Distinct patterns of histone methylation during human cell cycle progression are described. 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. These data suggest that H4 Lys 20 methylation by PR-Set7 during mitosis acts to antagonize H4 Lys 16 acetylation and to establish a mechanism by which this mark is epigenetically transmitted (Rice, 2002).
To determine histone methyltransferase activity during the human cell cycle, HeLa cells were arrested by treatment with thymidine followed by mimosine. Every 2.5 h following release from the G1 arrest, synchronized cells were isolated for analysis, and the cell cycle phase was determined by fluorescence-activated cell sorting (FACS). Nuclei were isolated from the cells at these various time points for the in nucleo HMT assay and for Western blot analysis (Rice, 2002).
The in nucleo assay was used to provide a preliminary indication of alterations in nuclear methyltransferase activity during specific phases in the cell cycle. The assay was performed by incubating synchronized nuclei in the presence of a radiolabeled methyl donor (3H-S-adenosyl-methionine), followed by SDS-PAGE and autoradiography. No apparent changes in the H3 HMT activity occur during the cell cycle. In contrast, the enzymatic methylation of histone H4 in this assay is greatly increased during mid-S-phase through mitosis and returned to levels observed during G1. Little, if any, H4 HMT activity is detected outside of this time frame. It is interesting to note that histones H3 and H4 were the only nuclear proteins in this assay that were detectably methylated. There are several potential limitations to this assay including occupancy of preexisting methylation sites, complications resulting from neighboring histone modifications, and/or decreased detection of HMT activity caused by as-yet undiscovered histone demethylases. Nonetheless, this assay provides a preliminary indication that H4 methylation, as opposed to the H3 methylation, is highly regulated throughout the human cell cycle (Rice, 2002).
To further characterize the HMT activity from the in nucleo assay, integrated densitometry was used to quantitate the intensity of the radiolabeled histone bands and the intensity of the Coomassie-stained histone bands. Once these values were determined, the radiolabeled histone band was divided by the loading control Coomassie-stained histones, yielding an arbitrary number. These values were then standardized by assigning the G1 value to 1 such that the fold change at each of the time points could be determined. Although undetectable by the gels, the analysis indicates a moderate increase in H3 HMT activity that peaks during mid-S-phase (2.5-fold), declines through G2/M, and reaches a baseline at G1. In contrast, the analysis of the histone H4 bands indicates a significant increase in HMT activity during mid-S-phase to G2 (5-10 h) that abruptly declines during mitosis and the transition to G1 (12.5 h). The H4 HMT activity then reaches a baseline upon return to G1 (Rice, 2002).
These results show that the enzymatic methylation of histones H3 and H4 occurs during distinct phases in the human cell cycle under these assay conditions. Although there is a modest increase in histone H3 methyltransferase activity during S-phase, it has yet to be determined which Arg or Lys residues are being methylated during this time point. This will be difficult to dissect because many arginines and lysines in histone H3 are known to be methylated in vivo. In contrast, a dramatic increase in histone H4 methyltransferase activity is observed during mid-S-phase and G2/M in the HeLa cell cycle. Because Arg 3 and Lys 20 are the only histone H4 residues presently known to be methylated in vivo (Rice, 2001; Zhang, 2001), an investigation was carried out to determine which of these two residues are being methylated during these time points in the human cell cycle (Rice, 2002).
Histones from synchronized HeLa cells were fractionated by SDS-PAGE and Western-blotted using antibodies specific for the H4 Arg 3-methyl or Lys 20-methyl modifications. Methylation of H4 Arg 3 is readily detected in HeLa cells arrested in G1 (0 h). Upon entry into S-phase (0-2.5 h), there is a decrease in this modification that increases back to the observed G1 levels during the transition from mid- to late-S-phase (5-7.5 h) and remains constant through mitosis (Rice, 2002).
