InteractiveFly: GeneBrief

grappa: Biological Overview | References

Gene name - grappa

Synonyms - Dot1

Cytological map position - 83E6-83E7

Function - enzyme

Keywords - chromatin, modification of histone H3, wingless pathway, telomeric silencing

Symbol - gpp

FlyBase ID: FBgn0264495

Genetic map position - 3R: 2,232,598..2,271,735 [+]

Classification - histone methyltransferase Dot1

Cellular location - nuclear

NCBI link: EntrezGene

gpp orthologs: Biolitmine
Recent literature
Nil, Z., Deshwar, A. R., Huang, Y., ..., Mao, X., Wegner, D. J., Sisco, K., Shinawi, M., Wangler, M. F., Weksberg, R., Yamamoto, S., Costain, G., Bellen, H. J. (2023). Rare de novo gain-of-function missense variants in DOT1L are associated with developmental delay and congenital anomalies. American journal of human genetics, 110(11):1919-1937 PubMed ID: 37827158
Misregulation of histone lysine methylation is associated with several human cancers and with human developmental disorders. DOT1L is an evolutionarily conserved gene encoding a lysine methyltransferase (KMT) that methylates histone 3 lysine-79 (H3K79) and was not previously associated with a Mendelian disease in OMIM. This study has identified nine unrelated individuals with seven different de novo heterozygous missense variants in DOT1L through the Undiagnosed Disease Network (UDN), the SickKids Complex Care genomics project, and GeneMatcher. All probands had some degree of global developmental delay/intellectual disability, and most had one or more major congenital anomalies. To assess the pathogenicity of the DOT1L variants, functional studies were performed in Drosophila and human cells. The fruit fly DOT1L ortholog, grappa, is expressed in most cells including neurons in the central nervous system. The identified DOT1L variants behave as gain-of-function alleles in flies and lead to increased H3K79 methylation levels in flies and human cells. These results show that human DOT1L and fly grappa are required for proper development and that de novo heterozygous variants in DOT1L are associated with a Mendelian disease.

A novel gene, grappa (gpp) is the Drosophila ortholog of the Saccharomyces cerevisiae gene Dot1, a histone methyltransferase that modifies the lysine (K)79 residue of histone H3. gpp is an essential gene identified in a genetic screen for dominant suppressors of pairing-dependent silencing, a Polycomb-group (Pc-G)-mediated silencing mechanism necessary for the maintenance phase of Bithorax complex (BX-C) expression. Surprisingly, gpp mutants not only exhibit Pc-G phenotypes, but also display phenotypes characteristic of trithorax-group mutants. Mutations in gpp also disrupt telomeric silencing but do not affect centric heterochromatin. These apparent contradictory phenotypes may result from loss of gpp activity in mutants at sites of both active and inactive chromatin domains. Unlike the early histone H3 K4 and K9 methylation patterns, the appearance of methylated K79 during embryogenesis coincides with the maintenance phase of BX-C expression, suggesting that there is a unique role for this chromatin modification in development (Shanower, 2005).

Recent studies on telomeric silencing in S. cerevisiae have led to the identification of a histone methlylase, DOT1, which has a number of unusual properties (Singer, 1998; Feng, 2002; Lacoste, 2002; van Leeuwen, 2002). First, unlike the previously identified histone methylases, DOT1 does not have a canonical SET domain. Instead, the DOT1 protein resembles a family of S-adenosyl methione methyltransferases that modify arginine residues. DOT1 methylates histone H3 at lysine 79 only when it is assembled into nucleosomes and methylation strongly depends upon prior Rad6 dependent ubiquitination of histone H2B at K123 (Briggs, 2002). Second, in yeast, deletion or overexpression of Dot1 disrupts TPE and also silencing of the mating-type loci (Singer, 1998). In contrast, silencing in the yeast ribosomal gene cluster is disrupted only when DOT1 is overexpressed (Singer, 1998). Third, both telomeric and mating-type silencing are disrupted by mutations in the lysine 79 residue of histone H3. Fourth, methylation of K79 appears to influence the recruitment of the SIR silencing proteins to the telomeres (van Leeuwen, 2002; Ng, 2003a). The SIR silencing proteins appear to preferentially associate with chromatin that is deficient in K79 methylation, while the proteins are generally not associated with chromatin in which there is an enrichment for K79 methylated H3 (van Leeuwen, 2002; Ng, 2003a). Fifth, there is evidence that K79 methylation is coordinated with polymerase transcription via the COMPASS complex (Krogan, 2003). Consistent with the idea that K79 methylation might be coordinated with transcription, H3meK79 is enriched in transcribed sequences in yeast and mammals (Im, 2003; Ng, 2003b). Interestingly, the distribution of H3meK79 in the β-globin locus differs from H3meK4 in that it is not found at the locus control region (Im, 2003). These findings have led to a model in which H3meK79 serves as a marker for transcribed sequences where it functions to block the association of chromatin proteins that mediate transcriptional silencing (Shanower, 2005).

