Changes In Promoter Methylation Research Articles

Poly(ADP-ribose) polymerase inhibitor CEP-8983 synergizes with bendamustine in chronic lymphocytic leukemia cells in vitro

DNA repair aberrations and associated chromosomal instability is a feature of chronic lymphocytic leukemia (CLL). To evaluate if DNA repair insufficiencies are related to methylation changes, we examined the methylation of nine promoter regions of DNA repair proteins by bisulfide sequencing in 26 CLL primary samples and performed quantitative PCR on a subset of samples to examine BRCA1 expression. We also investigated if changes in cytogenetic or expression level of DNA repair proteins led to changes in sensitivity to a novel PARP inhibitor, CEP-8983, alone and in combination with bendamustine. No changes in promoter methylation were identified in BRCA1, BRCA2, FANC-C, FANC-F, FANC-L, ATM, MGMT, hMLH1 and H2AX except for two cases of minor BRCA1 hypermethylation. CLL samples appeared to have reduced BRCA1 mRNA expression uniformly in comparison to non-malignant lymphocytes irrespective of promoter hypermethylation. CEP-8983 displayed single agent cytotoxicity and the combination with bendamustine demonstrated synergistic cytotoxicity in the majority of CLL samples. These results were consistent across cytogenetic subgroups, including 17p deleted and previously treated patients. Our results provide rationale for further exploration of the combination of a PARP inhibitor and DNA damaging agents as a novel therapeutic strategy in CLL.

Methylation of the SLC6a2 Gene Promoter in Major Depression and Panic Disorder

Reduced function of the noradrenaline transporter (NET) has been demonstrated in patients with major depressive disorder (MDD) and panic disorder. Attempts to explain NET dysfunction in MDD and panic disorder by genetic variation in the NET gene SLC6a2 have been inconclusive. Transcriptional silencing of the SLC6a2 gene may be an alternative mechanism which can lead to NET dysfunction independent of DNA sequence. The objective of this study was to characterise the DNA methylation state of the SLC6a2 gene promoter in patients with MDD and panic disorder. SLC6a2 promoter methylation was also analysed before and after antidepressant treatment. This study was performed with DNA from blood, using bisulphite sequencing and EpiTYPER methylation analyses. Patients with MDD or panic disorder were not found to differ significantly from healthy controls in the pattern of methylation of the SLC6a2 gene promotor. While significant correlations between methylation levels at some CpG sites and physiological measures were identified, overall the variation in DNA methylation between patients was small, and the significance of this variation remains equivocal. No significant changes in SLC6a2 promoter methylation were observed in response to antidepressant treatment. Further in-depth analysis of alternative mechanisms of transcriptional regulation of the SLC6a2 gene in human health and disease would be of value.

Abstract A112: The role of microenvironment in age-related breast cancers

Abstract Aside from being a woman, age is the greatest risk factor for developing breast cancer, but there is little understanding how the aging process modifies cancer susceptibility. Multipotent mammary epithelial progenitors are putative roots of some breast cancers, thus expansion combined with increased vulnerability of these cells to transformation could potentiate cancer progression. We have shown in humans that with age, the proportion of multipotent progenitors accumulates, whereas myoepithelial cells decrease, and that these shifts have a basis in altered progenitor function. Progenitors from post-menopausal women gave rise to incompletely differentiated myoepithelial cells and to basal-like luminal cells that expressed markers normally associated with myoepithelial cells in young women. Because epithelial progenitors are thought to be cells-of-origin for a number of breast cancers, and myoepithelial cells are thought to be tumor suppressive, we hypothesize that these tissue-level changes make post-menopausal women more vulnerable to malignant progression. Age-related tissue changes arise in parallel with age-dependent gene expression patterns that have unknown functional consequences and unknown origins. Here we demonstrate that utilizing biomimetic substrata with tuned physical and molecular properties to probe primary human mammary progenitors from women aged 16 to 91 years can reveal age-related functional consequences. As increased breast density is associated with greater breast cancer risk and stiff mechanical microenvironments are known to exacerbate malignant cell behavior, we examined how normal progenitors responded to changes in elastic modulus. Age-related changes in Rho and HIPPO pathway mechano-transduction and transcriptional regulation contributed to accumulation of progenitors by preventing normal differentiation in response to mechanical stimuli. Young progenitors gave rise to more luminal cells on compliant substrata and to more myoepithelial cells on stiffer substrata. However, post-menopausal progenitors were insensitive to physiological mechanical changes and differentiated stochastically instead. To better understand the genesis of the gene expression patterns underlying these age-related functional states, molecular analyses of co-cultures established with primary myoepithelial and luminal epithelial lineages from combinations of young and old individuals were preformed. Age-related gene expression states in luminal cells were imposed through heterotypic cell-cell interactions. Changes in gene expression were coincident with changes in DNA promoter methylation and expression of histone modifying enzymes. We propose that aging results in a continuum of microenvironmental changes that impose epigenetically regulated metastable gene expression states, which are the basis of age-related cell functions that ultimately lead to vulnerable tissue-level phenotypes. Citation Format: Fanny A. Pelissier, Masaru Miyano, James C. Garbe, Martha R. Stampfer, Mark A. LaBarge. The role of microenvironment in age-related breast cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A112.

