SET domain containing 7
In human cells appropriate mono-methylation of histone H4 lysine20 by PrSet7/SET8 is important for the correct transcription of specific genes, and timely progression through the cell cycle. Over-methylation appears to be prevented through the interaction of PrSet7 with PCNA, which targets PrSet7 destruction via the CRL4cdt2 pathway, however the factors involved in positive regulation of its histone methylation remain undefined. This study presents biochemical and genetic evidence for a previously undocumented interaction between dPrSet7 and DNA polymerase-α in Drosophila. Depletion of the polymerase reduces H4K20 mono-methylation suggesting that it is required for the expression of dPrSet7 histone methylation activity. It was also shown that the interaction between PCNA and PrSet7 is conserved in Drosophila, but is only detectable in chromatin fractions. Consistent with this, S2 cells show a significant loss of chromatin bound dPrSet7 protein as S phase progresses. Based on these data it is suggested that interaction with the DNA polymerase represents an important route for the expression of PrSet7 histone methylase activity, by allowing loading of dPrSet7 onto chromatin or its subsequent activation (Sahashi, 2014).
Histone H4 methyltransferase activity is cell cycle-regulated, consistent with increased H4 Lys 20 methylation at mitosis. This increase closely follows the cell cycle-regulated expression of the H4 Lys 20 methyltransferase, PR-Set7. Localization of PR-Set7 to mitotic chromosomes and subsequent increase in H4 Lys 20 methylation were inversely correlated to transient H4 Lys 16 acetylation in early S-phase. To expand the observation made in mammalian cells that H4 Lys 20 methylation decreases in S-phase and peaks at mitosis, immunofluorescence studies were performed in Drosophila embryos. A recent report shows that the H4 Lys 20-methyl modification is present in Drosophila and is essential for Drosophila development and viability (Nishioka, 2002b). In addition, the embryos provide an excellent model to study H4 Lys 20 methylation during the cell cycle since they rapidly and repeatedly shift from S-phase to mitosis -- this can be easily determined by DAPI staining. Consistent with the above findings, H4 Lys 20 methylation is clearly detected on chromosomes during both metaphase and anaphase, whereas staining during S-phase results in a faint signal even upon overexposure. It is hypothesized that the faint signal during S-phase most likely reflects a combination of dilution of the modification by histone deposition as well as the decrease in chromatin condensation, which could contribute to a dispersion of the signal resulting in a decreased ability to detect the modification. Regardless, these data confirm that H4 Lys 20 methylation is decreased during S-phase and increased specifically during mitosis (Rice, 2002).
To gain insights into the function of H4-K20 methylation, the highly specific methyl H4-K20-specific antibodies were used to analyze the distribution of methylated H4-K20 on Drosophila polytene chromosomes and mouse embryonic fibroblasts (MEFs). Methylation of histone H4-K20 on polytene chromosomes coincides with condensed chromosomal regions, including chromocentric heterochromatin and numerous bands on the euchromatic arms. Competition experiments were performed to verify that the observed staining pattern was specific for methyl H4-K20. In these experiments, the staining was completely removed with peptides that contained the H4 tail methylated at lysine 20, but not with unmodified peptides or by peptides that contained the H3 tail methylated at lysine 4 or lysine 9. Moreover, costaining of polytene chromosomes with antibodies raised in different organisms, a rabbit polyclonal and a mouse monoclonal antibody to methyl H4-K20, shows complete overlap (Nishioka, 2002b).
Having a monoclonal antibody allowed for a direct comparison of the distribution of methylated H4-K20 with that of other modifications that occur on the H3 and H4 tails, using existing rabbit polyclonal antibodies. Comparison of the distribution of methyl H4-K20 and methyl H3-K9 on polytene chromosomes established that the H4-K20 methylation pattern is distinct from the predominantly chromocentric pattern of methylated H3-K9, a modification that has been associated with constitutive heterochromatin in various species (Nishioka, 2002b).
