CiteULike: blobbybirdman's library [382 articles]

A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes.

DH Haft — 2007-05-09T04:18:46-00:00

PLoS Comput Biol, Vol. 1, No. 6. (November 2005)

Clustered regularly interspaced short palindromic repeats (CRISPRs) are a family of DNA direct repeats found in many prokaryotic genomes. Repeats of 21-37 bp typically show weak dyad symmetry and are separated by regularly sized, nonrepetitive spacer sequences. Four CRISPR-associated (Cas) protein families, designated Cas1 to Cas4, are strictly associated with CRISPR elements and always occur near a repeat cluster. Some spacers originate from mobile genetic elements and are thought to confer "immunity" against the elements that harbor these sequences. In the present study, we have systematically investigated uncharacterized proteins encoded in the vicinity of these CRISPRs and found many additional protein families that are strictly associated with CRISPR loci across multiple prokaryotic species. Multiple sequence alignments and hidden Markov models have been built for 45 Cas protein families. These models identify family members with high sensitivity and selectivity and classify key regulators of development, DevR and DevS, in Myxococcus xanthus as Cas proteins. These identifications show that CRISPR/cas gene regions can be quite large, with up to 20 different, tandem-arranged cas genes next to a repeat cluster or filling the region between two repeat clusters. Distinctive subsets of the collection of Cas proteins recur in phylogenetically distant species and correlate with characteristic repeat periodicity. The analyses presented here support initial proposals of mobility of these units, along with the likelihood that loci of different subtypes interact with one another as well as with host cell defensive, replicative, and regulatory systems. It is evident from this analysis that CRISPR/cas loci are larger, more complex, and more heterogeneous than previously appreciated.

Combinatorial regulation of genes essential for Myxococcus xanthus development involves a response regulator and a LysR-type regulator

Poorna Viswanathan — 2008-04-29T15:42:17-00:00

Proceedings of the National Academy of Sciences, Vol. 104, No. 19. (8 May 2007), pp. 7969-7974.

Myxococcus xanthus is a bacterium that undergoes multicellular development. C-signaling influences gene expression and movement of cells into aggregates. Expression of the dev operon, which includes genes essential for efficient sporulation, depends in part on C-signaling and reaches its highest level in cells within aggregates, ensuring that spores form within fruiting bodies. Here, an upstream DNA element was found to be essential for dev promoter activity and was bound by FruA, a response regulator in the C-signaling pathway. A second positive regulatory element, located approx350 bp downstream of the dev transcriptional start site, was bound by LadA, a newly identified transcription factor in the LysR family. Typically, LysR-type transcription factors bind upstream of the promoter and activate transcription in response to a coinducer. LadA appears to activate transcription from an unusual location for a LysR family member and likely subjects dev transcription to a different cue than does FruA. A ladA mutant exhibited similar developmental defects as dev mutants, suggesting that LadA may be devoted to dev regulation, unlike FruA, which regulates many developmental genes. FruA and LadA act on a regulatory region spanning >400 bp to bring about proper temporal and spatial expression of the dev operon, resembling the regulation of developmental genes in multicellular eukaryotes. 10.1073/pnas.0701569104

Network motifs in the transcriptional regulation network of Escherichia coli

Shai Shen-Orr — 2005-08-30T18:22:20-00:00

Nat Genet, Vol. 31, No. 1. (May 2002), pp. 64-68.

THE EVOLUTION OF GENETIC REGULATORY SYSTEMS IN BACTERIA

Harley Mcadams — 2006-08-03T22:49:21-00:00

Nat Rev Genet, Vol. 5, No. 3. (March 2004), pp. 169-178.

Orthologous Transcription Factors in Bacteria Have Different Functions and Regulate Different Genes

Morgan Price — 2007-09-07T19:49:10-00:00

PLoS Computational Biology, Vol. 3, No. 9. (1 September 2007), e175.

Transcription factors (TFs) form large paralogous gene families and have complex evolutionary histories. Here, we ask whether putative orthologs of TFs, from bidirectional best BLAST hits (BBHs), are evolutionary orthologs with conserved functions. We show that BBHs of TFs from distantly related bacteria are usually not evolutionary orthologs. Furthermore, the false orthologs usually respond to different signals and regulate distinct pathways, while the few BBHs that are evolutionary orthologs do have conserved functions. To test the conservation of regulatory interactions, we analyze expression patterns. We find that regulatory relationships between TFs and their regulated genes are usually not conserved for BBHs in Escherichia coli K12 and Bacillus subtilis. Even in the much more closely related bacteria Vibrio cholerae and Shewanella oneidensis MR-1, predicting regulation from E. coli BBHs has high error rates. Using gene–regulon correlations, we identify genes whose expression pattern differs between E. coli and S. oneidensis. Using literature searches and sequence analysis, we show that these changes in expression patterns reflect changes in gene regulation, even for evolutionary orthologs. We conclude that the evolution of bacterial regulation should be analyzed with phylogenetic trees, rather than BBHs, and that bacterial regulatory networks evolve more rapidly than previously thought.

