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Chromatin is composed of DNA and histones protein, which the DNA winds around. Modifications to histones and DNA methylation play a role in gene regulation in eukaryotic cells.

Histones undergo posttranslational modifications that alter their interaction with DNA and nuclear proteins. The H3 and H4 histones have long tails protruding from the nucleosome, which can be covalently modified at several places. Modifications of the tail include methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, citrullination, and ADP-ribosylation. The core of the histones H2A and H2B can also be modified. Combinations of modifications are thought to constitute a code, the so-called "histone code".^[38][39] Histone modifications act in diverse biological processes such as gene regulation, DNA repair, chromosome condensation (mitosis) and spermatogenesis (meiosis).^[40]

The common nomenclature of histone modifications is:

• The name of the histone (e.g., H3)
• The single-letter amino acid abbreviation (e.g., K for Lysine) and the amino acid position in the protein
• The type of modification (Me: methyl, P: phosphate, Ac: acetyl, Ub: ubiquitin)
• The number of modifications (only Me is known to occur in more than one copy per residue. 1, 2 or 3 is mono-, di- or tri-methylation)

So H3K4me1 denotes the monomethylation of the 4th residue (a lysine) from the start (i.e., the N-terminal) of the H3 protein.

Examples of histone modifications in transcriptional regulation

Type of modification	Histone
	H3K4	H3K9	H3K14	H3K27	H3K79	H3K36	H4K20	H2BK5	H2BK20
mono-methylation	activation[41]	activation[42]		activation[42]	activation[42][43]		activation[42]	activation[42]
di-methylation		repression[44]		repression[44]	activation[43]
tri-methylation	activation[45]	repression[42]		repression[42]	activation,[43] repression[42]	activation		repression[44]
acetylation	activation[46]	activation[45]	activation[45]	activation[47]					activation

Functions of histone modifications [edit]

A huge catalogue of histone modifications have been described, but a functional understanding of most is still lacking. Collectively, it is thought that histone modifications may underlie a histone code, whereby combinations of histone modifications have specific meanings. However, most functional data concerns individual prominent histone modifications that are biochemically amenable to detailed study.

Chemistry of histone modifications[edit]

Lysine methylation[edit]

The addition of one, two, or many methyl groups to lysine has little effect on the chemistry of the histone; methylation leaves the charge of the lysine intact and adds a minimal number of atoms so steric interactions are mostly unaffected. However, proteins containing Tudor, chromo or PHD domains, amongst others, can recognise lysine methylation with exquisite sensitivity and differentiate mono, di and tri-methyl lysine, to the extent that, for some lysines (e.g.: H4K20) mono, di and tri-methylation appear to have different meanings. Because of this, lysine methylation tends to be a very informative mark and dominates the known histone modification functions.

Glutamine serotonylation[edit]

Recently it has been shown, that the addition of a serotonin group to the position 5 glutamine of H3, happens in serotonergic cells such as neurons. This is part of the differentiation of the serotonergic cells. This post-translational modification happens in conjunction with the H3K4me3 modification. The serotonylation potentiates the binding of the general transcription factor TFIID to the TATA box.^[48]

Arginine methylation[edit]

What was said above of the chemistry of lysine methylation also applies to arginine methylation, and some protein domains—e.g., Tudor domains—can be specific for methyl arginine instead of methyl lysine. Arginine is known to be mono- or di-methylated, and methylation can be symmetric or asymmetric, potentially with different meanings.

Arginine citrullination[edit]

Enzymes called peptidylarginine deiminases (PADs) hydrolyze the imine group of arginines and attach a keto group, so that there is one less positive charge on the amino acid residue. This process has been involved in the activation of gene expression by making the modified histones less tightly bound to DNA and thus making the chromatin more accessible.^[49] PADs can also produce the opposite effect by removing or inhibiting mono-methylation of arginine residues on histones and thus antagonizing the positive effect arginine methylation has on transcriptional activity.^[50]

Lysine acetylation[edit]

Addition of an acetyl group has a major chemical effect on lysine as it neutralises the positive charge. This reduces electrostatic attraction between the histone and the negatively charged DNA backbone, loosening the chromatin structure; highly acetylated histones form more accessible chromatin and tend to be associated with active transcription. Lysine acetylation appears to be less precise in meaning than methylation, in that histone acetyltransferases tend to act on more than one lysine; presumably this reflects the need to alter multiple lysines to have a significant effect on chromatin structure. The modification includes H3K27ac.

