Evolution of DNA Methylation Across Ecdysozoa.

DNA methylation is a crucial, abundant mechanism of gene regulation in vertebrates. It is less prevalent in many other metazoan organisms and completely absent in some key model species, such as Drosophila melanogaster and Caenorhabditis elegans. We report here a comprehensive study of the presence and absence of DNA methyltransferases (DNMTs) in 138 Ecdysozoa, covering Arthropoda, Nematoda, Priapulida, Onychophora, and Tardigrada. Three of these phyla have not been investigated for the presence of DNA methylation before. We observe that the loss of individual DNMTs independently occurred multiple times across ecdysozoan phyla. We computationally predict the presence of DNA methylation based on CpG rates in coding sequences using an implementation of Gaussian Mixture Modeling, MethMod. Integrating both analysis we predict two previously unknown losses of DNA methylation in Ecdysozoa, one within Chelicerata (Mesostigmata) and one in Tardigrada. In the early-branching Ecdysozoa Priapulus caudatus, we predict the presence of a full set of DNMTs and the presence of DNA methylation. We are therefore showing a very diverse and independent evolution of DNA methylation in different ecdysozoan phyla spanning a phylogenetic range of more than 700 million years.

Toward a mechanistic explanation of phenotypic evolution: The need for a theory of theory integration.

Reconciling different underlying ontologies and explanatory contexts has been one of the main challenges and impediments for theory integration in biology. Here, we analyze the challenge of developing an inclusive and integrative theory of phenotypic evolution as an example for the broader challenge of developing a theory of theory integration within the life sciences and suggest a number of necessary formal steps toward the resolution of often incompatible (hidden) assumptions. Theory integration in biology requires a better formal understanding of the structure of biological theories The strategy for integrating theories crucially depends on the relationships of the underlying ontologies.

Expansion of gene clusters, circular orders, and the shortest Hamiltonian path problem.

Clusters of paralogous genes such as the famous HOX cluster of developmental transcription factors tend to evolve by stepwise duplication of its members, often involving unequal crossing over. Gene conversion and possibly other mechanisms of concerted evolution further obfuscate the phylogenetic relationships. As a consequence, it is very difficult or even impossible to disentangle the detailed history of gene duplications in gene clusters. In this contribution we show that the expansion of gene clusters by unequal crossing over as proposed by Walter Gehring leads to distinctive patterns of genetic distances, namely a subclass of circular split systems. Furthermore, when the gene cluster was left undisturbed by genome rearrangements, the shortest Hamiltonian paths with respect to genetic distances coincide with the genomic order. This observation can be used to detect ancient genomic rearrangements of gene clusters and to distinguish gene clusters whose evolution was dominated by unequal crossing over within genes from those that expanded through other mechanisms.

Uncovering missing pieces: duplication and deletion history of arrestins in deuterostomes.

BACKGROUND: The cytosolic arrestin proteins mediate desensitization of activated G protein-coupled receptors (GPCRs) via competition with G proteins for the active phosphorylated receptors. Arrestins in active, including receptor-bound, conformation are also transducers of signaling. Therefore, this protein family is an attractive therapeutic target. The signaling outcome is believed to be a result of structural and sequence-dependent interactions of arrestins with GPCRs and other protein partners. Here we elucidated the detailed evolution of arrestins in deuterostomes. RESULTS: Identity and number of arrestin paralogs were determined searching deuterostome genomes and gene expression data. In contrast to standard gene prediction methods, our strategy first detects exons situated on different scaffolds and then solves the problem of assigning them to the correct gene. This increases both the completeness and the accuracy of the annotation in comparison to conventional database search strategies applied by the community. The employed strategy enabled us to map in detail the duplication- and deletion history of arrestin paralogs including tandem duplications, pseudogenizations and the formation of retrogenes. The two rounds of whole genome duplications in the vertebrate stem lineage gave rise to four arrestin paralogs. Surprisingly, visual arrestin ARR3 was lost in the mammalian clades Afrotheria and Xenarthra. Duplications in specific clades, on the other hand, must have given rise to new paralogs that show signatures of diversification in functional elements important for receptor binding and phosphate sensing. CONCLUSION: The current study traces the functional evolution of deuterostome arrestins in unprecedented detail. Based on a precise re-annotation of the exon-intron structure at nucleotide resolution, we infer the gain and loss of paralogs and patterns of conservation, co-variation and selection.