While Dot1 homologs have been identified in higher eukaryotes, little is known about their biological functions (Feng, 2002). This report characterized the Drosophila Dot1 ortholog gpp. The gpp transcription unit is >40 kb in length and it encodes a complex array of alternatively spliced transcripts that range in size from 6.5 to >9 kb and are expressed at different developmental stages. Consistent with the assignment of the gpp gene, P-element and X-ray mutations disrupt this large transcription unit and in at least one case lead to the production of truncated mRNAs. The gpp transcripts are predicted to encode 170- to 232-kD polypeptides that share a common N-terminal domain that corresponds to about two-thirds of the protein but have different C-terminal domains. The common N-terminal domain contains the Dot1 homology region including the MT methyltransferase fold required for methylation of histone H3 (Feng, 2002). Mutation of conserved glycine residues in the active site of both yeast and human DOT1 protein inactivates the enzyme (Feng, 2002; van Leeuwen, 2002). GPP also contains domains that are not present in DOT1 including a coiled-coil motif also found in the human, C. elegans, D. pseudoobscura, and A. gambia DOT1-like proteins. In yeast, K79 is mono-, di-, and trimethylated and Dot1 is responsible for all three modifications (Feng, 2002; van Leeuwen, 2002). The different methylated states of H3 at K79 suggest that multiple regulatory activities are conferred on these modified nucleosomes (Ng, 2002a; van Leeuwen, 2002). However, in fly tissue culture cells, the mono- and di- but not the trimethylated form is observed (Mckittrick, 2004). Since database searches indicate that gpp is the only fly Dot1 homolog, it should also be the sole fly protein in this class that methylates histone H3 on K79. Consistent with this suggestion, discs and other tissues isolated from gpp mutant larvae have little if any H3 mono- or dimethyl K79 (Shanower, 2005).

Like its yeast counterpart, gpp is required for the silencing of reporter transgenes inserted into telomeric heterochromatin. However suppression of silencing associated with pericentric heterochromatin is unaffected by mutations in gpp. While these observations point to a role of gpp in silencing specific for telomeric heterochromatin, antibody staining experiments indicate that there is a paucity of H3dmeK79 at telomeres in polytene chromosomes compared to many other chromosomal DNA segments. In this respect it is interesting that both telomeric and mating-type chromatin in yeast are hypomethylated on K79 compared to 'bulk' chromatin even though DOT1 is required for SIR silencing in each case (Ng, 2003a). It has been suggested that the meK79 modification in euchromatic nucleosomes blocks SIR protein association and that silencing is lost in the absence of DOT1 because the SIR proteins spread into euchromatin (van Leeuwen, 2002). In contrast, in flies, since many euchromatic domains in wild-type polytene chromosomes have only little H3meK79, it is difficult to see how telomeric silencing proteins would be restricted to telomeres by this modification even when gpp is fully active (Shanower, 2005).

gpp also has functions in flies besides telomeric silencing. Unlike Dot1, gpp is essential for viability. Although the underlying cause of lethality remains to be established, gpp mutant larvae grow more slowly than wild type and this potentially implicates gpp in pathways that control growth rates and size in flies. In addition, gpp mutants display defects that are characteristic of both Pc-G and trx-G genes. The first gpp alleles were recovered as dominant suppressors of mini-white silencing by two BX-C PREs. Consistent with a role in Pc-G silencing, gpp mutants enhance the segmentation defects of several Pc-G genes. In this context, it is interesting to note that several Pc-G genes have recently been shown to play a role not only in the repression of genes in the homeotic complexes but also in telomeric silencing. Thus, it is possible that gpp activity in telomeric silencing may be linked in some manner to its role in Pc-G silencing (Shanower, 2005).

gpp mutants also exhibit transformations in segment identity and genetic interactions with Abd-B that are characteristic of trx-G mutations. This would point to a role in promoting rather than repressing gene expression. Some function in transcription would be consistent with studies in other systems as well as with the enrichment of meK79 seen in many polytene interbands and puffs. However, this correlation is not complete. Thus, there are many puffs and interbands that have only little H3dmeK79. Conversely, H3dmeK79 is sometimes enriched in bands. These findings would argue that in Drosophila, meK79 is not a ubiquitous marker for transcriptionally active chromatin, but rather may have functions that are specific to particular chromatin domains. In this case, the disruptions in homeotic gene expression seen in gpp mutants could reflect a special requirement for H3meK79 in the transcription of these particular genes. Domain-specific requirements for gpp activity in transcription could also potentially account for the effects of gpp mutations on Pc-G and telomeric silencing. In this model, Pc-G and telomeric silencing would be disrupted in gpp mutants because the expression of one or more Pc-G (and/or telomeric heterochromatin) genes is downregulated when gpp activity is compromised (Shanower, 2005).

