Evolutionary growth process of highly conserved sequences in vertebrate genomes

Genome sequence comparison between evolutionarily distant species revealed ultraconserved elements (UCEs) among mammals under strong purifying selection. Most of them were also conserved among vertebrates. Because they tend to be located in the flanking regions of developmental genes, they would have fundamental roles in creating vertebrate body plans. However, the evolutionary origin and selection mechanism of these UCEs remain unclear. Here we report that UCEs arose in primitive vertebrates, and gradually grew in vertebrate evolution. We searched for UCEs in two teleost fishes, Tetraodon nigroviridis and Oryzias latipes, and found 554 UCEs with 100% identity over 100 bps. Comparison of teleost and mammalian UCEs revealed 43 pairs of common, jawed-vertebrate UCEs (jUCE) with high sequence identities, ranging from 83.1% to 99.2%. Ten of them retain lower similarities to the Petromyzon marinus genome, and the substitution rates of four non-exonic jUCEs were reduced after the teleost-mammal divergence, suggesting that robust conservation had been acquired in the jawed vertebrate lineage. Our results indicate that prototypical UCEs originated before the divergence of jawed and jawless vertebrates and have been frozen as perfect conserved sequences in the jawed vertebrate lineage. In addition, our comparative sequence analyses of UCEs and neighboring regions resulted in a discovery of lineage-specific conserved sequences. They were added progressively to prototypical UCEs, suggesting step-wise acquisition of novel regulatory roles. Our results indicate that conserved non-coding elements (CNEs) consist of blocks with distinct evolutionary history, each having been frozen since different evolutionary era along the vertebrate lineage.     

Fugu: a compact vertebrate reference genome

At 400 Mb, the Japanese pufferfish, Fugu rubripes, has the smallest vertebrate genome but has a similar gene repertoire to other vertebrates. Its genes are densely packed with short intergenic and intronic sequences devoid of repetitive elements. It likely has a mutational bias towards DNA elimination and is probably close to a 'minimal' vertebrate genome. As such it is a useful reference genome for gene discovery and gene validation in other vertebrates. Its usefulness in the discovery of conserved regulatory elements has already been demonstrated. The Fugu genome sequence is a good complement to genetic studies in other vertebrates.     

Decoding cis-regulatory systems in ascidians

Ascidians, or sea squirts, are lower chordates, and share basic gene repertoires and many characteristics, both developmental and physiological, with vertebrates. Therefore, decoding cis-regulatory systems in ascidians will contribute toward elucidating the genetic regulatory systems underlying the developmental and physiological processes of vertebrates. cis-Regulatory DNAs can also be used for tissue-specific genetic manipulation, a powerful tool for studying ascidian development and physiology. Because the ascidian genome is compact compared with vertebrate genomes, both intergenic regions and introns are relatively small in ascidians. Short upstream intergenic regions contain a complete set of cis-regulatory elements for spatially regulated expression of a majority of ascidian genes. These features of the ascidian genome are a great advantage in identifying cis-regulatory sequences and in analyzing their functions. Function of cis-regulatory DNAs has been analyzed for a number of tissue-specific and developmentally regulated genes of ascidians by introducing promoter-reporter fusion constructs into ascidian embryos. The availability of the whole genome sequences of the two Ciona species, Ciona intestinalis and Ciona savignyi, facilitates comparative genomics approaches to identify cis-regulatory DNAs. Recent studies demonstrate that computational methods can help identify cis-regulatory elements in the ascidian genome. This review presents a comprehensive list of ascidian genes whose cis-regulatory regions have been subjected to functional analysis, and highlights the recent advances in bioinformatics and comparative genomics approaches to cis-regulatory systems in ascidians.     

Computational approaches for the discovery of bacterial small RNAs

Recent work has uncovered a growing number of bacterial small RNAs (sRNAs), some of which have been shown to regulate critical cellular processes. Computational approaches, in combination with experiments, have played an important role in the discovery of these sRNAs. In this article, we first give an overview of different computational approaches for genome-wide prediction of sRNAs. These approaches have led to the discovery of several novel sRNAs, however the regulatory roles are not yet known for a majority of these sRNAs. By contrast, several recent studies have highlighted the inverse problem where the functional role of the sRNA is already known and the challenge is to identify its genomic location. The focus of this article is on computational tools and strategies for identifying these specific sRNAs which function as key components of known regulatory pathways.     