Ecological plant epigenetics: Evidence from model and non-model species, and the way forward.

Growing evidence shows that epigenetic mechanisms contribute to complex traits, with implications across many fields of biology. In plant ecology, recent studies have attempted to merge ecological experiments with epigenetic analyses to elucidate the contribution of epigenetics to plant phenotypes, stress responses, adaptation to habitat, and range distributions. While there has been some progress in revealing the role of epigenetics in ecological processes, studies with non-model species have so far been limited to describing broad patterns based on anonymous markers of DNA methylation. In contrast, studies with model species have benefited from powerful genomic resources, which contribute to a more mechanistic understanding but have limited ecological realism. Understanding the significance of epigenetics for plant ecology requires increased transfer of knowledge and methods from model species research to genomes of evolutionarily divergent species, and examination of responses to complex natural environments at a more mechanistic level. This requires transforming genomics tools specifically for studying non-model species, which is challenging given the large and often polyploid genomes of plants. Collaboration among molecular geneticists, ecologists and bioinformaticians promises to enhance our understanding of the mutual links between genome function and ecological processes.

The ancestor of modern Holozoa acquired the CCA-adding enzyme from Alphaproteobacteria by horizontal gene transfer.

Transfer RNAs (tRNAs) require the absolutely conserved sequence motif CCA at their 3'-ends, representing the site of aminoacylation. In the majority of organisms, this trinucleotide sequence is not encoded in the genome and thus has to be added post-transcriptionally by the CCA-adding enzyme, a specialized nucleotidyltransferase. In eukaryotic genomes this ubiquitous and highly conserved enzyme family is usually represented by a single gene copy. Analysis of published sequence data allows us to pin down the unusual evolution of eukaryotic CCA-adding enzymes. We show that the CCA-adding enzymes of animals originated from a horizontal gene transfer event in the stem lineage of Holozoa, i.e. Metazoa (animals) and their unicellular relatives, the Choanozoa. The tRNA nucleotidyltransferase, acquired from an α-proteobacterium, replaced the ancestral enzyme in Metazoa. However, in Choanoflagellata, the group of Choanozoa that is closest to Metazoa, both the ancestral and the horizontally transferred CCA-adding enzymes have survived. Furthermore, our data refute a mitochondrial origin of the animal tRNA nucleotidyltransferases.

The CHR site: definition and genome-wide identification of a cell cycle transcriptional element.

The cell cycle genes homology region (CHR) has been identified as a DNA element with an important role in transcriptional regulation of late cell cycle genes. It has been shown that such genes are controlled by DREAM, MMB and FOXM1-MuvB and that these protein complexes can contact DNA via CHR sites. However, it has not been elucidated which sequence variations of the canonical CHR are functional and how frequent CHR-based regulation is utilized in mammalian genomes. Here, we define the spectrum of functional CHR elements. As the basis for a computational meta-analysis, we identify new CHR sequences and compile phylogenetic motif conservation as well as genome-wide protein-DNA binding and gene expression data. We identify CHR elements in most late cell cycle genes binding DREAM, MMB, or FOXM1-MuvB. In contrast, Myb- and forkhead-binding sites are underrepresented in both early and late cell cycle genes. Our findings support a general mechanism: sequential binding of DREAM, MMB and FOXM1-MuvB complexes to late cell cycle genes requires CHR elements. Taken together, we define the group of CHR-regulated genes in mammalian genomes and provide evidence that the CHR is the central promoter element in transcriptional regulation of late cell cycle genes by DREAM, MMB and FOXM1-MuvB.

Algebraic Dynamic Programming over general data structures.