The developmental profile of H3dmeK79 also suggests that this modification cannot be a ubiquitous marker for either transcriptionally active or silenced chromatin. High levels of Pol II transcription in somatic nuclei begin in the precellular blastoderm stage around nuclear cycle 11/12. Concomitant with the activation of transcription, H3meK4 can be first be detected at this stage, and the level of meK4 then increases through cellularization. By contrast, little if any H3 mono- or dimethyl K79 is in either the transcriptionally active somatic nuclei or the transcriptionally quiescent pole cell nuclei. H3meK79 can first be readily detected only later in development in germband extended embryos. However, at this stage accumulation is restricted primarily to a subset of cells in the embryo, most of which seem to be in the process of cell division. High levels of H3meK79 are not observed until stages 13-15, long after the initial upregulation of transcription in the early zygote. This result also suggests that the homeotic transformations seen in gpp mutants are unlikely to be due to defects in the initial establishment of parasegment-specific patterns of homeotic gene expression by the gap and pair-rule genes. Rather, these transformations probably reflect a requirement for gpp activity later in development during the maintenance phase of homeotic gene regulation -- a phase that is dependent upon Pc-G and trx-G genes. In this respect it is curious that homeotic transformations are not observed in gpp embryos when they hatch as first instar larvae. Maternally derived gpp activity in homozygous mutant embryos maybe sufficient to maintain specific parasegmental patterns of homeotic gene expression through the end of embryogenesis. Alternatively, there may not be absolute requirement for H3meK79 in maintaining appropriate parasegmental patterns of homeotic expression during embryogenesis (Shanower, 2005).

The developmental profile of H3meK79 indicates that this modification is present at low levels in specific developmental stages and tissues (CNS) undergoing active cell division. In contrast, the highest levels of H3meK79 are observed in epidermal cells that have exited the cell cycle and are undergoing differentiation. Thus, it seems possible that this modification may be activated when specific chromatin configurations, active or inactive, need to be maintained for extended periods of time in the absence of de novo DNA synthesis/chromatin assembly. In this respect it is interesting that Mckittrick (2004) has reported that the highest levels of meK79 are found in a histone H3 variant, H3.3, which is assembled into chromatin by a replication-independent mechanism. Further studies of gpp in Drosophila will be required to understand the mechanisms governing the temporal and tissue-specific regulation of the K79 modification and how this relates to the functions of this particular histone modification during development. Understanding this aspect of the histone code in a multicellular organism such as Drosophila will lead to a better understanding of chromatin regulatory mechanisms during development (Shanower, 2005).

Linking H3K79 trimethylation to Wnt signaling through a novel Dot1-containing complex

Epigenetic modifications of chromatin play an important role in the regulation of gene expression. KMT4/Dot1 is a conserved histone methyltransferase capable of methylating chromatin on Lys79 of histone H3 (H3K79). This study reports the identification of a multisubunit Dot1 complex (DotCom), which includes several of the mixed lineage leukemia (MLL) partners in leukemia such as ENL, AF9/MLLT3, AF17/MLLT6, and AF10/MLLT10, as well as the known Wnt pathway modifiers TRRAP, Skp1, and β-catenin. The human DotCom is indeed capable of trimethylating H3K79 and, given the association of β-catenin, Skp1, and TRRAP, a role was sought for Dot1 in Wnt/Wingless signaling in an in vivo model system. Knockdown of Dot1 in Drosophila (Grappa) results in decreased expression of a subset of Wingless target genes. Furthermore, the loss of expression for the Drosophila homologs of the Dot1-associated proteins involved in the regulation of H3K79 shows a similar reduction in expression of these Wingless targets. From yeast to human, specific trimethylation of H3K79 by Dot1 requires the monoubiquitination of histone H2B by the Rad6/Bre1 complex. This study demonstrates that depletion of Bre1, the E3 ligase required for H2B monoubiquitination, leads specifically to reduced bulk H3K79 trimethylation levels and a reduction in expression of many Wingless targets. Overall, this study describes for the first time the components of DotCom and links the specific regulation of H3K79 trimethylation by Dot1 and its associated factors to the Wnt/Wingless signaling pathway (Mohan, 2010).

In eukaryotic organisms, gene expression patterns are spatiotemporally regulated in a manner that allows for specification of diverse cell types and their differentiation. This spatiotemporal expression is coordinated in part by transcription factors and chromatin modifiers, and by the activity of several signaling pathways, which contribute to gene expression by regulating the transcription factors. Understanding the relationship between chromatin events and signaling pathways is crucial to understanding gene regulation, development of the organism, and disease pathogenesis (Mohan, 2010).

The nucleosome, the basic unit of chromatin, consists of histones H2A, H2B, H3, and H4, and 146 base pairs (bp) of DNA. Crystal structure studies have demonstrated that the N-terminal tails of each histone protrude outward from the core of the nucleosome. These histone tails are subject to various post-translational modifications, including methylation, ubiquitination, ADP ribosylation, acetylation, phosphorylation, and sumoylation, and such modifications are involved in many biological processes involving chromatin such as transcription, genome stability, replication, and repair (Mohan, 2010).

Histones are methylated on either the lysine and/or arginine residues by different histone methyltransferases (HMTases). Histone lysine methylation can occur as mono-, di-, or trimethylated forms, and several lysine residues of histones have been shown to be multiply methylated. This includes methylation on Lys4, Lys9, Lys27, Lys36, and Lys79 of histone H3, and Lys20 of histone H4. Almost all of the lysine HMTases characterized to date contain a SET domain, named after Drosophila Su(var)3-9, Enhancer of zeste [E(z)], and trithorax (trx). SET domain-containing enzymes can catalyze the methylation of specific lysines on histones H3 and H4, and many SET domain-containing enzymes, such as Trithorax and Enhancer of zeste, are central players in epigenetic regulation and development (Mohan, 2010).

