Abstract

PR-Set7 substrates and effectors

Although other yet-to-be-identified substrates may exist, methylation of H4 is likely to be the major effector of PR-Set7 function and a crucial mediator of its impact on viability. As is potentially the case for other histone modifications, H4K20me could act directly through altering nucleosome structure or indirectly through specific binders that mediate downstream functions. These two possibilities are further complicated in the case of PR-Set7, since the loss of H4K20me1 also causes a decrease in H4K20me2/3. However, the phenotype of Suv4-20h1/2^−/− mice (Suv4-20 double knockout) is much less severe than PR-Set7^−/− mice, suggesting a unique function for H4K20me1 independent of H4K20me2/3 (Schotta et al. 2008).

A wealth of evidence has emerged that supports both direct and indirect functions for H4K20me1. In vitro studies have shown that H4K20 forms internucleosomal contacts with the H2A C terminus on adjacent nucleosomes, possibly to promote compaction, and H4K20 methylation may also directly affect higher-order chromatin structure due to its position at the junction of DNA and the nucleosome (Lu et al. 2008). Furthermore, acetylation of a neighboring residue, H4K16, has been demonstrated to regulate higher-order chromatin dynamics and may be affected by H4K20me (Nishioka et al. 2002; Shogren-Knaak et al. 2006; Pesavento et al. 2008; Kapoor-Vazirani et al. 2011). This role of H4K20me in higher-order chromatin structure was also demonstrated by in vivo experiments in which loss of PR-Set7 resulted in large-scale chromatin decondensation in the interphase nucleus and expanded chromosome territory, signifying less compact chromatin (Oda et al. 2009). Moreover, further analysis of the loss of PR-Set7 showed a large change in overall subnuclear morphology (H Oda, D Beck, and D Reinberg, unpubl.). Therefore, PR-Set7 alone, presumably due to its activity toward H4K20, may contribute directly to chromatin structure without other factors.

In addition, H4K20me1 likely conveys its functions through the proteins that bind to it. There are three extensively investigated H4K20me1 binders that each carry a unique binding domain and contribute to distinct cellular functions: L3MBTL1 (Li et al. 2007; Trojer et al. 2007), 53BP1 (Sanders et al. 2004; Oda et al. 2009; Spektor and Rice 2009), and Condensin II (Liu et al. 2010). L3MBTL1 binds to both mono- and dimethylated H4K20 residues through its MBT domains. It works as a transcriptional repressor and can cause direct compaction of chromatin (see also PR-Set7 and Transcription). 53BP1 recognizes methylated lysine residues through its tandem tudor domain and functions within the DNA damage response pathway (see below, Function in DNA Damage). Condensin II recognizes H4K20me1 through its N-CAPD3 and N-CAPG2 subunits via two HEAT domains and is involved in mitotic progression (see below, Role in Cell Cycle Progression). In addition, the LRWD1/ORCA-associated factor of the origin of replication complex (ORC) binds to H4K20me3 and may link regulation of H4K20me to initiation of replication (Shen et al. 2010; Vermeulen et al. 2010).

It is difficult to attribute the essential function of PR-Set7 to any single signaling pathway, as deletion of any one of the specific binders of H4K20me1 does not result in a phenotype similar to PR-Set7 deletion (Ward et al. 2003; Qin et al. 2010). Moreover, loss of H4K20me2/3 may also contribute to the PR-Set7^−/−phenotype. These findings suggest that PR-Set7 is functioning through multiple different pathways, thereby causing a compound phenotype. Thus, PR-Set7 and H4K20me1 operate at the convergence of important regulatory processes.

