Detection of DNA Methylation by MeDIP and MBDCap Assays: An Overview of Techniques.

DNA methylation has been characterized as the representative example of epigenetic modifications and implicated in numerous biological processes, such as genomic imprinting and X chromosome inactivation. It primarily occurs at CpG dinucleotides in mammals and plays a critical role in transcriptional regulations. Examination of DNA methylation patterns in gene(s) or across a genome is vital to understand the role of epigenetic modulation in the progress of development and tumorigenesis. Currently, lots of approaches have been developed to investigate DNA methylation patterns for either limited regions or genome-scale studies, but some of them rely on using restriction enzymes. In this chapter, we describe two commonly used protocols to detect enrichment of methylated DNA regions, namely methylated immunoprecipitation (MeDIP) and capture of methylated DNA by methyl-CpG binding domain-based (MBD) proteins (MBDCap). They are the most economical and effective methods to study DNA methylation in either single locus or genome-wide scale.

Detection of DNA methylation by MeDIP and MBDCap assays: an overview of techniques.

DNA methylation has been characterized as the representative example of epigenetic modifications and implicated in numerous biological processes, such as genomic imprinting and X chromosome inactivation. It primarily occurs at CpG dinucleotides in mammals and plays a critical role in transcriptional regulations. Examination of DNA methylation patterns in gene(s) or across a genome is vital to understand the role of epigenetic modulation in the progress of development and tumorigenesis. Currently, lots of approaches have been developed to investigate DNA methylation patterns for either limited regions or for genome-scale studies, but some of them rely on using restriction enzymes. In this chapter, we describe two commonly used protocols to detect enrichment of methylated DNA regions, namely, methylated DNA immunoprecipitation (MeDIP) and capture of methylated DNA by methyl-CpG binding domain-based (MBD) proteins (MBDCap). They are the most economical and effective methods to study DNA methylation either at a single locus or in genome-wide scale.

Black raspberry-derived anthocyanins demethylate tumor suppressor genes through the inhibition of DNMT1 and DNMT3B in colon cancer cells.

We previously reported that oral administration of black raspberry powder decreased promoter methylation of tumor suppressor genes in tumors from patients with colorectal cancer. The anthocyanins (ACs) in black raspberries are responsible, at least in part, for their cancer-inhibitory effects. In the present study, we asked if ACs are responsible for the demethylation effects observed in colorectal cancers. Three days of treatment of ACs at 0.5, 5, and 25 µg/ml suppressed activity and protein expression of DNMT1 and DNMT3B in HCT116, Caco2 and SW480 cells. Promoters of CDKN2A, and SFRP2, SFRP5, and WIF1, upstream of Wnt pathway, were demethylated by ACs. mRNA expression of some of these genes was increased. mRNA expression of ß-catenin and c-Myc, downstream of Wnt pathway, and cell proliferation were decreased; apoptosis was increased. ACs were taken up into HCT116 cells and were differentially localized with DNMT1 and DNMT3B in the same cells visualized using confocal laser scanning microscopy. Although it was reported that DNMT3B is regulated by c-Myc in mouse lymphoma, DNMT3B did not bind with c-Myc in HCT116 cells. In conclusion, our results suggest that ACs are responsible, at least in part, for the demethylation effects of whole black raspberries in colorectal cancers.

Activation of silenced tumor suppressor genes in prostate cancer cells by a novel energy restriction-mimetic agent.

BACKGROUND: Targeting tumor metabolism by energy restriction-mimetic agents (ERMAs) has emerged as a strategy for cancer therapy/prevention. Evidence suggests a mechanistic link between ERMA-mediated antitumor effects and epigenetic gene regulation. METHODS: Microarray analysis showed that a novel thiazolidinedione-derived ERMA, CG-12, and glucose deprivation could suppress DNA methyltransferase (DNMT)1 expression and reactivate DNA methylation-silenced tumor suppressor genes in LNCaP prostate cancer cells. Thus, we investigated the effects of a potent CG-12 derivative, CG-5, vis-à-vis 2-deoxyglucose, glucose deprivation and/or 5-aza-deoxycytidine, on DNMT isoform expression (Western blotting, RT-PCR), DNMT1 transcriptional activation (luciferase reporter assay), and expression of genes frequently hypermethylated in prostate cancer (quantitative real-time PCR). Promoter methylation was assessed by pyrosequencing analysis. SiRNA-mediated knockdown and ectopic expression of DNMT1 were used to validate DNMT1 as a target of CG-5. RESULTS: CG-5 and glucose deprivation upregulated the expression of DNA methylation-silenced tumor suppressor genes, including GADD45a, GADD45b, IGFBP3, LAMB3, BASP1, GPX3, and GSTP1, but also downregulated methylated tumor/invasion-promoting genes, including CD44, S100A4, and TACSTD2. In contrast, 5-aza-deoxycytidine induced global reactivation of these genes. CG-5 mediated these epigenetic effects by transcriptional repression of DNMT1, which was associated with reduced expression of Sp1 and E2F1. SiRNA-mediated knockdown and ectopic expression of DNMT1 corroborated DNMT1's role in the modulation of gene expression by CG-5. Pyrosequencing revealed differential effects of CG-5 versus 5-aza-deoxycytidine on promoter methylation in these genes. CONCLUSIONS: These findings reveal a previously uncharacterized epigenetic effect of ERMAs on DNA methylation-silenced tumor suppressor genes, which may foster novel strategies for prostate cancer therapy.