Histone H3 at Lys79 (H3K79) can be mono-, di-, and trimethylated by Dot1, which to date is the only characterized non-SET domain-containing lysine HMTase. Dot1 is conserved from yeast to humans (Feng, 2002; Lacoste, 2002 Ng, 2002a; van Leeuwen, 2002; Shilatifard 2006). In yeast, telomeric silencing is lost when Dot1 is overexpressed or inactivated, as well as when H3K79 is mutated. Unlike other histone methylation patterns, the pattern of di- and trimethylation of H3K79 in yeast appears to be nonoverlapping (Schulze, 2009). It was also first discovered in yeast that monoubiquitination of histone H2B on Lys123 (H2BK123) by the Rad6/Bre1 complex is required for proper H3K79 trimethylation by Dot1 (Ng, 2002b; Wood, 2003; Shilatifard, 2006; Schulze, 2009). In vivo analysis of the pattern of H2B monoubiquitination in yeast demonstrated that the H3K79 trimethylation pattern overlaps with that of H2B monoubiquitination, and that the H3K79 dimethylation pattern and H2B monoubiquitination appear to be nonoverlapping (Schulze, 2009). This observation resulted in the proposal that the recruitment of the Rad6/Bre1 complex and the subsequent H2B monoubiquitination could dictate diversity between H3K79 di- and trimethylation on chromatin on certain loci within the genome. In addition to a role in the regulation of telomeric silencing in yeast, Dot1 has also been shown to be involved in meiotic checkpoint control (San-Segundo, 2000) and in double-strand break repair via sister chromatid recombination (Conde, 2009). A relationship has been found between cell cycle progression and H3K79 dimethylation, but not trimethylation (Schulze, 2009), by Dot1. Consequently, to date, very little is known about a specific biological role of histone H3K79 trimethylation (Mohan, 2010).

In Drosophila, H3K79 methylation levels correlate with gene activity (Schubeler, 2004). Mutations in grappa, the Dot1 ortholog in Drosophila, show not only the loss of silencing, but also Polycomb and Trithorax-group phenotypes, indicating a key role for H3K79 methylation in the regulation of gene activity during development (Shanower, 2005). Similarly, Dot1 in mammals has been implicated in the embryonic development of mice, including a role in the structural integrity of heterochromatin (Jones, 2008). Genome-wide profiling studies in various mammalian cell lines have suggested that Dot1 as well as H3K79me2 and H3K79me3 localize to the promoter-proximal regions of actively transcribed genes, and correlate well with high levels of gene transcription (Steger, 2008). It has also been proposed that Dot1 HMTase activity is required for leukemia pathogenesis (Mohan, 2010 and references therein).

The highly conserved Wnt/Wingless (Wnt/Wg) signaling pathway is essential for regulating developmental processes, including cell proliferation, organogenesis, and body axis formation. Deregulation or ectopic expression of members of the Wnt pathway has been associated with the development of various types of cancers, including acute myeloid and B-cell leukemias. In the canonical Wnt/Wg pathway, a cytoplasmic multiprotein scaffold consisting of Glycogen synthase kinase 3-β (GSK3-β), Adenomatous polyposis coli (APC), Casein kinase 1 (CK1), Protein phosphatase 2A, and Axin constitutively marks newly synthesized β-catenin/Armadillo for degradation by phosphorylation at the key N-terminal Ser and Thr residues. Binding of the Wnt ligands to the seven-transmembrane domain receptor Frizzled (Fz) leads to recruitment of an adaptor protein, Disheveled (Dvl), from the cytoplasm to the plasma membrane. Axin is then sequestered away from the multiprotein Axin complex, resulting in inhibition of GSK3-β and subsequent stabilization of hypophosphorylated β-catenin levels in the cytoplasm. Stabilized β-catenin translocates into the nucleus and binds to members of the DNA-binding T-cell factor/lymphoid enhancer factor (TCF/LEF) family, resulting in the recruitment of several chromatin-modifying complexes, including transformation/transcription domain-associated protein (TRRAP)/HIV Tat-interacting 60-kDa protein complex (TIP60) histone acetyltransferase (HAT), ISWI-containing complexes, and the SET1-type HMTase mixed lineage leukemia 1/2 (MLL1/MLL2) complexes (Sierra, 2006), thereby activating the expression of Wnt/Wg target genes (Mohan, 2010).