Maintenance of transposon-free regions throughout vertebrate evolution

Cas Simons — 2007-12-20T15:29:40-00:00

BMC Genomics, Vol. 8 (20 December 2007), 470.

Defining the sequence-recognition profile of DNA-binding molecules

Christopher Warren — 2006-01-26T22:16:34-00:00

PNAS, Vol. 103, No. 4. (24 January 2006), pp. 867-872.

Determining the sequence-recognition properties of DNA-binding proteins and small molecules remains a major challenge. To address this need, we have developed a high-throughput approach that provides a comprehensive profile of the binding properties of DNA-binding molecules. The approach is based on displaying every permutation of a duplex DNA sequence (up to 10 positional variants) on a microfabricated array. The entire sequence space is interrogated simultaneously, and the affinity of a DNA-binding molecule for every sequence is obtained in a rapid, unbiased, and unsupervised manner. Using this platform, we have determined the full molecular recognition profile of an engineered small molecule and a eukaryotic transcription factor. The approach also yielded unique insights into the altered sequence-recognition landscapes as a result of cooperative assembly of DNA-binding molecules in a ternary complex. Solution studies strongly corroborated the sequence preferences identified by the array analysis.

A Systems Approach to Measuring the Binding Energy Landscapes of Transcription Factors

Sebastian Maerkl — 2007-01-12T15:17:06-00:00

Science, Vol. 315, No. 5809. (12 January 2007), pp. 233-237.

A major goal of systems biology is to predict the function of biological networks. Although network topologies have been successfully determined in many cases, the quantitative parameters governing these networks generally have not. Measuring affinities of molecular interactions in high-throughput format remains problematic, especially for transient and low-affinity interactions. We describe a high-throughput microfluidic platform that measures such properties on the basis of mechanical trapping of molecular interactions. With this platform we characterized DNA binding energy landscapes for four eukaryotic transcription factors; these landscapes were used to test basic assumptions about transcription factor binding and to predict their in vivo function. 10.1126/science.1131007

Non-additivity in protein-DNA binding.

RA O'Flanagan — 2006-09-07T09:13:40-00:00

Bioinformatics, Vol. 21, No. 10. (15 May 2005), pp. 2254-2263.

MOTIVATION: Localizing protein binding sites within genomic DNA is of considerable importance, but remains difficult for protein families, such as transcription factors, which have loosely defined target sequences. It is generally assumed that protein affinity for DNA involves additive contributions from successive nucleotide pairs within the target sequence. This is not necessarily true, and non-additive effects have already been experimentally demonstrated in a small number of cases. The principal origin of non-additivity involves the so-called indirect component of protein-DNA recognition which is related to the sequence dependence of DNA deformation induced during complex formation. Non-additive effects are difficult to study because they require the identification of many more binding sequences than are normally necessary for describing additive specificity (typically via the construction of weight matrices). RESULTS: In the present work we will use theoretically estimated binding energies as a basis for overcoming this problem. Our approach enables us to study the full combinatorial set of sequences for a variety of DNA-binding proteins, make a detailed analysis of non-additive effects and exploit this information to improve binding site predictions using either weight matrices or support vector machines. The results underline the fact that, even in the presence of significant deformation, non-additive effects may involve only a limited number of dinucleotide steps. This information helps to reduce the number of binding sites which need to be identified for successful predictions and to avoid problems of over-fitting. AVAILABILITY: The SVM software is available upon request from the authors.

Functional Specificity of a Hox Protein Mediated by the Recognition of Minor Groove Structure

Rohit Joshi — 2007-11-01T22:38:21-00:00

Cell, Vol. 131, No. 3. (2 November 2007), pp. 530-543.

Summary The recognition of specific DNA-binding sites by transcription factors is a critical yet poorly understood step in the control of gene expression. Members of the Hox family of transcription factors bind DNA by making nearly identical major groove contacts via the recognition helices of their homeodomains. In vivo specificity, however, often depends on extended and unstructured regions that link Hox homeodomains to a DNA-bound cofactor, Extradenticle (Exd). Using a combination of structure determination, computational analysis, and in vitro and in vivo assays, we show that Hox proteins recognize specific Hox-Exd binding sites via residues located in these extended regions that insert into the minor groove but only when presented with the correct DNA sequence. Our results suggest that these residues, which are conserved in a paralog-specific manner, confer specificity by recognizing a sequence-dependent DNA structure instead of directly reading a specific DNA sequence.

The genesis and evolution of homeobox gene clusters

Jordi Garcia-Fernàndez — 2005-12-01T20:09:36-00:00

Nature Reviews Genetics, Vol. 6, No. 12. (10 November 2005), pp. 881-892.