Comparative genomics using fugu: a tool for the identification of conserved vertebrate cis-regulatory elements

With the imminent completion of the whole genome sequence of humans, increasing attention is being focused on the annotation of cis-regulatory elements in the human genome. Comparative genomics approaches based on evolutionary conservation have proved useful in the detection of conserved cis-regulatory elements. The pufferfish, Fugu rubripes, is an attractive vertebrate model for comparative genomics, by virtue of its compact genome and maximal phylogenetic distance from mammals. Fugu has lost a large proportion of nonessential DNA, and retained single orthologs for many duplicate genes that arose in the fish lineage. Non-coding sequences conserved between fugu and mammals have been shown to be functional cis-regulatory elements. Thus, fugu is a model fish genome of choice for discovering evolutionarily conserved regulatory elements in the human genome. Such evolutionarily conserved elements are likely to be shared by all vertebrates, and related to regulatory interactions fundamental to all vertebrates. The functions of these conserved vertebrate elements can be rapidly assayed in mammalian cell lines or in transgenic systems such as zebrafish/medaka and Xenopus, followed by validation of crucial elements in transgenic rodents.     

Early Evolution of Conserved Regulatory Sequences Associated with Development in Vertebrates

Comparisons between diverse vertebrate genomes have uncovered thousands of highly conserved non-coding sequences, an increasing number of which have been shown to function as enhancers during early development. Despite their extreme conservation over 500 million years from humans to cartilaginous fish, these elements appear to be largely absent in invertebrates, and, to date, there has been little understanding of their mode of action or the evolutionary processes that have modelled them. We have now exploited emerging genomic sequence data for the sea lamprey, Petromyzon marinus, to explore the depth of conservation of this type of element in the earliest diverging extant vertebrate lineage, the jawless fish (agnathans). We searched for conserved non-coding elements (CNEs) at 13 human gene loci and identified lamprey elements associated with all but two of these gene regions. Although markedly shorter and less well conserved than within jawed vertebrates, identified lamprey CNEs are able to drive specific patterns of expression in zebrafish embryos, which are almost identical to those driven by the equivalent human elements. These CNEs are therefore a unique and defining characteristic of all vertebrates. Furthermore, alignment of lamprey and other vertebrate CNEs should permit the identification of persistent sequence signatures that are responsible for common patterns of expression and contribute to the elucidation of the regulatory language in CNEs. Identifying the core regulatory code for development, common to all vertebrates, provides a foundation upon which regulatory networks can be constructed and might also illuminate how large conserved regulatory sequence blocks evolve and become fixed in genomic DNA.     

A Reporter Assay in Lamprey Embryos Reveals Both Functional Conservation and Elaboration of Vertebrate Enhancers

The sea lamprey is an important model organism for investigating the evolutionary origins of vertebrates. As more vertebrate genome sequences are obtained, evolutionary developmental biologists are becoming increasingly able to identify putative gene regulatory elements across the breadth of the vertebrate taxa. The identification of these regions makes it possible to address how changes at the genomic level have led to changes in developmental gene regulatory networks and ultimately to the evolution of morphological diversity. Comparative genomics approaches using sea lamprey have already predicted a number of such regulatory elements in the lamprey genome. Functional characterisation of these sequences and other similar elements requires efficient reporter assays in lamprey. In this report, we describe the development of a transient transgenesis method for lamprey embryos. Focusing on conserved non-coding elements (CNEs), we use this method to investigate their functional conservation across the vertebrate subphylum. We find instances of both functional conservation and lineage-specific functional evolution of CNEs across vertebrates, emphasising the utility of functionally testing homologous CNEs in their host species.     

RNA 3D Modules in Genome-Wide Predictions of RNA 2D Structure

Recent experimental and computational progress has revealed a large potential for RNA structure in the genome. This has been driven by computational strategies that exploit multiple genomes of related organisms to identify common sequences and secondary structures. However, these computational approaches have two main challenges: they are computationally expensive and they have a relatively high false discovery rate (FDR). Simultaneously, RNA 3D structure analysis has revealed modules composed of non-canonical base pairs which occur in non-homologous positions, apparently by independent evolution. These modules can, for example, occur inside structural elements which in RNA 2D predictions appear as internal loops. Hence one question is if the use of such RNA 3D information can improve the prediction accuracy of RNA secondary structure at a genome-wide level. Here, we use RNAz in combination with 3D module prediction tools and apply them on a 13-way vertebrate sequence-based alignment. We find that RNA 3D modules predicted by metaRNAmodules and JAR3D are significantly enriched in the screened windows compared to their shuffled counterparts. The initially estimated FDR of 47.0% is lowered to below 25% when certain 3D module predictions are present in the window of the 2D prediction. We discuss the implications and prospects for further development of computational strategies for detection of RNA 2D structure in genomic sequence.     