BACKGROUND: Dynamic programming algorithms provide exact solutions to many problems in computational biology, such as sequence alignment, RNA folding, hidden Markov models (HMMs), and scoring of phylogenetic trees. Structurally analogous algorithms compute optimal solutions, evaluate score distributions, and perform stochastic sampling. This is explained in the theory of Algebraic Dynamic Programming (ADP) by a strict separation of state space traversal (usually represented by a context free grammar), scoring (encoded as an algebra), and choice rule. A key ingredient in this theory is the use of yield parsers that operate on the ordered input data structure, usually strings or ordered trees. The computation of ensemble properties, such as a posteriori probabilities of HMMs or partition functions in RNA folding, requires the combination of two distinct, but intimately related algorithms, known as the inside and the outside recursion. Only the inside recursions are covered by the classical ADP theory. RESULTS: The ideas of ADP are generalized to a much wider scope of data structures by relaxing the concept of parsing. This allows us to formalize the conceptual complementarity of inside and outside variables in a natural way. We demonstrate that outside recursions are generically derivable from inside decomposition schemes. In addition to rephrasing the well-known algorithms for HMMs, pairwise sequence alignment, and RNA folding we show how the TSP and the shortest Hamiltonian path problem can be implemented efficiently in the extended ADP framework. As a showcase application we investigate the ancient evolution of HOX gene clusters in terms of shortest Hamiltonian paths. CONCLUSIONS: The generalized ADP framework presented here greatly facilitates the development and implementation of dynamic programming algorithms for a wide spectrum of applications.

Chromatin computation: epigenetic inheritance as a pattern reconstruction problem.

Eukaryotic histones carry a diverse set of specific chemical modifications that accumulate over the life-time of a cell and have a crucial impact on the cell state in general and the transcriptional program in particular. Replication constitutes a dramatic disruption of the chromatin states that effectively amounts to partial erasure of stored information. To preserve its epigenetic state the cell reconstructs (at least part of) the histone modifications by means of processes that are still very poorly understood. A plausible hypothesis is that the different combinations of reader and writer domains in histone-modifying enzymes implement local rewriting rules that are capable of "recomputing" the desired parental modification patterns on the basis of the partial information contained in that half of the nucleosomes that predate replication. To test whether such a mechanism is theoretically feasible, we have developed a flexible stochastic simulation system (available at http://www.bioinf.uni-leipzig.de/Software/StoChDyn) for studying the dynamics of histone modification states. The implementation is based on Gillespie's approach, i.e., it models the master equation of a detailed chemical model. It is efficient enough to use an evolutionary algorithm to find patterns across multiple cell divisions with high accuracy. We found that it is easy to evolve a system of enzymes that can maintain a particular chromatin state roughly stable, even without explicit boundary elements separating differentially modified chromatin domains. However, the success of this task depends on several previously unanticipated factors, such as the length of the initial state, the specific pattern that should be maintained, the time between replications, and chemical parameters such as enzymatic binding and dissociation rates. All these factors also influence the accumulation of errors in the wake of cell divisions.

Complete HOX cluster characterization of the coelacanth provides further evidence for slow evolution of its genome.

The living coelacanth is a lobe-finned fish that represents an early evolutionary departure from the lineage that led to land vertebrates, and is of extreme interest scientifically. It has changed very little in appearance from fossilized coelacanths of the Cretaceous (150 to 65 million years ago), and is often referred to as a "living fossil." An important general question is whether long-term stasis in morphological evolution is associated with stasis in genome evolution. To this end we have used targeted genome sequencing for acquiring 1,612,752 bp of high quality finished sequence encompassing the four HOX clusters of the Indonesian coelacanth Latimeria menadoensis. Detailed analyses were carried out on genomic structure, gene and repeat contents, conserved noncoding regions, and relative rates of sequence evolution in both coding and noncoding tracts. Our results demonstrate conclusively that the coelacanth HOX clusters are evolving comparatively slowly and that this taxon should serve as a viable outgroup for interpretation of the genomes of tetrapod species.

Proteinortho: detection of (co-)orthologs in large-scale analysis.

BACKGROUND: Orthology analysis is an important part of data analysis in many areas of bioinformatics such as comparative genomics and molecular phylogenetics. The ever-increasing flood of sequence data, and hence the rapidly increasing number of genomes that can be compared simultaneously, calls for efficient software tools as brute-force approaches with quadratic memory requirements become infeasible in practise. The rapid pace at which new data become available, furthermore, makes it desirable to compute genome-wide orthology relations for a given dataset rather than relying on relations listed in databases. RESULTS: The program Proteinortho described here is a stand-alone tool that is geared towards large datasets and makes use of distributed computing techniques when run on multi-core hardware. It implements an extended version of the reciprocal best alignment heuristic. We apply Proteinortho to compute orthologous proteins in the complete set of all 717 eubacterial genomes available at NCBI at the beginning of 2009. We identified thirty proteins present in 99% of all bacterial proteomes. CONCLUSIONS: Proteinortho significantly reduces the required amount of memory for orthology analysis compared to existing tools, allowing such computations to be performed on off-the-shelf hardware.

