Monday, July 4, 2011

Do bacterial pathogens secrete protein methylases to modify eukaryotic chromatin?

Figure: Hartmanella grasping a Legionella (source microbe world).

In eukaryotes members of the DOT1 family rather exclusively modify H3K79, and processively methylate it to give rise to mono-, di-, and trimethylated forms. DOT1-catalyzed methylation is rather distinctive in that it is a modification that targets a residue right within the globular histone fold, rather than lysines in low-complexity tails. Unlike the histone methylations catalyzed by the PRMT family, methylation catalyzed by the DOT1 family appears to have a predominantly negative effect on gene expression across eukaryotes. Studies in mammals indicate that DOT1 is part of a large protein complex, including two pairs of paralogous proteins, all of which give rise to fusion proteins arising from chromosomal translocations in mixed lineage leukemia (MLL): (1) ENL and AF9/MLLT3, both similar to TAF14, with a N-terminal YEATS domain and a C-terminal BrC domain and (2) AF17/MLLT6 and AF10/MLLT10, both with two N-terminal PHD fingers and C-terminal AT-hook motifs. 

Studies in Saccharomyces cerevisiae, supported by studies in other eukaryotes, suggest distinct roles for the di- and trimethylated forms of H3 generated by Dot1, which occur on largely mutually exclusive sets of genes. H3K79me3 occurs predominantly within the gene body (i.e., protein-coding sequence), and is largely absent in promoters and intergenic regions. This form has been associated with genes that are transcriptionally less active, and is explicitly excluded from the nucleosomes associated with the most highly expressed genes: 50% of the genes generating just 1–4 mRNAs per hour are enriched in nucleosomes showing this modification, in contrast to just 2% of the genes giving rise to >50 mRNAs per hour. The increased processivity of DOT1 in catalyzing trimethylation appears to depend on prior monoubiquitination of histone H2BK123 by the Rad6/Bre1 ubiquitinating complex. Unlike H3K79me3 levels, which do not vary greatly over the cell cycle, H3K79me2 levels change significantly with the cell-cycle, being lowest in G1 and elevated during the G2/M progression. Further, H3K79me2 is not restricted to the gene bodies and is also seen in intergenic regions, including promoters. Moreover, the genes associated with this modification tend to be transcriptionally inactive during the G2/M phase, when its levels are elevated. In trypanosomes, which possess three DOT1 paralogs, two have been functionally characterized. The first, DOT1A, mainly catalyzes H3K79me2 formation in a cell-cycle dependent manner, whereas the other paralog DOT1B appears to be involved in subtelomeric gene-silencing associated with antigenic variation in trypanosomes. In mammals, DOT1 appears to regulate heterochromatin formation at telomeric and centromeric regions, consistent with the observations in yeast and trypanosomes. In addition to its role in silencing and heterochromatin organization, other observations suggest that DOT1 methylation regulates multiple aspects of DNA repair, such as base excision repair, Rad9-mediated checkpoint function, and negative regulation of the action of the translesion repair polymerases.

A big question to us has been when and how this big player in chromatin modification emerged in eukaryotes. With the exception of the basal eukaryotes Trichomonas and Giardia, DOT1 orthologs are present in all other major eukaryotic lineages for which genome sequences are available. However, within those lineages there are certain notable instances of gene loss—while the basal plant lineages such as the chlorophyte algae and lycopodiophytes have one or more DOT1 paralogs, they have been completely lost in the crown-group land plants such as angiosperms. Within animals and fungi, typically only a single DOT1 paralog is seen and they display a largely vertical pattern of evolution. However, in the caenorhabditiform nematodes there has been a notable lineage-specific expansion (LSE) of DOT1, with at least five paralogs in Caenorhabditis elegans. It seems important to study the potential functional compartmentalization of these newly emergent DOT1 versions in this organism. Phylogenetic analysis also suggests that the precursor of DOT1A and DOT1B in trypanosomes appears to have been acquired via lateral transfer from the animal lineage (click here to access a tree from our Supplementary material). Following this transfer, it appears to have acquired an N-terminal Zn-chelating domain with four conserved cysteines, and was then duplicated to yield two functionally distinct paralogs. In microbial eukaryotes such as chlorophyte algae, stramenopiles, apicomplexans, ciliates, and trypanosomes, there appear to have been multiple lateral transfer events that have disseminated DOT1 paralogs between distantly related lineages. Consequently, some of these eukaryotes have multiple DOT1 paralogs, with particularly notable complements of three or more paralogs seen in certain stramenopiles and trypanosomes (the third trypanosome DOT1 paralog is distinct from the previously studied DOT1A and DOT1B). This presence of multiple DOT1 paralogs is rather different from the situation seen in most animals and fungi, raising the possibility that some of them might have evolved distinct substrate specificities or may regulate H3K79 methylation in alternative signaling or developmental contexts.

