Dna methylation analysis in development

Summary

Recent advances in DNA methylation analysis based on bisulphite conversion enable quantitative, large-scale, single-base resolution mapping of DNA methylation states in desired regions of complex genomes, providing the molecular details on how epigenetic marks are established, propagated and maintained. DNA methylation presents an essential and versatile epigenetic contribution to genome integrity and function. Being essential for proper invertebrate development and critical for mammalian and plant imprinting, it plays an important role in maintaining genomic stability through species. DNA methylation patterns are susceptible to changes in response to environmental cues such as diet or toxins, rendering the epigenome most vulnerable during early in utero development. Aberrant methylation is associated with disease progression and is a common feature of cancer genomes. Improvements in the 'gold standard' bisulphite protocol as well as development of various post-bisulphite analysis methods allow acquisition of highly reliable data crucial for detecting epigenetic changes that occur during development.

Keywords: DNA methylation; bisulphite conversion; species distribution; development; genomic imprinting

Introduction

Epigenetic inheritance defined as phenotypic changes at a cellular and organism level without accompanying alterations in genome sequence, are mediated by a variety of molecular mechanisms including histone modifications, ATP-dependent chromatin remodeling complexes, small and other non-coding RNAs and DNA methylation.(1) These diverse molecular mechanisms are closely intertwined and stabilize each other to ensure the faithful propagation of an epigenetic state over time and especially through cell division.

DNA methylation is the only genetically programmed DNA modification in vertebrates, occurring predominantly on the cytosine within CG dinucleotides (referred to as CpG).(2) Other types of methylation such as methylation of cytosines in the context of CpNpG or CpA sequences have been detected in mouse embryonic stem cells and plants, being rare in somatic mammalian tissues. CpG modification is propagated via a maintenance methyltransferase, DNMT1,(3) which preferentially recognizes and modifies hemi-methylated CpGs.(4) It is located at the replication fork during the S phase of the cell cycle and methylates the newly synthesized DNA strand using the parent strand as a template. Consequently, it passes the epigenetic information through cell generations. While the vast majority of CpGs are methylated in differentiated mammalian cells,(5) most methylation undergoes waves of erasure and reestablishment during gametogenesis and preimplantation development.(6) The reestablishment of methylation is carried out by de novo methyltransferases, DNMT3A and DNMT3B.(7) These enzymes have certain preferences for specific targets (e.g., DNMT3A methylates maternal imprinted genes and DNMT3B localizes at minor satellite repeats) but also work cooperatively to methylate the genome. Possible trigger mechanisms to initiate de novo methylation include preferred target DNA sequences, RNA interference, certain chromatin structures induced by histone modifications and other protein-protein interactions.(8,9)

Although CpG dinucleotides are significantly underrepresented in mammalian genomes, probably because they act as mutation hotspots, certain regions are relatively rich in CpGs, called CpG islands (CGIs).(10) While CGIs are found throughout the genome, they are often associated with promoter regions and first exons of many genes, with >70% of annotated genes having CGI-related promoters.(11) They are mostly unmethylated corresponding to the maintenance of an open chromatin structure and a potentially active state of transcription. However, a growing number of CGIs have been recently identified that are methylated in nonpathological somatic tissues.(12,13) Promoter CGIs of genes differ in their susceptibility to become methylated during normal development as well as during carcinogenesis, which might be due to intrinsic sequence properties.(14) This regulation is controlled in a tissue- and developmental-stage-specific manner and is maintained throughout the life of an individual.

Sites of DNA methylation recruit another group of proteins called DNA-methyl-binding domain (MBD1-4) proteins or methyl CpG-binding proteins (MeCP2) that recognize and bind to methylated DNA.(15,16) These associate with transcriptional corepressors such as histone deacetylating complexes, polycomb proteins, and chromatin remodeling complexes and attract chromodomain-binding proteins. Besides the structurally related MBD proteins, methylated DNA can also be bound by some zinc-finger proteins such as Kaiso and the more recently discovered ZBTB4 and ZBTB38 proteins that are also able to repress transcription in a methylation-dependent manner.(17,18)

DNA methylation in higher eukaryotes is usually associated with a repressed chromatin environment, while in the prokaryote kingdom both cytosine and adenine methylation have been described as a part of the host restriction system. Unravelling the events leading to the establishment, maintenance and plasticity of epigenetic states among species may prove crucial to elucidate the mechanisms associated with cellular differentiation and homeostasis.

