BIOLOGICAL OVERVIEW

A human histone H4 lysine 20 methyltransferase was purified based on its ability to methylate histone H4, and the encoding gene, PR/SET07, was cloned. The Drosophila homolog, Pr-Set7, is involved in chromatin function; therefore, to put this protein in a context, a brief discussion of chromatin follows.

In the nuclei of eukaryotic cells, DNA is stored in the form of chromatin; this structure is repressive to most, if not all, processes that require access of proteins to DNA. The basic building block of chromatin is the nucleosome, which is composed of two copies of each of four core histone proteins (H2A, H2B, H3, and H4) wrapped by 146 base pairs of DNA. The N-terminal tails of the core histone proteins protrude from the nucleosome. These histone tails appear to be unstructured and are believed to participate in the formation of higher order chromatin structure by mediating internucleosomal interactions, as well as contact with DNA that is positioned between the nucleosomes (linker DNA) (Nishioka, 2002b and references therein).

Longstanding cytological studies have generally defined two types of chromatin: euchromatin, which appears as an extended structure and is transcriptionally active, and heterochromatin, a compacted, transcriptionally silent structure. The mechanisms by which euchromatin is converted to heterochromatin and vice versa are poorly defined. However, an extensive literature documents that histone tails undergo a variety of posttranslational modifications, including acetylation, phosphorylation, methylation, and ubiquitination. Recent work has provided compelling evidence that these alterations affect chromatin structure and its functional properties. For example, acetylation of core histone tails correlates with the opening of the chromatin structure to allow transcription (Nishioka, 2002b and references therein).

Drosophila Pr-Set7 function was characterized based on the the availability of a P-element disruption of the 5' UTR region of the corresponding gene. A mutation in Drosophila pr-set7 is lethal: second instar larval death coincides with the loss of Histone H4 lysine 20 methylation, indicating a fundamental role for PR-Set7 in development. Transcriptionally competent regions lack H4 lysine 20 methylation, but the modification coincides with condensed chromosomal regions on polytene chromosomes, including chromocenter and euchromatic arms. The Drosophila male X chromosome, which is hyperacetylated at H4 lysine 16, shows significantly decreased levels of lysine 20 methylation compared to that of females. In vitro, methylation of lysine 20 and acetylation of lysine 16 on the H4 tail are competitive. Taken together, these results support the hypothesis that methylation of H4 lysine 20 maintains silent chromatin, in part, by precluding neighboring acetylation on the H4 tail (Nishioka, 2002b).

Other types of covalent modifications of chromatin components, including methylation of histones H3 and H4, also appear to play critical roles. First described in 1964, histones have long been known to be substrates for methylation. Early studies that used metabolic labeling followed by amino acid sequencing of bulk histones showed that several lysine residues, including lysines 4, 9, 27, and 36 of H3 and lysine 20 of H4, are preferred sites of methylation (Nishioka, 2002b and references therein).

An important breakthrough in the identification of enzymes that carry out histone-lysine methylation came from studies of suppressors of position effect variegation (PEV) in Drosophila. Suppressors of PEV, such as the Su(var)3-9, the polycomb-group protein Enhancer of zeste, and the trithorax-group protein Trithorax, all contain an evolutionarily conserved sequence motif termed the SET domain. The SET domain of the human homolog of Drosophila Su(var)3-9 (Suv39h1) was later found to share sequence similarity with several previously identified SET domain-containing methyltransferases from plants. This observation led to the discovery that Suv39h1 and its S. pombe homolog Clr4 each contain an intrinsic histone methyltransferase activity that specifically methylates histone H3 at lysine 9 (H3-K9). Mutagenesis studies with Suv39h1 have revealed that the SET domain and two adjacent cysteine-rich regions (the pre-SET and post-SET domains) are required for enzymatic activity. Subsequent studies resulted in the isolation of another enzyme, G9a, which displays substrate specificity similar to that of Suv39h1 but also methylates H3-K27. Recently, enzymes that methylate H3-K4 have also been identified in yeast and higher eukaryotes. However, it has been difficult to identify enzymes that methylate the tail of histone H4. Recent studies have demonstrated that the arginine-specific methyltransferase, PRMT1, methylates H4-R3, but the enzyme that methylates H4-K20 has not been identified (Nishioka, 2002b and references).

A mammalian histone methyltransferase (HMT) has been identified that is specific for lysine 20 of histone H4. This enzyme, PR-Set7, resides as a single polypeptide and is highly specific for nucleosomal histones. It was also shown that methylation of H4-K20 is associated with silent, transcriptionally inactive regions within euchromatin. Methylation of histone H4-K20 may maintain this higher order chromatin structure by inhibiting the acetylation of histone H4-K16. Taken together, these studies help to shed light on mechanisms that regulate chromatin structure through a series of concerted enzymatic reactions that ultimately 'mark' functionally distinct chromatin domains (Nishioka, 2002b).