Correlation between miRNA-targeted-gene promoter methylation and miRNA regulation of target genes

Background miRNA regulation of target genes and promoter methylation are known to be the primary mechanisms underlying the epigenetic regulation of gene expression. However, how these two processes cooperatively regulate gene expression has not been extensively studied.Methods Gene expression and promoter methylation profiles of 270 distinct human cell lines were obtained from Gene Expression Omnibus. P-values that describe both miRNA-targeted-gene promoter methylation and miRNA regulation of target genes were computed using the MiRaGE method proposed recently by the author.Results Significant changes in promoter methylation were associated with miRNA targeting. It was also found that miRNA-targeted-gene promoter hypomethylation was related to differential target gene expression; the genes with miRNA-targeted-gene promoter hypomethylation were downregulated during cell senescence and upregulated during cellular differentiation. Promoter hypomethylation was especially enhanced for genes targeted by miR-548 miRNAs, which are non-conserved, primate-specific miRNAs that are typically expressed at lower levels than the frequently investigated conserved miRNAs. miRNA-targeted-gene promoter methylation may also be related to the seed region features of miRNA.Conclusions It was found that promoter methylation was correlated to miRNA targeting. Furthermore, miRNA-targeted-gene promoter hypomethylation was especially enhanced in promoters of genes targeted by miRNAs that are not strongly expressed (e.g., miR-548 miRNAs) and was suggested to be highly related to some seed region features of miRNAs.

Expression and promoter methylation changes of the P15INK4b during ex vivo cord blood CD34+ cell expansion following co-culture with mesenchymal stromal cells

BackgroundBecause of the insufficient number of cord blood hematopoietic stem cells (CB-HSC), expansion of these cells seems to be important for clinical application in adults. Cell cycle inhibitors are important regulators in normal hematopoietic regeneration. In this study, mRNA expression and promoter methylation status of p15INK4b were evaluated during CB-HSC ex vivo expansion using cytokines and in co-culture system with a mesenchymal stem cells (MSCs) feeder layer.MethodsEx vivo cultures of CB-HSCs were performed in three culture conditions for 14 days: cytokines with an MSCs feeder layer, cytokines without a MSCs feeder layer, and co-culture with MSCs without cytokines. After expansion, measuring the total number of cells, CD34+ cells, and CFU assay was performed. Methylation status of the p15INK4b gene promoter was analyzed using methylation-specific polymerase chain reaction and p15 mRNA expression was evaluated by real-time reverse transcriptase polymerase chain reaction.ResultsMaximum CB-HSC expansion was observed on day 10 of expansion. The data showed that after 10 days, p15 mRNA expression in the expanded cells in all the three culture conditions was higher than in CD34+ fresh cells (P < 0.01). p15 gene promoter of expanded CD34+ cells remained in an unmethylated form just like fresh CD34+ cells in all the three culture conditions at days 5, 10, and 14 of culture.ConclusionsExpression of p15INK4b in HSCs was not decreased during ex vivo expansion. Also, no methylation of p15 promoter was observed, otherwise it would be capable of initiating some leukemic cell progression or disruption in hematopoietic regeneration.