Distribution and functional role of DNA methylation amongst species

In many eukaryotes, DNA methylation is thought to control gene expression by modulation of DNA-protein interactions.(19) However, DNA methylation is not essential in all eukaryotes. The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe and the nematode Caenorhabditis elegans appear to have lost the DNA methylation machinery as no DNA methyltransferase genes are present in their genomes and no DNA methylation has been detected by various methods.(20)

Very low levels of DNA methylation have been reported during development in Drosophila melanogaster and therefore display a relatively high mutation rate due to the vulnerability of their genome to genomic transposition.(21) Two potential DNA methyltransferase genes have been discovered in Drosophila. In contrast to the vertebrate DNMT2, which has mostly been associated with RNA methyltransferase activity,(22) silencing of DNMT2 did not have detectable effects on embryonic development and viability. However, over-expression of DNMT2 resulted in an extended fly life span and in the over-expression of several genes, suggesting that DNA methylation could enhance the expression of fly genes instead of acting as an on/off switch for gene transcription.(23)

Additionally, a single homolog of the mammalian MBD2 and MBD3 proteins has also been discovered in Drosophila with its functional role being under investigation.(24) It is possible that DNA methylation only plays an auxiliary role, for example, during development since the 5-methylcytosine (m5C) content of the Drosophila genome showed the highest signal in the early embryo.(25)

A functional DNA methylation system containing Dnmt1 and Dnmt3 as well as a functional ortholog of the mammalian MBD family has been described in the honeybee, Apis melifera.(26) In honeybee, DNA methylation plays an important role in the organization of social structures as well as labor division.(27) In bee communities, young worker bees feed a privileged subset of larvae with a substance called royal jelly. Larvae nurtured with royal jelly develop into queens, while other bees of the same clonal origin become worker bees. The larvae treated with small interfering RNA targeting a de novo DNA methyltransferase, Dnmt3, developed into queens with fully functional ovaries, while a control RNA did not induce that effect.(27) This finding highlights the function of DNA methylation in phenotypic plasticity and also shows an elegant way of how epigenomes respond to environmental signals in order to determine different developmental fates.

Within the filamentous fungi, DNA methylation has been detected in many species but it is not universally present, e.g., Aspergillus nidulans has no detectable DNA methylation while very low levels have been reported from the closely related A. flavus.(28) Two fungi that have been used extensively for DNA methylation research, Neurospora crassa and Ascobolus immersus, exhibit heterogeneous distribution of m5C in all possible sequence contexts. In these species, DNA methylation is thought to be involved in "genome defense", e.g., by silencing invading transposable elements, and is not essential for survival. In Neurospora, DNA methylation is catalyzed by a single DNMT, DIM-2, not essential for the organism viability.(29) This stands in contrast to mammalian organisms that utilize four DNMTs and require DNA methylation for embryonic development. Nevertheless, the Neurospora genome methylation level (2-3%) is comparable to mammals and the availability of a DNMT mutant, combined with the comparatively low genome complexity, has provided an opportunity to study the biological function of DNA methylation. A study mapping DNA methylation in the Neurospora genome revealed that most of the methylated sequences correspond to transposon relics,(30) in line with a role for DNA methylation in preventing the reactivation of parasitic genomic sequences in eukaryotes.

In plants, such as Arabidopsis thaliana, DNA methylation similarly silences the expression of transposable elements. Upon loss of DNA methylation, transposons reactivate and integrate in various regions of the genome, causing pleiotropic effects on development as has been demonstrated by use of ddm1 mutants.(31) Additionally, in plants, DNA methylation has a central role in genomic imprinting, the monoallelic expression of a gene from either the maternal or the paternal copy.(32,33) Loss-of-function of the maintenance DNA methyltransferase, MET1, the Arabidopsis ortholog of Dnmt1, leads to developmental abnormalities such as delayed flowering and reduced fertility, which become very severe when additional methyltransferase genes (CMT3 and/or DRM2) are mutated.(34,35) Either the loss or gain of methylation at specific genes (FWA, SUP) can also lead to developmental abnormalities in Arabidopsis.(32, 36,37)

In vertebrates (including representatives of fish, amphibia, reptiles, birds and mammals), there is abundant evidence that aberrant DNA methylation can preclude normal development. Knockout mutations of any one of the three mouse genes that encode DNA methyltransferases (Dnmt1, Dnmt3a and Dnmt3b) are lethal.(33) Depletion of Dnmt1 in zebrafish embryos causes defects in terminal differentiation of the intestine, exocrine pancreas and retina.(38) Genomic imprinting and X-chromosome inactivation in female mammals is also dependent on DNA methylation.(39) The high failure rate of cloning by somatic nuclear transfer has been attributed to improper reprogramming of DNA methylation patterns in the donor nucleus.(40)