Estrogen-mediated epigenetic repression of the imprinted gene cyclin-dependent kinase inhibitor 1C in breast cancer cells.

While tumor suppressor genes frequently undergo epigenetic silencing in cancer, how the instructions directing this transcriptional repression are transmitted in cancer cells remain largely unclear. Expression of cyclin-dependent kinase inhibitor 1C (CDKN1C), an imprinted gene on chromosomal band 11 p15.5, is reduced or lost in the majority of breast cancers. Here, we report that CDKN1C is suppressed by estrogen through epigenetic mechanisms involving the chromatin-interacting noncoding RNA KCNQ1OT1 and CCCTC-binding factor (CTCF). Activation of estrogen signaling reduced CDKN1C expression 3-fold (P < 0.001) and established repressive histone modifications at the 5' regulatory region of the locus. These events were concomitant with induction of KCNQ1OT1 expression as well as increased recruitment of CTCF to both the distal KCNQ1OT1 promoter-associated imprinting control region (ICR) and the CDKN1C locus. Transient depletion of CTCF by small interfering RNA increased CDKN1C expression and significantly reduced the estrogen-mediated repression of CDKN1C. Further studies in breast cancer cell lines indicated that the epigenetic silencing of CDKN1C occurs in part as the result of genetic loss of the inactive methylated 11p15.5 ICR allele (R(2) = 0.612, P < 0.001). We also found a novel cis-encoded antisense transcript, CDKN1C-AS, which is induced by estrogen signaling following pharmacologic inhibition of DNA methyltransferase and histone deacetylase activity. Forced expression of CDKN1C-AS was capable of repressing endogenous CDKN1C in vivo. Our findings suggest that in addition to promoter hypermethylation, epigenetic repression of tumor suppressor genes by CTCF and noncoding RNA transcripts could be more common and important than previously understood.

Epigenetic silencing mediated through activated PI3K/AKT signaling in breast cancer.

Trimethylation of histone 3 lysine 27 (H3K27me3) is a critical epigenetic mark for the maintenance of gene silencing. Additional accumulation of DNA methylation in target loci is thought to cooperatively support this epigenetic silencing during tumorigenesis. However, molecular mechanisms underlying the complex interplay between the two marks remain to be explored. Here we show that activation of PI3K/AKT signaling can be a trigger of this epigenetic processing at many downstream target genes. We also find that DNA methylation can be acquired at the same loci in cancer cells, thereby reinforcing permanent repression in those losing the H3K27me3 mark. Because of a link between PI3K/AKT signaling and epigenetic alterations, we conducted epigenetic therapies in conjunction with the signaling-targeted treatment. These combined treatments synergistically relieve gene silencing and suppress cancer cell growth in vitro and in xenografts. The new finding has important implications for improving targeted cancer therapies in the future.

Epigenetic influences of low-dose bisphenol A in primary human breast epithelial cells.

Substantial evidence indicates that exposure to bisphenol A (BPA) during early development may increase breast cancer risk later in life. The changes may persist into puberty and adulthood, suggesting an epigenetic process being imposed in differentiated breast epithelial cells. The molecular mechanisms by which early memory of BPA exposure is imprinted in breast progenitor cells and then passed onto their epithelial progeny are not well understood. The aim of this study was to examine epigenetic changes in breast epithelial cells treated with low-dose BPA. We also investigated the effect of BPA on the ERα signaling pathway and global gene expression profiles. Compared to control cells, nuclear internalization of ERα was observed in epithelial cells preexposed to BPA. We identified 170 genes with similar expression changes in response to BPA. Functional analysis confirms that gene suppression was mediated in part through an ERα-dependent pathway. As a result of exposure to BPA or other estrogen-like chemicals, the expression of lysosomal-associated membrane protein 3 (LAMP3) became epigenetically silenced in breast epithelial cells. Furthermore, increased DNA methylation in the LAMP3 CpG island was this repressive mark preferentially occurred in ERα-positive breast tumors. These results suggest that the in vitro system developed in our laboratory is a valuable tool for exposure studies of BPA and other xenoestrogens in human cells. Individual and geographical differences may contribute to altered patterns of gene expression and DNA methylation in susceptible loci. Combination of our exposure model with epigenetic analysis and other biochemical assays can give insight into the heritable effect of low-dose BPA in human cells.

