Histone H3 lysine 9 methylation

Establishment and removal of the H3K9 methylation mark

In mammals the histone methyltransferases G9a and G9a-like protein GLP monomethylate lysine 9 of histone H3 [73]. G9a is implicated in downregulation of euchromatic gene regions most probably by methylation of H3K9 [74]. However, G9a expressed in cell-culture is distributed to heterochromatic regions [75]. In Drosophila G9a has a suppression effect in position-effect variagation experiments and is required for gene silencing [76]. Therefore it is likely that G9a has an influence on H3K9 monomethylation of heterochromatin.

Suv39h1/h2 as well as ESET/SETDB1 histone methyltransferase mediate di- or trimethylation of histone H3 lysine 9 [23, 77]. These mechanisms include interaction of the histone methyltransferases with DNA-binding proteins as well as with small RNAs [78].

Suv39-like enzymes are mainly located at heterochromatin and are obviously responsible for heterochromatic-specific H3K9 marks in animals [79, 80]. Heterochromatic foci of human double null Suv39h1^-/Suv39h2^- mice failed to show H3K9 trimethylation [81] and in Drosophila a gain-of-function mutation of Su(var)3-9 lead to ectopic heterochromatinization [82].

In contrast, SETDB1 was mainly found in euchromatic regions, where it participates in gene silencing [83]. Without SETDB1, the relative concentration of H3K9 methylation at heterochromatic regions remain unchanged [84]. Therefore it is likely that G9a as well as Suv39h1/h2 are the main HMTs establishing H3K9 methylation at heterochromatic regions.

Several demethylating enzyme ‘erasers’ of the H3K9 methylation have been described. For example, in a complex with the androgen receptor LSD1 demethylates H3K9me and activates transcription [44]. H3K9 can also be demethylated by the jumonji proteins JHDM2A, JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1 and JMJD2D [23] but the mechanism and occurrences of these events are not well understood so far.

Localization of H3K9 methylation

H3K9me1 occurs at insulator or enhancer regions of genes. Additionally H3K9me1 can be detected at active gene regions predominantly in close proximity to the transcriptional start site and the 5’ coding region [65]. H3K9me2/3 is a classic mark of constitutive heterochromatin associating with gene deserts, imprinted domains, repetitive elements, centromeric and pericentromeric regions as shown in Figure 1-4C. Nevertheless, H3K9me3 can also be found in active gene regions [85].

Thus, three localizations of H3K9me exist, which (a) define heterochromatic regions, (b) are involved in silencing of euchromatic loci by modification of the promotor regions or (c) might take part in repression of unintentional transcription inside of active transcriptional units [86].

The H3K9 metylation seem to be a very ancient mark of heterochromatin. Evolutionary, three of the five basal groups of eukaryotes (unikonts, plants, chromalveolates) show heterochromatin-associated H3K9 methylation [86]. In contrast, other heterochromatic methylation marks such as H4K20me3, which is used only in animals to establish heterochromatin, seem to be more or less lineage-specific [87].

Biology of H3K9 methylation

Besides DNA methylation, H3K9 methylation seems to be an ancient feature of heterochromatic regions of most eukaryotes. In Drosophila the knockout of the H3K9 histone methyltransferase Su(var)3-9 leads to defects in differentiation and to impaired heterochromatin stability [88, 89]. These data are consistent with observations of knockouts of the homologous Suv39h1/h2 genes in mice. Suv39h1/h2 deficient mice have an impaired viability and severely reduced genome stability [90]. Therefore H3K9 methylation might play not only a role in heterochromatin formation and maintenance but might also function in cellular memory and epigenetic pathways [27, 57, 87, 91].

Epigenetic changes are changes in phenotype or gene expression caused by mechanisms other than modulation of the DNA sequence. Importantly, these changes are heritable. Stable propagation of DNA methylation was directly demonstrated [92]. But the mechanism of transmission of H3K9me from one cell generation to the next is still under debate. During replication the assembly of the nucleosomal core particle seems to include two steps. The (H3-H4)2 tetramer is deposited on DNA followed by H2A-H2B dimer association. Recently it was suggested that instead of (H3-H4)2 tetramer one newly synthesized H3-H4 dimer are paired with H3-H4 dimer from the mother strand, which would lead to an even segregation of parental nucleosomes [93]. Nevertheless, both pathways could provide the inheritance of

histone modification marks. Interestingly, the propagation of the silencing H3K9 methylation mark is dependent on the RNAi machinery and DNA recognition factors [94]. In addition it was shown, that H3K9 methylation could direct DNA methylation [87]. Thus, H3K9 methylation is implicated in the epigenetic pathways of the cell.

Regulation of H3K9me3 mark

Lysine 9 of histone H3 is embedded in an ARKS motif, which occurs also in other histone and non-histone proteins [95]. Interestingly PTMs of the neighboring arginine (methylation) as well as the neighboring serine (phosphorylation) has been described [96, 97]. These additional PTMs establish another layer of regulation as shown for phosphorylation of serine 10 and HP1. At the onset of mitosis Aurora B phosphorylates serine 10. This phosphorylation destroys the interaction of the HP1 chromodomain with H3K9me3 due to blocking of an essential hydrogen bond (compare also with 1.2.3/chromodomains) and displaces HP1 from heterochromatin [27]. This PTM correlates strongly with the initial condensation of chromatin during mitosis and has recruitment potential for chromosomal condensation factors [50].

H3K9me2/3 readout

In higher eukaryotes heterochromatin protein 1 isoforms HP1, HP1 and HP1 bind to the H3K9 methylation mark [55, 98] with their chromodomains and mediate the heterochromatinization of genetic regions in Drosophila [99]. The chromodomain of Chp1, a protein of S. pombe, also recognizes H3K9me3 and is critical for efficient establishment of centromeric heterochromatin [100]. The ankyrin repeats of G9a were also shown to interact with H3K9me1/2 at least in vitro [73]. Recently also ICBP90, a ubiquitin-E3-ligase, interacting with the DNA-methyltransferase DNMT1 has been described as H3K9 methylation binding protein [57, 58].

Because of the many different functions of H3K9 methylation it is likely that also other heterochromatin effector proteins recognize H3K9me2/me3. Recently, a new protein family harboring a putative histone methylation-binding module, a chromodomain, was identified, the CDY family (chromodomain on the Y). Several findings such as its association with the CoREST complex or interaction with HDAC1 and HDAC2 indicate a heterochromatin association and function [62, 101, 102]. But until now, it is not known if and how CDY family proteins interact and function on heterochromatin.