Mining of putative cis-acting elements for chromatin mediated regulation of Hox genes in mammals by in-silico analysis.

Hemant Bengani — 2007-03-22T23:13:37-00:00

J Exp Zoolog B Mol Dev Evol (14 March 2007)

The remarkable conservation in developmental strategies across phyla is well reflected in the conservation of the homeotic gene complexes responsible for establishing the body plan in embryonic development. On the other hand, changes in the strategy of transcription regulation are believed to form one of the major factors in the evolution of developmental mechanisms and phenotypic evolution of species. Apart from transcription regulation by gene specific transcription factors, the role of regulators mediating modifications of chromatin proteins, especially of HOX gene clusters in Drosophila is well documented. By comparative genomics we have identified novel motifs conserved in mouse, chimpanzee and human in the noncoding upstream/intronic sequences of Hox genes by in silico analysis. These motifs lack the binding sites for known transcription factors and are significantly over represented in the target genes of one of the core components of Polycomb Repressive Complex namely Supressor of zeste 12 (SUZ12) in human embryonic cells reported by Lee et al. [2006a. Cell 125:301-313]. Therefore, we predict that they could be the sites of interaction of chromatin modifying complexes for epigenetic regulation. J. Exp. Zool. (Mol. Dev. Evol.) 308B, 2007. (c) 2007 Wiley-Liss, Inc.

Hox transcription factors and their elusive mammalian gene targets

T Svingen — 2006-05-25T21:12:39-00:00

Heredity, Vol. aop, No. current.

Conservation of regulatory sequences and gene expression patterns in the disintegrating Drosophila Hox gene complex

Barbara Negre — 2007-06-15T08:22:22-00:00

Genome Res., Vol. 15, No. 5. (1 May 2005), pp. 692-700.

Homeotic (Hox) genes are usually clustered and arranged in the same order as they are expressed along the anteroposterior body axis of metazoans. The mechanistic explanation for this colinearity has been elusive, and it may well be that a single and universal cause does not exist. The Hox-gene complex (HOM-C) has been rearranged differently in several Drosophila species, producing a striking diversity of Hox gene organizations. We investigated the genomic and functional consequences of the two HOM-C splits present in Drosophila buzzatii. Firstly, we sequenced two regions of the D. buzzatii genome, one containing the genes labial and abdominal A, and another one including proboscipedia, and compared their organization with that of D. melanogaster and D. pseudoobscura in order to map precisely the two splits. Then, a plethora of conserved noncoding sequences, which are putative enhancers, were identified around the three Hox genes closer to the splits. The position and order of these enhancers are conserved, with minor exceptions, between the three Drosophila species. Finally, we analyzed the expression patterns of the same three genes in embryos and imaginal discs of four Drosophila species with different Hox-gene organizations. The results show that their expression patterns are conserved despite the HOM-C splits. We conclude that, in Drosophila, Hox-gene clustering is not an absolute requirement for proper function. Rather, the organization of Hox genes is modular, and their clustering seems the result of phylogenetic inertia more than functional necessity. 10.1101/gr.3468605

Animal Evolution: When Did the `Hox System' Arise?

Chris Amemiya — 2006-09-25T15:02:21-00:00

Current Biology, Vol. 16, No. 14. (25 July 2006), pp. R546-R548.

The origins of the Hox gene clusters and their coordinated activities during development have long been of considerable interest to biologists. In a recent paper in Current Biology, the Hox-like genes of two cnidarians are interpreted as evidence that the `Hox system', sensu stricto, originated after the split from the lineage leading to bilaterian animals and that it was not requisite for complex axial patterning.

Genomic evolution of Hox gene clusters.

D Lemons — 2006-10-02T12:33:45-00:00

Science, Vol. 313, No. 5795. (29 September 2006), pp. 1918-1922.

The family of Hox genes, which number 4 to 48 per genome depending on the animal, control morphologies on the main body axis of nearly all metazoans. The conventional wisdom is that Hox genes are arranged in chromosomal clusters in colinear order with their expression patterns on the body axis. However, recent evidence has shown that Hox gene clusters are fragmented, reduced, or expanded in many animals-findings that correlate with interesting morphological changes in evolution. Hox gene clusters also contain many noncoding RNAs, such as intergenic regulatory transcripts and evolutionarily conserved microRNAs, some of whose developmental functions have recently been explored.

Comparative analysis of Hox downstream genes in Drosophila

Stefanie Hueber — 2007-01-18T21:50:06-00:00

Development, Vol. 134, No. 2. (15 January 2007), pp. 381-392.

