*2.2. Histone Modification*

Histones are important proteins responsible for maintaining the structure of chromatin and play a role in the dynamic and long term regulation of genes. The *N*-terminal tail of histone 3 (H3), one of the five histones found in eukaryotic nuclei, is subject to methylation or acetylation of lysine and arginine residues as well as phosphorylation of serine and threonine residues [41]. Histones are acetylated by histone acetyltransferases (HAT) and deacytelated by histone deacetylases (HDAC). These modifications may have opposing effects based on which residue is modified or which moiety is added. For example, methylation of H3 lysine 4 (H3K4) is a hallmark of actively transcribed DNA while methylation of H3 lysine 9 (H3K9) is associated with repressed gene expression [42,43]. Acetylation of histones is associated with transcriptionally active DNA, as the *N*-terminal of the histone tails become neutralized and their affinity for DNA reduced, so loosening the conformation of the chromatin [44]. Interestingly, while methylation at H3K9 allows for the binding of heterochromodoman protein 1 (HP1), leading to the repression of transcription, methylation at H3K4 blocks the binding of transcriptional repressor, nucleosome remodeling and deacetylase (NuRD), leading to transcription. Acetylation of lysines allows for the binding of bromodomain proteins, such as histone acetyltransferase, GCN5 [43].

The most common histone modification assay is the chromatin immunoprecipitation (ChIP), which uses antibodies against site-specific epigenetic marks to identify histone-DNA complexes with that mark [45]. Following fragmentation, histone-DNA complexes that showcase specific modifications are immunoprecipated using an antibody against that modification. Enriched and purified DNA fragments can then be detected by quantitative PCR (ChIP-qPCR), microarray analysis (ChIP-on-chip) or deep sequencing (ChIP-seq) [46]. ChIP-seq uses next-generation sequencing instead of amplifying purified DNA that was hybridized to a DNA microarray [47]. Importantly, ChIP-seq allows for mapping of the newly sequenced DNA to the reference genome, enabling researchers to determine the genome-wide distribution of particular modifications [46].

Conventional epigenomic analyses require large amounts of starting material due to the low DNA recovery efficiency after bisulfite conversion and enzyme digestion [48]. However, using large sample sizes masks cell to cell heterogeneity which may be important to the disease state. As molecular technology progresses to the single-cell level, many new approaches are being developed to study the single-cell epigenome, albeit with limited efficiency and scalability [39,48], including: single-cell bisulfite sequencing [49], single-cell reduced representation bisulfite sequencing [50], DNase I sequencing [51], single-cell DamID sequencing [52] and single-cell ATAC-seq [53]. Drawbacks of these methods include low coverage of CpG islands as compared to bulk methods, but the methodology is expected to grow to meet these challenges [54]. One such recent development is single cell combinatorial indexing for methylation analysis (sci-MET) which boasts of a 69% alignment rate to bulk methods [55].