Although much is known about Dot1 as an H3K79 HMTase, biochemical studies isolating to homogeneity a Dot1-containing complex have not been successful during the past decade. This study reports the first biochemical isolation of a multisubunit complex associated with Dot1, which has been called DotCom. DotCom is comprised of Dot1, AF10, AF17, AF9, ENL, Skp1, TRRAP, and β-catenin. This complex is enzymatically active and can catalyze H3K79 dimethylation and trimethylation. Indeed, nucleosomes containing monoubiquitinated H2B are a better substrate for DotCom in the generation of trimethylated H3K79. Given the association of Skp1, TRRAP, and β-catenin with DotCom, and the fact that these factors have been linked to the Wnt signaling pathway in previous studies, this study investigated the role of the Drosophila homolog of Dot1, dDot1 (Grappa), for the regulation of Wg target genes. RNAi of dDot1 leads to a reduced expression of a subset of Wg target genes, including senseless, a high-threshold Wingless target gene. Furthermore, reduction by RNAi in the levels of the Drosophila homologs of other components of DotCom that regulate the pattern of H3K79 methylation in humans also showed a similar reduction in senseless expression and other Wg target genes. Importantly, DotCom requires monoubiquitination of H2B for H3K79 trimethylation, and, in Drosophila, the loss of Bre1, the E3 ubiquitin ligase, leads to reduction of H3K79 trimethylation and decreased expression of the senseless gene. Taken together, these data support a model in which monoubiquitinated H2B provides a regulatory platform for a novel Dot1 complex to mediate H3K79 trimethylation, which is required for the proper transcriptional control of Wnt/Wg target genes (Mohan, 2010).

Although H3K79 methylation is a ubiquitous mark associated with actively transcribed genes, and its presence is a clear indicator for the elongating form of RNA polymerase II (Krogan, 2003; Steger, 2008), Dot1 itself has a very low abundance and is very hard to detect in cells. This indicates that Dot1 is an active enzyme with a very high specific activity toward its substrate, H3K79. Due to the low abundance of Dot1 in cells, its molecular isolation and biochemical purification have been hindered for the past decade. This study reports the biochemical isolation of a Dot1-containing complex (DotCom) and demonstrate a specific link between H3K79 trimethylation by DotCom and the Wnt signaling pathway. The study reports (1) the identification and biochemical isolation of a large macromolecular complex (~2 MDa) containing human Dot1, in association with human AF10, AF17, AF9, ENL, Skp1, TRRAP, and β-catenin; (2) the biochemical demonstration that the human DotCom is capable of trimethylating H3K79, and the analysis of the role of histone H2B monoubiquitination in the enhancement of this H3K79 trimethylase activity of the human DotCom; (3) identification of the role of the components of DotCom in the regulation of its H3K79 methylase activity; (4) demonstration of a role for the Drosophila homolog of Dot1 and its associated factors in the Wnt signaling pathway; and, finally, (5) the identification of a specific requirement of H3K79 trimethylation, but not mono- or dimethylation, in the regulation of Wnt target transcription, thereby linking H3K79 trimethylation to Wnt signaling (Mohan, 2010).

Dot1 was initially isolated from yeast, and these studies demonstrated that the enzyme is capable of mono-, di-, and trimethylating H3K79. Subsequent molecular and biochemical studies demonstrated that prior H2B monoubiquitination by the Rad6/Bre1 complex is required for proper H3K79 trimethylation by yeast Dot1 (Briggs, 2002; Ng, 2002b; Wood, 2003; Schulze, 2009). A recent analysis of the human homolog of Dot1 suggested that its HMTase domain is not capable of trimethylating H3K79, and that this enzyme can only dimethylate its substrate (McGinty, 2008). McGinty, (2008) also demonstrated that reconstitution of monoubiquitinated H2B into chemically defined nucleosomes, followed by enzymatic treatment with Dot1, resulted only in dimethylation of H3K79. Since these observations are in contrast with the published studies in yeast, this study tested the enzymatic activity of purified human DotCom toward monoubiquitinated and nonmonoubiquitinated nucleosomes. The studies demonstrate that the human DotCom can indeed trimethylate H3K79, and that monoubiquitination of histone H2B enhances this enzymatic property of the human DotCom. Since the enzymatic studies employ antibodies generated toward mono-, di-, and trimethylated H3K79 to identify the products of the enzymatic reactions containing human Dot1, it was important to make certain that the observations are not the result of cross-reactivity between these antibodies. Therefore recombinant nucleosomes were generated and treated with human Dot1 in the presence and absence of SAM, and the products were analyzed by MS. The chemical analysis of the products from this enzymatic reaction confirmed that human Dot1 is capable of trimethylating H3K79. The hDot1-treated nucleosome samples were digested with Endoproteinase Arg-C because previous unpublished work on analyzing yeast histone modifications by MudPIT had shown that the trimethylated peptide containing H3K79 was not detected when digesting with trypsin. Notably, McGinty (2008) performed their digestions with trypsin, which might explain their failure to detect this modification by MS (Mohan, 2010).

These studies identified several factors—including ENL, AF9, AF17, AF10, SKP1, TRA1/TRAPP, and β-catenin—as components of the human DotCom. To test the role of these factors in regulating Dot1’s catalytic activity, their levels were reduced via RNAi. These studies demonstrated that AF10 functions with Dot1 to regulate its catalytic properties in vivo. Significant differences in Dot1’s H3K79 HMTase activity were not detected in vivo when reducing the levels of ENL, AF9, and AF17. Factors that significantly alter the H3K79 methylation pattern by Dot1 are also linked to its transcriptional regulatory functions at Wnt target genes (Mohan, 2010).

