Histone modification assays The cellular processes of transcription, DNA replication and DNA repair involve the interaction between genomic DNA and nuclear proteins. It had been known that certain regions within chromatin were extremely susceptible to DNAse I digestion, which cleaves DNA in a low sequence specificity manner. Such hypersensitive sites were thought to be transcriptionally active regions, as evidenced by their association with RNA polymerase and topoisomerases I and II. It is now known that sensitivity to DNAse I regions correspond to regions of chromatin with loose DNA-histone association. Hypersensitive sites most often represent promoters regions, which require for DNA to be accessible for DNA binding transcriptional machinery to function. ChIP-Chip and ChIP-Seq Histone modification was first detected on a genome wide level through the coupling of chromatin immunoprecipitation (ChIP) technology with DNA microarrays, termed ChIP-Chip. However instead of isolating a DNA-binding transcription factor or enhancer protein through chromatin immunoprecipitation, the proteins of interest are the modified histones themselves. First, histones are cross-linked to DNA in vivo through light chemical treatment (e.g., formaldehyde). The cells are next lysed, allowing for the chromatin to be extracted and fragmented, either by sonication or treatment with a non-specific restriction enzyme (e.g., micrococcal nuclease). Modification-specific antibodies in turn, are used to immunoprecipitate the DNA-histone complexes. Following immunoprecipitation, the DNA is purified from the histones, amplified via PCR and labeled with a fluorescent tag (e.g., Cy5, Cy3). The final step involves hybridization of labeled DNA, both immunoprecipitated DNA and non-immunoprecipitated onto a microarray containing immobilized gDNA. Analysis of the relative signal intensity allows the sites of histone modification to be determined. ChIP-chip was used extensively to characterize the global histone modification patterns of yeast. From these studies, inferences on the function of histone modifications were made; that transcriptional activation or repression was associated with certain histone modifications and by region. While this method was effective providing near full coverage of the yeast epigenome, its use in larger genomes such as humans is limited. In order to study histone modifications on a truly genome level, other high-throughput methods were coupled with the chromatin immunoprecipitation, namely: SAGE: serial analysis of gene expression (ChIP-SAGE), PET: paired end ditag sequencing (ChIP-PET) and more recently, next-generation sequencing (ChIP-Seq). ChIP-seq follows the same protocol for chromatin immunoprecipitation but instead of amplification of purified DNA and hybridization to a microarray, the DNA fragments are directly sequenced using next generation parallel re-sequencing. It has proven to be an effective method for analyzing the global histone modification patterns and protein target sites, providing higher resolution than previous methods. DNA methylation assays Techniques for characterizing primary DNA sequences could not be directly applied to methylation assays. For example, when DNA was amplified in PCR or bacterial cloning techniques, the methylation pattern was not copied and thus the information lost. The DNA hybridization technique used in DNA assays, in which radioactive probes were used to map and identify DNA sequences, could not be used to distinguish between methylated and non-methylated DNA. Restriction endonuclease based methods Non genome-wide approaches The earliest methylation detection assays used methylation modification sensitive restriction endonucleases. Genomic DNA was digested with both methylation-sensitive and insensitive restriction enzymes recognizing the same restriction site. The idea being that whenever the site was methylated, only the methylation insensitive enzyme could cleave at that position. By comparing restriction fragment sizes generated from the methylation-sensitive enzyme to those of the methylation-insensitive enzyme, it was possible to determine the methylation pattern of the region. This analysis step was done by amplifying the restriction fragments via PCR, separating them through gel electrophoresis and analyzing them via southern blot with probes for the region of interest. This technique was used to compare the DNA methylation modification patterns in the human adult and hemoglobin gene loci. Different regions of the gene (gamma delta beta globin) were known to be expressed at different stages of development. Consistent with a role of DNA methylation in gene repression, regions that were associated with high levels of DNA methylation were not actively expressed. This method was limited not suitable for studies on the global methylation pattern, or ‘methylome’. Even within specific loci it was not fully representative of the true methylation pattern as only those restriction sites with corresponding methylation sensitive and insensitive restriction assays could provide useful information. Further complications could arise when incomplete digestion of DNA by restriction enzymes generated false negative results. Genome wide approaches DNA methylation profiling on a large scale was first made possible through the Restriction