PR-Set7 interacts with a number of nuclear proteins, most notably PCNA, a processivity factor involved in DNA replication (Jorgensen et al. 2007; Huen et al. 2008). This interaction recruits PR-Set7 to sites of DNA damage and replication and is also essential for the CRL4^Cdt2-dependent degradation of PR-Set7 (Abbas et al. 2010; Centore et al. 2010; Oda et al. 2010). PCNA-dependent recruitment of PR-Set7 to sites of DNA damage is required for recruitment of 53BP1 (Oda et al. 2010; see below, Regulation of PR-Set7 Expression Level, Activity, and Function; and Function in DNA Damage). Besides its ability to bind to monomethylated lysine residues such as H4K20me1, L3MBTL1 also interacts directly with PR-Set7 (Kalakonda et al. 2008). One attractive possibility is that this latter interaction could serve to propagate the H4K20me1 mark. Furthermore, PR-Set7 has been shown to interact with LEF3 (Z Li et al. 2011), RNA polymerase II (Y Li et al. 2011), ER (Y Li et al. 2011), and TWIST (Yang et al. 2011), and these interactions may direct PR-Set7 activity to specific genes during transcription, although there is limited evidence for the potential functions of these associations.

Besides H4K20, PR-Set7 methylates p53 at lysine residue 382, preventing p53 promoter binding and thereby suppressing transcriptional activation of p21 and PUMA (Shi et al. 2007). This event results in an inhibition of apoptosis (Shi et al. 2007). Thus, PR-Set7 may also regulate the DNA damage response and cell cycle progression via methylation of p53, a key checkpoint protein (Sherr 2004). However, the DNA damage and cell cycle defects caused by PR-Set7 knockdown are not entirely p53-dependent, and p53^−/− cells do not show massive DNA damage, suggesting that the major impact of PR-Set7 is independent of p53 (Donehower et al. 1992; Tardat et al. 2007; Houston et al. 2008). Moreover, in stark contrast to PR-Set7^−/− cells, p53^−/− cells are viable (Donehower et al. 1992). For these reasons, we focus our attention here on H4K20me when discussing PR-Set7 function.

Regulation of PR-Set7 expression level, activity, and function

PR-Set7 protein expression is tightly regulated during the cell cycle; it is highest during G2/M and early G1 and is absent during S phase. (Fig. 2; Oda et al. 2009). This regulation is also reflected by a similar oscillation in the levels of H4K20mel during the different phases of the cell cycle. PR-Set7 levels are controlled primarily through post-translational modification by multiple enzymes, including kinases, SUMO, and ubiquitin ligases (Abbas et al. 2010; Centore et al. 2010; Oda et al. 2010; Tardat et al. 2010; Spektor et al. 2011). Yet recent evidence supports that PR-Set7 are also regulated at the transcriptional level (Wakabayashi et al. 2009). We speculate that there are additional, as-yet-unidentified factors that further modulate PR-Set7 activity and recruitment to chromosomes and specific loci and that these proteins may provide key insights into the regulation and function of H4K20me1.

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Figure 2.

PR-Set7 is highly regulated during, and is important for multiple aspects of, the cell cycle. PR-Set7 levels oscillate—as shown by YFP-PR-Set7 (green) expression in the outer circle—during the different stages of the cell cycle (mCherry-PCNA). PR-Set7 is post-translationally regulated by multiple different enzymes during the cell cycle, including CRL4-Cdt2, SCF-Skp2, APC-Cdh1, and Cyclin B1/Cdk1. The various functions of PR-Set7 and H4K20me1 are listed within the cell cycle circle.

Role in cell cycle progression

PR-Set7 is unique among histone methyltransferases in that its protein levels are regulated during the cell cycle. This oscillation reflects the specific roles of PR-Set7 and H4K20me1 during different phases of the cell cycle that are essential for proper cell cycle progression. Loss-of-function studies established a role for PR-Set7 in proper mitotic condensation, S-phase progression, and genome integrity. Further insights have emerged from determining that PR-Set7 is degraded through PCNA-coupled CRL4^Cdt2-mediated ubiquitination prior to S phase. These studies have implicated a role for PR-Set7 and H4K20me1 in DNA replication. Moreover, these efforts have defined PR-Set7 as an important controller of both S and G2/M phase and potentially link these two processes together.