Since Dot1 also appears to interact with β-catenin, and given the known role for β-catenin, Skp1, and TRRAP in the Wnt signaling pathway, the role for Dot1 and the components of its complex were tested in Wnt signaling. Drosophila is an outstanding model system for the study of the Wnt signaling pathway. Given the power of genetics and biochemistry in Drosophila, the role of dDot1 and the members of its complex in wingless signaling were tested. From this study, it was learned that down-regulation of Drosophila Dot1 and Drosophila AF10 had the most significant effects in the regulation in the expression of the Wg target senseless. Given the fact that the molecular studies demonstrated that Dot1 and AF10 have the strongest effect in the regulation of H3K79 methylation in vivowe wanted to determine whether a specific form of H3K79 methylation is required for Wnt target gene expression was tested (Mohan, 2010).

Histone H2B monoubiquitination is required for proper H3K79 trimethylation (Shilatifard 2006; Schulze, 2009). The E2/E3 complex Rad6/Bre1 is required for the proper implementation of H2B monoubiquitination on chromatin, and this complex is highly conserved from yeast to humans. Deletion of the Drosophila homolog of Bre1 results in the loss of H2B monoubiquitination and the specific loss of H3K79 trimethylation. Interestingly, reduction in the levels of H3K79 trimethylation results in a defect in expression of one of the Wnt target genes, senseless, although the H3K79 mono- and dimethylation in this mutant background appear to be normal. In addition to senseless, the role of H3K79 methylation at other Wnt targets was tested, and the same effect was observed for Notum and CG6234. Overall, these studies demonstrate a link between H3K79 trimethylation by the DotCom and the Wnt signaling pathway (Mohan, 2010).

Wnt/Wg signaling serves a critical role in tissue development, proliferation of progenitor cells, and many human cancers. The key player in the Wnt pathway is β-catenin, which is shuttled into the nucleus at the onset of activation of the pathway. Various proteins that interact with β-catenin in the nucleus—such as CBP/p300, TRRAP, MLL1/MLL2, Brg1, telomerase, Hyrax, Pygopus, and CDK8—modulate the transcriptional output of Wg/Wnt target genes (Willert, 2006; Carrera, 2008; Firestein, 2008; Park, 2009). These proteins probably provide the context specificity to Wnt response directing proliferation or differentiation effects of Wnt signaling. The finding that dDotCom is required for expression of a subset of Wg targets suggests that dDotCom might also facilitate Wg-regulated programs of transcriptional regulation in specific contexts. As most human cancers have elevated levels of Wnt signaling and require Wnt signaling for continued proliferation, DotCom might play a role in supporting the high rate of expression of Wnt target genes in such cancers (Mohan, 2010).

Several studies have found interactions between Dot1 and many translocation partners of MLL. While these associations suggest a link between Dot1 methylation and leukemogenesis, it was not clear how Dot1 methylation would participate in this process. Recently, GSK3, a regulator of β-catenin and Wnt signaling, was found to be essential for proliferation of MLL-transformed cells and for progression of a mouse model of MLL-based leukemia (Wang, 2008). These studies linking Dot1 H3K79me3 with Wnt signaling provide insight into the role of Wnt signaling and Dot1 methylation in MLL translocation-based leukemia (Mohan, 2010).

Overexpression of grappa encoding a histone methyltransferase enhances stress resistance in Drosophila

Histone deacetylases, such as silent information regulator 2 (Sir2) and Rpd3 are involved in chromatin silencing and implicated in lifespan determination in several organisms. The yeast Dot1 gene encoding a histone methyltransferase affects localization of silencing proteins including Sir2, and plays an essential role in the repair of damaged DNA. However, it is not known whether an alteration of a histone methyltransferase activity influences lifespan or stress resistance, which is often associated with extended lifespan. This study investigated whether the Drosophila grappa (gpp) gene, a Dot1 homolog influences lifespan and stress resistance using transgenic flies overexpressing gpp and those bearing a partial loss-of-function mutation. Overexpression of gpp throughout the adult stage did not extend the lifespan, but significantly enhanced resistances when they were kept on medium containing 1% H2O2, or those with poor nutrients. As well, gpp-overexpressing flies were behaviourally more active than control flies. Whether gpp overexpression induced anti-oxidant genes, Catalase, Sod, Sod2, GstD2, dhd, TrxT and Trx-2, was investigated. However, none of these genes was induced. A partial loss-of-function mutations in gpp dramatically reduced the lifespan under oxidative and caloric stresses. Taken together, these results demonstrated that gpp is required for normal lifespan and stress resistance, and that its overexpression increases stress resistance in Drosophila, without obvious induction of representative anti-oxidant genes (List, 2009).

The Mcp element mediates stable long-range chromosome-chromosome interactions in Drosophila

Chromosome organization inside the nucleus is not random but rather is determined by a variety of factors, including interactions between chromosomes and nuclear components such as the nuclear envelope or nuclear matrix. Such interactions may be critical for proper nuclear organization, chromosome partitioning during cell division, and gene regulation. An important, but poorly documented subset, includes interactions between specific chromosomal regions. Interactions of this type are thought to be involved in long-range promoter regulation by distant enhancers or locus control regions and may underlie phenomena such as transvection. This study used an in vivo microscopy assay based on Lac Repressor/operator recognition to show that Mcp, a polycomb response element from the Drosophila bithorax complex, is able to mediate physical interaction between remote chromosomal regions. These interactions are tissue specific, can take place between multiple Mcp elements, and seem to be stable once established. It is speculated that this ability to interact may be part of the mechanism through which Mcp mediates its regulatory function in the bithorax complex (Vazquez, 2006).

