The transposable element-derived transcript of LIN28B has a placental origin and is not specific to tumours.

Transposable elements (TEs) are genetic elements that have evolved as crucial regulators of human development and cancer, functioning as both genes and regulatory elements. When TEs become dysregulated in cancer cells, they can serve as alternate promoters to activate oncogenes, a process known as onco-exaptation. This study aimed to explore the expression and epigenetic regulation of onco-exaptation events in early human developmental tissues. We discovered co-expression of some TEs and oncogenes in human embryonic stem cells and first trimester and term placental tissues. Previous studies identified onco-exaptation events in various cancer types, including an AluJb SINE element-LIN28B interaction in lung cancer cells, and showed that the TE-derived LIN28B transcript is associated with poor patient prognosis in hepatocellular carcinoma. This study further characterized the AluJb-LIN28B transcript and confirmed that its expression is restricted to the placenta. Targeted DNA methylation analysis revealed differential methylation of the two LIN28B promoters between placenta and healthy somatic tissues, indicating that some TE-oncogene interactions are not cancer-specific but arise from the epigenetic reactivation of developmental TE-derived regulatory events. In conclusion, our findings provide evidence that some TE-oncogene interactions are not limited to cancer and may originate from the epigenetic reactivation of TE-derived regulatory events that are involved in early development. These insights broaden our understanding of the role of TEs in gene regulation and suggest the potential importance of targeting TEs in cancer therapy beyond their conventional use as cancer-specific markers.

Germline mutations and somatic inactivation of TRIM28 in Wilms tumour.

Wilms tumour is a childhood tumour that arises as a consequence of somatic and rare germline mutations, the characterisation of which has refined our understanding of nephrogenesis and carcinogenesis. Here we report that germline loss of function mutations in TRIM28 predispose children to Wilms tumour. Loss of function of this transcriptional co-repressor, which has a role in nephrogenesis, has not previously been associated with cancer. Inactivation of TRIM28, either germline or somatic, occurred through inactivating mutations, loss of heterozygosity or epigenetic silencing. TRIM28-mutated tumours had a monomorphic epithelial histology that is uncommon for Wilms tumour. Critically, these tumours were negative for TRIM28 immunohistochemical staining whereas the epithelial component in normal tissue and other Wilms tumours stained positively. These data, together with a characteristic gene expression profile, suggest that inactivation of TRIM28 provides the molecular basis for defining a previously described subtype of Wilms tumour, that has early age of onset and excellent prognosis.

Global demethylation in loss of imprinting subtype of Wilms tumor.

Epigenetic abnormalities at the IGF2/H19 locus play a key role in the onset of Wilms tumor. These tumors can be classified into three molecular subtypes depending on the events occurring at this locus: loss of imprinting (LOI), loss of heterozygosity (LOH), or retention of imprinting (ROI). As IGF2 LOI is a consequence of aberrant methylation, we hypothesized that this subtype of Wilms tumors might display global abnormalities of methylation. We therefore analyzed the methylation status of satellite DNA, as a surrogate for global methylation in 50 Wilms tumor patients. Satellite methylation was quantified by a methylation-sensitive quantitative PCR. We confirmed hypomethylation of both satellite α (Sat α) and satellite 2 (Sat 2) DNA in Wilms tumor samples compared with normal kidney. In addition, we found that LOI tumors, unlike ROI or LOH ones, showed concordant hypomethylation of both Sat α and Sat 2 DNA. This would suggest that the LOI subtype of Wilms tumor, which unlike other subtypes results from an epimutation, has a global deregulation of methylation mechanisms.

Targeted DNA Methylation Editing Using an All-in-One System Establishes Paradoxical Activation of EBF3.

Cutaneous melanoma is rapidly on the rise globally, surpassing the growth rate of other cancers, with metastasis being the primary cause of death in melanoma patients. Consequently, understanding the mechanisms behind this metastatic process and exploring innovative treatments is of paramount importance. Recent research has shown promise in unravelling the role of epigenetic factors in melanoma progression to metastasis. While DNA hypermethylation at gene promoters typically suppresses gene expression, we have contributed to establishing the newly understood mechanism of paradoxical activation of genes via DNA methylation, where high methylation coincides with increased gene activity. This mechanism challenges the conventional paradigm that promoter methylation solely silences genes, suggesting that, for specific genes, it might actually activate them. Traditionally, altering DNA methylation in vitro has involved using global demethylating agents, which is insufficient for studying the mechanism and testing the direct consequence of gene methylation changes. To investigate promoter hypermethylation and its association with gene activation, we employed a novel approach utilising a CRISPR-SunTag All-in-one system. Here, we focused on editing the DNA methylation of a specific gene promoter segment (EBF3) in melanoma cells using the All-in-one system. Using bisulfite sequencing and qPCR with RNA-Seq, we successfully demonstrated highly effective methylation and demethylation of the EBF3 promoter, with subsequent gene expression changes, to establish and validate the paradoxical role of DNA methylation. Further, our study provides novel insights into the function of the EBF3 gene, which remains largely unknown. Overall, this study challenges the conventional view of methylation as solely a gene-silencing mechanism and demonstrates a potential function of EBF3 in IFN pathway signalling, potentially uncovering new insights into epigenetic drivers of malignancy and metastasis.