Function in DNA damage

Spontaneous DNA damage in the absence of PR-Set7

As described earlier, deletion of PR-Set7 in ES cells results in massive DNA damage, as shown by immunofluorescence staining with γ-H2AX antibodies (Oda et al. 2009). Moreover, in an unbiased RNAi screen, PR-Set7 was identified as a factor whose depletion leads to an increase in γ-H2AX foci in HeLa cells (Paulsen et al. 2009). It is unlikely that this increase in spontaneous DNA damage upon loss of PR-Set7 is the result solely of an impaired DNA damage pathway, since there is normally very little spontaneous damage in the cell (Vilenchik and Knudson 2003). Instead, the massive damage seen after loss of PR-Set7 is more likely attributable to increased genomic instability due to defects during DNA replication or mitosis (Fig. 3). In the absence of PR-Set7, DNA damage accumulates only if the cells are allowed to enter S phase, which suggests that the defect occurs during DNA replication (Jorgensen et al. 2007). Furthermore, this damage can be suppressed by depletion of RAD51, a key regulator of homologous recombination, placing PR-Set7 as a potential effector of this type of repair (Jorgensen et al. 2007). Interestingly, the increase of γ-H2AX foci was not observed in PR-Set7 knockout embryos even though they exhibited the same G2/M arrest as their ES cell counterparts. This finding suggests that checkpoint and DNA damage signaling in the early embryonic stages is different from that in later stage ES cells or differentiated cells.

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Figure 3.

Role of PR-Set7 in genome integrity. PR-Set7 has multiple functions that are important for proper genome stability and DNA damage response. (Top left) PR-Set7 methylates H4K20me1, which is important for recruiting 53BP1 recruitment to sites of DNA damage. (Top right) PR-Set7 alters p53-dependent gene expression, potentially regulating checkpoint and repair processes. (Bottom left) H4K20me alters chromatin compaction and mitotic condensation, thereby affecting higher-order chromatin structure. Finally, PR-Set7 and H4K20me are required for proper DNA replication, possibly through regulation of fork stability.

PR-Set7 and transcription

Despite numerous efforts, the role of PR-Set7 and H4K20me1 in gene transcription has remained elusive. Removal of PR-Set7 causes various cellular changes that can indirectly affect gene expression, making it difficult to determine its direct functions. For this reason, we are currently dependent on correlative studies, which have been inconclusive and imply complex roles for these factors in transcription. This is apparent when comparing the conclusions reached from initial subcellular localization studies suggesting a role for H4K20me1 in transcriptional repression with those from more recent genome-wide ChIP analysis implicating H4K20me1 in transcriptional activation.

Supporting a role in repression, H4K20me1 could operate directly by promoting chromatin compaction (Lu et al. 2008; Oda et al. 2009) or indirectly through its progression toward H4K20me2/3 and/or recruitment of modification-specific binders such as L3MBTL1, a transcriptional repressor (Trojer et al. 2007). Moreover, insights into a potential role for PR-Set7 in repression of transcription were initially made in Drosophila, wherein H4K20me1 localizes exclusively to regions on polytene chromosomes that are devoid of active transcription (Nishioka et al. 2002). Furthermore, deletion of PR-Set7 causes a suppression of the variegation phenotype in Drosophila, suggesting that PR-Set7 acts to silence gene expression (Karachentsev et al. 2005). However, this result is complicated in that Suv4-20 is also a suppressor of variegation, and the suppression of variegation phenotype identified after loss of PR-Set7me may be secondary to that of decreased H4K20me2/3 levels. For example, H4K20me3 has a well-established role in gene repression, specifically at repetitive elements (Schotta et al. 2004, 2008; Fodor et al. 2010). In addition to these results from Drosophila, localization studies in mouse cells showed an enrichment of H4K20me1 on the inactive X chromosome, a classic example of facultative heterochromatin (Kohlmaier et al. 2004).

Several additional studies support a role for PR-Set7 in gene repression. In-depth ChIP studies on a large subset of the genome in HeLa cells demonstrated that H4K20me1 localized primarily within gene bodies. Loss of PR-Set7 causes increased expression of H4K20me1-enriched genes, implying a role for PR-Set7 in gene repression (Congdon et al. 2010). In addition, genomic regions enriched for H4K20me1 can repress luciferase expression in an artificial promoter system (Congdon et al. 2010). Still other studies showed that H4K20me1 localized to repressed genes regulated by E2F (Abbas et al. 2010). Finally, knockdown of PHF8, which demethylates H4K20me1, causes increased repression of E2F-responsive genes due to increased H4K20me1 and L3MBTL1 binding (Liu et al. 2010).