The isolation of mutants that suppress Mcp-dependent silencing of mini-white could potentially uncover chromosomal proteins that play a role in chromosome-chromosome interaction. One such mutation, grappa (gpp), has been described in detail previously (Shanower, 2005). gpp encodes the Drosophila homologue of the yeast Dot1p, a Histone H3 methyltransferase that modulates chromatin structure and gene silencing in yeast. In Drosophila, the dominant grappa allele gpp1A is homozygous viable. When tested on various double recombinant P[Mcp, mini-white] chromosomes, long-distance Mcp-mediated mini-white silencing is often (but not always) suppressed in heterozygous gpp1A flies. If occurring, suppression is always enhanced in a homozygous gpp1A background. Therefore, the colocalization of inserts OM4 and OM7 were examined in gpp1A/gpp1A flies. Mcp elements were paired in >90% of the nuclei, a frequency similar to that observed in flies wild-type for grappa. Although limited, these results suggest that gpp1A does not prevent the establishment or maintenance of chromosome-chromosome interactions (Vazquez, 2006).

This suggests that pairing may be an initial necessary step in the regulatory process mediated by Mcp and that grappa acts subsequently to induce chromatin changes required for silencing. In the absence of additional data, however, other possibilities cannot be excluded. For example, the timing of pairing could be critical to allow developmentally regulated factors to associate to, and repress transcription around the Mcp element. In such a model, gpp1A could be delaying the onset of pairing, resulting in reduced levels of silencing. Additional studies will be necessary to establish the series of events that lead to pairing-dependent silencing of Mcp-associated genes (Vazquez, 2006).

Methylation of H3-lysine 79 is mediated by a new family of HMTases without a SET domain

The N-terminal tails of core histones are subjected to multiple covalent modifications, including acetylation, methylation, and phosphorylation. Similar to acetylation, histone methylation has emerged as an important player in regulating chromatin dynamics and gene activity. Histone methylation occurs on arginine and lysine residues and is catalyzed by two families of proteins, the protein arginine methyltransferase family and the SET-domain-containing methyltransferase family. This study reports that lysine 79 (K79) of H3, located in the globular domain, can be methylated. K79 methylation occurs in a variety of organisms ranging from yeast, Drosophila, chicken and human. In budding yeast, K79 methylation is mediated by the silencing protein DOT1. Consistent with conservation of K79 methylation, DOT1 homologs can be found in a variety of eukaryotic organisms. A human DOT1-like (DOT1L) protein has been identified, and this protein has been identified to possesses intrinsic H3-K79-specific histone methyltransferase (HMTase) activity in vitro and in vivo. Furthermore, K79 methylation level is regulated throughout the cell cycle. Thus, these studies reveal a new methylation site and define a novel family of histone lysine methyltransferase (Feng, 2002).


Search PubMed for articles about Drosophila Grappa

Briggs, S. D., T. Xiao, Z. W. Sun, J. A. Caldwell, J. Shabanowitz et al., 2002. Gene silencing: trans-histone regulatory pathway in chromatin. Nature 418: 498. PubMed ID: 12152067

Carrera, I., Janody, F., Leeds, N., Duveau, F. and Treisman, J. E. (2008). Pygopus activates Wingless target gene transcription through the mediator complex subunits Med12 and Med13. Proc. Natl. Acad. Sci. 105: 6644-6649. PubMed ID: 18451032

Conde, F., et al. (2009). The Dot1 histone methyltransferase and the Rad9 checkpoint adaptor contribute to cohesin-dependent double-strand break repair by sister chromatid recombination in Saccharomyces cerevisiae. Genetics 182: 437-446. PubMed ID: 19332880

Feng, Q., Wang, H., Ng, H.H., Erdjument-Bromage, H., Tempst, P., Struhl, K. and Zhang, Y. (2002). Methylation of H3-lysine 79 is mediated by a new family of HMTases without a SET domain. Curr. Biol. 12(12): 1052-1058. PubMed ID: 12123582

Firestein, R., et al. (2008). CDK8 is a colorectal cancer oncogene that regulates β-catenin activity. Nature 455: 547-551. PubMed ID: 18794900

Im, H., et al. (2003). Dynamic regulation of histone H3 methylated at lysine 79 within a tissue-specific chromatin domain. J. Biol. Chem. 278: 18346-18352. PubMed ID: 12604594

Jones, B., et al. (2008). The histone H3K79 methyltransferase Dot1L is essential for mammalian development and heterochromatin structure. PLoS Genet. 4: e1000190. PubMed ID: 18787701

Krogan, N. J., et al. (2003). Methylation of histone H3 by Set2 in Saccharomyces cerevisiae is linked to transcriptional elongation by RNA polymerase II. Mol. Cell. Biol. 23: 4207-18. PubMed ID: 12773564