Technical considerations for reduced representation bisulfite sequencing with multiplexed libraries.

Reduced representation bisulfite sequencing (RRBS), which couples bisulfite conversion and next generation sequencing, is an innovative method that specifically enriches genomic regions with a high density of potential methylation sites and enables investigation of DNA methylation at single-nucleotide resolution. Recent advances in the Illumina DNA sample preparation protocol and sequencing technology have vastly improved sequencing throughput capacity. Although the new Illumina technology is now widely used, the unique challenges associated with multiplexed RRBS libraries on this platform have not been previously described. We have made modifications to the RRBS library preparation protocol to sequence multiplexed libraries on a single flow cell lane of the Illumina HiSeq 2000. Furthermore, our analysis incorporates a bioinformatics pipeline specifically designed to process bisulfite-converted sequencing reads and evaluate the output and quality of the sequencing data generated from the multiplexed libraries. We obtained an average of 42 million paired-end reads per sample for each flow-cell lane, with a high unique mapping efficiency to the reference human genome. Here we provide a roadmap of modifications, strategies, and trouble shooting approaches we implemented to optimize sequencing of multiplexed libraries on an a RRBS background.

Editing of DNA Methylation Patterns Using CRISPR-Based Tools.

DNA methylation is an epigenetic modification with an established role in both normal cellular function and mammalian disease. Despite well-characterized associations between aberrant DNA methylation changes and gene expression, evidence for a causal relationship in this context has been difficult to obtain. Early techniques for interrogating the role of DNA methylation in the regulation of gene transcription lack specificity and, where more specific techniques such and ZNFs and TALEs have been developed, they are limited by their extensive cost and labor requirements. However, the recent advent of CRISPR-based technologies has revolutionized our potential for site-specific epigenomic editing. Here, we provide a detailed protocol for the design, construction, and utilization of a transient, CRISPR-based DNA methylation-editing system in mammalian cells.

Next-Generation Bisulfite Sequencing for Targeted DNA Methylation Analysis.

Bisulfite sequencing is the "gold-standard" technique for DNA methylation analysis. By combining bisulfite sequencing with high-throughput, next-generation sequencing technology, we can document methylation from many thousands of individual reads (equivalent to alleles or "cells"), for multiple target regions and from many samples simultaneously. Here, we describe a next-generation bisulfite-sequencing assay for targeted DNA methylation analysis which offers scope for the simultaneous interrogation of multiple genomic loci across numerous samples.

Epigenetic basis and targeting of cancer metastasis.

Despite the development of novel therapeutic approaches and improved clinical management, survival from metastatic disease remains poor. Indeed, metastasis accounts for the vast majority of cancer-related deaths. The metastatic cascade comprises a complex range of molecular events that cannot be explained by genetic aberrations alone; dynamic, epigenetic regulatory mechanisms are now being implicated as key drivers of successful metastasis. With the emergence of CRISPR-based epigenomic editing, it is now possible to investigate the direct role of locus-specific epigenetic alterations in metastatic progression. Here, we review the role of epigenetic mechanisms in cancer metastasis, explore recent developments in technologies for epigenomic investigation, and highlight the emerging applications of epigenomic editing technologies in the clinical management of cancer.

Hypomethylation of functional retrotransposon-derived genes in the human placenta.

DNA hypomethylation is assumed to be a feature of the mammalian placenta; however, its role in regulating placental gene expression is not well defined. In this study, MeDIP and Sequenom MassARRAY were used to identify hypomethylated gene promoters in the human placenta. Among the genes identified, the hypomethylation of an alternative promoter for KCNH5 was found to be restricted to the placenta and chorion. Complete methylation of this promoter correlates with a silenced KCNH5 transcript in embryonic tissues, including the amnion. Unusually, this hypomethylated promoter and the alternative first exon are derived from a SINE (AluY) retrotransposon. Examination of additional retrotransposon-derived gene promoters in the placenta confirmed that retrotransposon hypomethylation permits the placenta-specific expression of these genes. Furthermore, the lineage-specific methylation displayed by KCNH5, INSL4, and ERVWE1 revealed that dichotomous methylation establishes differential retrotransposon silencing between the extra-embryonic and embryonic lineages. The hypomethylation of the retrotransposons that regulate these genes, each of which arose during recent primate evolution, is consistent with these genes having functional roles that are unique to the invasive haemochorial placentas of humans and recent primates.

