In eukaryotes, nuclear DNA exists in complex with a particular class of proteins known as histones. A DNA sequence of 147 bp wraps around the nucleosome core particle in 1.7 supercoiled turns. The core nucleosome is comprised of two histones 3-histone 4 (H3-H4) and two histone 2A-histone 2B (H2A-H2B) dimers. A 10-80 bp DNA linker associated with the linker histone 1 (H1) separates nucleosomes, by promoting
greater compaction results in its condensed appearance. This complex of DNA and nucleosome folds into a 10 nm diameter fibre. In vitro studies have shown that in vivo this fibre forms a helical fibre containing 6-11 nucleosomes per turn. This in turn folds further to make higher order chromatin fibres in interphase, and a 200-300 nm structures during condensation of mitotic chromosomes (reviewed in Felsenfeld et.
al[177]).
Histone modifications lead to changes in nucleosome occupancy and regulatory potential. These are post-translation modifications that have been reported to affect over 60 different amino acid residues on histones, instigated by protein-modifying enzymes, many of which also have non-histone substrates[178]. These include:
acetylation, methylation, phosphorylation, ubiquitylation, propionylation, butyrylation, formylation among others[179, 180]. Enhancer-bound TFs have recently been shown to actively recruit histone-modifying enzymes through direct interactions with histone tails, and ATP-dependent remodellers of chromatin that disrupt nucleosome-DNA contacts and allow nucleosome displacement along the DNA, and its removal or exchange[40, 89, 181, 182]. Histone modifications are critical for regulation of transcription. For example, the acetylation of lysine residues on histone tails neutralises their positive charge and changes chromatin overall charge[183, 184].
Similarly, lysine methylation may lead to an increase in the binding affinity on the DNA-binding domains on a number of factors believed to act upon chromatin packaging[185]. Disruptions to the histone modification process have been associated with a number of disease phenotypes[186-188]. This is not unlikely given their function as transducers of intrinsic signals from the cell to the genome[189].
The ‘histone code’ refers to the combinations of modifications required to instigate downstream events[165, 190] (Figure 1.4). It is now commonplace to refer to some of the modifications as ‘activator’ or ‘repressor’ marks depending on the outcome of the event they regulate[191]. There are currently over 150 described histone modifications, and a single nucleosome could carry multiple modifications at the same time, alas only a small number of these modification patterns have been discovered [67, 192]. Several groups have been working on profiling and mapping different histone marks genome-wide to identify the underlying code and how it associates with gene activation/repression, or other genomic features such as promoters, enhancers and insulators[21, 56, 193-195].
Figure 1.4: Histone modifications "code" for cis-regulatory elements
Various histone modification combinations dictate the regulatory potential of promoters (a) and enhancers (b). Figure adapted from Zhou et al. [196].
As discussed earlier in 1.1.1, most promoters colocalise with regions of high GC content, CGIs, and are known as high GC promoters (HCPs), in contrast to their counterparts in low-CGI regions, the low GC promoters (LCPs). Studies have shown that the histone mark H3K4me3 coincided with HCPs, and were characterised with increased chromatin accessibility, histone acetylation, binding of histone H3.3 and marked DNase I hypersensitivity[197-199] (Figure 1.4a). Similar to the HCPs, these accessible H3K4me3-marked regions were also hypomethylated at the DNA level[200].
On the contrary, LCPs appeared inactive by default, and they lacked any measurable enrichment with either H3K4me3 or H3K4me2 in ESCs or adult cell lines. However, a minor subset of LCPs bore the H3K4me3 mark and were highly expressed compared to the unmarked LCPs[21, 200] (Figure 1.4a).
Repressed promoters display a unique histone modification pattern that reflect their transcriptionally inactive state. They are usually marked by the tri-methylation of lysine 27 of its histone 3 (H3K27me3), which is also the prototypical mark of the Polycomb repressor complex. Polycomb repressor complexes, PRC1 and PRC2, inhibit transcription to maintain cell-type-specific gene expression patterns[201]. A large number of HCPs are targeted by Polycomb in mammalian genomes. For example, about 20% of HCPs in ESCs are bound by PRC2 and marked with its associated mark, H3K27me3[202-204]. Interestingly, these promoters also carry the H3K4me3 activator mark , thus are capable of ‘bivalent’ characteristics, being both activated and repressed[205]. ESC bivalent promoters show very low levels of gene expression[206], but later studies have identified some RNAP II enrichment[56, 207]. The tri-methylation of lysine 9 of histone 3 (H3K9me3) is another repressed promoter mark that correlates with constitutive heterochromatin and DNA hypermethylation[196].
Whereas mapping promoters is somewhat straightforward, histone
unbiased fashion[196]. Enhancers are characterised by the presence of particular histone marks in addition to the binding of TFs and other co-activators such as p300[75]. Analysis have shown enhancers to be enriched for marks such as H3K27ac, H3K4me2, H3K9me1, H3K27me1, H2BK5me1 and H3K36me1, indicating a degree of redundancy in the histone code[199] (Figure 1.4b). Nevertheless, an enhancer histone code could also be fine-tuned by acetylation of H2A.Z, resulting in corresponding differences in downstream gene activation[198]. Although enhancers are commonly marked with H3K4me1 and H3K27ac, they could also be marked with H3K4m3, the active promoter mark, if an enhancer is highly transcribed[195, 208, 209]. Therefore, putative active enhancers are identified by a cohort of criteria including measuring the ratio of H3K4me1 to H3K4me3, along with the presence of H3K27ac, the replacement of histones with the variant H2A.Z, the binding of coactivators p300/CBP and cooperative binding of master TFs[16, 210-215]. Poised enhancers, on the other hand, are characterised by the noted absence of the H3K27ac mark and the enrichment of H3K27me3 and/or H3K9me3, an epigenetic feature later found to be common a large number of enhancers with tissue specificity. This poised state could, however, be readily reversed when the histone mark H3K27me3 is modified and replaced with H3K27ac[209].