The computational challenges of applying comparative-based computational methods to whole genomes

The explosion in genomic sequence available in public databases has resulted in an unprecedented opportunity for computational whole genome analyses. A number of promising comparative-based approaches have been developed for gene finding, regulatory element discovery and other purposes, and it is clear that these tools will play a fundamental role in analysing the enormous amount of new data that is currently being generated. The synthesis of computationally intensive comparative computational approaches with the requirement for whole genome analysis represents both an unprecedented challenge and opportunity for computational scientists. We focus on a few of these challenges, using by way of example the problems of alignment, gene finding and regulatory element discovery, and discuss the issues that have arisen in attempts to solve these problems in the context of whole genome analysis pipelines.     

The lens in focus: a comparison of lens development in Drosophila and vertebrates

The evolution of the eye has been a major subject of study dating back centuries. The advent of molecular genetics offered the surprising finding that morphologically distinct eyes rely on conserved regulatory gene networks for their formation. While many of these advances often stemmed from studies of the compound eye of the fruit fly, Drosophila melanogaster, and later translated to discoveries in vertebrate systems, studies on vertebrate lens development far outnumber those in Drosophila. This may be largely historical, since Spemann and Mangold's paradigm of tissue induction was discovered in the amphibian lens. Recent studies on lens development in Drosophila have begun to define molecular commonalities with the vertebrate lens. Here, we provide an overview of Drosophila lens development, discussing intrinsic and extrinsic factors controlling lens cell specification and differentiation. We then summarize key morphological and molecular events in vertebrate lens development, emphasizing regulatory factors and networks strongly associated with both systems. Finally, we provide a comparative analysis that highlights areas of research that would help further clarify the degree of conservation between the formation of dioptric systems in invertebrates and vertebrates.     

Comparative structure analysis of vertebrate ribonuclease P RNA.

Ribonuclease P cleaves 5'-precursor sequences from pre-tRNAs. All cellular RNase P holoenzymes contain homologous RNA elements; the eucaryal RNase P RNA, in contrast to the bacterial RNA, is catalytically inactive in the absence of the protein component(s). To understand the function of eucaryal RNase P RNA, knowledge of its structure is needed. Considerable effort has been devoted to comparative studies of the structure of this RNA from diverse organisms, including eucaryotes, primarily fungi, but also a limited set of vertebrates. The substantial differences in the sequences and structures of the vertebrate RNAs from those of other organisms have made it difficult to align the vertebrate sequences, thus limiting comparative studies. To expand our understanding of the structure of diverse RNase P RNAs, we have isolated by PCR and sequenced 13 partial RNase P RNA genes from 11 additional vertebrate taxa representing most extant major vertebrate lineages. Based on a recently proposed structure of the core elements of RNase P RNA, we aligned the sequences and propose a minimum consensus secondary structure for the vertebrate RNase P RNA.     

Discovery of cell-type specific regulatory elements in the human genome using differential chromatin modification analysis

Chromatin modifications have been comprehensively illustrated to play important roles in gene regulation and cell diversity in recent years. Given the rapid accumulation of genome-wide chromatin modification maps across multiple cell types, there is an urgent need for computational methods to analyze multiple maps to reveal combinatorial modification patterns and define functional DNA elements, especially those are specific to cell types or tissues. In this current study, we developed a computational method using differential chromatin modification analysis (dCMA) to identify cell-type-specific genomic regions with distinctive chromatin modifications. We then apply this method to a public data set with modification profiles of nine marks for nine cell types to evaluate its effectiveness. We found cell-type-specific elements unique to each cell type investigated. These unique features show significant cell-type-specific biological relevance and tend to be located within functional regulatory elements. These results demonstrate the power of a differential comparative epigenomic strategy in deciphering the human genome and characterizing cell specificity.