On the other hand, results from genome-wide ChIP analyses argue against a role in repression. In T lymphocytes, H4K20me1 peaks were similarly found to localize downstream from the transcription start site (TSS), yet in contrast to the above-mentioned HeLa cell study, H4K20me1 correlates with highly transcribed genes (Barski et al. 2007; Cui et al. 2009). Further analysis revealed that H4K20me1 is one of the histone modifications most highly correlated with active transcription (Wang et al. 2008). This correlation between H4K20me1 and active transcription had previously been shown at a limited set of loci in murine erythroleukemia cells, adipocytes, and HeLa cells (Talasz et al. 2005; Vakoc et al. 2006; Wakabayashi et al. 2009). These findings suggest that PR-Set7 and H4K20me1 may function during active transcription as well as transcriptional repression.

Further evidence from gene-specific studies showed that H4K20me1 levels increased at estrogen-responsive genes after their activation (Y Li et al. 2011). In another focused analysis, H4K20me1 and PR-Set7 were enriched specifically at wnt3a-responsive gene promoters after wnt3a stimulation (Z Li et al. 2011). Upon PR-Set7 knockdown, wnt3a-dependent activation of wnt response genes was abolished. This regulation was shown to be physiologically relevant, since PR-Set7 participates in the wnt developmental pathway in zebrafish (Z Li et al. 2011). These results are slightly complicated by the fact that PR-Set7 was found to both stimulate and repress the wnt3a response genes potentially by cooperating with Lef3, a known transcriptional activator and repressor. A similar type of dual transcriptional regulation was demonstrated for PR-Set7 cooperation with TWIST, an important regulator of epithelial-to-mesenchymal transition (Yang et al. 2011).

Thus, despite numerous studies examining the role of H4K20me1, this modification still has an inconclusive role in gene expression, with evidence supporting a correlation with both gene expression and repression. Some gene-specific analyses, along with all genome-wide ChIP studies to date, have correlated H4K20me1 with active transcription, while a subset of non-genome-wide ChIP and biochemical studies have correlated H4K20me1 with gene repression.

To better decipher the role of H4K20me1 in regulation of transcription, we embarked on a comparative analysis of recently released ENCODE (Encyclopedia of DNA Elements) data sets, established as a means to identify important functional genomic elements (The ENCODE Project Consortium 2004; Ernst et al. 2011). This analysis allows for the first cross-comparison of data sets between different cell lineages that, importantly, were generated using similar experimental procedures and data analysis. After comparing H4K20me1 ChIP sequencing data from the nine human cell lines examined by the ENCODE project, we found that H4K20me1 is specifically enriched in intragenic regions in all data sets (Fig. 4A). Supporting earlier observations, H4K20me1 also correlates with highly expressed genes and is found predominantly downstream from the TSS (Fig. 4B), although the magnitude of these peaks varies between cell lines (Fig. 4C,D). We further analyzed the gene ontologies enriched with H4K20me1 that are unique or conserved between three ENCODE cell types: H1 ES, HepG2 hepatocellular carcinoma, and K562 erythroleukemia cells. Genes with H4K20me1 peaks that were conserved across all three cell lines were primarily highly expressed metabolic and cellular housekeeping genes. In contrast, genes with H4K20me1 peaks unique to one specific cell line tended to be highly expressed tissue-specific genes functionally inherent to that lineage; i.e., development for H1, carbohydrate metabolism for HepG2, and cellular structure for K562 (Fig. 4). These combined results suggest that H4K20me1 correlates with highly expressed genes, independent of the lineage, and does not seem to be very specific to any functional subset of genes. Although these findings do not directly demonstrate a function of PR-Set7 and H4K20me1 in transcription, they suggest that these factors may participate in active transcription.