To identify and analyze HMTs present in human cells that specifically methylate histone H4, nuclear extracts from HeLa cells were fractionated on several chromatographic resins. Fractions from the columns were assayed for HMT activity using as substrates either core histone polypeptide or mono- and oligo-nucleosomes, in the presence and absence of histone H1. The separation of proteins in the DEAE-cellulose flowthrough (unbound) fraction on a negatively charged column (phosphocellulose) resulted in the resolution of two HMT activities, each with a different substrate and histone specificity. The histone H3-specific activity was eluted from the column at a lower salt concentration and was able to methylate core histone polypeptides as well as oligonucleosomes. This activity was specific for the K9 residue of H3 and was identified as Suv39h1. The other major HMT activity was eluted from the phosphocellulose column at a higher salt concentration and exclusively methylated nucleosomal histone H4. Further separation of the H4-specific HMT on a gel filtration column demonstrated that the activity had an apparent native mass of approximately 70 kDa. The final step of the purification scheme, fractionation on a Heparin agarose column, showed that the H4 HMT activity correlated with the appearance of a single polypeptide of approximately 40 kDa. It was later found by gel-filtration analysis that the enzymatically active 40 kDa protein resides as a homodimeric complex (Nishioka, 2002b).

The purified native enzyme was subjected to further analysis, in order to more clearly define its substrate specificity. Assays were conducted with known substrates for several previously characterized protein methyltransferases, and it was found that the newly purified enzyme was highly specific for nucleosomal histone H4. A reaction mixture that contained nucleosomal histone H4, ³H-labeled S-adenosyl methionine (SAM), and the purified enzyme was then subjected to Edman degradation, and this analysis demonstrated that the target site for methylation is lysine 20. Moreover, when an HMT assay was carried out using nucleosomes reconstituted with an H4 species that contained an alanine in place of a lysine at position 20 (K20A), the newly purified HMT was unable to methylate the substrate, demonstrating further that this enzyme is specific for H4-K20 (Nishioka, 2002b).

Mass spectrometric analysis of peptides derived from the protein that coeluted with the nucleosomal H4-specific HMT activity allowed probes to be generated with which to isolate a full-length cDNA clone. cDNA sequence analysis revealed that the activity was encoded by a gene that is absent in lower eukaryotes but is present in worms, flies, and vertebrates. The cDNA sequence matched perfectly with a sequence deposited in GenBank referred to as PR/SET domain containing protein 07 (accession number AAL40879). For simplicity, the enzyme was termed PR-Set7 (Nishioka, 2002b).

Because a PR-Set7 homolog is present in Drosophila as a gene product of CG3307 (see Figure S1A), and because methylation of H4-K20 can be detected in the fruit fly, Drosophila was chosen as a model system to analyze the biological significance of this modification. The catalytic SET domain of Drosophila pr-set7 is about 40% identical in amino acid composition to that of human PR-Set7 (Nishioka, 2002b).

Drosophila and mammalian PR-Set7 specifically methylate lysine 20 of histone H4 exclusively within a nucleosomal context. Although histone proteins have long been recognized to be methylated at specific residues in vivo, the enzymes that catalyze the modification reaction and the functions of these modifications have only recently begun to be revealed (Zhang, 2001). Prior to this study, the function(s) of lysine methylated histone H4 was obscure, but was largely believed to be associated with transcriptionally active rather than repressed genes. However, this study has established that methylated H4-K20 is associated with silent chromatin. In support of the 'histone code hypothesis' (Strahl, 2000) methylation at H4-K20 inhibits acetylation of H4-K16 and vice versa. Consistent with the notion that an enzyme that alters the establishment of silent chromatin should have a tremendous impact on gene expression, these studies establish that the absence of methyl H4-K20 in vivo impairs the development and viability of a multicellular organism. Based upon the available evidence, the view is favored that a lack of, or diminishment of, H4-K20 methylation may alter patterns of gene expression, by perturbing a generally repressive, higher order chromatin structure that critically depends upon H4-K20 methylation (Nishioka, 2002b).