Functional diversification of body parts is dependent on the formation of specialized structures along the various body axes. In animals, region-specific morphogenesis along the anteroposterior axis is controlled by a group of conserved transcription factors encoded by the Hox genes. Although it has long been assumed that Hox proteins carry out their function by regulating distinct sets of downstream genes, only a small number of such genes have been found, with very few having direct roles in controlling cellular behavior. We have quantitatively identified hundreds of Hox downstream genes in Drosophila by microarray analysis, and validated many of them by in situ hybridizations on loss- and gain-of-function mutants. One important finding is that Hox proteins, despite their similar DNA-binding properties in vitro, have highly specific effects on the transcriptome in vivo, because expression of many downstream genes respond primarily to a single Hox protein. In addition, a large fraction of downstream genes encodes realizator functions, which directly affect morphogenetic processes, such as orientation and rate of cell divisions, cell-cell adhesion and communication, cell shape and migration, or cell death. Focusing on these realizators, we provide a framework for the morphogenesis of the maxillary segment. As the genomic organization of Hox genes and the interaction of Hox proteins with specific co-factors are conserved in vertebrates and invertebrates, and similar classes of downstream genes are regulated by Hox proteins across the metazoan phylogeny, our findings represent a first step toward a mechanistic understanding of morphological diversification within a species as well as between species. 10.1242/dev.02746

HOM-C evolution in Drosophila: is there a need for Hox gene clustering?

Barbara Negre — 2007-01-17T03:43:30-00:00

Trends in Genetics, Vol. In Press, Corrected Proof

The conservation of Homeotic (Hox) gene clustering and colinearity in many metazoans indicates that functional constraints operate on this genome organization. However, several studies have questioned its relevance in Drosophila. Here, we analyse the genomic organization of Hox and Hox-derived genes in 13 fruitfly species and the mosquito Anopheles gambiae. We found that at least seven different Homeotic complex (HOM-C) arrangements exist among Drosophila species, produced by three major splits, five microinversions and six gene transpositions. This dynamism contrasts with the stable organization of the complex in many other taxa. Although there is no evidence of an absolute requirement for Hox gene clustering in Drosophila, we found that strong functional constraints act on the individual genes.

Kinetics of Protein-DNA Interaction: Facilitated Target Location in Sequence-Dependent Potential

Michael Slutsky — 2008-01-24T16:36:20-00:00

Biophys. J., Vol. 87, No. 6. (1 December 2004), pp. 4021-4035.

Recognition and binding of specific sites on DNA by proteins is central for many cellular functions such as transcription, replication, and recombination. In the process of recognition, a protein rapidly searches for its specific site on a long DNA molecule and then strongly binds this site. Here we aim to find a mechanism that can provide both a fast search (1-10 s) and high stability of the specific protein-DNA complex (Kd = 10-15-10-8 M). Earlier studies have suggested that rapid search involves sliding of the protein along the DNA. Here we consider sliding as a one-dimensional diffusion in a sequence-dependent rough energy landscape. We demonstrate that, despite the landscape's roughness, rapid search can be achieved if one-dimensional sliding is accompanied by three-dimensional diffusion. We estimate the range of the specific and nonspecific DNA-binding energy required for rapid search and suggest experiments that can test our mechanism. We show that optimal search requires a protein to spend half of its time sliding along the DNA and the other half diffusing in three dimensions. We also establish that, paradoxically, realistic energy functions cannot provide both rapid search and strong binding of a rigid protein. To reconcile these two fundamental requirements we propose a search-and-fold mechanism that involves the coupling of protein binding and partial protein folding. The proposed mechanism has several important biological implications for search in the presence of other proteins and nucleosomes, simultaneous search by several proteins, etc. The proposed mechanism also provides a new framework for interpretation of experimental and structural data on protein-DNA interactions. 10.1529/biophysj.104.050765

Predicting prokaryotic ecological niches using genome sequence analysis.

G Suen — 2007-10-02T09:26:27-00:00

PLoS ONE, Vol. 2, No. 1. (2007)

Automated DNA sequencing technology is so rapid that analysis has become the rate-limiting step. Hundreds of prokaryotic genome sequences are publicly available, with new genomes uploaded at the rate of approximately 20 per month. As a result, this growing body of genome sequences will include microorganisms not previously identified, isolated, or observed. We hypothesize that evolutionary pressure exerted by an ecological niche selects for a similar genetic repertoire in those prokaryotes that occupy the same niche, and that this is due to both vertical and horizontal transmission. To test this, we have developed a novel method to classify prokaryotes, by calculating their Pfam protein domain distributions and clustering them with all other sequenced prokaryotic species. Clusters of organisms are visualized in two dimensions as 'mountains' on a topological map. When compared to a phylogenetic map constructed using 16S rRNA, this map more accurately clusters prokaryotes according to functional and environmental attributes. We demonstrate the ability of this map, which we term a "niche map", to cluster according to ecological niche both quantitatively and qualitatively, and propose that this method be used to associate uncharacterized prokaryotes with their ecological niche as a means of predicting their functional role directly from their genome sequence.

