DNA methylation profiling in cancer: from single nucleotides towards methylome

Genomic DNA methylation pattern (methylome) represents epigenetic program of a cell. It controls expression of genetic information. In tumor cells, significant alterations in DNA methylation take place, which can be identified as one of the earliest and most consistent features of tumorigenesis. Detailed survey of methylcytosines' distribution in genome is extremely important for understanding of real tumor etiology and early diagnostics. Progress in the field has been hampered by the unavailability of methods for large-scale determination of methylation patterns. Nowadays, variety of techniques is in development that allow for highly parallel regime of samples analysis (high-throughput analysis) or large loci DNA profiling (large-scale analysis). Aim of the work is to consider the main trends in the field of new methods development. The principles of the most frequently used approaches to DNA methylation studies are reviewed as well as their application and results. Most attention is paid to DNA microarrays as a technology of choice for epigenetic tumor analysis (oligonucleotide microarrays, BAC-arrays etc.). Alternative DNA sequencing based techniques are discussed, which can soon take on the leadership. Results of a large-scale analysis can be used for identification of new epigenetic markers and epigenetic classification of neoplasia.     

DNA methylation profiling in cancer: From single nucleotides towards the methylome

The genomic DNA methylation pattern (methylome) is a cell epigenetic program that controls the expression of genetic information. The methylation pattern substantially changes in early carcinogenesis. A detailed survey of the methylcytosine distribution in the genome in norm and pathology is of immense importance for a better understanding of the etiology of cancer and its early diagnosis. The techniques available make it possible to simultaneously examine many samples (high-throughput analysis) and to examine large genome loci or even the total methylome (large-scale analysis). The review considers the main trends in the development of new approaches to DNA methylation and describes the techniques most commonly used in the field, their application, and results. Emphasis is placed on the use of various DNA microarrays (oligonucleotide microarrays, BAC arrays, etc.) as a method of choice for epigenetic analysis of tumors. Alternative sequence-based techniques of methylation analysis are discussed. The use of large-scale analysis to identify new epigenetic markers and to develop an epigenetic classification of neoplasms is considered.     

Drugging the methylome: DNA methylation and memory

Over the past decade, since epigenetic mechanisms were first implicated in memory formation and synaptic plasticity, dynamic DNA methylation reactions have been identified as integral to long-term memory formation, maintenance, and recall. This review incorporates various new findings that DNA methylation mechanisms are important regulators of non-Hebbian plasticity mechanisms, suggesting that these epigenetic mechanisms are a fundamental link between synaptic plasticity and metaplasticity. Because the field of neuroepigenetics is so young and the biochemical tools necessary to probe gene-specific questions are just now being developed and used, this review also speculates about the direction and potential of therapeutics that target epigenetic mechanisms in the central nervous system and the unique pharmacokinetic and pharmacodynamic properties that epigenetic therapies may possess. Mapping the dynamics of the epigenome in response to experiential learning, even a single epigenetic mark in isolation, remains a significant technical and bioinformatic hurdle facing the field, but will be necessary to identify changes to the methylome that govern memory-associated gene expression and effectively drug the epigenome.     

Whole DNA methylome profiling in mice exposed to secondhand smoke

Aberration of DNA methylation is a prime epigenetic mechanism of carcinogenesis. Aberrant DNA methylation occurs frequently in lung cancer, with exposure to secondhand smoke (SHS) being an established risk factor. The causal role of SHS in the genesis of lung cancer, however, remains elusive. To investigate whether SHS can cause aberrant DNA methylation in vivo, we have constructed the whole DNA methylome in mice exposed to SHS for a duration of 4 mo, both after the termination of exposure and at ensuing intervals post-exposure (up to 10 mo). Our genome-wide and gene-specific profiling of DNA methylation in the lung of SHS-exposed mice revealed that all groups of SHS-exposed mice and controls share a similar pattern of DNA methylation. Furthermore, the methylation status of major repetitive DNA elements, including long-interspersed nuclear elements (LINE L1), intracisternal A particle long-terminal repeat retrotransposons (IAP-LTR), and short-interspersed nuclear elements (SINE B1), in the lung of all groups of SHS-exposed mice and controls remains comparable. The absence of locus-specific gain of DNA methylation and global loss of DNA methylation in the lung of SHS-exposed mice within a timeframe that precedes neoplastic-lesion formation underscore the challenges of lung cancer biomarker development. Identifying the initiating events that cause aberrant DNA methylation in lung carcinogenesis may help improve future strategies for prevention, early detection and treatment of this highly lethal disease.     