Estrogen-mediated epigenetic repression of large chromosomal regions through DNA looping.

The current concept of epigenetic repression is based on one repressor unit corresponding to one silent gene. This notion, however, cannot adequately explain concurrent silencing of multiple loci observed in large chromosome regions. The long-range epigenetic silencing (LRES) can be a frequent occurrence throughout the human genome. To comprehensively characterize the influence of estrogen signaling on LRES, we analyzed transcriptome, methylome, and estrogen receptor alpha (ESR1)-binding datasets from normal breast epithelia and breast cancer cells. This "omics" approach uncovered 11 large repressive zones (range, 0.35 approximately 5.98 megabases), including a 14-gene cluster located on 16p11.2. In normal cells, estrogen signaling induced transient formation of multiple DNA loops in the 16p11.2 region by bringing 14 distant loci to focal ESR1-docking sites for coordinate repression. However, the plasticity of this free DNA movement was reduced in breast cancer cells. Together with the acquisition of DNA methylation and repressive chromatin modifications at the 16p11.2 loci, an inflexible DNA scaffold may be a novel determinant used by breast cancer cells to reinforce estrogen-mediated repression.

Promoter hypermethylation of CIDEA, HAAO and RXFP3 associated with microsatellite instability in endometrial carcinomas.

OBJECTIVE: DNA promoter methylation is an epigenetic phenomenon for long-term gene silencing during tumorigenesis. The purpose of this study is to identify novel hypermethylated loci associated with clinicopathologic variables in endometrioid endometrial carcinomas. METHODS: To find hypermethylated promoter loci, we used differential methylation hybridization coupling with microarray and further validated by combined bisulfite restriction analysis and MassARRAY assay. Methylation levels of candidate loci were corrected with clinicopathologic factors of endometrial carcinomas. RESULTS: Increased promoter methylation of CIDE, HAAO and RXFP3 was detected in endometrial carcinomas compared with adjacent normal tissues, and was associated with decreased gene expression of all three genes. In a clinical cohort, promoter hypermethylation on CIDEA, HAAO and RXFP3 was detected in 85, 63 and 71% of endometrial carcinomas, respectively (n=118, P<0.001) compared with uninvolved normal endometrium. Methylation status of CIDEA, HAAO and RXFP3 had significant association with microsatellite instability in tumors (P<0.001). Furthermore, methylation levels of HAAO were further found to relate to disease-free survivals (P=0.034). CONCLUSIONS: Hypermethylation of CIDEA, HAAO and RXFP3 promoter regions appears to be a frequent event in endometrial carcinomas. Hypermethylation at these loci is strongly associated with microsatellite instability status. Moreover, HAAO methylation predicts disease-free survival in this cohort of patients with endometrioid endometrial cancer.

Methylated DNA immunoprecipitation and microarray-based analysis: detection of DNA methylation in breast cancer cell lines.

The methylated DNA immunoprecipitation microarray (MeDIP-chip) is a genome-wide, high-resolution approach to detect DNA methylation in whole genome or CpG (cytosine base followed by a guanine base) islands. The method utilizes anti-methylcytosine antibody to immunoprecipitate DNA that contains highly methylated CpG sites. Enriched methylated DNA can be interrogated using DNA microarrays or by massive parallel sequencing techniques. This combined approach allows researchers to rapidly identify methylated regions in a genome-wide manner, and compare DNA methylation patterns between two samples with diversely different DNA methylation status. MeDIP-chip has been applied successfully for analyses of methylated DNA in the different targets including animal and plant tissues. Here we present a MeDIP-chip protocol that is routinely used in our laboratory, illustrated with specific examples from MeDIP-chip analysis of breast cancer cell lines. Potential technical pitfalls and solutions are also provided to serve as workflow guidelines.

Xenoestrogen-induced epigenetic repression of microRNA-9-3 in breast epithelial cells.