Landmark Genome Scanning (RLGS) technique. Like the locus-specific DNA methylation assay, the technique identified methylated DNA via its digestion methylation sensitive enzymes. However it was the use of two-dimensional gel electrophoresis that allowed be characterized on a broader scale. However it was not until the advent of microarray and next generation sequencing technology when truly high resolution and genome-wide DNA methylation became possible. As with RLGS, the endonuclease component is retained in the method but it is coupled to new technologies. One such approach is the differential methylation hybridization (DMH), in which one set of genomic DNA is digested with methylation-sensitive restriction enzymes and a parallel set of DNA is not digested. Both sets of DNA are subsequently amplified and each labelled with fluorescent dyes and used in two-colour array hybridization. The level of DNA methylation at a given loci is determined by the relative intensity ratios of the two dyes. Adaptation of next generation sequencing to DNA methylation assay provides several advantages over array hybridization. Sequence-based technology provides higher resolution to allele specific DNA methylation, can be performed on larger genomes, and does not require creation of DNA microarrays which require adjustments based on CpG density to properly function. Bisulfite sequencing Bisulfite sequencing relies on chemical conversion of unmethylated cytosines exclusively, such that they can be identified through standard DNA sequencing techniques. Sodium bisulfate and alkaline treatment does this by converting unmethylated cytosine residues into uracil while leaving methylated cytosine unaltered. Subsequent amplification and sequencing of untreated DNA and sodium bisulphite treated DNA allows for methylated sites to be identified. Bisulfite sequencing, like the traditional restriction based methods, was historically limited to methylation patterns of specific gene loci, until whole genome sequencing technologies became available. However, unlike traditional restriction based methods, bisulfite sequencing provided resolution on a nucleotide level. Limitations of the bisulfite technique include the incomplete conversion of cytosine to uracil, which is a source of false positives. Further, bisulfite treatment also causes DNA degradation and requires an additional purification step to remove the sodium bisulfite. Next-generation sequencing is well suited in complementing bisulfite sequencing in genome-wide methylation analysis. While this now allows for methylation pattern to be determined on the highest resolution possible, on the single nucleotide level, challenges still remain in the assembly step because of reduced sequence complexity in bisulphite treated DNA. Increases in read length seek to address this challenge, allowing for whole genome shotgun bisulphite sequencing (WGBS) to be performed. The WGBS approach using an Illumina Genome Analyzer platform and has already been implemented in Arabidopsis thaliana. Reduced representation genomic methods based on bisulfite sequencing exist as well, and they are particularly suitable for species with large genome sizes. Chromatin accessibility assays Chromatin accessibility is the measure of how "accessible" or "open" a region of genome is to transcription or binding of transcription factors. The regions which are inaccessible (i.e. because they're bound by nucleosomes) are not actively transcribed by the cell while open and accessible regions are actively transcribed. Changes in chromatin accessibility are important epigenetic regulatory processes that govern cell- or context-specific expression of genes. Assays such as MNase-seq, DNase-seq, ATAC-seq or FAIRE-seq are routinely used to understand the accessible chromatin landscape of cells. The main feature of all these methods is that they're able to selectively isolate either the DNA sequences that are bounded to the histones, or those that are not. These sequences are then compared to a reference genome that allows to identify their relative position. MNase-seq and DNase-seq both follow the same principles, as they employ lytic enzymes that target nucleic acids to cut the DNA strands unbounded by nucleosomes or other proteic factors, while the bounded pieces are sheltered, and can be retrieved and analysed. Since active, unbound regions are destroyed, their detection can only be indirect, by sequencing with a Next Generation Sequencing technique and comparison with a reference. MNase-seq utilises a micrococcal nuclease that produces a single strand cleavage on the opposite strand of the target sequence. DNase-seq employs DNase I, a non-specific double strand-cleaving endonuclease. This technique has been used to such an extent that nucleosome-free regions have been labelled as DHSs, DNase I hypersensitive sites, and has been ENCODE consortium's election method for genome wide chromatin accessibility analyses. The main issue of this technique is that the cleavage distribution can be biased, lowering the quality of the results. FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements) requires as its first step crosslinking of the DNA with nucleosomes, then DNA shearing by sonication. The free and linked fragments are
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