Functional genome annotation through phylogenomic mapping

Balaji Srinivasan — 2005-06-07T08:20:31-00:00

Nature Biotechnology, Vol. 23, No. 6. (06 June 2005), pp. 691-698.

Complete genome sequence of the myxobacterium Sorangium cellulosum

Susanne Schneiker — 2007-11-09T06:46:58-00:00

Nature Biotechnology, Vol. 25, No. 11. (28 October 2007), pp. 1281-1289.

Bacterial response regulators: versatile regulatory strategies from common domains

Rong Gao — 2008-01-21T16:43:28-00:00

Trends in Biochemical Sciences, Vol. 32, No. 5. (May 2007), pp. 225-234.

Response regulators (RRs) comprise a major family of signaling proteins in prokaryotes. A modular architecture that consists of a conserved receiver domain and a variable effector domain enables RRs to function as phosphorylation-regulated switches that couple a wide variety of cellular behaviors to environmental cues. Recently, advances have been made in understanding RR functions both at genome-wide and molecular levels. Global techniques have been developed to analyze RR input and output, expanding the scope of characterization of these versatile components. Meanwhile, structural studies have revealed that, despite common structures and mechanisms of function within individual domains, a range of interactions between receiver and effector domains confer great diversity in regulatory strategies, optimizing individual RRs for the specific regulatory needs of different signaling systems.

MazF, an mRNA Interferase, Mediates Programmed Cell Death during Multicellular Myxococcus Development.

H Nariya — 2008-01-21T16:49:45-00:00

Cell, Vol. 132, No. 1. (11 January 2008), pp. 55-66.

In prokaryotes, the toxin-antitoxin systems are thought to play important roles in growth regulation under stress conditions. In the E. coli MazE-MazF system, MazF toxin functions as an mRNA interferase cleaving mRNAs at ACA sequences to inhibit protein synthesis leading to cell growth arrest. Myxococcus xanthus is a bacterium displaying multicellular fruiting body development during which approximately 80% of cells undergo obligatory cell lysis. Here, we demonstrate that M. xanthus has a solitary mazF gene that lacks a cotranscribed antitoxin gene. The mazF deletion results in elimination of the obligatory cell death during development causing dramatic reduction in spore formation. Surprisingly, MrpC, a key developmental regulator, functions as a MazF antitoxin and a mazF transcription activator. Transcription of mrpC and mazF is negatively regulated via MrpC phosphorylation by a Ser/Thr kinase cascade. These findings reveal the regulated deployment of a toxin gene for developmental programmed cell death in bacteria.

Understanding of bat wing evolution takes flight

Kimberly Cooper — 2008-01-22T11:40:56-00:00

Genes Dev., Vol. 22, No. 2. (15 January 2008), pp. 121-124.

10.1101/gad.1639108

Regulation of the Myxococcus xanthus C-Signal-Dependent Omega4400 Promoter by the Essential Developmental Protein FruA

Deborah Yoder-Himes — 2008-01-22T02:54:24-00:00

J. Bacteriol., Vol. 188, No. 14. (15 July 2006), pp. 5167-5176.

The bacterium Myxococcus xanthus employs extracellular signals to coordinate aggregation and sporulation during multicellular development. Extracellular, contact-dependent signaling that involves the CsgA protein (called C-signaling) activates FruA, a putative response regulator that governs a branched signaling pathway inside cells. One branch regulates cell movement, leading to aggregation. The other branch regulates gene expression, leading to sporulation. C-signaling is required for full expression of most genes induced after 6 h into development, including the gene identified by Tn5 lac insertion Omega4400. To determine if FruA is a direct regulator of Omega4400 transcription, a combination of in vivo and in vitro experiments was performed. Omega4400 expression was abolished in a fruA mutant. The DNA-binding domain of FruA bound specifically to DNA upstream of the promoter -35 region in vitro. Mutations between bp -86 and -77 greatly reduced binding. One of these mutations had been shown previously to reduce Omega4400 expression in vivo and make it independent of C-signaling. For the first time, chromatin immunoprecipitation (ChIP) experiments were performed on M. xanthus. The ChIP experiments demonstrated that FruA is associated with the Omega4400 promoter region late in development, even in the absence of C-signaling. Based on these results, we propose that FruA directly activates Omega4400 transcription to a moderate level prior to C-signaling and, in response to C-signaling, binds near bp -80 and activates transcription to a higher level. Also, the highly localized effects of mutations between bp -86 and -77 on DNA binding in vitro, together with recently published footprints, allow us to predict a consensus binding site of GTCG/CGA/G for the FruA DNA-binding domain. 10.1128/JB.00318-06

Chipper: discovering transcription-factor targets from chromatin immunoprecipitation microarrays using variance stabilization

Francis Gibbons — 2008-01-17T20:38:57-00:00

Genome Biology, Vol. 6, No. 11. (2005)

Chromatin immunoprecipitation combined with microarray technology (Chip2) allows genome-wide determination of protein-DNA binding sites. The current standard method for analyzing Chip2 data requires additional control experiments that are subject to systematic error. We developed methods to assess significance using variance stabilization, learning error-model parameters without external control experiments. The method was validated experimentally, shows greater sensitivity than the current standard method, and incorporates false-discovery rate analysis. The corresponding software ('Chipper') is freely available. The method described here should help reveal an organism's transcription-regulatory 'wiring diagram'.