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Figure 4.

Genome-wide distribution and features of H4K20me1 from the ENCODE project. Bioinformatic analysis (MACS) was used to analyze H4K20me1 genome-wide distribution from ENCODE data sets from nine different human cell lines: H1 (human ES cells), K562 (erythrocytic leukemia), GM12878 (B-lymphoblastoid), HepG2 (hepatocellular carcinoma), NHEK (normal human epidermal keratinocytes), HSMM (primary human skeletal muscle myoblasts), NHLF (normal human lung fibroblasts), Huvec (primary human umbilical vein endothelial cells), and HMEC (primary human mammary epithelial cells). (A) H4K20me1 is enriched on intragenic features in H1 cells, representative of other cell lines. (B) H4K20me1 is enriched downstream from the TSS in H1 cells, representative of other cell lines. (C) H4K20me1 is found within the gene body and is correlated best with the highly expressed genes (Q100) and less so with the lower expressed genes in H1 cells, representative of other cell lines. (D) Comparison across all nine cell lines; Q100 genes show a similar distribution, with differences in the level of enrichment. (E) Venn diagram comparing gene ontology term analysis of genes within 3 kb of an H4K20me1 peaks. Conserved genes with H4K20me1 peaks across all three cell lines are housekeeping genes required in all cell types, whereas cell-specific genes enriched with H4K20me1 are genes that are only highly expressed in that specific lineage.

These apparently conflicting experimental results concerning PR-Set7 function in gene expression may be explained by multiple, potentially confounding biological and technical variables. One major variable may be the proliferation rates of different cell lines, given the cell cycle-dependent variation in PR-Set7 and H4K20me1 levels. The role of H4K20me1 may also be context-dependent. For example, neighboring modifications on the H4 tail may affect H4K20me1 function. The presence of H4K20me1 at highly expressed genes may potentially be affected by the co-occurrence of neighboring H4K16 acetylation (H4K16ac), which has an established role in activating gene expression. H4K16ac could potentially override a subset of H4K20me1 function, creating a different role for H4K20me1 in gene expression in this context. Other subsets of H4K20me1, specifically at E2F target genes, may be important for gene repression and are demethylated by PHF8 during the cell cycle (Liu et al. 2010). Still other H4K20me1 may be destined to progress to H4K20me2/3, which repress repetitive DNA elements (Schotta et al. 2004, 2008). Finally, the H4K20me1 that is not sterically masked may be regulating transcription through L3MBTL1 recruitment (Trojer et al. 2007). Detailed investigation of H4K20me1 and specific binders of H4K20me1 and H4K16Ac in synchronized cells will be necessary to parse out the meaning of the differential localization patterns of H4K20me1 and determine the exact correlation between gene expression and cell cycle progression. These combined possibilities reveal a plethora of potential functions for H4K20me1. An outstanding resolution, although unlikely and difficult to prove, is that the modification may not have a specific role in gene expression even though potential correlations abound.

Final thoughts

Over the last 10 years, we have made great strides toward understanding the function of PR-Set7, yet we still have far to go. A major question remains as to why PR-Set7 and independent regulation of H4K20me1 emerged during evolution. To date, the least-characterized function of PR-Set7 and H4K20me1 is in transcription and gene expression. Clarification of how this modification is regulated genome-wide and the relevance of its distribution may better explain the major functions of PR-Set7 in cell cycle progression and in the DNA damage response. Recently, PR-Set7 was shown to potentially have physiological importance in cancer progression, making understanding the function of H4K20me1 even more important (Yang et al. 2011).

Although our understanding of the function of H4K20me1 has become more comprehensive, we still have not elucidated the mechanism by which one modification can have such a vast array of functions. The answer to these questions will help characterize a unique and essential modification and provide key insights into how a single modification can regulate so many cellular functions. Hopefully, further analyses, including knockout studies, will lead to a detailed understanding of the role of each modification binder in regulating gene expression and cell cycle progression. This key information will help decipher the contributions of PR-Set7 and H4K20me1 that are determinant to cell viability.

Acknowledgments

Footnotes

References