Locus-Specific DNA Methylation Editing in Melanoma Cell Lines Using a CRISPR-Based System.

DNA methylation is a key epigenetic modification implicated in the pathogenesis of numerous human diseases, including cancer development and metastasis. Gene promoter methylation changes are widely associated with transcriptional deregulation and disease progression. The advent of CRISPR-based technologies has provided a powerful toolkit for locus-specific manipulation of the epigenome. Here, we describe a comprehensive global workflow for the design and application of a dCas9-SunTag-based tool for editing the DNA methylation locus in human melanoma cells alongside protocols for downstream techniques used to evaluate subsequent methylation and gene expression changes in methylation-edited cells. Using transient system delivery, we demonstrate both highly efficacious methylation and demethylation of the EBF3 promoter, which is a putative epigenetic driver of melanoma metastasis, achieving up to a 304.00% gain of methylation and 99.99% relative demethylation, respectively. Furthermore, we employ a novel, targeted screening approach to confirm the minimal off-target activity and high on-target specificity of our designed guide RNA within our target locus.

Hairpin-bisulfite sequencing of cells exposed to decitabine documents the process of DNA demethylation.

Although the mechanism of DNA demethylating drugs has been understood for many years, the direct effect of these drugs on methylation of the complementary strands of DNA has not been formally demonstrated. By using hairpin-bisulphite sequencing, we describe the kinetics and pattern of DNA methylation following treatment of cells by the DNA methyltransferase 1 (DNMT1) inhibitor, decitabine. As expected, we demonstrate complete loss of methylation on the daughter strand following S-phase in selected densely methylated genes in synchronized Jurkat cells. Thereafter, cells showed a heterogeneous pattern of methylation reflecting replication of the unmethylated strand and restoration of methylation.

Identification and validation of DNA methylation changes in pre-eclampsia.

INTRODUCTION: Pre-eclampsia (PE) is a dangerous placental condition that can lead to premature labour, seizures and death of mother and infant. Several studies have identified altered placental DNA methylation in PE; however, there is widespread inconsistency between studies and most findings have not been replicated. This study aimed to identify and validate consistent differences in methylation across multiple PE cohorts. METHODS: Seven publicly available 450K methylation array datasets were analysed to identify consistent differentially methylated positions (DMPs) in PE. DMPs were identified based on methylation difference (≥10%) and significance (p-value ≤ 1 × 10-7). Targeted deep bisulfite sequencing was then performed to validate a subset of DMPs in an additional independent PE cohort. RESULTS: Stringent analysis of the seven 450K datasets identified 25 DMPs (associated with 11 genes) in only one dataset. Using more relaxed criteria confirmed 19 of the stringent 25 DMPs in at least four of the remaining six datasets. Targeted deep bisulfite sequencing of eight DMPs (associated with three genes; CMIP, ST3GAL1 and DAPK3) in an independent PE cohort validated two DMPs in the CMIP gene. Seven additional CpG sites in CMIP were found to be significantly differentially methylated in PE. DISCUSSION: The identification and validation of significant differential methylation in CMIP suggests that the altered DNA methylation of this gene may be associated with the pathogenesis of PE, and may have the potential to serve as diagnostic biomarkers for this dangerous condition of pregnancy.

Silencing of TESTIN by dense biallelic promoter methylation is the most common molecular event in childhood acute lymphoblastic leukaemia.