Evidence for the Adaptive Evolution of Mutation Rates

David Metzgar — 2007-06-08T13:10:19-00:00

Cell, Vol. 101, No. 6. (9 June 2000), pp. 581-584.

Mutation rates in mammalian genomes

Sudhir Kumar — 2007-10-29T16:46:28-00:00

Proceedings of the National Academy of Sciences, Vol. 99, No. 2. (22 January 2002), pp. 803-808.

Knowledge of the rate of point mutation is of fundamental importance, because mutations are a vital source of genetic novelty and a significant cause of human diseases. Currently, mutation rate is thought to vary many fold among genes within a genome and among lineages in mammals. We have conducted a computational analysis of 5,669 genes (17,208 sequences) from species representing major groups of placental mammals to characterize the extent of mutation rate differences among genes in a genome and among diverse mammalian lineages. We find that mutation rate is approximately constant per year and largely similar among genes. Similarity of mutation rates among lineages with vastly different generation lengths and physiological attributes points to a much greater contribution of replication-independent mutational processes to the overall mutation rate. Our results suggest that the average mammalian genome mutation rate is 2.2 x 10[-]9 per base pair per year, which provides further opportunities for estimating species and population divergence times by using molecular clocks. 10.1073/pnas.022629899

Noncoding RNA genes.

SR Eddy — 2005-03-09T21:12:08-00:00

Curr Opin Genet Dev, Vol. 9, No. 6. (December 1999), pp. 695-699.

Some genes produce RNAs that are functional instead of encoding proteins. Noncoding RNA genes are surprisingly numerous. Recently, active research areas include small nucleolar RNAs, antisense riboregulator RNAs, and RNAs involved in X-dosage compensation. Genome sequences and new algorithms have begun to make systematic computational screens for noncoding RNA genes possible.

Mapping the C. elegans noncoding transcriptome with a whole-genome tiling microarray

Housheng He — 2007-09-12T08:47:56-00:00

Genome Res. (4 September 2007), gr.6611807.

The number of annotated protein coding genes in the genome of Caenorhabditis elegans is similar to that of other animals, but the extent of its non-protein-coding transcriptome remains unknown. Expression profiling on whole-genome tiling microarrays applied to a mixed-stage C. elegans population verified the expression of 71% of all annotated exons. Only a small fraction (11%) of the polyadenylated transcription is non-annotated and appears to consist of [~]3200 missed or alternative exons and 7800 small transcripts of unknown function (TUFs). Almost half (44%) of the detected transcriptional output is non-polyadenylated and probably not protein coding, and of this, 70% overlaps the boundaries of protein-coding genes in a complex manner. Specific analysis of small non-polyadenylated transcripts verified 97% of all annotated small ncRNAs and suggested that the transcriptome contains [~]1200 small (<500 nt) unannotated noncoding loci. After combining overlapping transcripts, we estimate that at least 70% of the total C. elegans genome is transcribed. 10.1101/gr.6611807

A Computational Pipeline for High- Throughput Discovery of cis-Regulatory Noncoding RNA in Prokaryotes

Zizhen Yao — 2007-07-08T12:17:39-00:00

PLoS Computational Biology, Vol. 3, No. 7. (1 July 2007), e126.

Noncoding RNAs (ncRNAs) are important functional RNAs that do not code for proteins. We present a highly efficient computational pipeline for discovering cis-regulatory ncRNA motifs de novo. The pipeline differs from previous methods in that it is structure-oriented, does not require a multiple-sequence alignment as input, and is capable of detecting RNA motifs with low sequence conservation. We also integrate RNA motif prediction with RNA homolog search, which improves the quality of the RNA motifs significantly. Here, we report the results of applying this pipeline to Firmicute bacteria. Our top-ranking motifs include most known Firmicute elements found in the RNA family database (Rfam). Comparing our motif models with Rfam's hand-curated motif models, we achieve high accuracy in both membership prediction and base-pair–level secondary structure prediction (at least 75% average sensitivity and specificity on both tasks). Of the ncRNA candidates not in Rfam, we find compelling evidence that some of them are functional, and analyze several potential ribosomal protein leaders in depth.

Comparative genomics at the vertebrate extremes.

D Boffelli — 2007-04-04T20:07:25-00:00

Nat Rev Genet, Vol. 5, No. 6. (June 2004), pp. 456-465.