The distribution of the methyl H4-K20 was analyzed with respect to transcriptionally active or competent genes. To accomplish this, the staining pattern on Drosophila polytene chromosomes obtained with the polyclonal antibody to methyl H4-K20 was compared to that observed with monoclonal antibody to the transcription-engaged form of RNA polymerase II. This analysis demonstrated nonoverlapping patterns for each of the antibodies in the entire chromosomes, except at regions that were not fully spread; this led to the conclusion that methylated H4-K20 was very low or absent from transcriptionally competent regions. Similar results were obtained when the staining pattern of the antibody to methyl H4-K20 was compared to that of the transcriptionally active form of RNA polymerase II at heat shock loci under heat shock (transcriptionally permissive) conditions (Nishioka, 2002b).
To further analyze the association of the methyl H4-K20 mark with transcriptionally silent chromatin, the methyl H4-K20 staining pattern was compared to that obtained with antibodies specific to methyl H3-K4, a mark that has been correlated with transcriptionally competent genes in higher eukaryotes. Consistent with the results with RNAPII staining, nonoverlapping patterns for methyl H3-K4 and methyl H4-K20 were observed in the entire polytene chromosomes, except at regions that were not fully spread. Thus, it is concluded that the methylated H4-K20 modification marks transcriptionally silent chromatin (Nishioka, 2002b).
Previous studies have established that interplay occurs between acetylation and methylation of residues within the H3 and H4 histone tails (Rea, 2000, Wang, 2001a, 2001b; Nishioka, 2002a). The H4 tail can be acetylated at lysines 5, 8, 12, and 16; acetylation of lysines 5 and 12 correlates with histone deposition of newly synthesized histones in Drosophila and human cells (Sobel, 1995). Acetylation of lysine 16 is observed in the transcriptionally hyperactive male X chromosome in Drosophila (Turner, 1992) and is enriched in transcriptionally active chromatin in human cells (Johnson, 1998). In light of the results suggesting that methyl H4-K20 is associated with transcriptionally silent chromatin, tests were performed to see whether methyl H4-K20 affects acetylation of H4-K16. Given that hyperacetylated H4-K16 is a hallmark of the hyperactive Drosophila male X chromosome (Turner, 1992), the presence of methylated H4-K20 and acetylated H4-K16 on this chromosome was analyzed. Significant staining with antibody specific to acetylated H4-K16 is detected on the Drosophila male X chromosome and not on the female X. In contrast, low levels of methylated H4-K20 are detected on the male X chromosome, which is comparable to that of female. Thus, there is an inverse correlation in the number and intensity of bands containing methyl H4-K20 and acetyl H4-K16. These data suggest a negative interplay between methylation of H4-K20 and acetylation of H4-K16 (Nishioka, 2002b).
In the Drosophila gene disruption project, a single, homozygous lethal P element insertion into the 5'-untranslated region of the first exon of CG3307 [l(3)neo41] was isolated. This P element is deleted by Df(3R)red31 and is lethal over the deficiency. This mutant was crossed with a green fluorescent protein (GFP)-tagged TM3 balancer chromosome in order to identify homozygous mutants (pr-set7-/-). Although heterozygous mutants (pr-set7+/-) generated no obvious phenotype, homozygote mutants die as late second instar larvae (Nishioka, 2002b).
In order to determine if the homozygote mutant Drosophila larvae lack methylation on H4-K20, wild-type, homozygote mutant and heterozygote, GFP-positive sibling second instar larvae were collected. Western blot analysis was carried out on homozygote mutant and wild-type extract using anti-methyl H4-K20 antibodies. The results demonstrate that the methyl H4-K20 modification clearly exists in the wild-type (pr-set7+/+) larval extract as well as in their heterozygous siblings (pr-set7+/-). In contrast, the homozygous (pr-set7-/-) larval extract yielded no detectable methyl H4-K20. These observations demonstrate that Drosophila pr-set7 encodes the major H4-K20 HMT in Drosophila and that methylation at this residue is essential for development and viability (Nishioka, 2002b).