Serine/threonine/tyrosine phosphorylation[edit]

Addition of a negatively charged phosphate group can lead to major changes in protein structure, leading to the well-characterised role of phosphorylation in controlling protein function. It is not clear what structural implications histone phosphorylation has, but histone phosphorylation has clear functions as a post-translational modification, and binding domains such as BRCT have been characterised.

Functions in transcription[edit]

Most well-studied histone modifications are involved in control of transcription.

Actively transcribed genes[edit]

Two histone modifications are particularly associated with active transcription:

Trimethylation of H3 lysine 4 (H3K4me3)

This trimethylation occurs at the promoter of active genes^[51][52][53] and is performed by the COMPASS complex.^[54][55][56] Despite the conservation of this complex and histone modification from yeast to mammals, it is not entirely clear what role this modification plays. However, it is an excellent mark of active promoters and the level of this histone modification at a gene's promoter is broadly correlated with transcriptional activity of the gene. The formation of this mark is tied to transcription in a rather convoluted manner: early in transcription of a gene, RNA polymerase II undergoes a switch from initiating’ to ‘elongating’, marked by a change in the phosphorylation states of the RNA polymerase II C terminal domain (CTD). The same enzyme that phosphorylates the CTD also phosphorylates the Rad6 complex,^[57][58] which in turn adds a ubiquitin mark to H2B K123 (K120 in mammals).^[59] H2BK123Ub occurs throughout transcribed regions, but this mark is required for COMPASS to trimethylate H3K4 at promoters.^[60][61]

Trimethylation of H3 lysine 36 (H3K36me3)

This trimethylation occurs in the body of active genes and is deposited by the methyltransferase Set2.^[62] This protein associates with elongating RNA polymerase II, and H3K36Me3 is indicative of actively transcribed genes.^[63] H3K36Me3 is recognised by the Rpd3 histone deacetylase complex, which removes acetyl modifications from surrounding histones, increasing chromatin compaction and repressing spurious transcription.^[64][65][66] Increased chromatin compaction prevents transcription factors from accessing DNA, and reduces the likelihood of new transcription events being initiated within the body of the gene. This process therefore helps ensure that transcription is not interrupted.

Repressed genes[edit]

Three histone modifications are particularly associated with repressed genes:

Trimethylation of H3 lysine 27 (H3K27me3)

This histone modification is depositied by the polycomb complex PRC2.^[67] It is a clear marker of gene repression,^[68] and is likely bound by other proteins to exert a repressive function. Another polycomb complex, PRC1, can bind H3K27me3^[68] and adds the histone modification H2AK119Ub which aids chromatin compaction.^[69][70] Based on this data it appears that PRC1 is recruited through the action of PRC2, however, recent studies show that PRC1 is recruited to the same sites in the absence of PRC2.^[71][72]

Di and tri-methylation of H3 lysine 9 (H3K9me2/3)

H3K9me2/3 is a well-characterised marker for heterochromatin, and is therefore strongly associated with gene repression. The formation of heterochromatin has been best studied in the yeast Schizosaccharomyces pombe, where it is initiated by recruitment of the RNA-induced transcriptional silencing (RITS) complex to double stranded RNAs produced from centromeric repeats.^[73] RITS recruits the Clr4 histone methyltransferase which deposits H3K9me2/3.^[74] This process is called histone methylation. H3K9Me2/3 serves as a binding site for the recruitment of Swi6 (heterochromatin protein 1 or HP1, another classic heterochromatin marker)^[75][76] which in turn recruits further repressive activities including histone modifiers such as histone deacetylases and histone methyltransferases.^[77]

Trimethylation of H4 lysine 20 (H4K20me3)

This modification is tightly associated with heterochromatin,^[78][79] although its functional importance remains unclear. This mark is placed by the Suv4-20h methyltransferase, which is at least in part recruited by heterochromatin protein 1.^[78]

Bivalent promoters[edit]

Analysis of histone modifications in embryonic stem cells (and other stem cells) revealed many gene promoters carrying both H3K4Me3 and H3K27Me3, in other words these promoters display both activating and repressing marks simultaneously. This peculiar combination of modifications marks genes that are poised for transcription; they are not required in stem cells, but are rapidly required after differentiation into some lineages. Once the cell starts to differentiate, these bivalent promoters are resolved to either active or repressive states depending on the chosen lineage.^[80]

Other functions[edit]

DNA damage[edit]

Marking sites of DNA damage is an important function for histone modifications. It also protects DNA from getting destroyed by ultraviolet radiation of sun.