One possible explanation for the observed decrease in the H4 Arg 3-methyl modification is the deposition of newly synthesized histone H4, which occurs immediately following DNA replication. Thus, the apparent decrease may be caused by a dilution due to the deposition of newly synthesized and unmethylated histone H4 proteins that ultimately become methylated during later points in the cell cycle, most likely mediated by the H4 Arg 3-specific HMT, PRMT1 (Strahl, 2001). In addition, the methylation of H4 Arg 3 during mid-S-phase suggests that this modification may be associated with euchromatin, or transcriptionally active regions, which are replicated early in S-phase. This would be consistent with a recent finding (Wang, 2001a) that H4 Arg 3 methylation plays a role in transcriptional activation of nuclear hormone receptors (Rice, 2002).
Although the increase in H4 Arg 3 methylation accounts for the observed increase in H4 HMT activity during S-phase (2.5-7.5 h), it does not explain the increased activity during G2/M. Because Lys 20 is the only other histone H4 residue known to be methylated, it was hypothesized that Lys 20 methylation is increased during this phase in the cell cycle. Blot analysis shows that H4 Lys 20 methylation also decreases during S-phase, albeit later in the cell cycle compared with H4 Arg 3 methylation. During the transition from early- to mid-S-phase (2.5-5 h), H4 Lys 20 methylation drops and remains low through late-S-phase (7.5 h) and G2 (10 h), which may be caused, again, by dilution of this modification by the deposition of newly synthesized and unmethylated histone H4 proteins. The observed decrease in H4 Lys 20 methylation in mid-S-phase suggests that this modification is associated with heterochromatin, which is known to replicate later in S-phase compared to euchromatin. This theory is supported by a recent report showing that the H4 Lys 20-methyl modification is associated with transcriptionally silent regions of the genome (Nishioka, 2002; Rice, 2002).
During mitosis (12.5 h), H4 Lys 20 methylation returns to levels similar to those observed in G1 and stay constant through the remainder of the cell cycle. Although FACS analysis shows approximately equal numbers of cells in mitosis as G1 at the 12.5-h time point, the single appearance of the mitotic-specific phosphorylation of histone H3 serine 28 indicates that H4 Lys 20 methylation occurs during mitosis rather than during the transition to G1. Furthermore, because histone H3 serine 28 is phosphorylated specifically in the early phases of mitosis and is dephosphorylated abruptly during the metaphase-to-anaphase transition, the results suggest that the methylation of H4 Lys 20 also begins early in mitosis (Rice, 2002).
To further expand the observation 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. 2002). 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).
It was recently reported that histone H4 Lys 20 methylation inhibits acetylation of H4 Lys 16, and vice versa (Nishioka, 2002b). Based on this, it was predicted that these two modifications would occur at distinct points in the cell cycle. Western blot analysis showed that H4 Lys 16 acetylation is low during G1 (0 hours) when H4 Lys 20 methylation is the highest. However, H4 Lys 16 acetylation significantly increases and peaks during mid-S-phase, the time when H4 Lys 20 methylation is the lowest. During mitosis (12.5 h), the acetylation of H4 Lys 16 dramatically decreases just as H4 Lys 20 methylation peaks. These observations show that H4 Lys 20 methylation inhibits H4 Lys 16 acetylation and vice versa (Rice, 2002).
Immunofluorescence studies were performed on mitotic HeLa cells to provide a qualitative estimate of the relative phase during mitosis in which H4 Lys 20 methylation increases. The specific phases of mitosis were determined by staining of DNA with DAPI. At prophase, the H4 Lys 20-methyl modification displays a more punctate and less intense staining pattern compared with interphase cells that have high levels of H4 Lys 20 methylation. This suggests that the observed decrease in H4 Lys 20 methylation during S-phase and G2 persists through the early stages of mitosis. It is unlikely that the observed decreased staining at prophase is a consequence of chromatin condensation since H4 Lys 20 methylation is clearly detected at other phases of mitosis when chromatin is even more condensed. In contrast to prophase, there was a visually pronounced increase in H4 Lys 20 methylation at metaphase that coincides directly with alignment of chromosomes on the metaphase plate. This suggests that the increase in H4 Lys 20 methylation occurs prior to or during metaphase. The H4 Lys 20-methyl modification persists through the rest of mitosis. Taken together, these results document the mitotic-specific enzymatic methylation of H4 Lys 20 (Rice, 2002).