DNA methylation plays also important role in the biology of prokaryotes. Bacterial DNA contains in addition to m5C, two additional methylated bases, namely N6-methyladenine (m6A), and a more recently discovered minor base N4-methylcytosine (m4C).(41) These modified bases are involved in the protection of bacterial DNA from the action of specific endonucleases via the host specific restriction-modification system which is regarded as a defense mechanism against bacteriophage infection. In addition, the roles of m6A are multiple and include for example the regulation of virulence and the control of many bacterial DNA functions such as the replication, repair, expression and transposition of DNA.(42)

Despite the clear importance of DNA methylation, the extent to which changes in somatic DNA methylation are involved in mammalian gene regulation is unclear.(33, 43) This is largely owing to our limited knowledge of DNA methylation patterns. A recent study estimated that DNA methylation of less than 0.1% of the human genome has been analyzed in detail.(44) In order to understand further the biology that promotes methylation changes in normal development, accurate and reproducible methods to analyse and quantify the DNA methylation sequence in detail are required.

DNA methylation analysis: Improvements in bisulphite conversion method

Various methods have been developed for determining the distribution of m5C in DNA.(45-47) These can be divided into three broad groups: (I) differential enzymatic cleavage of DNA using restriction endonuclease(s) whose activity is influenced by methylation of the recognition site, (II) selective chemical cleavage of DNA using chemical reactions that modify either cytosine or m5C, and (III) differential sensitivity to chemical conversion using a protein that has different affinity to methylated and non methylated DNA.

The major advance in DNA methylation analysis has been the development of bisulfite modification of DNA to convert unmethylated cytosines to uracil, leaving methylated cytosines unchanged.(48) This method aims at distinguishing methylated from unmethylated DNA via PCR amplification and analysis of the PCR products. During PCR amplification, unmethylated cytosines amplify as thymine and methylated cytosines amplify as cytosine. The key to the bisulphite protocol for determining DNA methylation is based on the selective chemical reaction of sodium bisulphite with cytosine versus m5C residues. The reaction is highly single-strand specific and cannot be performed on double-stranded DNA. The deamination of cytosine by sodium bisulphite and subsequent PCR involves five critical steps: denaturation of the DNA into single strands; reaction of bisulphite with the 5-6 double bond of cytosine to give a cytosine sulphonate derivative; hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil sulphonate derivative; removal of the sulphonate group by a subsequent alkali treatment to give uracil and PCR amplification.

Because the conversion of cytosines to uracils creates non complementary strands (i.e., uracils opposite guanines), DNA must be amplified with separate pairs of primers that are specific for either the top or bottom DNA strands. Following PCR amplification, the uracils are amplified as thymines, whereas m5C residues are amplified as cytosines. To determine methylation at single-nucleotide resolution, the PCR amplicon can either be sequenced directly or cloned and sequenced. DNA methylation in the PCR target region is then read by scoring, the remaining cytosine resides in the sequence.

However, the conventional bisulfite treatment requiring hours of exposure to low-molarity, low-temperature bisulfite and, sometimes, thermal denaturation, may lead to two types of conversion errors: inappropriate conversion of m5C to thymine and failure to convert unmethylated cytosine to uracil.(49)

Inappropriate conversion occurs when a methylated cytosine is deaminated, yielding thymine.(50) Like uracils that result from deamination of cytosines, thymines that arise through inappropriate conversion of m5C will pair with adenine during PCR. As a result, m5C that undergo inappropriate conversion will be misinterpreted as unmethylated. When inappropriate conversion occurs and is ignored in data analysis, it will lead to underestimates of genomic methylation densities. In contrast, when inappropriate conversion occurs and its frequency is known, it can be included as a parameter in the data analysis.(50)

Failed conversion is said to occur when an unmethylated cytosine fails to be deaminated, and thus appears in resulting data as if it had been methylated. Because m5C in somatic cells of mammals occurs exclusively or almost exclusively at CpG cytosines,(51) the failed-conversion frequency for bisulfite treatment of mammalian DNA is indicated by the fraction of non-CpG cytosines that appear as cytosines in sequence data. When not explicitly incorporated as a parameter in data analysis, failed conversion can inflate estimates of methylation densities, and can undermine efforts to determine the sequence motif preferences of DNA methyltransferases. The failed conversion frequency can typically be reduced by increasing the duration of bisulfite treatment,(50) by increasing the number of thermal denaturation steps used during conversion,(52) or both.