Early exposure to xenoestrogens may predispose to breast cancer risk later in adult life. It is likely that long-lived, self-regenerating epithelial progenitor cells are more susceptible to these exposure injuries over time and transmit the injured memory through epigenetic mechanisms to their differentiated progeny. Here, we used progenitor-containing mammospheres as an in vitro exposure model to study this epigenetic effect. Expression profiling identified that, relative to control cells, 9.1% of microRNAs (82 of 898 loci) were altered in epithelial progeny derived from mammospheres exposed to a synthetic estrogen, diethylstilbestrol. Repressive chromatin marks, trimethyl Lys27 of histone H3 (H3K27me3) and dimethyl Lys9 of histone H3 (H3K9me2), were found at a down-regulated locus, miR-9-3, in epithelial cells preexposed to diethylstilbestrol. This was accompanied by recruitment of DNA methyltransferase 1 that caused an aberrant increase in DNA methylation of its promoter CpG island in mammosphere-derived epithelial cells on diethylstilbestrol preexposure. Functional analyses suggest that miR-9-3 plays a role in the p53-related apoptotic pathway. Epigenetic silencing of this gene, therefore, reduces this cellular function and promotes the proliferation of breast cancer cells. Promoter hypermethylation of this microRNA may be a hallmark for early breast cancer development, and restoration of its expression by epigenetic and microRNA-based therapies is another viable option for future treatment of this disease.

Ethanol inhibition of angiotensin II-stimulated Tyr705 and Ser727 STAT3 phosphorylation in cultured rat hepatocytes: relevance to activation of p42/44 mitogen-activated protein kinase.

Angiotensin (Ang) II-stimulated phosphorylation of signal transducer and activator transcription (STAT) 3 in rat hepatocytes and the effects of ethanol on this activation were investigated. Angiotensin II (100 nM) stimulated Tyr705 and Ser727 phosphorylation of STAT3 and formation of sis-inducing factor complexes. In the presence of U-0126 (10microM), a p42/44 mitogen-activated protein kinase (MAPK) kinase inhibitor, Ang II further increased Tyr705 phosphorylation of STAT3 but completely abrogated Ser727 phosphorylation of STAT3. Inhibition of p42/44MAPK also increased STAT3 DNA-binding activity. Pretreatment with ethanol (100mM) for 24h resulted in decrease in Tyr705 phosphorylation of STAT3 by ethanol alone and inhibition of Tyr705 phosphorylation of STAT3 stimulated by Ang II. Although ethanol potentiates Ang II stimulated p42/44 MAPK activation in hepatocytes, ethanol inhibited Ser727 phosphorylation of STAT3 stimulated by Ang II. Angiotensin II-stimulated STAT3-binding activity was not significantly affected by ethanol treatment. These results suggest a negative regulation of Ang II-stimulated STAT3 tyrosine phosphorylation and STAT3-binding activity through p42/44 MAPK activation in hepatocytes. However, ethanol modulation of Ang II-stimulated STAT3 phosphorylation occurs by MAPK independent mechanisms. Ethanol potentiation of MAPK signaling without suppression of STAT3 function may modulate the course of alcoholic liver injury.

Effects of chronic ethanol treatment on the angiotensin II-mediated p42/p44 mitogen-activated protein kinase and phosphorylase a activation in rat hepatocytes.

We have demonstrated previously that 24 h of ethanol treatment potentiates angiotensin II (ANG II)-stimulated p42/p44 mitogen-activated protein kinase (MAPK) activity in hepatocytes. This potentiation of p42/p44 MAPK by ethanol exhibited agonist selectivity. To compare the effects of acute (24 h) versus chronic (6 weeks) ethanol treatment, ANG II-induced intracellular signaling was examined in (1) rat hepatocytes treated with ethanol for 24 h and (2) hepatocytes obtained from rats fed ethanol for 6 weeks. In hepatocytes obtained from rats fed ethanol for 6 weeks, ANG II-stimulated phosphorylase a was reduced, and this activity was calcium dependent and p42/p44 MAPK independent. Surprisingly, ANG II-stimulated p42/p44 MAPK activation was not affected in hepatocytes obtained from rats fed ethanol chronically (6 weeks). However, chronic (6 weeks) ethanol treatment decreased ethanol potentiation of p42/p44 MAPK by about 56.3% +/- 3.6% for p42 MAPK and 61.3% +/- 11.7% for p44 MAPK. Furthermore, ethanol had no effect on the expression of angiotensinogen and c-myc mRNA in hepatocytes. A decrease in ANG II-activated phosphorylase a, but not in p42/p44 MAPK activation, after chronic (6 weeks) ethanol treatment leads to the conclusion that they may not be dependent on each other.