A novel histone H4 Lys 20-specific methyltransferase, PR-Set7, has been identified in HeLa cells (Nishioka, 2002b). It was hypothesized that PR-Set7 expression would increase simultaneously with H4 Lys 20 methylation during cell cycle progression. Northern blot analysis in synchronized HeLa cells indicates that PR-Set7 mRNA expression is greatly increased during late S-phase and G2/M and declines during transition to G1. To determine if PR-Set7 protein expression is also increased during these times, a polyclonal PR-Set7 antibody was developed and confirmed to be specific for PR-Set7. The antibodies detect recombinant as well as endogenous PR-Set7. Consistent with the RNA expression findings, Western blot analysis shows that PR-Set7 protein is not detected during G1. The PR-Set7 protein levels elevate steadily beginning at early S-phase through G2 (10 h) and peak during mitosis (12.5 h). The increase during mitosis was confirmed by the appearance of the mitotic-specific phosphorylation of histone H3 serine 28. Moreover, Western blot analysis in G1-arrested cells confirms that the PR-Set7 protein is undetectable, whereas in mitotic-arrested cells there are significantly abundant levels of PR-Set7. Subsequent to mitosis, the PR-Set7 protein abruptly decreases and continues to decrease as more cells entered G1 (15-20 h). These findings indicate that PR-Set7 RNA and protein expression are up-regulated during cell cycle progression, consistent with the observed increase in H4 HMT activity and H4 Lys 20 methylation (Rice, 2002).
Immunofluorescence studies in HeLa cells were performed to determine the localization of PR-Set7 during different phases in the cell cycle. At metaphase and anaphase PR-Set7 is clearly associated with mitotic chromosomes. PR-Set7 is also detected at prometaphase, although the localization was relatively dispersed compared with metaphase and anaphase (Rice, 2002).
These data indicate that PR-Set7 expression is cell cycle-regulated and that the PR-Set7 protein is localized to mitotic chromosomes, coincident with the increase in H4 Lys 20 methylation. The steady increase in PR-Set7 expression during cell cycle progression is directly correlated with the observed increase in H4 HMT activity during these same times. Although these data indicate that there is sufficient enzymatically active PR-Set7 in the nucleus during S-phase and G2, the methylation of H4 Lys 20 is delayed prior to metaphase. It is presently unknown what mechanisms account for this delay; however, it is clear that PR-Set7 expression and the PR-Set7 protein must be tightly regulated by, as yet, uncharacterized mechanisms to prevent the premature methylation of H4 Lys 20. Consistent with this theory, studies performed with Xenopus egg extracts showed that the Xenopus PR-Set7 protein is phosphorylated during mitosis (Georgi, 2002). Although phosphorylation of human PR-Set7 is not essential for its HMT activity in vitro, phosphorylation of the enzyme in vivo may serve to regulate its association with mitotic chromosomes (Rice, 2002).
The cell cycle-regulated methylation of H4 Lys 20 suggests that this modification is localized to specific regions in the genome and inherited in an epigenetic fashion. Using telomere position effect variegation as a model for epigenetic silencing, data in yeast suggest that this repressive chromatin state is disassembled during S-phase and reassembled by G2/M. This coincides with the decrease in H4 Lys 20 methylation during S-phase and its increase during mitosis. Once established following replication, telomeric silent chromatin is relatively stable, much like histone methylation. The similarities between these two suggest that the H4 Lys 20-methyl modification may serve as a stable epigenetic mark that aides in the establishment of discrete chromosomal regions involved in specific chromatin-mediated events. The association of PR-Set7 with mitotic chromosomes may represent a mechanism through which the H4 Lys 20-methyl mark is epigenetically transmitted. It is likely that the localization of PR-Set7 to mitotic chromosomes allows recognition of this mark on the parent chromosomes, which are then duplicated to the daughter chromosomes (Rice, 2002).
