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. 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. 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. 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 separated with a traditional phenol-chloroform extraction, since the proteic fraction is stuck in the interphase while the unlinked DNA shifts to the aqueous phase and can be analysed with various methods. Sonication produces random breaks, and therefore is not subject to any kind of bias, and is also the bigger length of the fragments (200-700 nt) makes this technique suitable for wider regions, while it's unable to resolve the single nucleosome. ATAC-seq is based on the activity of Tn5 transposase. The transposase is used to insert tags in the genome, with higher frequency on regions not covered by proteic factors. The tags are then used as adapters for PRC or other analytical tools. Spatial epigenomic methods extend chromatin accessibility and histone-modification assays to intact tissue sections by combining in situ transposition or antibody-based profiling with spatial barcoding strategies. One such approach, deterministic barcoding in tissue (DBiT-seq), uses orthogonal microfluidic channels to deliver positional barcodes and enables high-spatial-resolution mapping of chromatin state and gene expression in fixed tissues. Commercial implementations of DBiT-seq, such as the AtlasXomics platform, provide microfluidic chips and reagent kits for spatial ATAC-seq and CUT&Tag on fresh-frozen and formalin-fixed paraffin-embedded tissues, and have been used to map chromatin accessibility and histone marks in human dorsal root ganglion and other mammalian tissues.
Direct detection Polymerase sensitivity in
single-molecule real-time sequencing made it possible for scientists to directly detect epigenetic marks such as methylation as the polymerase moves along the DNA molecule being sequenced. Several projects have demonstrated the ability to collect genome-wide epigenetic data in bacteria.
Nanopore sequencing is based on changes of electrolytic current signals according to base modifications (e.g. Methylation). A
polymerase mediates the entrance of
ssDNA in the pore: the ion-current variation is modulated by a section of the pore and the consequently generated difference is recorded revealing the position of
CpG. Discrimination between hydroxymethylation and
methylation is possible thanks to solid-state
nanopores even if the current while passing through the high-field region of the pore may be slightly influenced in it. As a reference amplified DNA is used which will not present copied methylationed sites after the
PCR process. The Oxford Nanopore Technologies
MinION sequencer is a technology where, according to a hidden
Markov model, it is possible to distinguish unmethylated cytosine from the methylated one even without chemical treatment that acts to enhance the signal of that modification. The data are registered commonly in picoamperes during established time. Other devices are the Nanopolish and the SignaAlign: the former expresses the frequency of a methylation in a read while the latter gives a probability of it derived from the sum of all the reads.
Single-molecule real-time sequencing (SMRT) is a single-molecule
DNA sequencing method. Single-molecule real-time sequencing utilizes a
zero-mode waveguide (ZMW). A single DNA polymerase enzyme is bound to the bottom of a ZMW with a single molecule of DNA as a template. Each of the four DNA bases is attached to one of four different
fluorescent dyes. When a nucleotide is incorporated by the DNA polymerase, the fluorescent tag is cleaved off and the detector detects the fluorescent signal of the nucleotide incorporation. As the sequencing occurs, the polymerase enzyme kinetics shift when it encounters a region of methylation or any other base modification. When the enzyme encounters chemically modified bases, it will slow down or speed up in a uniquely identifiable way. Fluorescence pulses in SMRT sequencing are characterized not only by their emission spectra but also by their duration and by the interval between successive pulses. These metrics, defined as
pulse width and interpulse duration (IPD), add valuable information about DNA polymerase kinetics. Pulse width is a function of all kinetic steps after nucleotide binding and up to fluorophore release, and IPD is determined by the kinetics of nucleotide binding and polymerase translocation. In 2010 a team of scientists demonstrated the use of single-molecule real-time sequencing for direct detection of modified nucleotide in the DNA template including
N6-methyladenosine,
5-methylcytosine and
5-hydroxylcytosine. These various modifications affect polymerase kinetics differently, allowing discrimination between them. In 2017, another team proposed a combined bisulfite conversion with third-generation single-molecule real-time sequencing, it is called single-molecule real-time bisulfite sequencing (SMRT-BS), which is an accurate targeted CpG methylation analysis method capable of a high degree of multiplying and long read lengths (1.5 kb) without the need for PCR amplicon sub-cloning. ==Theoretical modeling approaches==