BACKGROUND: Aberrant promoter DNA methylation has been reported in childhood acute lymphoblastic leukaemia (ALL) and has the potential to contribute to its onset and outcome. However, few reports demonstrate consistent, prevalent and dense promoter methylation, associated with tumour-specific gene silencing. By screening candidate genes, we have detected frequent and dense methylation of the TESTIN (TES) promoter. RESULTS: Bisulfite sequencing showed that 100% of the ALL samples (n = 20) were methylated at the TES promoter, whereas the matched remission (n = 5), normal bone marrow (n = 6) and normal PBL (n = 5) samples were unmethylated. Expression of TES in hyperdiploid, TEL-AML+, BCR-ABL+, and E2A-PBX+ subtypes of B lineage ALL was markedly reduced compared to that in normal bone marrow progenitor cells and in B cells. In addition TES methylation and silencing was demonstrated in nine out of ten independent B ALL propagated as xenografts in NOD/SCID mice. CONCLUSION: In total, 93% of B ALL samples (93 of 100) demonstrated methylation with silencing or reduced expression of the TES gene. Thus, TES is the most frequently methylated and silenced gene yet reported in ALL. TES, a LIM domain-containing tumour suppressor gene and component of the focal adhesion complex, is involved in adhesion, motility, cell-to-cell interactions and cell signalling. Our data implicate TES methylation in ALL and provide additional evidence for the involvement of LIM domain proteins in leukaemogenesis.

Epigenetic silencing in non-neoplastic epithelia identifies E-cadherin (CDH1) as a target for chemoprevention of lobular neoplasia.

Invasive lobular carcinoma (ILC) of the breast is believed to develop from in situ lesions, atypical lobular hyperplasia (ALH), and lobular carcinoma in situ (LCIS). Down-regulation of the cell-cell adhesion protein E-cadherin is a defining feature of lobular breast cancer (LBC) and already occurs in ALH and LCIS. Apart from mutational mechanisms, epigenetic silencing of the E-cadherin gene (CDH1) is thought to be involved in E-cadherin down-regulation and has been observed at a high frequency in ILC. Whether CDH1 promoter methylation is already present in in situ lesions and thus contributes to the initiation of LBC is not established. We thus examined microdissected archived tissue from 20 LBCs by methylation-specific PCR to determine the CDH1 methylation status of lobular lesions. Nineteen of the 20 LBCs had a hypermethylated CDH1 promoter, including 13/14 ILCs and 13/13 ALHs or LCIS. Bisulphite sequencing indicated that methylation was complete within the investigated promoter fragment. Intriguingly, CDH1 methylation was likewise present in 8/8 adjacent non-neoplastic epithelia, but not in 6/6 mammary epithelia from healthy subjects. E-cadherin protein and mRNA were down-regulated in in situ lesions relative to adjacent epithelia. Together, these results indicate that CDH1 promoter methylation occurs in LBC prior to E-cadherin down-regulation and neoplastic formation. We thus propose that epigenetic silencing represents the first of the two hits required to silence both CDH1 alleles for LBC to develop. Because promoter methylation is in principle reversible, our findings suggest that chemoprevention of LBC by epigenetic drugs should be feasible. Furthermore, the presence of CDH1 methylation in pre-neoplastic epithelia suggests the existence of mammary regions with increased disease susceptibility, providing an explanation for the often multifocal presentation of LBC.

Promoter DNA Hypermethylation and Paradoxical Gene Activation.

DNA methylation is a stable epigenetic modification that contributes to the spatiotemporal regulation of gene expression. The manner in which DNA methylation contributes to transcriptional control is dependent on the biological context, including physiological state and the properties of the DNA itself. Classically, dense promoter DNA methylation is associated with transcriptional repression. However, growing evidence suggests that this association may not always hold true, and promoter hypermethylation now also appears to be associated with high transcriptional activity. Furthermore, in a selection of contexts, increasing levels of promoter methylation correlate directly with increased gene expression. These findings postulate a context-dependent model whereby epigenetic contributions to transcriptional regulation occur in a more complex and dynamic manner. We present current evidence documenting promoter hypermethylation and high levels of gene expression, offer insights into the possible mechanisms by which this occurs, and discuss the potential implications for both research and clinical applications.

Silencing of Testin expression is a frequent event in spontaneous lymphomas from Trp53-mutant mice.

The tumour suppressor gene, TES, is frequently methylated in many human tumours. Previously, we demonstrated that TES promoter methylation and transcriptional silencing was the most common molecular abnormality detected in childhood acute lymphoblastic leukaemia (ALL). Trp53-mutant mouse models predominantly develop B- and T-cell lymphomas, which are widely considered equivalent to childhood T and B ALL. In this study, we examined expression of Tes transcript and Testin protein in spontaneous tumours obtained from three Trp53-mutant mouse models. Using immunohistochemistry, we report that 47% of lymphomas lacked Testin protein compared to only 7% of non-lymphoid tumours. Further examination of the lymphomas from Trp53-null and Trp53-mΔpro homozygous mutant mice revealed that 63% and 69% respectively of the isolated lymphomas were Testin negative, which is similar to reported rates in childhood T-ALL. Surprisingly, lymphomas from Trp53-Δ122 mice were frequently Testin positive (> 60%), suggesting that the presence of the Trp53-Δ122 protein appeared to mitigate the requirement for Tes silencing in lymphomagenesis. Quantitative RT-PCR results confirmed that this lack of Testin protein was due to Tes transcriptional silencing, although bisulfite sequencing demonstrated that this was not due to promoter methylation. These results are consistent with the Testin protein having lymphoid tumour suppressor activity in both mice and humans.