Drosophila PR-Set7 or SET8 is a histone methyltransferase that specifically monomethylates histone H4 lysine 20 (H4K20). L(3)MBT has been identified as a reader of methylated H4K20. It contains several conserved domains including three MBT repeats binding mono- and dimethylated H4K20 peptides. Depletion of PR-Set7 was found to block de novo H4K20me1 resulting in the immediate activation of the DNA damage checkpoint, an increase in the size of interphase nuclei, and drastic reduction of cell viability. L(3)mbt, in contrast, stabilizes the monomethyl mark, as L(3)mbt-depleted S2 cells show a reduction of more than 60% of bulk monomethylated H4K20 (H4K20me1) while viability is barely affected. Ploidy and basic chromatin structure show only small changes in PR-Set7-depleted cells, but higher order interphase chromatin organization is significantly affected presumably resulting in the activation of the DNA damage checkpoint. In the absence of any other known functions of PR-Set7, the setting of the de novo monomethyl mark appears essential for cell viability in the presence or absence of the DNA damage checkpoint, but once newly assembled chromatin is established the monomethyl mark, protected by L(3)mbt, is dispensable (Sakaguchi, 2012).
In these studies it was established that PR-Set7 sets the H4K20 monomethyl mark in vivo and that at least in S2 cells K20 is the only amino acid that is methylated. It was further found that depleting PR-Set7 in Drosophila S2 cells leads to the activation of the DNA damage checkpoint and within about 10 days to cell death. When the DNA damage checkpoint is abrogated by double knock-down of PR-Set7 and the checkpoint genes mei-41 or grp, the half/life of the cells is increased by 1 to two days, but ultimately the cells still die, suggesting that whatever is perturbed in the absence of PR-Set7 cannot be repaired. No double strand breaks were observed when staining for anti-phosphorylated histone H2A. This does not agree with results observed in vertebrate cells and may be because, in Drosophila, H2Av is not phosphorylated in the absence of H4K20me1 or the specific epitope is obscured. Alternatively, double strand breaks may not exist and the checkpoint is activated because of abnormal chromatin organization or because protein complexes are not removed in a timely manner as observed in Saccharomyces cerevisiae (Sakaguchi, 2012).
In this context it is interesting to note that in vertebrates H4K20 me2 is implicated in double strand break repair. Because H4K20me1 is the likely substrate for Suv4-20H1 and H2, the di- and trimethyltransferases, an additional link between H4K20 methylation and double strand breaks seems to exist. However, besides potentially setting the monomethyl mark at double strand breaks, PR-Set7 would have to have additional functions, because in both flies and vertebrates PR-Set7 mutants have a substantially stronger phenotype than the loss of the Suv4-20 enzymes (Sakaguchi, 2012).
The increase in nuclear volume, together with the changes in the number of FISH signals per nucleus observed in interphase cells following PR-Set7 RNAi would be consistent with a role for PR-Set7 in chromosome compaction and higher-order chromatin organization. Interestingly, mass spectrometry experiments show that the H4K20 monomethyl mark is set at the G2/M transition well after newly synthesized histone H4 is incorporated into chromatin in S phase. These findings suggest that the abnormalities in chromosome compaction and organization evident in interphase nuclei might be due to defects arising during the G2/M transition. Consistent with this possibility, cells depleted for only Pr-Set7 appear to arrest mostly in early mitosis. But unlike what is observed in larval brains, there is also a subset of cells that arrest in S phase; these may represent cells that despite the abnormalities in higher order chromatin organization are able to continue through the cell cycle until a checkpoint is activated during S. The discrepancy between the brain and tissue culture cells may be a reflection of differences in their cell cycle and developmental potential (Sakaguchi, 2012).
Results from several laboratories suggest that PR-Set7 function is coupled to DNA replication based on its targeting to the dividing fork via its interaction with PCNA. The current findings indicate that while abnormalities in chromatin organization and compaction appear to accumulate after growth without Pr-Set7 activity, these defects are inconsistent with massive disruptions in de novo nucleosome assembly during replication. Instead, the DNA damage checkpoint activation must arise from more subtle abnormalities in chromatin or DNA structure (Sakaguchi, 2012).
As for the l(3)mbt, its functional requirement does not appear to overlap with that of PR-Set7, neither in tissue culture as shown in this study, nor in flies. In larvae the loss of l(2)mbt results in an expansion of the neuroblast pool and subsequent tumorous overgrowth of the optic lobe while in PR-Set7 mutants the cell cycle of neuroblasts arrests in early mitosis resulting in fewer cells. PR-Set7 is essential for de novo methylation of H4K20. While the loss of H4K20me1 could occur either because in the absence of L(3)mbt protection the H4K20me1 is lost, or it could be a secondary effect. Consistent with the latter explanation, recent results show that L(3)mbt binds to DNA boundary elements and affects the level of transcription of Salvador-Wart-Hippo pathway genes both positively and negatively. That L(3)mbt possibly controls expression of many genes is also supported by the observation that the transcription level of all genes tested was reduced compared to wild type (Sakaguchi, 2012).