Diet-dependent Alterations of Hepatic Scd1 Expression are Accompanied by Differences in Promoter Methylation

Obesity and alterations of lipid homeostasis are hallmarks of the metabolic syndrome and largely influenced by the dietary conditions of the individual. Although heritability is considered to be a major risk factor, the almost 40 candidate genes identified by genome-wide association studies (GWAS) so far account for only 5-10% of the observed variance in BMI in human subjects. Alternatively, diet-induced changes of epigenetic gene regulation might be involved in disturbed lipid homeostasis and weight development. The aim of this study was to investigate how a high-carbohydrate diet (HCD; 70 kcal% from carbohydrates, 10 kcal% from fat) or a high-fat diet (HFD; 20 kcal% from carbohydrates, 60 kcal% from fat) affects hepatic expression of genes involved in fatty acid metabolism and if these alterations are correlated to changes in promoter methylation. Expression of stearoyl-CoA desaturase 1 (Scd1) was lower in livers from HFD-fed C57BL/6 J mice compared to HCD-fed animals and correlated inversely with the degree of DNA methylation at 2 distinct, adjacent CpG sites in the Scd1 promoter. In contrast, expression of transcription factors peroxisome proliferator activated receptor alpha and gamma (Ppara, Pparg), and sterol regulatory element binding transcription factor 1 (Srebf1) was not affected. The degree of hepatic Scd1 promoter methylation at these CpG sites correlated positively to fat mass and serum leptin levels, whereas serum ghrelin levels were inversely correlated with methylation at both CpG sites. Taken together, hepatic expression of Scd1 is differentially affected by carbohydrate- and lipid content of the diet. These differences in Scd1 expression are associated with altered promoter methylation, indicating that diets affect lipid metabolism in the liver via epigenetic mechanisms.

Infection with influenza A viruses causes changes in promoter DNA methylation of inflammatory genes

Replication of influenza virus in the host cells results in production of immune mediators like cytokines. Excessive secretion of cytokines (hypercytokinemia) has been observed during highly pathogenic avian influenza virus (HPAI-H5N1) infections resulting in high fatality rates. The exact mechanism of hypercytokinemia during influenza virus infection is still not known completely. As promoter DNA methylation changes are linked with expression changes in genes, we intend to identify whether changes in promoter DNA methylation have any role in expression of cytokines during influenza A virus infection. A panel of 24 cytokine genes and genes known to be involved in inflammatory response were analyzed for their promoter DNA methylation changes during influenza A virus infections. Four different strains of influenza A viruses, viz. H5N1, H1N1, pandemic (2009) H1N1, and a vaccine strain of H5N1, were used for the study. We found seven of the total 24 inflammatory genes studied, showing significant changes in their promoter methylation levels in response to virus infection. These genes included proinflammatory cytokines CXCL14, CCL25, CXCL6, and interleukines IL13, IL17C, IL4R. The changes in DNA methylation levels varied across different strains of influenza viruses depending upon their virulence. Significant promoter hypomethylation in IL17C and IL13 genes was observed in cells infected with HPAI-H5N1 virus compared with other influenza viruses. This decrease in methylation was found to be positively correlating with the increased expression of these genes. Analysis of IL17C promoter region using bisulfite sequencing resulted in identification of a CpG site within Retinoid X receptor-alpha (RXR-α) transcription factor binding site undergoing demethylation specifically in H5N1-infected cells but not in other influenza-infected cells. Thus, the study could demonstrate that changes in promoter methylation in certain specific cytokine genes actually have a possible role in their expression changes during influenza A virus infection.

Abstract 3643: Fetal exposure to diethylstilbestrol and DNA methylation in adult women.