Whole DNA methylome profiling in mice exposed to secondhand smoke

Aberration of DNA methylation is a prime epigenetic mechanism of carcinogenesis. Aberrant DNA methylation occurs frequently in lung cancer, with exposure to secondhand smoke (SHS) being an established risk factor. The causal role of SHS in the genesis of lung cancer, however, remains elusive. To investigate whether SHS can cause aberrant DNA methylation in vivo, we have constructed the whole DNA methylome in mice exposed to SHS for a duration of 4 mo, both after the termination of exposure and at ensuing intervals post-exposure (up to 10 mo). Our genome-wide and gene-specific profiling of DNA methylation in the lung of SHS-exposed mice revealed that all groups of SHS-exposed mice and controls share a similar pattern of DNA methylation. Furthermore, the methylation status of major repetitive DNA elements, including long-interspersed nuclear elements (LINE L1), intracisternal A particle long-terminal repeat retrotransposons (IAP-LTR), and short-interspersed nuclear elements (SINE B1), in the lung of all groups of SHS-exposed mice and controls remains comparable. The absence of locus-specific gain of DNA methylation and global loss of DNA methylation in the lung of SHS-exposed mice within a timeframe that precedes neoplastic-lesion formation underscore the challenges of lung cancer biomarker development. Identifying the initiating events that cause aberrant DNA methylation in lung carcinogenesis may help improve future strategies for prevention, early detection and treatment of this highly lethal disease.     

DNA methylation microarray uncovers a permissive methylome for cardiomyocyte differentiation in human mesenchymal stem cells

Differentiation of Wharton's Jelly-Mesenchymal Stem cells (WJ-MSCs) into cardiomyocytes (CMs) in vitro has been reported widely although contradictions remain regarding the maturation of differentiated MSCs into fully functioning CMs. Studies suggest that use of epigenetic modifiers like 5′Azacytidine (5-AC) in MSCs de-methylates DNA and results in expression of cardiac-specific genes (CSGs). However, only partial expression of the CSG set leads to incomplete differentiation of WJ-MSCs to CMs. We used the Agilent 180 K human DNA methylation microarray on WJ-MSCs, 5-AC treated WJ-MSCs and human cardiac tissue (hCT) to analyze differential DNA methylation profiles which were then validated by bisulfite sequencing PCR (BSP). BSP confirmed that only a limited number of CSGs were de-methylated by 5-AC in WJ-MSCs. It also revealed that hCT displays a methylation profile similar to promoter regions of CSG in untreated WJ-MSCs. Thus, the presence of hypo-methylated CSGs indicates that WJ-MSCs are ideal cell types for cardiomyogenic differentiation.     

Transcript profiling in Arabidopsis reveals complex responses to global inhibition of DNA methylation and histone deacetylation

Blocking histone deacetylation with trichostatin A (TSA) or blocking cytosine methylation using 5-aza-2'-deoxycytosine (aza-dC) can derepress silenced genes in multicellular eukaryotes, including animals and plants. We questioned whether DNA methylation and histone deacetylation overlap in the regulation of endogenous plant genes by monitoring changes in expression of approximately 7800 Arabidopsis thaliana genes following treatment with azadC, TSA, or both chemicals together. RNA levels for approximately 4% of the genes were reproducibly changed 3-fold or more by at least one treatment. Distinct subsets of genes are up-regulated or down-regulated in response to aza-dC, TSA, or simultaneous treatment with both chemicals, with little overlap among subsets. Surprisingly, the microarray data indicate that TSA and aza-dC are often antagonistic rather than synergistic in their effects. Analysis of green fluorescent protein transgenic plants confirmed this finding, showing that TSA can block the up-regulation of silenced green fluorescent protein transgenes in response to aza-dC or a ddm1 (decrease in DNA methylation 1) mutation. Our results indicate that global inhibition of DNA methylation or histone deacetylation has complex, nonredundant effects for the majority of responsive genes and suggest that activation of some genes requires one or more TSA-sensitive deacetylation events in addition to cytosine demethylation.     

Mouse olfactory bulb methylome and hydroxymethylome maps reveal noncanonical active turnover of DNA methylation

Hydroxylation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by TET enzymes presents a particular regulatory mechanism in the mammalian brain. However, although methylation and hydroxymethylation of cytosines in non-CpG contexts have been reported, these mechanisms remain poorly understood. Here, we applied TAB-seq and oxBS-seq selectively to detect 5hmC and 5mC at base resolution in olfactory bulb derived from female mice. We found that active turnover of 5mC to 5hmC occurred in both CpG and non-CpG contexts. Strikingly, we identified a different sequence preference for 5mC and 5hmC in a CH context, in which H = A, C, or T, TNCA/TC for 5mC and NNCA/T/CN for 5hmC. More importantly, we found that genes showing 5mC to 5hmC turnover showed only limited overlap in CpG and CH contexts, and that olfactory receptor genes were marked with higher turnover of 5mC to 5hmC in non-CpG context. Collectively, we identified an unexpected sequence preference for non-CpG hydroxymethylation and distinct target genes regulated by the turnover of 5mC to 5hmC in CpG and CH contexts.     

Statistical approaches for the analysis of DNA methylation microarray data

Following the rapid development and adoption in DNA methylation microarray assays, we are now experiencing a growth in the number of statistical tools to analyze the resulting large-scale data sets. As is the case for other microarray applications, biases caused by technical issues are of concern. Some of these issues are old (e.g., two-color dye bias and probe- and array-specific effects), while others are new (e.g., fragment length bias and bisulfite conversion efficiency). Here, I highlight characteristics of DNA methylation that suggest standard statistical tools developed for other data types may not be directly suitable. I then describe the microarray technologies most commonly in use, along with the methods used for preprocessing and obtaining a summary measure. I finish with a section describing downstream analyses of the data, focusing on methods that model percentage DNA methylation as the outcome, and methods for integrating DNA methylation with gene expression or genotype data.     

Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA methylation profiling

DNA methylation is an important epigenetic modification involved in gene regulation, which can now be measured using whole-genome bisulfite sequencing. However, cost, complexity of the data, and lack of comprehensive analytical tools are major challenges that keep this technology from becoming widely applied. Here we present BSmooth, an alignment, quality control and analysis pipeline that provides accurate and precise results even with low coverage data, appropriately handling biological replicates. BSmooth is open source software, and can be downloaded from http://rafalab.jhsph.edu/bsmooth.     

DNA methylome profiling identifies novel methylated genes in African American patients with colorectal neoplasia

The identification of genes that are differentially methylated in colorectal cancer (CRC) has potential value for both diagnostic and therapeutic interventions specifically in high-risk populations such as African Americans (AAs). However, DNA methylation patterns in CRC, especially in AAs, have not been systematically explored and remain poorly understood. Here, we performed DNA methylome profiling to identify the methylation status of CpG islands within candidate genes involved in critical pathways important in the initiation and development of CRC. We used reduced representation bisulfite sequencing (RRBS) in colorectal cancer and adenoma tissues that were compared with DNA methylome from a healthy AA subject’s colon tissue and peripheral blood DNA. The identified methylation markers were validated in fresh frozen CRC tissues and corresponding normal tissues from AA patients diagnosed with CRC at Howard University Hospital. We identified and validated the methylation status of 355 CpG sites located within 16 gene promoter regions associated with CpG islands. Fifty CpG sites located within CpG islands—in genes ATXN7L1 (2), BMP3 (7), EID3 (15), GAS7 (1), GPR75 (24), and TNFAIP2 (1)—were significantly hypermethylated in tumor vs. normal tissues (P < 0.05). The methylation status of BMP3, EID3, GAS7, and GPR75 was confirmed in an independent, validation cohort. Ingenuity pathway analysis mapped three of these markers (GAS7, BMP3 and GPR) in the insulin and TGF-β1 network—the two key pathways in CRC. In addition to hypermethylated genes, our analysis also revealed that LINE-1 repeat elements were progressively hypomethylated in the normal-adenoma-cancer sequence. We conclude that DNA methylome profiling based on RRBS is an effective method for screening aberrantly methylated genes in CRC. While previous studies focused on the limited identification of hypermethylated genes, ours is the first study to systematically and comprehensively identify novel hypermethylated genes, as well as hypomethylated LINE-1 sequences, which may serve as potential biomarkers for CRC in African Americans. Our discovered biomarkers were intimately linked to the insulin/TGF-B1 pathway, further strengthening the association of diabetic disorders with colon oncogenic transformation.     

DNA methylome profiling identifies novel methylated genes in African American patients with colorectal neoplasia

The identification of genes that are differentially methylated in colorectal cancer (CRC) has potential value for both diagnostic and therapeutic interventions specifically in high-risk populations such as African Americans (AAs). However, DNA methylation patterns in CRC, especially in AAs, have not been systematically explored and remain poorly understood. Here, we performed DNA methylome profiling to identify the methylation status of CpG islands within candidate genes involved in critical pathways important in the initiation and development of CRC. We used reduced representation bisulfite sequencing (RRBS) in colorectal cancer and adenoma tissues that were compared with DNA methylome from a healthy AA subject’s colon tissue and peripheral blood DNA. The identified methylation markers were validated in fresh frozen CRC tissues and corresponding normal tissues from AA patients diagnosed with CRC at Howard University Hospital. We identified and validated the methylation status of 355 CpG sites located within 16 gene promoter regions associated with CpG islands. Fifty CpG sites located within CpG islands—in genes ATXN7L1 (2), BMP3 (7), EID3 (15), GAS7 (1), GPR75 (24), and TNFAIP2 (1)—were significantly hypermethylated in tumor vs. normal tissues (P < 0.05). The methylation status of BMP3, EID3, GAS7, and GPR75 was confirmed in an independent, validation cohort. Ingenuity pathway analysis mapped three of these markers (GAS7, BMP3 and GPR) in the insulin and TGF-β1 network—the two key pathways in CRC. In addition to hypermethylated genes, our analysis also revealed that LINE-1 repeat elements were progressively hypomethylated in the normal-adenoma-cancer sequence. We conclude that DNA methylome profiling based on RRBS is an effective method for screening aberrantly methylated genes in CRC. While previous studies focused on the limited identification of hypermethylated genes, ours is the first study to systematically and comprehensively identify novel hypermethylated genes, as well as hypomethylated LINE-1 sequences, which may serve as potential biomarkers for CRC in African Americans. Our discovered biomarkers were intimately linked to the insulin/TGF-B1 pathway, further strengthening the association of diabetic disorders with colon oncogenic transformation.