNA expression. However, we cannot rule out additional mechanisms of Aza-dC action, as Aza-dC also causes a decrease in SUR1 expression and lower doses of Aza-dC do not alter the unmethylated DNA fraction but do elicit a small increase in SUR2 expression. The conclusion that DNA methylation alone is not the only regulator of SUR subunit expression is also consistent with observations in native myocytes, where the CpG islands of both SUR genes are essentially unmethylated in both atrial and ventricular myocytes. Collectively, these data demonstrate the potential for CpG methylation to regulate SURx subunit expression and raises the possibility that regulated or aberrant CpG methylation might play a role in controlling channel structure and function under different physiological conditions or different species.

Acetyl-l-carnitine prevents homocysteine-induced suppression of Nrf2/Keap1 mediated antioxidation in human lens epithelial cells.

Previous studies have revealed that high levels of serum homocysteine (Hcy) are closely associated with the development of juvenile and age-related cataracts. An increased concentration of Hcy is likely to induce gene specific demethylation in DNA promoter regions. The aim of the present study was to prevent this demethylation by administering acetyl-l-carnitine (ALCAR) to human lens epithelial cells (HLECs). Different concentrations of Hcy were used to treat HLECs for 3, 6, 12 and 24 h and the findings were used to determine the optimum dose to induce endoplasmic reticulum (ER) stress. Similarly, the concentration of ALCAR was standardized. The production of reactive oxygen species (ROS) and the percentage of cells undergoing cell death were measured. The levels of antioxidants, ER stress-associated proteins, mRNA levels of nuclear factor erythroid-2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1) and promoter DNA methylation of the Keap1 gene were also assessed. Hcy was observed to induce ER stress, produce ROS and lead to cell death. However, administration of ALCAR prevented these effects to a significant degree. Additionally, western blot analysis revealed that ALCAR increased the levels of antioxidant proteins, including catalase, superoxide dismutase, glutathione peroxidase, Nrf2, Keap1 and glutathione. Similarly, the reverse transcription-quantitative polymerase chain reaction experiments on Nrf2 and Keap1, as well as the bisulfite genomic DNA sequencing analysis revealed a preventive effect of ALCAR against Hcy-induced ER stress. The ER stress-induced activation of the unfolded protein response is responsible for demethylation of Keap1 promoter DNA to activate the expression of the Keap1 protein, which then increases the targeting of Nrf2 for proteosomal degradation. This decrease in Nrf2 activity represses the transcription of numerous antioxidant enzyme genes and alters the redox-balance towards lens oxidation. However, treatment with ALCAR led to significant protection from these effects. The present results suggested that ALCAR either prevents or ameliorates the actions of the antioxidant system in HLECs at the level of the protein and the gene. Further advanced studies are required for the development of ALCAR as an anti-cataract agent.

The Significance of Megakaryocytic Transcription Factor Fli1 and Erythroid Transcription Factor EKLF in the Ribosomopathies: 5q Minus Syndrome and Diamond-Blackfan Anemia. the Role of Fli1 in p53 Regulation and in 5q Minus Syndrome Megakaryopoiesis,

Abstract 3825 Introduction:5q- syndrome and DBA belong to ribosomopathies. 5q- syndrome patients (pts) have haploinsufficient RPS14 gene and approximately 50% DBA pts have mutated some of genes for ribosomal proteins. These two disorders share similar erythroid and megakaryocytic characteristics: macrocytic anemia, decreased erythropoiesis, normal platelet count and they differ in: frequent neutropenia in 5q- syndrome, rare thrombocytemia and absence of hypolobulated megakaryocytes in DBA. We recently studied two transcription factors Fli1(Friend leukemia virus integration 1) and EKLF (Erythroid Krüppel like factor, also named KLF1) involved in MEP (common megakaryocytic and erythroid progenitor) differentiation in MDS 5q-syndrome pts. Now we present the examination of these factors in DBA pts.There exists cross-antagonism between Fli1 and EKLF with probable dominance of EKLF. Fli1 mRNA is target for microRNA-l45 (miR-145) localized in common deleted region of 5q. Haploinsuffciency of miR-145 in 5q- syndrome stabilizes Fli1 mRNA and increases Fli1 (Kumar et al. Blood 2009;114 abstr.947). Fli1 is also increased by interkleukin 6. IL-6 is induced by haploinsufficiency of miR-145 and miR-146a. Fli1 gene promoter is upregulated by Fli1 itself. Transcription factor PU.1 is positive regulator of Fli1 gene expression. In mice Fli1 regulates p53 via MDM2 (mouse double minute 2 or HDM2 in humans), an E3 ubiquitin ligase, which promotes p53 degradation in proteasomes (Truong et al. Oncogene 2005;24:962–69). Patients and Methods:Mononuclear cells were isolated from blood of 31 pts with 5q- syndrome, 26 MDS low risk pts with normal chromosome 5, 16 healthy controls and 10 DBA pts (7 with mutation of RP and 3 without proved mutation). Further, mononuclear cells were obtained from bone marrow of 17 pts with 5q-syndrome, 15 MDS low risk pts with normal chromosome 5, and 8 healthy controls. In total RNA of blood and bone marrow mononucklear cells Fli1, EKLF, p53, HDM2 and PU.1 mRNA levels were determined by quantitative real-time PCR. Related levels of mRNAs were calculated to the level of housekeeping GAPDH mRNA. We examined methylation status of EKLF promoter region in 5q- syndrome pts by bisulfite genomic DNA sequencing. All MDS pts involved in this study were examined by FISH and in all 5q- syndrome pts the deletion of 5q31 was confirmed. Results:In 5q- syndrome pts íncreased levels of Fli1 mRNA and decreased EKLF mRNA were detected both in blood and bone marrow mononuclear cells in comparison with healthy controls. In MDS low risk pts with normal chromosome 5 the increased Fli1 mRNA was found only in peripheral blood. There was no difference between EKLF mRNA levels in pts and in controls.In 8 of 10 DBA pts EKLF mRNA was decreased in blood in comparison to healthy controls, while Fli1 mRNA levels did not differ from controls with one pt exception. Methylation of EKLF gene promoter region is not responsible for the decrease EKLF mRNA in 5q- syndrome. Significantly increased level of p53 mRNA was found only in bone marrow of 5q- pts but not in blood of 5q- or DBA pts. There was no significant difference in HDM2 and PU.1 mRNA levels in blood and bone marrow of both groups of MDS pts and also in blood of DBA pts. Discussion:Significant EKLF decrease in all pts with 5q- syndrome and in 8 of 10 DBA pts corresponds to anemia and decreased erythropoiesis in both groups, but the cause of the EKLF decrease remains unknown. In 5q- syndrome low EKLF might be explained by the cross-antagonism between Fli1 and EKLF.Most significant is high Fli1 in all 5q- pts. This finding is important for the explanation of effective megakaryopoiesis contrary to defective erythropoiesis. The situation is different in DBA pts, where Fli1 mRNA is not increased with one exception (pt with atypical RPL5 mutation).Increased Fli1 might be the clue for the maintenance of effective megakaryopoiesis in 5q- syndrome. In erythroid cells ribosomal proteins inhibit HDM2, p53 is not degraded and increased p53 enhances apoptosis of erythroid cells. High Fli1 in megakaryocytic cells transcriptionally stimulates HDM2. In addition, IL-6 stimulates not only Fli1 but also DNA methylase 1, which methylates TP53 promoter and silences TP53 expression. These processes prevent apoptosis of megakaryocytic cells. For effective megakaryopoiesis in DBA pts we have not explanation so far. Disclosures:No relevant conflicts of interest to declare.