Thus our studies raise two key issues:

1) While DOT1 seems to perform immensely important roles across the model eukaryotes, it was probably not present in the earliest branches of eukarya, and was acquired only prior to the separation of the parabasalids and diplomonads from other eukaryotes.

2) In many eukaryotes the multiplicity of DOT1s suggests that histone methylations catalyzed by the multiple paralogous forms might have a much richer contextual “meaning” than what is seen in model systems.

This leads to the question: So, after all where did DOT1 come from?
We discovered that DOT1 is nested within a vast radiation of bacterial methylases that are involved in the synthesis of secondary metabolites such as mycolic acids in mycobacteria (including the mycolic acid cyclopropane synthases), polyether antibiotics such as nigericin (e.g., NigE of Streptomyces sp. DSM4137) and as yet uncharacterized compounds in Micromonospora (gi: 288794127; in the same operon as a SnoaL-like polyketide cyclase). Further, gene neighborhood analysis suggests that several members in this bacterial radiation (e.g., gi: 294507034 from Salinibacter ruber) are specified by a conserved operon along with an amino acid transporter. It is conceivable that these versions are involved in the utilization of particular amino acids, or metabolites derived from them (Click here to access the operons). Thus, it appears the DOT1-like group arose as part of the radiation of methylases involved in generating diversity among secondary metabolites by adding of specific methyl groups to these metabolites – a common strategy used in the arms race between antibiotic producers and their intended victims. Alternative some of them were used by bacteria probably as a strategy to utilize amines or amino acids by methylating them. Some of these bacterial forms, such as those seen in Legionella, myxobacteria, and Protochlamydia, are particular close to the eukaryotic forms and share conserved sequence motifs in both the N-terminal extended element and in the loop between strand-6 and strand-7 (e.g., the conserved aromatic residue, click here [svg] or here [txt] to access an alignment of Dot1 and its homologs). Most interestingly, of the bacterial versions closest to the eukaryotic forms, some are encoded by intracellular pathogens or endosymbionts: These include the causative agent of Legionnaires' disease and Pontiac fever, i.e. Legionella.  In addition to infecting animals Legionella is a very versatile endoparasite that infects amoebozoans like Hartmannella and Acanthamoeba, heteroloboseans like Naegleria and ciliates like Tetrahymena. DOT1 homologs are also seen in Protochlamydia a dedicated bacterial endosymbiont of Acanthamoeba. Given that it was laterally exchanged between distantly related endo- symbionts/parasites points to its importance for this mode of life. 

This raises the issue of whether DOT1 is used to regulate the eukaryotic host’s behavior by modifying its histones. In support of this contention we found that these versions have signal peptides that are likely to allow their secretion into the host cells. Second, unlike the other bacterial versions they also lack operonic associations with secondary metabolite biosynthesis. Hence, it would be of great interest for experimental works to test our prediction to see if these bacterial DOT1s play a role in regulating host behavior via histone methylation comparable to the endogenous DOT1.  Importantly, this observation also suggests that DOT1 was originally acquired by eukaryotes from their intracellular bacterial symbionts/parasites. The other bacteria with such DOT1 homologs are the myxobacteria – we suggest that these might enable them to play in the “big league” i.e. compete with environmental eukaryotes by deploying this secreted DOT1, among other proteins as a potential toxin. You can read a detailed account of protein methylases here. Also feel free to browse the extensive supplement.