H4K20me3 is abundant at heterochromatin regions that are rich in repetitive sequences, such as satellite repeats and transposable elements, among others. Heterochromatin is typically associated with reduced transcription, and H4K20me3 enrichment at heterochromatin is known to contribute to gene silencing. H4K20me3 is particularly enriched at repetitive elements, preventing their transcription to maintain genomic stability. However, recent research suggests that H4K20me3 can also regulate transcription in other genomic regions. For instance, it negatively regulates ribosomal RNA (rRNA) transcription during quiescence and growth arrest, as well as the long-non-coding RNA LINC01510 in non-small cell lung cancer. Repressed gene transcription in heterochromatin is believed to be mediated through chromatin compaction, thereby preventing the binding of DNA factors necessary for transcription. Nevertheless, in embryonic stem cells, the association of H4K20me3 with the active transcription methylation mark H3K4me3 suggests a more complex mechanism that requires further investigation. In summary, the specific localization, and interactions of H4K20me3 in different genomic contexts influence its impact on gene expression.
Furthermore, H4K20me3 methylation is mediated by multiple events, including catalysis by various methyltransferases and their preceding PMTs. These events can be summarized through the following steps. First, unmethylated H4K20 is converted to H4K20 mono-methylation (H4K20me1) by the methyltransferase SET8 or KMT5A. H4K20me1 can also be demethylated and converted to H4K20me0 by the PHD and Jumonji C (JmjC) domain-containing protein PHF8. This mono-methylation plays a critical role in cell cycle regulation and in genomic integrity. Following mono-methylation, KMT5B, also known as the drosophila homologue SUV420H1 (Suppressor of variegation 4–20 homolog 1), catalyzes the formation of H4K20 di-methylation (H4K20me2) which has an important role in DNA damage response, DNA replication, and in cell cycle regulation. H4K20me2 can be converted into its H4K20me1 state by the dosage compensation complex (DCC) subunit DPY-21, a Jumonji demethylase found in both C. elegans and mammals. Third, KMT5C or SUV420H2 catalyzes H4K20 trimethylation (H4K20me3), which is the primary H4K20 PTM involved in heterochromatin silencing. H4K20, in all its methylation states (me1/2/3), has been reported to be demethylated by hHR23A/B, two human homologues of RAD23 in yeast. Moreover, reports have shown that H4K20me1 can be used as a substrate of KMT5B and KMT5C, directly generating H4K20me2 or H4K20me3 respectively. Indeed, due to the sequence and structural similarity of KMT5B and KMT5C in their catalytic domain, KMT5B and KMT5C have overlapping functions. Nevertheless, knockout studies indicate that KMT5B loss leads to a 60% reduction of H4K20me2 with no change in H4K20me3, while KMT5C depletion eliminated H4K20me3 without any significant impact on H4K20me2 in primary mouse embryonic fibroblasts (MEFs).
While less is known about the events preceding H4K20me3 at facultative heterochromatin, at constitutive heterochromatic regions, another histone PTM has been reported to facilitate H4K20me3 formation. This PTM, H3K9me3, catalyzed by the KMT1A/SUV39H1 methyltransferase, serves as a docking site for Heterochromatin Protein 1 (HP1). HP1 binds to H3K9me3 though its chromodomain which then leads to the recruitment of KMT5C. Once recruited, KMT5C catalyzes H4K20me3. Previously, it was believed that KMT5C interacted with all HP1 isoforms (HP1α, HP1β, and HP1γ) and that each HP1 isoform interacted with different regions of the KMT5C C-terminus. However, a recent report identified a far more intricate mechanism. While both HP1α and HP1β are enriched at pericentric heterochromatin (PCH) regions, they seem to have opposing roles in chromatin compaction. Loss of HP1β leads to global decompaction of chromatin through its direct functional link with H4K20me3 and KMT5C. On the other hand, loss HP1α leads to hyper-compaction of chromatin. Furthermore, when both HP1α and HP1β are lost, HP1γ localization at PCH regions is disrupted. Thus, the interaction between HP1β and KMT5C appears to be key for H4K20me3 formation at constitutive heterochromatin regions.
Since H4K20me3 is enriched at telomeric regions leading to their compaction and stabilization, its loss has significant implications on telomeric structure and lenght. Cells deficient for KMT5C methyltransferase, or for both KMT5B/C, have reduced levels of H4K20me3 in both telomeres and subtelomeric regions. This loss is associated with lengthening of telomeres and subtelomeric regions. Loss also results in increased sister chromatid recombination globally and at telomeric regions. These changes are specific to loss of H4K20me2/3 as H3K9me3 levels were not affected, lending further support that H3K9me3 is not dependent on H4K20me2/3. And, while telomere length was altered, there was no evidence of defective telomere capping. A similar telomer lengthening defect was observed following depletion of Retinoblastoma1 (RB1), which is known to interact with KMT5C. In this work, loss of RB1 led to reduced H4K20me3 and subsequent telomere lengthening in primary mouse embryonic fibroblasts (MEFs). However, whether the increase in telomere length when H4K20me3 is reduced is due to more accessibility to telomerase, or if it is through an alternative lengthening of telomeres mechanism (ALT), which relies on the recombination between telomeric sequences to maintain telomeric length was not determined. In contrast to these findings, PWP1, a chromatin binding protein important for controlling growth downstream of mTOR, has been reported to regulate H4K20me3 levels through binding to and stabilizing the shelterin complex in mouse embryonic stem cells, leading to telomere shortening. Reduced expression of PWP1 correlated with reduced levels of H4K20me3 at specific telomeric and subtelomeric regions. PWP1 depletion was also shown to induce telomere shortening and therefore increased DNA damage in telomeric regions. Additionally, restoration of telomere length in PWP1 depleted cells was only achieved by overexpressing PWP1 along with KMT5C. In support of this association, PWP1 was found to interact with KMT5C along with the shelterin complex, providing regulation of chromatin length. In addition to proteins such as RB1 and PWP1 that are directly involved in growth, major epigenetic modifiers are also correlated with H4K20me3 levels. In Drosophila, loss of the DNA methyltransferase DNMT2, has been correlated with loss of H4K20me3 at retrotransposons and subtelomeric repeats. An additional study reported that in telomerase-deficient mice with short telomeres, H3K9me3 and H4K20me3 levels were reduced in telomeric and subtelomeric chromatin, accompanied by increase acetylation of H3 and H4 at these regions. Hence, loss of telomeric repeats appears to lead to loss of heterochromatin features, including H4K20me3. Whether the loss of H4K20me3 in telomeric regions leads to telomere lengthening or shortening is debated and seems to depend on the context. Loss of KMT5C leads to increased telomere length, but loss of PWP1, a protein involved in shelterin complex stabilization, reduces H4K20me3 and decreases telomere length. Contradicting reports, expose the complexity of telomere length regulation which could also be due to differences in experimental design and model systems.
The H4K20me3 mark is also highly enriched in constitutive heterochromatin, including pericentric regions, and thus alterations in H4K20me3 directly impact pericentric chromatin structure and chromocenter structure. Similar to telomeric regions, pericentric regions are also gene-poor and require proper silencing to maintain cell homeostasis. Silencing is reported to be through both H3K9me3 and H4K20me3.