Ultraconserved elements in the human genome.

G Bejerano — 2005-03-03T10:51:51-00:00

Science, Vol. 304, No. 5675. (28 May 2004), pp. 1321-1325.

There are 481 segments longer than 200 base pairs (bp) that are absolutely conserved (100% identity with no insertions or deletions) between orthologous regions of the human, rat, and mouse genomes. Nearly all of these segments are also conserved in the chicken and dog genomes, with an average of 95 and 99% identity, respectively. Many are also significantly conserved in fish. These ultraconserved elements of the human genome are most often located either overlapping exons in genes involved in RNA processing or in introns or nearby genes involved in the regulation of transcription and development. Along with more than 5000 sequences of over 100 bp that are absolutely conserved among the three sequenced mammals, these represent a class of genetic elements whose functions and evolutionary origins are yet to be determined, but which are more highly conserved between these species than are proteins and appear to be essential for the ontogeny of mammals and other vertebrates.

Repeated morphological evolution through cis-regulatory changes in a pleiotropic gene

Benjamin Prud'homme — 2006-04-20T00:45:36-00:00

Nature, Vol. 440, No. 7087., pp. 1050-1053.

Gene duplication and the adaptive evolution of a classic genetic switch

Chris Hittinger — 2007-10-10T19:21:12-00:00

Nature, Vol. 449, No. 7163. (11 October 2007), pp. 677-681.

Colloquium Papers: Emerging principles of regulatory evolution.

B Prud'homme — 2007-06-14T22:46:27-00:00

Proc Natl Acad Sci U S A, Vol. 104 Suppl 1 (15 May 2007), pp. 8605-8612.

Understanding the genetic and molecular mechanisms governing the evolution of morphology is a major challenge in biology. Because most animals share a conserved repertoire of body-building and -patterning genes, morphological diversity appears to evolve primarily through changes in the deployment of these genes during development. The complex expression patterns of developmentally regulated genes are typically controlled by numerous independent cis-regulatory elements (CREs). It has been proposed that morphological evolution relies predominantly on changes in the architecture of gene regulatory networks and in particular on functional changes within CREs. Here, we discuss recent experimental studies that support this hypothesis and reveal some unanticipated features of how regulatory evolution occurs. From this growing body of evidence, we identify three key operating principles underlying regulatory evolution, that is, how regulatory evolution: (i) uses available genetic components in the form of preexisting and active transcription factors and CREs to generate novelty; (ii) minimizes the penalty to overall fitness by introducing discrete changes in gene expression; and (iii) allows interactions to arise among any transcription factor and downstream CRE. These principles endow regulatory evolution with a vast creative potential that accounts for both relatively modest morphological differences among closely related species and more profound anatomical divergences among groups at higher taxonomical levels.

The regulatory content of intergenic DNA shapes genome architecture.

CE Nelson — 2006-07-20T14:14:26-00:00

Genome Biol, Vol. 5, No. 4. (2004)

BACKGROUND: Factors affecting the organization and spacing of functionally unrelated genes in metazoan genomes are not well understood. Because of the vast size of a typical metazoan genome compared to known regulatory and protein-coding regions, functional DNA is generally considered to have a negligible impact on gene spacing and genome organization. In particular, it has been impossible to estimate the global impact, if any, of regulatory elements on genome architecture. RESULTS: To investigate this, we examined the relationship between regulatory complexity and gene spacing in Caenorhabditis elegans and Drosophila melanogaster. We found that gene density directly reflects local regulatory complexity, such that the amount of noncoding DNA between a gene and its nearest neighbors correlates positively with that gene's regulatory complexity. Genes with complex functions are flanked by significantly more noncoding DNA than genes with simple or housekeeping functions. Genes of low regulatory complexity are associated with approximately the same amount of noncoding DNA in D. melanogaster and C. elegans, while loci of high regulatory complexity are significantly larger in the more complex animal. Complex genes in C. elegans have larger 5' than 3' noncoding intervals, whereas those in D. melanogaster have roughly equivalent 5' and 3' noncoding intervals. CONCLUSIONS: Intergenic distance, and hence genome architecture, is highly nonrandom. Rather, it is shaped by regulatory information contained in noncoding DNA. Our findings suggest that in compact genomes, the species-specific loss of nonfunctional DNA reveals a landscape of regulatory information by leaving a profile of functional DNA in its wake.

Mining ChIP-chip data for transcription factor and cofactor binding sites.