Although methylation does not affect the overall charge of the histone tail, it does increase the hydrophobicity and basicity of the lysine residue, suggesting an increased attraction for the negatively charged DNA. However, a more likely function for this modification is that it serves as a recognition motif for the binding of chromatin-associated proteins that mediate changes in higher-order chromatin structure during mitosis, similar to HP1 binding of methylated H3 Lys 9 to establish heterochromatic regions. Alternatively, methylation of H4 Lys 20 may serve to prevent the association of factors to the H4 tail, similar to H3 Lys 4 methylation, which prevents the association of the NuRD complex with the H3 tail. Based on these findings, a likely candidate would be a histone acetyltransferase that modifies H4 Lys 16 (Rice, 2002).
For biochemical characterization, a His-tagged, full-length, wild-type recombinant PR-Set7 (rPR-Set7) protein was produced in bacteria, as well as a mutant version that contained a single arginine-to-glycine substitution (rPR-Set7-R265G). Arginine 265 corresponds to a key conserved residue within the SET domain that has been shown to be required for HMT activity. The wild-type and mutant recombinant proteins were then tested for HMT activity using both octamers and nucleosomes as substrates, and, as expected, only the wild-type rPR-Set7 was active. Much like the native protein, rPR-Set7 preferentially methylates nucleosomal substrates rather than core histone polypeptides. The specific site of the modification by rPR-Set7 was further verified using nucleosomes that had been reconstituted with a mutant form of histone H4 where lysine 20 was replaced by alanine (K20A); as predicted, the activity of rPR-Set7 was significantly reduced. Therefore, it is concluded that PR-Set7 is a histone H4-K20-specific HMT and that the SET domain is responsible for the catalytic activity of the enzyme, as observed with other lysine-specific HMTs (Nishioka, 2002b).
Interestingly and unexpectedly, the strict nucleosomal substrate specificity of PR-Set7 was lost upon the generation of a recombinant PR-Set7 protein lacking the first 14 amino acids at the N-terminal end of the protein (DN14-rPR-Set7). In contrast to rPR-Set7, DN14-rPR-Set7 is able to methylate nucleosomes as well as octamer and the H4 polypeptide. It is currently unclear if the deletion of the first 14 amino acids of PR-Set7, the cloning of DN14-rPR-Set7 into a different vector, the differences in purification schemes, or all of these directly contributed to the observed alterations in substrate specificity. Regardless, this mutant form of rPR-Set7 methylates histone H4 polypeptides exclusively at lysine 20, as was previously observed with the native protein. It was confirmed by a gel-filtration analysis that this relaxed substrate recognition is not due to aggregation of the mutant enzyme. These results demonstrate striking alterations in substrate recognition (i.e., free histone versus nucleosome) of known HMTs resulting from deletions in the protein far removed in primary sequence from the catalytic SET domain (Nishioka, 2002b).
Obvious PR-Set7 homologs are absent from yeast. This observation prompted a search for the presence of methylated H4-K20 in a variety of organisms, including the yeast Saccharomyces cerevisiae. Toward this end, polyclonal antibody that specifically recognized methyl H4-K20 (a-methyl H4-K20) was generated. This antibody is highly specific for methylated H4-K20 and fails to recognize an unmodified H4 polypeptide as well as other well known methylated lysine residues, including histone H3 lysine 4 or lysine 9. Western blot analyses with the antibody were then performed with acid-extracted histones derived from several organisms. Interestingly, the H4-K20 modification is detectable only on histone H4 derived from higher eukaryotes such as fly, frog, and human. The modification was not found in yeast histone H4. Because yeast histone H4 has replaced an isoleucine for a valine at position 21, it was possible that the antibody is unable to recognize methylated yeast histone H4 for this reason. rPR-Set7 was therefore used to methylate yeast nucleosomal H4 in vitro and the modified H4 was subjected to Western blotting using the a-methyl H4-K20 antibody. Indeed, the antibody is able to recognize the in vitro-methylated K20 in yeast H4. Therefore, these findings strongly suggest that H4 from S. cerevisiae is not methylated at position K20 in vivo (Nishioka, 2002b).
