Susan J. Clark

Bisulfite genomic sequencing: systematic investigation of critical experimental parameters

Bisulfite genomic sequencing is the method of choice for the generation of methylation maps with single-base resolution. The method is based on the selective deamination of cytosine to uracil by treatment with bisulfite and the sequencing of subsequently generated PCR products. In contrast to cytosine, 5-methylcytosine does not react with bisulfite and can therefore be distinguished. In order to investigate the potential for optimization of the method and to determine the critical experimental parameters, we determined the influence of incubation time and incubation temperature on the deamination efficiency and measured the degree of DNA degradation during the bisulfite treatment. We found that maximum conversion rates of cytosine occurred at 55 degrees C (4-18 h) and 95 degrees C (1 h). Under these conditions at least 84-96% of the DNA is degraded. To study the impact of primer selection, homologous DNA templates were constructed possessing cytosine-containing and cytosine-free primer binding sites, respectively. The recognition rates for cytosine (>/=97%) and 5-methylcytosine (>/=94%) were found to be identical for both templates.

miRNA‐dependent gene silencing involving Ago2‐mediated cleavage of a circular antisense RNA

MicroRNAs (miRNAs) are ∼22 nt non‐coding RNAs that typically bind to the 3′ UTR of target mRNAs in the cytoplasm, resulting in mRNA destabilization and translational repression. Here, we report that miRNAs can also regulate gene expression by targeting non‐coding antisense transcripts in human cells. Specifically, we show that miR‐671 directs cleavage of a circular antisense transcript of the Cerebellar Degeneration‐Related protein 1 (CDR1) locus in an Ago2‐slicer‐dependent manner. The resulting downregulation of circular antisense has a concomitant decrease in CDR1 mRNA levels, independently of heterochromatin formation. This study provides the first evidence for non‐coding antisense transcripts as functional miRNA targets, and a novel regulatory mechanism involving a positive correlation between mRNA and antisense circular RNA levels.

Coordinated epigenetic repression of the miR-200 family and miR-205 in invasive bladder cancer.

MicroRNAs (miRNA) are small noncoding RNAs commonly deregulated in cancer. The miR‐200 family (miR‐200a, ‐200b, ‐200c, ‐141 and ‐429) and miR‐205 are frequently silenced in advanced cancer and have been implicated in epithelial to mesenchymal transition (EMT) and tumor invasion by targeting the transcriptional repressors of E‐cadherin, ZEB1 and ZEB2. ZEB1 is also known to repress miR‐200c‐141 transcription in a negative feedback loop, but otherwise little is known about the transcriptional regulation of the miR‐200 family and miR‐205. Recently, miR‐200 silencing was also reported in cancer stem cells, implying that miR‐200 deregulation is a key event in multiple levels of tumor biology. However, what prevents miR‐200 expression remains largely unanswered. Here we report concerted transcriptional regulation of the miR‐200 and miR‐205 loci in bladder tumors and bladder cell lines. Using a combination of miRNA expression arrays, qPCR assays and mass spectrometry DNA methylation analyses, we show that the miR‐200 and miR‐205 loci are specifically silenced and gain promoter hypermethylation and repressive chromatin marks in muscle invasive bladder tumors and undifferentiated bladder cell lines. Moreover, we report that miR‐200c expression is significantly correlated with early stage T1 bladder tumor progression, and propose miR‐200 and miR‐205 silencing and DNA hypermethylation as possible prognostic markers in bladder cancer. In addition, we observe that the mesoderm transcription factor TWIST1 and miR‐200 expression are inversely correlated in bladder tumor samples and cell lines. TWIST1 associates directly with the miR‐200 and miR‐205 promoters, and may act as a repressor of miR‐200 and miR‐205 expression.

Identification and resolution of artifacts in bisulfite sequencing

Bisulfite sequencing has become the most widely used application to detect 5-methylcytosine (5-MeC) in DNA, and provides a reliable way of detecting any methylated cytosine at single-molecule resolution in any sequence context. The process of bisulfite treatment exploits the different sensitivity of cytosine and 5-MeC to deamination by bisulfite under acidic conditions, in which cytosine undergoes conversion to uracil while 5-MeC remains unreactive. In this article, we address the more commonly encountered experimental artifacts associated with bisulfite sequencing, and provide methods for the detection and elimination of these artifacts. In particular, we focus on conditions that inhibit complete bisulfite-mediated conversion of cytosines in a target sequence, and demonstrate the necessity of complete protein removal from DNA samples prior to bisulfite treatment. We also include a brief summary of the experimental protocol for bisulfite treatment and tips for designing polymerase chain reaction (PCR) primers to amplify from bisulfite-treated DNA.

CpNpG methylation in mammalian cells

In vertebrate DMA, 3% to 5% of cytosine residues are present as 5–methylcytosine, and it is generally accepted that essentially all of this methylation occurs at cytosines which are contained in the symmetrical dinucleotide CpG. In this report we demonstrate, using bisulphite genomic sequencing, that the methylation machinery of mammalian cells is capable of both maintenance and de novo methylation at CpNpG sites. The existence of inherited CpNpG methylation in mammalian cells has important implications in gene regulation and in the aetiology of disease

DNA methylation and gene silencing in cancer: which is the guilty party?

The DNA methylation pattern of a cell is exquisitely controlled during early development resulting in distinct methylation patterns. The tight control of DNA methylation is released in the cancer cell characterized by a reversal of methylation states. CpG island associated genes, in particular tumour suppressor or related genes, are often hypermethylated and this is associated with silencing of these genes. Therefore methylation is commonly convicted as a critical causal event in silencing this important class of genes in cancer. In this review, we argue that methylation is not the initial guilty party in triggering gene silencing in cancer, but that methylation of CpG islands is a consequence of prior gene silencing, similar to the role of methylation in maintaining the silencing of CpG island genes on the inactive X chromosome. We propose that gene silencing is the critical precursor in cancer, as it changes the dynamic interplay between de novo methylation and demethylation of the CpG island and tilts the balance to favour hypermethylation and chromatin inactivation.