Lacoste, N., et al. (2002). Disruptor of telomeric silencing-1 is a chromatin-specific histone H3 methyltransferase. J. Biol. Chem 277: 30421-30424. PubMed ID: 12097318

McGinty, R. K., et al. (2008). Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation. Nature 453: 812-816. PubMed ID: 18449190

Mckittrick, E., Gafken, P., Ahmad, K. and Henikoff, S. (2004). Histone H3.3 is enriched in covalent modifications associated with active chromatin. Proc. Natl. Acad. Sci. 101: 1525-1530. PubMed ID: 14732680

Mohan, M., et al. (2010). Linking H3K79 trimethylation to Wnt signaling through a novel Dot1-containing complex (DotCom). Genes Dev. 24(6): 574-89. PubMed ID: 20203130

Ng, H. H., et al. (2002a). Lysine methylation within the globular domain of histone H3 by Dot1 is important for telomeric silencing and Sir protein association. Genes Dev. 16: 1518-1527. PubMed ID: 12080090

Ng, H. H., Xu, R. M, Zhang, Y. and Struhl, K. (2002b). Ubiquitination of histone H2B by Rad6 is required for efficient Dot1-mediated methylation of histone H3 lysine 79. J. Biol. Chem. 277: 34655-34657. PubMed ID: 12167634

Ng, H. H., et al. (2003a). Lysine-79 of histone H3 is hypomethylated at silenced loci in yeast and mammalian cells: a potential mechanism for position-effect variegation. Proc. Natl. Acad. Sci. 100: 1820-1825. PubMed ID: 12574507

Ng, H. H., Dole, S. and Struhl, K. (2003b). The Rtf1 component of the Paf1 transcriptional elongation complex is required for ubiquitination of histone H2B. J. Biol. Chem. 278: 33625-33628. PubMed ID: 12876293

List, O., et al. (2009). Overexpression of grappa encoding a histone methyltransferase enhances stress resistance in Drosophila. Hereditas 146(1): 19-28. PubMed ID: 19348653

Park, J. I., et al. (2009). Telomerase modulates Wnt signalling by association with target gene chromatin. Nature 460: 66-72. PubMed ID: 19571879

San-Segundo, P. A. and Roeder, G. S. (2000). Role for the silencing protein Dot1 in meiotic checkpoint control. Mol. Biol. Cell 11: 3601-3615. PubMed ID: 11029058

Schubeler, D., et al. (2004). The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote. Genes Dev. 18: 1263-1271. PubMed ID: 15175259

Schulze, J. M., et al. (2009). Linking cell cycle to histone modifications: SBF and H2B monoubiquitination machinery and cell-cycle regulation of H3K79 dimethylation. Mol. Cell 35: 626-641. PubMed ID: 19682934

Shanower, G. A., Muller, M., Blanton, J. L., Honti, V., Gyurkovics, H. and Schedl, P. (2005). Characterization of the grappa gene, the Drosophila histone H3 lysine 79 methyltransferase. Genetics 169: 173-184. PubMed ID: 15371351

Shilatifard, A. (2006). Chromatin modifications by methylation and ubiquitination: Implications in the regulation of gene expression. Annu. Rev. Biochem 75: 243-269. PubMed ID: 16756492

Sierra, J., Yoshida, T., Joazeiro, C. A. and Jones, K. A. (2006). The APC tumor suppressor counteracts β-catenin activation and H3K4 methylation at Wnt target genes. Genes Dev. 20: 586-600. PubMed ID: 16510874

Singer, M. S., et al. (1998). Identification of high-copy disruptors of telomeric silencing in Saccharomyces cerevisiae. Genetics 150: 613-632. PubMed ID: 9755194

Steger, D. J., et al. (2008). DOT1L/KMT4 recruitment and H3K79 methylation are ubiquitously coupled with gene transcription in mammalian cells. Mol. Cell Biol. 28: 2825-2839. PubMed ID: 18285465

Vazquez, J., Müller, M., Pirrotta, V. and Sedat, J. W. (2006). The Mcp element mediates stable long-range chromosome-chromosome interactions in Drosophila. Mol. Biol. Cell 17(5): 2158-65. PubMed ID: 16495335

van Leeuwen, F., Gafken, P. R. and Gottschling, D. E. (2002). Dot1p modulates silencing in yeast by methylation of the nucleosome core. Cell 109: 745-756. PubMed ID: 12086673

Wang, Z., et al. (2008). Glycogen synthase kinase 3 in MLL leukaemia maintenance and targeted therapy. Nature 455: 1205-1209. PubMed ID: 18806775

Willert, K., and Jones, K. A. (2006). Wnt signaling: Is the party in the nucleus? Genes Dev. 20: 1394-1404. PubMed ID: 16751178

Wood, A., et al. (2003) Bre1, an E3 ubiquitin ligase required for recruitment and substrate selection of Rad6 at a promoter. Mol. Cell 11: 267-274. PubMed ID: 12535539

Biological Overview

date revised: 25 April 2024

Home page: The Interactive Fly © 2009 Thomas Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.