The presence of methylated H4-K20 was not detected in the ciliated protozoan Tetrahymena. The sequence of the Tetrahymena H4 tail is significantly different from that of other eukaryotes including the loss of an arginine at position 3. However, by insertion of a novel serine at position 18, Tetrahymena has established a lysine residue at position 20. The inability to detect H4-K20 methylation in Tetrahymena may be due to inability of the antibody to recognize this sequence variation. In addition, neither rPR-Set7 nor DN14-rPR-Set7 was able to methylate Tetrahymena histone H4, indicating that either Tetrahymena is not methylated at H4-K20 or that PR-Set7 possesses a strict amino acid specificity for enzymatic activity that is not present in the Tetrahymena sequence. Therefore, it remains an open question whether methylation at H4-K20 occurs in Tetrahymena (Nishioka, 2002b).
Because H3-K9 methylation is associated with constitutive heterochromatin in various species, the staining pattern of methyl H4-K20 was examined in MEFs lacking the known H3-K9 HMTs Suv39h1 and Suv39h2. The localization of methyl H4-K20 in wild-type MEFs was to euchromatin arms and was absent in the constitutive heterochromatin. Double knockout MEFs demonstrated unchanged localization of methyl H4-K20, corroborating the findings in Drosophila that methyl H4-K20 and methyl H3-K9 are separate and independent modifications. These data suggest that the methyl H3-K9 and methyl H4-K20 modifications may direct the formation of distinct types of silent chromatin (Nishioka, 2002b).
To determine whether there is a biochemical link between these modifications, the ability of the histone acetyltransferase (HAT), p300, to acetylate H4-K16 in the presence or absence of methyl H4-K20 was tested. An unmodified and a methyl H4-K20-containing peptide from residues 9 to 25 were created and used as substrates in the assay. It was found that methylation of H4-K20 inhibits acetylation by p300 by 50%-60% compared to the unmodified peptide. Since this peptide set contains lysines 12 and 16, both of which are known targets of p300 HAT activity (Schiltz, 1999), the question of which lysine residue is affected by methyl H4-K20 had to be resolved. Dot blot analysis using site-specific acetyl-H4 antibodies has demonstrated that the p300-mediated acetylation of lysine 16, but not acetylation of lysine 12, is inhibited by methylation of lysine 20. Using the same experimental approach, it was discovered that the acetylation of H4-K16 inhibits the DN14-rPR-Set7-mediated methylation of H4-K20. Taken together, these data indicate that acetylation of H4-K16 and methylation of H4-K20 are mutually restrictive both in vivo and in vitro, indicating that methyl H4-K20 maintains silent chromatin, in part, by precluding neighboring acetylation on the histone H4 tail (Nishioka, 2002b).
SET8 (also known as PR-SET7) is a histone H4-Lys-20-specific methyltransferase that is implicated in cell-cycle-dependent transcriptional silencing and mitotic regulation in metazoans. This study reports the crystal structure of human SET8 (hSET8) bound to a histone H4 peptide bearing Lys-20 and the product cofactor S-adenosylhomocysteine. Histone H4 intercalates in the substrate-binding cleft as an extended parallel beta-strand. Residues preceding Lys-20 in H4 engage in an extensive array of salt bridge, hydrogen bond, and van der Waals interactions with hSET8, while the C-terminal residues bind through predominantly hydrophobic interactions. Mutational analysis of both the substrate-binding cleft and histone H4 reveals that interactions with residues in the N and C termini of the H4 peptide are critical for conferring substrate specificity. Finally, analysis of the product specificity indicates that hSET8 is a monomethylase, consistent with its role in the maintenance of Lys-20 monomethylation during cell division (Couture, 2005).
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