Hox clusters of the bichir (Actinopterygii, Polypterus senegalus) highlight unique patterns of sequence evolution in gnathostome phylogeny.

Teleost fishes have extra Hox gene clusters owing to shared or lineage-specific genome duplication events in rayfinned fish (actinopterygian) phylogeny. Hence, extrapolating between genome function of teleosts and human or even between different fish species is difficult. We have sequenced and analyzed Hox gene clusters of the Senegal bichir (Polypterus senegalus), an extant representative of the most basal actinopterygian lineage. Bichir possesses four Hox gene clusters (A, B, C, D); phylogenetic analysis supports their orthology to the four Hox gene clusters of the gnathostome ancestor. We have generated a comprehensive database of conserved Hox noncoding sequences that include cartilaginous, lobe-finned, and ray-finned fishes (bichir and teleosts). Our analysis identified putative and known Hox cis-regulatory sequences with differing depths of conservation in Gnathostoma. We found that although bichir possesses four Hox gene clusters, its pattern of conservation of noncoding sequences is mosaic between outgroups, such as human, coelacanth, and shark, with four Hox gene clusters and teleosts, such as zebrafish and pufferfish, with seven or eight Hox gene clusters. Notably, bichir Hox gene clusters have been invaded by DNA transposons and this trend is further exemplified in teleosts, suggesting an as yet unrecognized mechanism of genome evolution that may explain Hox cluster plasticity in actinopterygians. Taken together, our results suggest that actinopterygian Hox gene clusters experienced a reduction in selective constraints that surprisingly predates the teleost-specific genome duplication.

The challenges and scope of theoretical biology.

Scientific theories seek to provide simple explanations for significant empirical regularities based on fundamental physical and mechanistic constraints. Biological theories have rarely reached a level of generality and predictive power comparable to physical theories. This discrepancy is explained through a combination of frozen accidents, environmental heterogeneity, and widespread non-linearities observed in adaptive processes. At the same time, model building has proven to be very successful when it comes to explaining and predicting the behavior of particular biological systems. In this respect biology resembles alternative model-rich frameworks, such as economics and engineering. In this paper we explore the prospects for general theories in biology, and suggest that these take inspiration not only from physics, but also from the information sciences. Future theoretical biology is likely to represent a hybrid of parsimonious reasoning and algorithmic or rule-based explanation. An open question is whether these new frameworks will remain transparent to human reason. In this context, we discuss the role of machine learning in the early stages of scientific discovery. We argue that evolutionary history is not only a source of uncertainty, but also provides the basis, through conserved traits, for very general explanations for biological regularities, and the prospect of unified theories of life.

"Hypothesis for the modern RNA world": a pervasive non-coding RNA-based genetic regulation is a prerequisite for the emergence of multicellular complexity.

The transitions to multicellularity mark the most pivotal and distinctive events in life's history on Earth. Although several transitions to "simple" multicellularity (SM) have been recorded in both bacterial and eukaryotic clades, transitions to complex multicellularity (CM) have only happened a few times in eukaryotes. A large number of cell types (associated with large body size), increased energy consumption per gene expressed, and an increment of non-protein-coding DNA positively correlate with CM. These three factors can indeed be understood as the causes and consequences of the regulation of gene expression. Here, we discuss how a vast expansion of non-protein-coding RNA (ncRNAs) regulators rather than large numbers of novel protein regulators can easily contribute to the emergence of CM. We also propose that the evolutionary advantage of RNA-based gene regulation derives from the robustness of the RNA structure that makes it easy to combine genetic drift with functional exploration. We describe a model which aims to explain how the evolutionary dynamic of ncRNAs becomes dominated by the accessibility of advantageous mutations to innovate regulation in complex multicellular organisms. The information and models discussed here outline the hypothesis that pervasive ncRNA-based regulatory systems, only capable of being expanded and explored in higher eukaryotes, are prerequisite to complex multicellularity. Thereby, regulatory RNA molecules in Eukarya have allowed intensification of morphological complexity by stabilizing critical phenotypes and controlling developmental precision. Although the origin of RNA on early Earth is still controversial, it is becoming clear that once RNA emerged into a protocellular system, its relevance within the evolution of biological systems has been greater than we previously thought.