Abstract In utero diethylstilbestrol (DES) exposure has long term consequences including increased risk of vaginal and breast cancer. Epigenetic factors are suspected to contribute to some of these increased risks and animal studies show DES-mediated effects on DNA methylation in both uterine tissue and blood. However, there are currently no studies examining the effect of fetal DES exposure on DNA methylation in humans. Therefore, we investigated differences in DNA methylation between women exposed to DES in utero compared to unexposed women. All our study subjects were participants in the Sister Study -a prospective cohort focused on environmental and familial risk factors for breast cancer and other diseases. Within this study 1,156 women (2.3%) said that they were definitely or probably exposed to DES in utero. In a special sub study (including both women who reported positive exposure and women who reported no exposure), we confirmed exposure history with the woman's mother and from those with matching mother daughter reports we randomly selected 100 exposed and 100 unexposed women. We analyzed DNA methylation patterns in blood using the Illumina 450k array to assess methylation at 473,844 CpG sites. Primary analysis did not reveal any differentially methylated CpGs (dmCpGs) below the false discovery rate (FDR, q&amp;lt;0.05), nonetheless there were about 8000 dmCpGs with p-values &amp;lt;0.05. Stratification of women into subgroups -based on menopausal status, fertility related diagnoses or hormone intake- did not alter the results. Previous work using mouse models shows gene specific changes in gene expression and DNA methylation following perinatal exposure to DES. When we compared these expression changes in ten genes to changes in promoter methylation in our data we found an inverse correlation between gene expression in mouse and the direction of methylation in our samples. This was especially evident in the ERBB2 promoter region. We also examined the subset of genes known to directly interact with DES: in four of the seven direct DES targets (AR, TTR, AFP, and GLI3) we found a majority of the 5’ CpGs had lower methylation levels in exposed women, although these differences were not statistically significant. This is the first study investigating genome wide methylation changes in women subjected to an unusual and very potent prenatal exposure that is known to have severe consequences into adulthood. Study weaknesses include lack of information regarding dose and exact timing of exposure as well as use of whole blood instead of uterine target tissue, but we still observe changes at p&amp;lt;0.05. If DNA methylation changes are detectable in blood decades after exposure this strengthens the idea that DES exposure leads to epigenetic changes and should be possible to verify even further using samples from uterine or vaginal tissue. Citation Format: S Sophia Harlid, Zongli Xu, Vijayalakshmi Panduri, Aimee D'Aloisio, Lisa DeRoo, Dale Sandler, Jack A. Taylor. Fetal exposure to diethylstilbestrol and DNA methylation in adult women. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3643. doi:10.1158/1538-7445.AM2013-3643

Effects of folylpolyglutamate synthase modulation on CpG promoter DNA methylation and gene expression in human colon cancer cells

Folylpolyglutamate synthase (FPGS) facilitates intracellular retention of folate by polyglutamylation, thereby playing a critical role in folate homeostasis. We investigated whether FPGS modulation would affect CpG promoter methylation and gene expression profiles in human HCT116 colon cancer cells. Illumina HT‐12 and Infinium Methylation assay were used, and functional analysis was performed by Ingenuity Pathway Analysis. We validated mRNA expression of selected genes by qRT‐PCR. 2897 genes (most involved pathways: cell cycle and DNA replication, recombination, and repair) were differentially expressed in the FPGS‐overexpressed cells, while 359 genes (most involved pathways: cell death and cell cycle) were differentially expressed in the FPGS‐inhibited cells. 864 and 626 genes showed altered CpG promoter methylation associated with FPGS overexpression and inhibition, respectively. An integrated analysis revealed 65 and 6 differentially expressed genes involved in cell cycle, cell death, and cell‐to‐cell signaling and interaction, expression of which was regulated by CpG promoter methylation changes associated with FPGS overexpression and inhibition, respectively. FPGS modulation can affect differential gene expression and CpG promoter methylation involved in important biological pathways, and some of the observed differential gene expression appear to be epigenetically regulated.Grant Funding Source: CIHR Fund 14126

Effects of γ‐glutamyl hydrolase modulation on CpG promoter DNA methylation and gene expression in human colon cancer cells

γ‐Glutamyl hydrolase (GGH) plays an important role in folate homeostasis by catalyzing hydrolysis of polyglutamylated folate into monoglutamates, thereby facilitating export of folate from cell. We investigated whether GGH modulation would affect CpG promoter methylation and gene expression profiles in human HCT116 colon cancer cells. Illumina HT‐12 and Infinium Methylation assay were performed in the GGH‐modulated HCT116 cells, and Ingenuity Pathway Analysis was used for functional analysis. The mRNA expression of selected genes, expression of which was epigenetically regulated was confirmed by qRT‐PCR. We identified 153 differentially expressed genes (most involved pathways: cellular movement and cell death) associated with GGH overexpression, and 321 differentially expressed genes (most involved pathways: cell death and cell cycle) associated with GGH inhibition. 905 and 1869 genes showed altered CpG promoter methylation associated with GGH overexpression and inhibition, respectively. An integrated analysis revealed 5 and 16 genes, expression of which was regulated by CpG promoter methylation changes associated with GGH overexpression and inhibition, respectively. Thus, GGH modulation can affect differential gene expression and CpG promoter DNA methylation involved in important biological pathways and some of the observed altered gene expression appear to be epigenetically regulated.Grant Funding Source: CIHR Fund 14126