Phosphorylation of H2AX at serine 139 (γH2AX)

Phosphorylated H2AX (also known as gamma H2AX) is a marker for DNA double strand breaks,^[81] and forms part of the response to DNA damage.^[36][82] H2AX is phosphorylated early after detection of DNA double strand break, and forms a domain extending many kilobases either side of the damage.^[81][83][84] Gamma H2AX acts as a binding site for the protein MDC1, which in turn recruits key DNA repair proteins^[85] (this complex topic is well reviewed in^[86]) and as such, gamma H2AX forms a vital part of the machinery that ensures genome stability.

Acetylation of H3 lysine 56 (H3K56Ac)

H3K56Acx is required for genome stability.^[87][88] H3K56 is acetylated by the p300/Rtt109 complex,^[89][90][91] but is rapidly deacetylated around sites of DNA damage. H3K56 acetylation is also required to stabilise stalled replication forks, preventing dangerous replication fork collapses.^[92][93] Although in general mammals make far greater use of histone modifications than microorganisms, a major role of H3K56Ac in DNA replication exists only in fungi, and this has become a target for antibiotic development.^[94]

DNA repair[edit]

Trimethylation of H3 lysine 36 (H3K36me3)

H3K36me3 has the ability to recruit the MSH2-MSH6 (hMutSα) complex of the DNA mismatch repair pathway.^[95] Consistently, regions of the human genome with high levels of H3K36me3 accumulate less somatic mutations due to mismatch repair activity.^[96]

Chromosome condensation[edit]

Phosphorylation of H3 at serine 10 (phospho-H3S10)

The mitotic kinase aurora B phosphorylates histone H3 at serine 10, triggering a cascade of changes that mediate mitotic chromosome condensation.^[97][98]Condensed chromosomes therefore stain very strongly for this mark, but H3S10 phosphorylation is also present at certain chromosome sites outside mitosis, for example in pericentric heterochromatin of cells during G2. H3S10 phosphorylation has also been linked to DNA damage caused by R-loop formation at highly transcribed sites.^[99]

Phosphorylation H2B at serine 10/14 (phospho-H2BS10/14)

Phosphorylation of H2B at serine 10 (yeast) or serine 14 (mammals) is also linked to chromatin condensation, but for the very different purpose of mediating chromosome condensation during apoptosis.^[100][101] This mark is not simply a late acting bystander in apoptosis as yeast carrying mutations of this residue are resistant to hydrogen peroxide-induced apoptotic cell death.

Addiction[edit]

Epigenetic modifications of histone tails in specific regions of the brain are of central importance in addictions.^{[102][103][104]} Once particular epigenetic alterations occur, they appear to be long lasting "molecular scars" that may account for the persistence of addictions.^[102]

Cigarette smokers (about 15% of the US population) are usually addicted to nicotine.^[105] After 7 days of nicotine treatment of mice, acetylation of both histone H3 and histone H4 was increased at the FosB promoter in the nucleus accumbens of the brain, causing 61% increase in FosB expression.^[106] This would also increase expression of the splice variant Delta FosB. In the nucleus accumbens of the brain, Delta FosB functions as a "sustained molecular switch" and "master control protein" in the development of an addiction.^[107][108]

About 7% of the US population is addicted to alcohol. In rats exposed to alcohol for up to 5 days, there was an increase in histone 3 lysine 9 acetylation in the pronociceptin promoter in the brain amygdala complex. This acetylation is an activating mark for pronociceptin. The nociceptin/nociceptin opioid receptor system is involved in the reinforcing or conditioning effects of alcohol.^[109]

Methamphetamine addiction occurs in about 0.2% of the US population.^[110] Chronic methamphetamine use causes methylation of the lysine in position 4 of histone 3 located at the promoters of the c-fos and the C-C chemokine receptor 2 (ccr2) genes, activating those genes in the nucleus accumbens (NAc).^[111] c-fos is well known to be important in addiction.^[112] The ccr2 gene is also important in addiction, since mutational inactivation of this gene impairs addiction.^[111]