Genomic organization of eukaryotic tRNAs.

BACKGROUND: Surprisingly little is known about the organization and distribution of tRNA genes and tRNA-related sequences on a genome-wide scale. While tRNA gene complements are usually reported in passing as part of genome annotation efforts, and peculiar features such as the tandem arrangements of tRNA gene in Entamoeba histolytica have been described in some detail, systematic comparative studies are rare and mostly restricted to bacteria. We therefore set out to survey the genomic arrangement of tRNA genes and pseudogenes in a wide range of eukaryotes to identify common patterns and taxon-specific peculiarities. RESULTS: In line with previous reports, we find that tRNA complements evolve rapidly and tRNA gene and pseudogene locations are subject to rapid turnover. At phylum level, the distributions of the number of tRNA genes and pseudogenes numbers are very broad, with standard deviations on the order of the mean. Even among closely related species we observe dramatic changes in local organization. For instance, 65% and 87% of the tRNA genes and pseudogenes are located in genomic clusters in zebrafish and stickleback, resp., while such arrangements are relatively rare in the other three sequenced teleost fish genomes. Among basal metazoa, Trichoplax adherens has hardly any duplicated tRNA gene, while the sea anemone Nematostella vectensis boasts more than 17000 tRNA genes and pseudogenes. Dramatic variations are observed even within the eutherian mammals. Higher primates, for instance, have 616 +/- 120 tRNA genes and pseudogenes of which 17% to 36% are arranged in clusters, while the genome of the bushbaby Otolemur garnetti has 45225 tRNA genes and pseudogenes of which only 5.6% appear in clusters. In contrast, the distribution is surprisingly uniform across plant genomes. Consistent with this variability, syntenic conservation of tRNA genes and pseudogenes is also poor in general, with turn-over rates comparable to those of unconstrained sequence elements. Despite this large variation in abundance in Eukarya we observe a significant correlation between the number of tRNA genes, tRNA pseudogenes, and genome size. CONCLUSIONS: The genomic organization of tRNA genes and pseudogenes shows complex lineage-specific patterns characterized by an extensive variability that is in striking contrast to the extreme levels of sequence-conservation of the tRNAs themselves. The comprehensive analysis of the genomic organization of tRNA genes and pseudogenes in Eukarya provides a basis for further studies into the interplay of tRNA gene arrangements and genome organization in general.

Evolution of vault RNAs.

Vault RNAs (vtRNAs) are small, about 100 nt long, polymerase III transcripts contained in the vault particles of eukaryotic cells. Presumably due to their enigmatic function, they have received little attention compared with most other noncoding RNA (ncRNA) families. Their poor sequence conservation makes homology search a complex and tedious task even within vertebrates. Here we report on a systematic and comprehensive analysis of this rapidly evolving class of ncRNAs in deuterostomes, providing a comprehensive collection of computationally predicted vtRNA genes. We find that all previously described vtRNAs are located at a conserved genomic locus linked to the protocadherin gene cluster, an association that is conserved throughout gnathostomes. Lineage-specific expansions to small vtRNA gene clusters are frequently observed in this region. A second vtRNA locus is syntenically conserved across eutherian mammals. The vtRNAs at the two eutherian loci exhibit substantial differences in their promoter structures, explaining their differential expression patterns in several human cancer cell lines. In teleosts, expression of several paralogous vtRNA genes, most but not all located at the syntenically conserved protocadherin locus, was verified by reverse transcriptase-polymerase chain reaction.

Innovation in gene regulation: the case of chromatin computation.

Chromatin regulation is understood to be one of the fundamental modes of gene regulation in eukaryotic cells. We argue that the basic proteins that determine the chromatin architecture constitute an evolutionary ancient layer of transcriptional regulation common to all three domains of life. We explore phylogenetically, sources of innovation in chromatin regulation, focusing on protein domains related to chromatin structure and function, demonstrating a step-wise increase of complexity in chromatin regulation. Building upon the highly conserved use of variants of chromosomal architectural proteins to distinguish chromosomal states, Eukarya secondarily acquired mechanisms for "writing" chemical modifications onto chromatin that constitute persistent signals. The acquisition of reader domains enabled decoding of these complex, signal combinations and a decoupling of the signal from immediate biochemical effects. We show how the coupling of reading and writing, which is most prevalent in crown-group Eukarya, could have converted chromatin into a powerful computational device capable of storing and processing more information than pure cis-regulatory networks.