Correlation between miRNA-targeted-gene promoter methylation and miRNA regulation of target genes

Background miRNA regulation of target genes and promoter methylation are known to be the primary mechanisms underlying the epigenetic regulation of gene expression. However, how these two processes cooperatively regulate gene expression has not been extensively studied. Methods Gene expression and promoter methylation profiles of 271 distinct human cell lines were obtained from gene expression omnibus. P-values that describe both miRNA-targeted-gene promoter methylaion and miRNA regulation of target genes were computed using the MiRaGE method proposed recently by the author.Results Significant changes in promoter methylation were associated with miRNA targeting. It was also found that miRNA-targeted-gene promoter hypomethylation was related to differential target gene expression; the genes with miRNA-targeted-gene promoter hypomethylation were downregulated during cell senescence and upregulated during cellular differentiation. Promoter hypomethylation was especially enhanced for genes targeted by miR-548 miRNAs, which are non-conserved, primate-specific miRNAs that are typically expressed at lower levels than the frequently investigated conserved miRNAs.Conclusions It was found that promoter methylation was affected by miRNA targeting. Furthermore, miRNA-targeted-gene promoter hypomethylation is suggested to facilitate gene regulation by miRNAs that are not strongly expressed (e.g., miR-548 miRNAs).

Decreased cortical muscarinic M1 receptors in schizophrenia are associated with changes in gene promoter methylation, mRNA and gene targeting microRNA

Many studies have shown decreased cortical muscarinic M1 receptors (CHRM1) in schizophrenia (Sz), with one study showing Sz can be separated into two populations based on a marked loss of CHRM1 (∼75%) in ∼25% of people (Def-Sz) with the disorder. To better understand the mechanism contributing to the loss of CHRM1 in Def-Sz, we measured specific markers of gene expression in the cortex of people with Sz as a whole, people differentiated into Def-Sz and people with Sz that do not have a deficit in cortical CHRM1 (Non-Def-Sz) and health controls. We now report that cortical CHRM1 gene promoter methylation and CHRM1 mRNA are decrease in Sz, Def-Sz and Non-Def-Sz but levels of the micro RNA (miR)-107, a CHRM1 targeting miR, are increased only in Def-Sz. We also report in vitro data strongly supporting the notion that miR-107 levels regulate CHRM1 expression. These data suggest there is a reversal of the expected inverse relationship between gene promoter methylation and CHRM1 mRNA in people with Sz and that a breakdown in gene promoter methylation control of CHRM1 expression is contributing to the global pathophysiology of the syndrome. In addition, our data argues that increased levels of at least one miR, miR-107, is contributing to the marked loss of cortical CHRM1 in Def-Sz and this may be a differentiating pathophysiology. These latter data continue to support the hypothesis that microRNAs (miRNA) have a role in the underlying neurobiology of Sz but argue they are differentially affected in subsets of people within that syndrome.

Correlation between miRNA-targeting-specific promotermethylation and miRNA regulation of target genes

Background miRNA regulation of target genes and promoter methylation were known to be the primary mechanisms underlying the epigenetic regulation of gene expression. However, how these two processes cooperatively regulate gene expression has not been extensively studied. Methods Gene expression and promoter methylation profiles of 271 distinct human cell lines were obtained from gene expression omnibus. P-values that describe both miRNA-targeting-specific promoter methylation and miRNA regulation of target genes were computed with the MiRaGE method proposed recently by the author. Results We found that promoter methylation was miRNA-targeting-specific. In other words, changes in promoter methylation were associated with miRNA binding at target genes. It was also found that miRNA-targeting-specific promoter hypomethylation was related to miRNA regulation; the genes with miRNA-targeting-specific promoter hypomethylation were downregulated during cell senescence and upregulated during cellulardierentiation. Promoter hypomethylation was especially enhanced for genes targeted by miR-548 miRNAs, which are non-conserved, and primate-specific miRNAs that are typically expressed at lower levels than the frequently investigated conserved miRNAs. Conclusions It was found that promoter methylation was affected by miRNA targeting. Furthermore, miRNA-targeting-specific promoter hypomethylation was suggested to facilitate gene regulation by miRNAs that are not strongly expressed (e.g., miR-548 miRNAs).