Transcriptional Gene Silencing Promotes DNA Hypermethylation through a Sequential Change in Chromatin Modifications in Cancer Cells

It is well established that DNA hypermethylation of tumor suppressor and tumor-related genes can occur in cancer cells and that each cancer subtype has specific gene sets that are commonly susceptible to methylation and silencing. Glutathione S-transferase (GSTP1) is one example of a gene that is hypermethylated and inactivated in the majority of prostate cancers. We previously reported that hypermethylation of the GSTP1 CpG island promoter in prostate cancer cells is initiated by a combination of transcriptional gene silencing (by removal of the Sp1 sites) and seeds of methylation that, instead of being constantly removed because of demethylation associated with transcription, acts as a catalyst for the spread of methylation across the CpG island. In this study, we now demonstrate that the seeds of DNA methylation also play an important role in initiating chromatin modification. Our results address a number of central questions about the temporal relationship between gene expression, DNA hypermethylation, and chromatin modification in cancer cells. We find that for the GSTP1 gene, (a) histone acetylation is independent of gene expression, (b) histone deacetylation is triggered by seeds of DNA methylation, (c) the spread of DNA hypermethylation across the island is linked to MBD2 and not MeCP2 binding, and (d) histone methylation occurs after histone deacetylation and is associated with extensive DNA methylation of the CpG island. These findings have important implications for understanding the biochemical events underlying the mechanisms responsible for abnormal hypermethylation of CpG island-associated genes in cancer cells.

Detailed methylation analysis of the glutathione S-transferase π ( GSTP1 ) gene in prostate cancer

Glutathione-S-Transferases (GSTs) comprise a family of isoenzymes that provide protection to mammalian cells against electrophilic metabolites of carcinogens and reactive oxygen species. Previous studies have shown that the CpG-rich promoter region of the π-class gene GSTP1 is methylated at single restriction sites in the majority of prostate cancers. In order to understand the nature of abnormal methylation of the GSTP1 gene in prostate cancer we undertook a detailed analysis of methylation at 131 CpG sites spanning the promoter and body of the gene. Our results show that DNA methylation is not confined to specific CpG sites in the promoter region of the GSTP1 gene but is extensive throughout the CpG island in prostate cancer cells. Furthermore we found that both alleles are abnormally methylated in this region. In normal prostate tissue, the entire CpG island was unmethylated, but extensive methylation was found outside the island in the body of the gene. Loss of GSTP1 expression correlated with DNA methylation of the CpG island in both prostate cancer cell lines and cancer tissues whereas methylation outside the CpG island in normal prostate tissue appeared to have no effect on gene expression.

Epigenetics and human obesity

Background:Recent technological advances in epigenome profiling have led to an increasing number of studies investigating the role of the epigenome in obesity. There is also evidence that environmental exposures during early life can induce persistent alterations in the epigenome, which may lead to an increased risk of obesity later in life.Method:This paper provides a systematic review of studies investigating the association between obesity and either global, site-specific or genome-wide methylation of DNA. Studies on the impact of pre- and postnatal interventions on methylation and obesity are also reviewed. We discuss outstanding questions, and introduce EpiSCOPE, a multidisciplinary research program aimed at increasing the understanding of epigenetic changes in emergence of obesity.Results:An electronic search for relevant articles, published between September 2008 and September 2013 was performed. From the 319 articles identified, 46 studies were included and reviewed. The studies provided no consistent evidence for a relationship between global methylation and obesity. The studies did identify multiple obesity-associated differentially methylated sites, mainly in blood cells. Extensive, but small, alterations in methylation at specific sites were observed in weight loss intervention studies, and several associations between methylation marks at birth and later life obesity were found.Conclusions:Overall, significant progress has been made in the field of epigenetics and obesity and the first potential epigenetic markers for obesity that could be detected at birth have been identified. Eventually this may help in predicting an individual’s obesity risk at a young age and opens possibilities for introducing targeted prevention strategies. It has also become clear that several epigenetic marks are modifiable, by changing the exposure in utero, but also by lifestyle changes in adult life, which implies that there is the potential for interventions to be introduced in postnatal life to modify unfavourable epigenomic profiles.

Hypermethylation trigger of the glutathione-S-transferase gene (GSTP1) in prostate cancer cells

Understanding what triggers hypermethylation of tumour suppressor genes in cancer cells is critical if we are to discern the role of methylation in the oncogenic process. CpG sites in CpG island promoters, that span most tumour suppressor genes, remain unmethylated in the normal cell, despite the fact that CpG sites are the prime target for de novo methylation by the DNA methyltransferases. The CpG island-associated with the GSTP1 gene is an intriguing example of a CpG rich region which is susceptible to hypermethylation in the majority of prostate tumours and yet is unmethylated in the normal prostate cell. In this study we evaluate a number of factors purported to be involved in hypermethylation to test their role in triggering hypermethylation of GSTP1 in prostate cancer DU145 and LNCaP cells. We find that hypermethylation is not associated with (1) elevated expression of the DNA methyltranferases, or (2) removal of Sp1 transcription factor binding sites in the CpG island or (3) removal of CpG island boundary elements or (4) prior gene silencing. Instead our results support a model that requires a combination of prior gene silencing and random ‘seeds’ of methylation to trigger hypermethylation of the GSTP1 gene in the prostate cancer cell. We propose that the GSTP1 gene is initially silenced in the prostate cancer and random sites of methylation accumulate that result in subsequent hypermethylation and chromatin remodelling.