Gene-Specific Targeting of DNA Methylation in the Mammalian Genome.

DNA methylation is the most widely-studied epigenetic modification, playing a critical role in the regulation of gene expression. Dysregulation of DNA methylation is implicated in the pathogenesis of numerous diseases. For example, aberrant DNA methylation in promoter regions of tumor-suppressor genes has been strongly associated with the development and progression of many different tumors. Accordingly, technologies designed to manipulate DNA methylation at specific genomic loci are very important, especially in the context of cancer therapy. Traditionally, epigenomic editing technologies have centered around zinc finger proteins (ZFP)- and transcription activator-like effector protein (TALE)-based targeting. More recently, however, the emergence of clustered regulatory interspaced short palindromic repeats (CRISPR)-deactivated Cas9 (dCas9)-based editing systems have shown to be a more specific and efficient method for the targeted manipulation of DNA methylation. Here, we describe the regulation of the DNA methylome, its significance in cancer and the current state of locus-specific editing technologies for altering DNA methylation.

DNA methylation of hepatic iron sensing genes and the regulation of hepcidin expression.

Production of the iron regulatory peptide hepcidin is tightly controlled by a network of proteins in hepatocytes that sense levels of iron in the circulation (as diferric-transferrin) and in tissues (in ferritin). Human studies show high variability in the normal range of serum hepcidin levels. We have postulated that this may, in part, be related to inter-individual variability in the expression of genes in the iron sensing pathway, potentially governed by epigenetic factors. Here, we have investigated whether genes encoding hepatic iron sensing proteins and hepcidin are regulated by DNA methylation. Experiments were performed on two human hepatoma cell lines, HepG2 cells and Huh7 cells. Basal expression of TFR2 and HAMP was significantly lower in Huh7 cells compared with HepG2 cells. Analysis of bisulphite-converted DNA from Huh7 cells revealed partial methylation of TFR2 (alpha transcript), which could result in gene silencing. Demethylation using 5-aza-2'-deoxycitidine (AZA) increased TFR2 mRNA expression in Huh7. PCR analysis of bisulphite-converted HAMP promoter DNA, using methylation-specific primers, revealed no differences between cell lines. However, HAMP mRNA expression in Huh7 was increased by AZA treatment, suggesting that methylation of one or more iron sensing genes may indirectly influence HAMP expression. Our study provides evidence that DNA methylation might control expression of HAMP and other hepatic iron sensing genes, and indicates that epigenetic influences on iron homeostasis warrant further investigation.

The role of DNA methylation in human trophoblast differentiation.

The placenta is a vital fetal exchange organ connecting mother and baby. Specialised placental epithelial cells, called trophoblasts, are essential for adequate placental function. Trophoblasts transform the maternal vasculature to allow efficient blood flow to the placenta and facilitate adequate nutrient uptake. Placental development is in part regulated by epigenetic mechanisms. However, our understanding of how DNA methylation contributes to human trophoblast differentiation is limited. To better understand how genome-wide methylation differences affect trophoblast differentiation, reduced representation bisulfite sequencing (RRBS) was conducted on four matched sets of trophoblasts; side-population trophoblasts (a candidate human trophoblast stem cell population), cytotrophoblasts (an intermediate progenitor population), and extravillous trophoblasts (EVT, a terminally differentiated population) each isolated from the same first trimester placenta. Each trophoblast population had a distinct methylome. In line with their close differentiation relationship, the methylation profile of side-population trophoblasts was most similar to cytotrophoblasts, whilst EVT had the most distinct methylome. In comparison to mature trophoblast populations, side-population trophoblasts exhibited differential methylation of genes and miRNAs involved in cell cycle regulation, differentiation, and regulation of pluripotency. A combined methylomic and transcriptomic approach was taken to better understand cytotrophoblast differentiation to EVT. This revealed methylation of 41 genes involved in epithelial to mesenchymal transition and metastatic cancer pathways, which likely contributes to the acquisition of an invasive EVT phenotype. However, the methylation status of a gene did not always predict gene expression. Therefore, while CpG methylation plays a role in trophoblast differentiation, it is likely not the only regulatory mechanism involved in this process.