SynBlast: assisting the analysis of conserved synteny information.

MOTIVATION: In the last years more than 20 vertebrate genomes have been sequenced, and the rate at which genomic DNA information becomes available is rapidly accelerating. Gene duplication and gene loss events inherently limit the accuracy of orthology detection based on sequence similarity alone. Fully automated methods for orthology annotation do exist but often fail to identify individual members in cases of large gene families, or to distinguish missing data from traceable gene losses. This situation can be improved in many cases by including conserved synteny information. RESULTS: Here we present the SynBlast pipeline that is designed to construct and evaluate local synteny information. SynBlast uses the genomic region around a focal reference gene to retrieve candidates for homologous regions from a collection of target genomes and ranks them in accord with the available evidence for homology. The pipeline is intended as a tool to aid high quality manual annotation in particular in those cases where automatic procedures fail. We demonstrate how SynBlast is applied to retrieving orthologous and paralogous clusters using the vertebrate Hox and ParaHox clusters as examples. SOFTWARE: The SynBlast package written in Perl is available under the GNU General Public License at http://www.bioinf.uni-leipzig.de/Software/SynBlast/.

The amphioxus Hox cluster: characterization, comparative genomics, and evolution.

The amphioxus Hox cluster is often viewed as "archetypal" for the chordate lineage. Here, we present a descriptive account of the 448 kb region spanning the Hox cluster of the amphioxus Branchiostoma floridae from Hox14 to Hox1. We provide complete coding sequences of all 14 previously described amphioxus sequences and give a detailed analysis of the conserved noncoding regulatory sequence elements. We find that the posterior part of the Hox cluster is so highly derived that even the complete genomic sequence is insufficient to decide whether the posterior Hox genes arose by independent duplications or whether they are true orthologs of the corresponding gnathostome paralog groups. In contrast, the anterior region is much better conserved. The amphioxus Hox cluster strongly excludes repetitive elements with the exception of two repeat islands in the posterior region. Repeat exclusion is also observed in gnathostomes, but not protostome Hox clusters. We thus hypothesize that the much shorter vertebrate Hox clusters are the result of extensive resolution of the redundancy of regulatory DNA after the genome duplications rather than the consequence of a selection pressure to remove nonfunctional sequence from the Hox cluster.

Computational RNomics of drosophilids.

BACKGROUND: Recent experimental and computational studies have provided overwhelming evidence for a plethora of diverse transcripts that are unrelated to protein-coding genes. One subclass consists of those RNAs that require distinctive secondary structure motifs to exert their biological function and hence exhibit distinctive patterns of sequence conservation characteristic for positive selection on RNA secondary structure. The deep-sequencing of 12 drosophilid species coordinated by the NHGRI provides an ideal data set of comparative computational approaches to determine those genomic loci that code for evolutionarily conserved RNA motifs. This class of loci includes the majority of the known small ncRNAs as well as structured RNA motifs in mRNAs. We report here on a genome-wide survey using RNAz. RESULTS: We obtain 16 000 high quality predictions among which we recover the majority of the known ncRNAs. Taking a pessimistically estimated false discovery rate of 40% into account, this implies that at least some ten thousand loci in the Drosophila genome show the hallmarks of stabilizing selection action of RNA structure, and hence are most likely functional at the RNA level. A subset of RNAz predictions overlapping with TRF1 and BRF binding sites [Isogai et al., EMBO J. 26: 79-89 (2007)], which are plausible candidates of Pol III transcripts, have been studied in more detail. Among these sequences we identify several "clusters" of ncRNA candidates with striking structural similarities. CONCLUSION: The statistical evaluation of the RNAz predictions in comparison with a similar analysis of vertebrate genomes [Washietl et al., Nat. Biotech. 23: 1383-1390 (2005)] shows that qualitatively similar fractions of structured RNAs are found in introns, UTRs, and intergenic regions. The intergenic RNA structures, however, are concentrated much more closely around known protein-coding loci, suggesting that flies have significantly smaller complement of independent structured ncRNAs compared to mammals.