Correlation between miRNA-targeting-specific promotermethylation and miRNA regulation of target genes

Background miRNA regulation of target genes and promoter methylation were known to be the primary mechanisms underlying the epigenetic regulation of gene expression. However, how these two processes cooperatively regulate gene expression has not been extensively studied. Methods Gene expression and promoter methylation profiles of 271 distinct human cell lines were obtained from gene expression omnibus. P-values that describe both miRNA-targeting-specific promoter methylation and miRNA regulation of target genes were computed with the MiRaGE method proposed recently by the author. Results We found that promoter methylation was miRNA-targeting-specific. In other words, changes in promoter methylation were associated with miRNA binding at target genes. It was also found that miRNA-targeting-specific promoter hypomethylation was related to miRNA regulation; the genes with miRNA-targeting-specific promoter hypomethylation were downregulated during cell senescence and upregulated during cellulardierentiation. Promoter hypomethylation was especially enhanced for genes targeted by miR-548 miRNAs, which are non-conserved, and primate-specific miRNAs that are typically expressed at lower levels than the frequently investigated conserved miRNAs. Conclusions It was found that promoter methylation was affected by miRNA targeting. Furthermore, miRNA-targeting-specific promoter hypomethylation was suggested to facilitate gene regulation by miRNAs that are not strongly expressed (e.g., miR-548 miRNAs).

Genome-wide analysis of DNA methylation differences in muscle and fat from monozygotic twins discordant for type 2 diabetes.

BackgroundMonozygotic twins discordant for type 2 diabetes constitute an ideal model to study environmental contributions to type 2 diabetic traits. We aimed to examine whether global DNA methylation differences exist in major glucose metabolic tissues from these twins.Methodology/Principal FindingsSkeletal muscle (n = 11 pairs) and subcutaneous adipose tissue (n = 5 pairs) biopsies were collected from 53–80 year-old monozygotic twin pairs discordant for type 2 diabetes. DNA methylation was measured by microarrays at 26,850 cytosine-guanine dinucleotide (CpG) sites in the promoters of 14,279 genes. Bisulfite sequencing was applied to validate array data and to quantify methylation of intergenic repetitive DNA sequences. The overall intra-pair variation in DNA methylation was large in repetitive (LINE1, D4Z4 and NBL2) regions compared to gene promoters (standard deviation of intra-pair differences: 10% points vs. 4% points, P<0.001). Increased variation of LINE1 sequence methylation was associated with more phenotypic dissimilarity measured as body mass index (r = 0.77, P = 0.007) and 2-hour plasma glucose (r = 0.66, P = 0.03) whereas the variation in promoter methylation did not associate with phenotypic differences. Validated methylation changes were identified in the promoters of known type 2 diabetes-related genes, including PPARGC1A in muscle (13.9±6.2% vs. 9.0±4.5%, P = 0.03) and HNF4A in adipose tissue (75.2±3.8% vs. 70.5±3.7%, P<0.001) which had increased methylation in type 2 diabetic individuals. A hypothesis-free genome-wide exploration of differential methylation without correction for multiple testing identified 789 and 1,458 CpG sites in skeletal muscle and adipose tissue, respectively. These methylation changes only reached some percentage points, and few sites passed correction for multiple testing.Conclusions/SignificanceOur study suggests that likely acquired DNA methylation changes in skeletal muscle or adipose tissue gene promoters are quantitatively small between type 2 diabetic and non-diabetic twins. The importance of methylation changes in candidate genes such as PPARGC1A and HNF4A should be examined further by replication in larger samples.