The enzymatic activity of PR-Set7 is contained within a single polypeptide of ~40 kDa that appears to exist as a homodimer, because the native protein elutes from a gel filtration column with an apparent mass of ~70 kDa. The results demonstrate that PR-Set7 is an H4-K20-specific HMT, since the enzyme does not methylate any other residue on histone H4 or on any other histone. In agreement with previous studies demonstrating that the SET domain can be a signature for lysine-HMTs, PR-Set7 contains a SET domain, and a single substitution of a conserved arginine to glycine within the SET domain abolishes its enzymatic activity. Interestingly, PR-Set7 is devoid of the Pre- and Post-SET domains, demonstrating that these domains, although important for the functions of other HMTs, are not absolutely required for HMT activity. PR-Set7 is highly specific for nucleosomes, since no activity could be demonstrated when histones were used as a substrate. This lack of activity on nonnucleosomal histones is not likely to be due to the absence of the Pre- and Post-SET domains, because an HMT has been isolated with specificity for H3-K4 that exclusively methylates free histones and lacks both of these domains (Nishioka, 2002b).

Enzymes that affect the formation of silent chromatin are expected to be present in all organisms capable of compacting their DNA. However, these studies indicate that both the homolog of PR-Set7 and methyl H4-K20 are absent from the yeast S. cerevisiae. An explanation for this apparently disparate finding may reside in the way that nucleosomes are packaged in yeast. Recent crystallographic studies have established that internucleosomal interactions are different in higher eukaryotes (frog) and yeast. In nucleosomes of higher eukaryotes, a patch of basic residues, amino acids 16-25, within the histone H4 tail has been reported to interact with a patch of acidic residues present on the surface of the H2A-H2B dimer in the neighboring nucleosome. Therefore, enzymes that can modify residues within these regions, including acetylation of H4-K16 and methylation of H4-K20, may likely alter or control higher order chromatin structure. Importantly, structural studies of yeast nucleosome revealed that the histone H4 tail does not interact with the patch of acidic residues in the neighboring nucleosome (White, 2001). These observations, combined with the fact that yeast exhibits more relaxed and less condensed chromatin, provide a plausible explanation to the finding that H4-K20 methylation is not detectable in yeast (Nishioka, 2002b).

These data show that methylated H4-K20 and acetylated H4-K16 are mutually restrictive modifications and that methyl H4-K20 is localized in transcriptionally silent chromatin. According to previous observations by chromatin immunoprecipitation analysis, acetylated H4-K16 is enriched in transcriptionally active chromatin (Johnson, 1998). The ATPase activity of Drosophila ISWI, a protein present in complexes, has been suggested to be involved in maintenance higher order chromatin structure. Taken together with the results obtained from structural studies of nucleosomes, these observations strongly suggest that methylated H4-K20 not only renders chromatin 'tightly closed' by inhibiting the acetylation of histone H4-K16 but may provide an appropriate substrate for ISWI to generate regularly spaced higher order chromatin structure. Moreover, and related to these observations, previous studies have established that the localization of ISWI on polytene chromosomes is predominantly associated with regions that are not highly transcribed and thus would be expected to overlap the pattern of methylated H4-K20. Although specific binding of human ISWI to methyl H4-K20-containing peptides or alterations of the ISWI ATPase activity by methylated H4-K20 have not been detected, it is tempting to speculate that ISWI is recruited to chromatin containing methylated H4-K20 (Nishioka, 2002b).

It is known that histone H4 is temporally hyperacetylated during (or shortly after) DNA replication and is deacetylated after nucleosome deposition, although some parts of the chromosomes remain hyperacetylated during mitosis. A hypothesis has been put forth that acetylation of histone H4 could provide a mechanism for propagating the appropriate chromatin structure, presumably transcription-competent chromatin, to newly synthesized DNA (Jeppesen, 1997). However, a counterregulatory machinery that maintains the integrity of transcriptionally silent chromatin has not been discovered. Particularly, none of the histone deacetylase activity targeting 'nucleosomal H4-K16' has been identified. The data presented here indicate that methylated H4-K20 and acetylated H4-K16 are mutually restrictive modifications, and it is suggested that these modifications are coordinately regulated during the cell cycle. In other words, it is speculated that during mitosis, PR-Set7 marks H4-K20 residues with methyl groups according to the map of nonacetylated H4-K16 on mitotic chromosomes, and then the methyl H4-K20 can counteract acetylation of H4-K16 during the next cell cycle. Thus, methylated H4-K20 could represent an epigenetic mark of silent chromatin that is propagated during cell division (Nishioka, 2002b).

GENE STRUCTURE

cDNA clone length - 3323

Bases in 5' UTR - 289

Exons - 3

Bases in 3' UTR - 958

PROTEIN STRUCTURE

Amino Acids - 691

Structural Domains

Sequence analysis of Human PR-Set7 has shown that it contains an evolutionarily conserved SET domain, a motif that is shared by all other known lysine-directed HMTs. In contrast to other HMTs, except for Set9, PR-Set7 is devoid of the Pre- and Post-SET domains, which were thought to be required for enzymatic activity (Nishioka, 2002b).

date revised: 11 June 2022

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