Couture, J.-F., et, al. (2005). Structural and functional analysis of SET8, a histone H4 Lys-20 methyltransferase. Genes Dev. 19: 1455-1465. 15933070
Georgi, A. B., Stukenberg, P. T., and Kirschner, M. W. (2002). Timing of events in mitosis. Curr. Biol. 12: 105-114. 11818060
Jeppesen P. (1997) Histone acetylation: a possible mechanism for the inheritance of cell memory at mitosis. Bioessays 19: 67-74. 9008418
Johnson, C. A., et al. (1998). Distinctive patterns of histone H4 acetylation are associated with defined sequence elements within both heterochromatic and euchromatic regions of the human genome. Nucleic Acids Res. 26: 994-1001. 9461459
Nishioka, K., et al. (2002a). Set9, a novel histone H3 methyltransferase that facilitates transcription by precluding histone tail modifications required for heterochromatin formation. Genes Dev. 16(4): 479-89. 11850410
Nishioka, K., et al. (2002b). PR-Set7 is a nucleosome-specific methyltransferase that modifies lysine 20 of histone H4 and is associated with silent chromatin. Molec. Cell 9: 1201-1213. 12086618
Rea S., et al. (2000). Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406: 593-599. 10949293
Rice, J. C. and Allis, C. D. (2001). Histone methylation versus histone acetylation: New insights into epigenetic regulation. Curr. Opin. Cell Biol. 13: 263-273. 11343896
Rice, J. C., et al. (2002). Mitotic-specific methylation of histone H4 Lys 20 follows increased PR-Set7 expression and its localization to mitotic chromosomes. Genes Dev. 16: 2225-2230. 12208845
Sahashi, R., Crevel, G., Pasko, J., Suyari, O., Nagai, R., Saura, M. M., Yamaguchi, M. and Cotterill, S. (2014). DNA polymerase alpha interacts with PrSet7 and mediates H4K20 monomethylation in Drosophila. J Cell Sci 127(Pt 14):3066-78. PubMed ID: 24806961
Sakaguchi, A., Joyce, E., Aoki, T., Schedl, P. and Steward, R. (2012). The histone H4 lysine 20 monomethyl mark, set by PR-Set7 and stabilized by L(3)mbt, is necessary for proper interphase chromatin organization. PLoS One 7: e45321. PubMed ID:23024815
Schiltz, R. L., et al. (1999). Overlapping but distinct patterns of histone acetylation by the human coactivators p300 and PCAF within nucleosomal substrates. J. Biol. Chem. 274 :1189-1192. 9880483
Sobel, R. E., et al. (1995). Conservation of deposition-related acetylation sites in newly synthesized histones H3 and H4. Proc. Natl. Acad. Sci. 92: 1237-1241. 7862667
Strahl, B. D. and Allis, C. D. (2000). The language of covalent histone modifications. Nature 403: 41-45. 10638745
Strahl, B. D., et al. (2001). Methylation of histone H4 at arginine 3 occurs in vivo and is mediated by the nuclear receptor coactivator PRMT1. Curr. Biol. 11: 996-1000. 11448779
Turner, B. M., Birley, A. J. and Lavender, J. (1992). Histone H4 isoforms acetylated at specific lysine residues define individual chromosomes and chromatin domains in Drosophila polytene nuclei. Cell 69: 375-384. 1568251
Wang, H., et al. (2001a). Methylation of histone H4 at arginine 3 facilitating transcriptional activation by nuclear hormone receptor. Science 293: 853-857. 11387442
Wang, H., et al. (2001b). Purification and functional characterization of a histone H3-lysine 4-specific methyltransferase. Mol. Cell 8: 1207-1217. 11779497
White, C. L., Suto, R. K. and Luger, K. (2001). Structure of the yeast nucleosome core particle reveals fundamental changes in internucleosome interactions. EMBO J. 20: 5207-5218. 11566884
Zhang, Y. and Reinberg, D. (2001). Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails. Genes Dev. 15: 2343-2360. 11562345
date revised: 23 July 2014
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