Ethanol‐Induced Htr3a Promoter Methylation Changes in Mouse Blood and Brain

Abnormal DNA methylation has been observed in promoter regions of a number of genes in human alcoholics. It is unclear whether DNA methylation changes in alcoholics result directly from alcohol consumption or predated the occurrence of alcohol abuse or dependence and whether altered DNA methylation influences gene expression. We investigated ethanol (EtOH)-induced DNA methylation changes in mouse serotonin receptor 3a gene (Htr3a). A 5-day drinking-in-the-dark paradigm was applied to 28 male outbred CD-1 mice (15 EtOH-drinking and 13 water-drinking). The Sequenom MassARRAY approach was used to quantify methylation levels of 8 CpGs around Htr3a transcription start site in trunk blood and 9 brain regions (dorsomedial prefrontal cortex [DMPFC], ventromedial prefrontal cortex, ventral tegmental area, dorsolateral striatum, dorsomedial striatum [DMSTR], ventral striatum, amygdala, hippocampus [HIPPO], and cerebellum). DNA methylation differences between the 2 groups of mice (EtOH- and water-drinking) were analyzed using multivariate analysis of covariance with consideration of EtOH consumption amount. Expression levels of Htr3a in the DMSTR were measured by real-time PCR in 14 EtOH-drinking and 14 water-drinking male CD-1 mice. EtOH drinking increased methylation levels of specific Htr3a promoter CpGs in mouse blood (CpG-27: p = 0.028; CpG+54: p = 0.044) and HIPPO (CpG+151: p = 0.012) but reduced methylation levels of specific Htr3a promoter CpGs in mouse DMSTR (CpG-96: p = 0.020; CpG-27: p = 0.035) and DMPFC (CpG+138: p = 0.011; CpG+151: p = 0.040). Nevertheless, methylation levels of Htr3a promoter CpGs in 6 other brain regions were not significantly altered by EtOH consumption. Additionally, the expression level of Htr3a in the DMSTR was 1.43-fold higher in alcohol-drinking mice than in water-drinking mice (p = 0.044). Our findings indicate that alcohol consumption may induce tissue-specific DNA methylation changes and further suggest that Htr3a promoter methylation levels may be reversely correlated with Htr3a expression levels in specific brain regions such as DMSTR.

Transcriptional Regulation of the Protocadherin β Cluster during Her-2 Protein-induced Mammary Tumorigenesis Results from Altered N-Glycan Branching

Changes in the levels of N-acetylglucosaminyltransferase V (GnT-V) can alter the function of several types of cell surface receptors and adhesion molecules by causing altered N-linked glycan branching. Using a her-2 mammary tumor mouse model, her-2 receptor signaling was down-regulated by GnT-V knock-out, resulting in a significant delay in the onset of her-2-induced mammary tumors. To identify the genes that contributed to this GnT-V regulation of early events in tumorigenesis, microarray analysis was performed using her-2 induced mammary tumors from wild-type and GnT-V-null mice. We found that 142 genes were aberrantly expressed (>2.0-fold) with 64 genes up-regulated and 78 genes down-regulated after deletion of GnT-V. Among differentially expressed genes, the expression of a subgroup of the cadherin superfamily, the protocadherin β (Pcdhβ) cluster, was up-regulated in GnT-V-null tumors. Altered expression of the Pcdhβ cluster in GnT-V-null tumors was not due to changes in promoter methylation; instead, impaired her-2-mediated signaling pathways were implicated at least in part resulting from reduced microRNA-21 expression. Overexpression of Pcdhβ genes inhibited tumor cell growth, decreased the proportion of tumor-initiating cells, and decreased tumor formation in vivo, demonstrating that expression of the Pcdhβ gene cluster can serve as an inhibitor of the transformed phenotype. Our results suggest the up-regulation of the Pcdhβ gene cluster as a mechanism for reduced her-2-mediated tumorigenesis resulting from GnT-V deletion.

Dietary protein restriction and excess of pregnant German Landrace sows induce changes in hepatic gene expression and promoter methylation of key metabolic genes in the offspring