Role of PKC and MAPK in cytosolic PLA2 phosphorylation and arachadonic acid release in primary murine astrocytes.

Although Group IV cytosolic phospholipase A2 (cPLA2) in astrocytes has been implicated in a number of neurodegenerative diseases, mechanisms leading to its activation and release of arachidonic acid (AA) have not been clearly elucidated. In primary murine astrocytes, phorbol myristate acetate (PMA) and ATP stimulated phosphorylation of ERK1/2 and cPLA2 as well as evoked AA release. However, complete inhibition of phospho-ERK by U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK), did not completely inhibit PMA-stimulated cPLA2 and AA release. Epidermal growth factor (EGF) also stimulated phosphorylation of ERK1/2 and cPLA2[largely through a protein kinase C (PKC)-independent pathway], but EGF did not evoke AA release. These results suggest that phosphorylation of cPLA2 due to phospho-ERK is not sufficient to evoke AA release. However, complete inhibition of ATP-induced cPLA2 phosphorylation and AA release was observed when astrocytes were treated with GF109203x, a general PKC inhibitor, together with U0126, indicating the important role for both PKC and ERK in mediating the ATP-induced AA response. There is evidence that PMA and ATP stimulated AA release through different PKC isoforms in astrocytes. In agreement with the sensitivity of PMA-induced responses to PKC down-regulation, prolonged treatment with PMA resulted in down-regulation of PKCalpha and epsilon in these cells. Furthermore, PMA but not ATP stimulated rapid translocation of PKCalpha from cytosol to membranes. Together, our results provided evidence for an important role of PKC in mediating cPLA2 phosphorylation and AA release in astrocytes through both ERK1/2-dependent and ERK1/2-independent pathways.

Phosphotidylethanol mimics ethanol modulation of p42/44 mitogen-activated protein kinase signalling in hepatocytes.

AIMS: Although long-term exposure of hepatocytes to ethanol results in agonist-selective potentiation of p42/44 mitogen-activated protein kinase (MAPK) activation, mediators of this effect of ethanol are not known. METHODS: We examined the role of phosphatidylethanol (PEth), a novel phospholipid formed exclusively in the presence of ethanol. RESULTS: PEth accumulated in primary cultures of rat hepatocytes treated with ethanol. Exogenously added PEth potentiated angiotensin II-stimulated p42/44 MAPK similarly to that observed with ethanol treatment of cells for 24 h, a condition where PEth accumulates. PEth levels remained elevated 2 h after ethanol removal subsequent to a 24-h exposure, and the potentiating effects of ethanol were also present. PEth did not potentiate p42/44 MAPK activation by either epidermal growth factor or vasopressin, thus further mimicking the known agonist selectivity for this ethanol effect. CONCLUSIONS: These results offer a novel role for PEth as a mediator in the ethanol modulation of p42/44 MAPK cascade in hepatocytes.

Angiotensin II activation of focal adhesion kinase and pp60c-Src in relation to mitogen-activated protein kinases in hepatocytes.

We have investigated signaling pathways leading to angiotensin II (Ang II) activation of mitogen-activated protein kinase (MAPK) in hepatocytes. MAPK activation by Ang II was abolished by the Ang II type 1 (AT1) receptor antagonist losartan, but not by the Ang II type 2 (AT2) receptor antagonist PD123319. Ang II (100 nM) induced a rapid phosphorylation of Src (peak approximately 2 min) and focal adhesion kinase (FAK, peak approximately 5 min) followed by a decrease to basal levels in 30 min. An increased association between FAK and Src in response to Ang II was detected after 1 min, which declined to basal levels after 30 min. Treatment with the Src kinase inhibitor PP-1 inhibited FAK phosphorylation. Downregulation of PKC, intracellular Ca2+ chelator BAPTA or inhibitors of PKC, Src kinase, MAPK kinase (MEK), Ca2+/calmodulin dependent protein kinase, phosphatidylinositol 3-kinase all blocked Ang II-induced MAPK phosphorylation. In contrast to other cells, there was no evidence for the role of EGF receptor transactivation in the activation of MAPK by Ang II. However, PDGF receptor phosphorylation is involved in the Ang II stimulated MAPK activation. Furthermore, Src/FAK and Ca/CaM kinase activation serve as potential links between the Ang II receptor and MAPK activation. These studies offer insight into the signaling network upstream of MAPK activation by AT1 receptor in hepatocytes.