An alternative protocol by Shiraishi and Hayatsu showed that a complete deamination of cytosine to uracil can also be achieved in shorter periods by using a highly concentrated bisulfite solution at an elevated temperature, therefore, reducing inappropriate conversion.(53) A recent study by Genereux et al (49) showed that this high-molarity and high temperature protocol yields greater homogeneity among sites and among molecules in conversion rates and thus yields more reliable data than the conventional low molarity approach. Currently there is one commercially available conversion kit, implementing the high temperature bisulphite conversion approach, the Imprint DNA modification kit from Sigma-Aldrich.

Another disadvantage of bisulphite conversion is the degradation of DNA due to the acidic conditions that develop during this process. The conversion of small samples typically available for developmental studies often yields very small template counts for PCR leading to an increased risk of product redundancy and contamination. A recent approach using molecular barcoding and batchstamping to label each genomic DNA template with an individual sequence tag, sample ID and analysis date prior to PCR amplification proved highly sensitive in identifying redundant and contaminant sequences, serving as reliable method for positive identification of desired sequences.(54)

The majority of new data on DNA methylation is now based on prior treatment of the DNA with bisulphite, followed by DNA amplification with target specific primers. However, the method of analysis of the amplified PCR fragments can vary considerably depending on the degree of specificity and detail of methylation required. The various post-bisulphite analysis options can be divided into either 'Selective Detection' for analysis of only methylated DNA or only unmethylated DNA (Table 1) or 'Non-selective Detection' for quantitative methylation analysis (Table 2).

Methods of selective amplification of methylated or unmethylated sequences from bisulphate-treated DNA include variations of methylation specific PCR (MSP). This is the most rapid and highly sensitive technique to screen for methylation.(55) Following bisulfite modification, PCR is performed using two sets of primers designed to amplify either methylated or unmethylated alleles. MSP has the advantages of being highly sensitive (able to detect one methylated allele in a population of more than 1,000 unmethylated alleles)(56) and can be used on DNA samples of limited quantity and quality. Variations of MSP called MethylLight(57) or quantitative analysis of methylated alleles(58) have been developed using real-time PCR for methylation detection (Table 1).

Furthermore, quantitative methylation analysis can be performed in bisulphite-treated DNA using a variety of new high-throughput technologies which allow investigation of thousands of CpG methylation sites across the genome, collecting tissue-specific as well as age- and sex-dependent DNA methylation signatures (Table 2). Bisulphite genomic sequencing analyses can give resolution at every cytosine site in the target sequence at the single-molecule level. Among these methods, hairpin-bisulphite PCR may prove particularly useful in the detection of hemimethylated states which are transitional states playing critical role in developmental processes since active demethylation or de novo methylation may sometimes be involved in gene reactivation or inactivation.(52) The development of ''hairpin-bisulfite PCR'' for analyzing patterns of cytosine methylation on complementary strands of individual DNA molecules has proved a powerful tool in estimating the fidelity with which epigenetic state of cytosine is transmitted. This method uses a hairpin linker, targeted and ligated to restriction-enzyme-cleaved genomic DNA, to maintain attachment of complementary strands during the subsequent denaturation steps required by bisulfite conversion(52) and PCR amplification.

Other analytical procedures can be less detailed but can give either a semi-quantitative or an average estimate of the methylation state of the amplified target sequence.

Conclusions

Continued rapid improvements in technology make the study of DNA methylation more accessible and exciting. Advances in DNA methylation analysis, making use of bisulfite conversion of m5C in non-repetitive regions of DNA in association with high throughput sequencing, are able to generate single-base resolution methylation profiles, representing the most promising, comprehensive high-resolution method for determining DNA methylation states. It has been recently applied in the elucidation of the methylation patterns of the filamentous fungus Neurospora crassa with potential application in mapping methylation patterns in the non-repetitive regions of higher eukaryotes, such as fungi, plants and animal genomes.(59,60) Studies on non-mammalian models have provided valuable insights into how eukaryotic genomes are organized, how gene clusters or regulons are co-ordinately regulated by epigenetic mechanisms and how DNA methylation affects the expression of specific genes. Application of this methodology in human methylome analysis and specifically in both differentiated as well as undifferentiated cells is currently in progress aiming at a better understanding of the function of DNA methylation in health and disease.

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