Maternal nutrition during gestation has important effects on offspring gene expression mediated by DNA methylation. In order to evaluate the effect of restricted and excess protein intake during gestation, hepatic gene expression and DNA methylation of key metabolic genes NR3C1, PPARα, HMGCR, PGC1α, INSR and CYP2C34 were investigated. Liver samples of German Landrace offspring were collected at Gestational Day 95, at birth, at weaning and from finisher pigs. Gene expression in foetal liver revealed significant differences between the control group (CO) and the low-protein group (LP) in HMGCR (P<.0001), INSR (P=.0003), NR3C1 (P=.020) and PGC1α (P=.003). At birth INSR (P=.032), PPARα (P=.0006) and CYP2C34 (P<.0001) showed significant differences between LP and CO. CYP2C34 was significantly increased in the high-protein group (HP) compared to CO (P=.001). At weaning, INSR was significantly higher expressed in LP than in CO (P=.018). HMGCR showed a significant decrease of transcript amount in HP compared to CO (P=.0006). Furthermore, we studied the question whether gene expression differences between distinct diet groups are a result of differential DNA methylation status. CpG sites in the 5′-flanking region of CYP2C34 showed a significant positive correlation with transcript amount in LP (nt −137: R=0.67, P<.0001; nt −112: R=0.54, P=.003). In NR3C1 methylation, differences in the CpG island were negatively correlated with gene expression data in LP (R=−0.34, P=.032). The mean of methylation of PPARα over CpG sites from nt −220 to −11 was significantly increased in the LP group compared with CO (P=.043). These data suggest an influence of DNA methylation in nutrient-dependent transcriptional regulation of NR3C1, PPARα and CYP2C34.

PSEN1 Promoter Demethylation in Hyperhomocysteinemic TgCRND8 Mice is the Culprit, not the Consequence

In recent years, in parallel with the growing awareness of the multifactorial nature of Late Onset Alzheimer's Disease, the possibility that epigenetic mechanisms could be involved in the onset and/or progression of the pathology assumed an increasingly intriguing and leading role in Alzheimer's research. Today, many scientific reports indicate the existence of an epigenetic drift during ageing, in particular in Alzheimer's subjects. At the same time, experimental evidences are provided with the aim to demonstrate the causative or consequential role of epigenetic mechanisms. Our research group was involved in the last ten years in studying DNA methylation, the main epigenetic modification, in relationship to altered one-carbon metabolism (namely high homocysteine and low B vitamins levels), in Alzheimer's experimental models. Our previous findings about the demethylation of Presenilin1 gene promoter in nutritionally-induced hyperhomocysteinemia in a transgenic mouse model clearly demonstrated that Presenilin1 is regulated by DNA methylation. One of the open questions raised by our studies was if the observed demethylation was solely due to the induced imbalance of one-carbon metabolism or could be a response to the massive deposition of amyloid plaques in transgenic mice. Here we analyzed old (10 months) mice under standard diet in order to evidence possible changes in Presenilin1 promoter methylation in transgenic (TgCRND8 mice, carrying a double-mutated human APP transgene) vs. wt mice (129Sv) after prolonged exposure to amyloid. We found no differences in Presenilin1 methylation despite a slight increase in gene expression; these results suggest that amyloid production is not responsible for Presenilin1 demethylation in TgCRND8 mice brain.

Sulforaphane Induction of p21Cip1 Cyclin-dependent Kinase Inhibitor Expression Requires p53 and Sp1 Transcription Factors and Is p53-dependent

Sulforaphane (SFN) is an important cancer preventive agent derived from cruciferous vegetables. We show that SFN treatment suppresses normal human keratinocyte proliferation via a mechanism that involves increased expression of p21(Cip1). SFN treatment produces a concentration-dependent increase in p21(Cip1) promoter activity via a mechanism that involves stabilization of the p53 protein leading to increased p53 binding to the p21(Cip1) promoter p53 response elements. The proximal p21(Cip1) promoter GC-rich Sp1 factor binding elements are also required, as the SFN-dependent increase is lost when these sites are mutated. SFN treatment increases Sp1 binding to these elements, and the response is enhanced in the presence of exogenous Sp1 and reduced in the presence of ΔN-Sp3. CpG island methylation alters p21(Cip1) promoter activity some systems; however, expression in SFN-treated keratinocytes does not involve changes in proximal promoter methylation. The promoter is minimally methylated, and the methylation level is not altered by SFN treatment. This study indicates that SFN increases p21(Cip1) promoter transcription via a mechanism that involves SFN-dependent stabilization of p53 and increased p53 and Sp1 binding to their respective response elements in the p21(Cip1) promoter. These results are in marked contrast to the mechanisms observed in skin cancer cell lines and suggest that SFN may protect normal keratinocytes from damage while causing cancer cells to undergo apoptosis.