AD Smith — 2006-10-02T17:30:12-00:00

Bioinformatics, Vol. 21 Suppl 1 (June 2005)

MOTIVATION: Identification of single motifs and motif pairs that can be used to predict transcription factor localization in ChIP-chip data, and gene expression in tissue-specific microarray data. RESULTS: We describe methodology to identify de novo individual and interacting pairs of binding site motifs from ChIP-chip data, using an algorithm that integrates localization data directly into the motif discovery process. We combine matrix-enumeration based motif discovery with multivariate regression to evaluate candidate motifs and identify motif interactions. When applied to the HNF localization data in liver and pancreatic islets, our methods produce motifs that are either novel or improved known motifs. All motif pairs identified to predict localization are further evaluated according to how well they predict expression in liver and islets and according to how conserved are the relative positions of their occurrences. We find that interaction models of HNF1 and CDP motifs provide excellent prediction of both HNF1 localization and gene expression in liver. Our results demonstrate that ChIP-chip data can be used to identify interacting binding site motifs. AVAILABILITY: Motif discovery programs and analysis tools are available on request from the authors.

Genomic cis-regulatory architecture and trans-acting regulators of a single interneuron-specific gene battery in C. elegans.

AS Wenick — 2007-07-02T23:27:56-00:00

Dev Cell, Vol. 6, No. 6. (June 2004), pp. 757-770.

Gene batteries are sets of coregulated genes with common cis-regulatory elements that define the differentiated state of a cell. The nature of gene batteries for individual neuronal cellular subtypes and their linked cis-regulatory elements is poorly defined. Through molecular dissection of the highly modular cis-regulatory architecture of individual neuronally expressed genes, we have defined a conserved 16 bp cis-regulatory motif that drives gene expression in a single interneuron subtype, termed AIY, in the nematode Caenorhabditis elegans. This motif is bound and activated by the Paired- and LIM-type homeodomain proteins CEH-10 and TTX-3. Using genome-wide phylogenetic footprinting, we delineated the location, distribution, and evolution of AIY-specific cis-regulatory elements throughout the genome and thereby defined a large battery of AIY-expressed genes, all of which represent direct Paired/LIM homeodomain target genes. The identity of these homeodomain targets provides novel insights into the biology of the AIY interneuron.

Functional Evolution of a cis-Regulatory Module

Michael Ludwig — 2007-09-23T23:02:44-00:00

PLoS Biology, Vol. 3, No. 4. (1 April 2005), e93.

Lack of knowledge about how regulatory regions evolve in relation to their structure–function may limit the utility of comparative sequence analysis in deciphering cis-regulatory sequences. To address this we applied reverse genetics to carry out a functional genetic complementation analysis of a eukaryotic cis-regulatory module—the even-skipped stripe 2 enhancer—from four Drosophila species. The evolution of this enhancer is non-clock-like, with important functional differences between closely related species and functional convergence between distantly related species. Functional divergence is attributable to differences in activation levels rather than spatiotemporal control of gene expression. Our findings have implications for understanding enhancer structure–function, mechanisms of speciation and computational identification of regulatory modules.

A regulatory code for neurogenic gene expression in the Drosophila embryo.

M Markstein — 2006-09-29T22:03:20-00:00

Development, Vol. 131, No. 10. (May 2004), pp. 2387-2394.

Bioinformatics methods have identified enhancers that mediate restricted expression in the Drosophila embryo. However, only a small fraction of the predicted enhancers actually work when tested in vivo. In the present study, co-regulated neurogenic enhancers that are activated by intermediate levels of the Dorsal regulatory gradient are shown to contain several shared sequence motifs. These motifs permitted the identification of new neurogenic enhancers with high precision: five out of seven predicted enhancers direct restricted expression within ventral regions of the neurogenic ectoderm. Mutations in some of the shared motifs disrupt enhancer function, and evidence is presented that the Twist and Su(H) regulatory proteins are essential for the specification of the ventral neurogenic ectoderm prior to gastrulation. The regulatory model of neurogenic gene expression defined in this study permitted the identification of a neurogenic enhancer in the distant Anopheles genome. We discuss the prospects for deciphering regulatory codes that link primary DNA sequence information with predicted patterns of gene expression.

An evolutionary constraint: Strongly disfavored class of change in DNA sequence during divergence of cis-regulatory modules

Andrew Cameron — 2007-10-15T11:53:00-00:00

Proceedings of the National Academy of Sciences, Vol. 102, No. 33. (16 August 2005), pp. 11769-11774.

The DNA of functional cis-regulatory modules displays extensive sequence conservation in comparisons of genomes from modestly distant species. Patches of sequence that are several hundred base pairs in length within these modules are often seen to be 80-95% identical, although the flanking sequence cannot even be aligned. However, it is unlikely that base pairs located between the transcription factor target sites of cis-regulatory modules have sequence-dependent function, and the mechanism that constrains evolutionary change within cis-regulatory modules is incompletely understood. We chose five functionally characterized cis-regulatory modules from the Strongylocentrotus purpuratus (sea urchin) genome and obtained orthologous regulatory and flanking sequences from a bacterial artificial chromosome genome library of a congener, Strongylocentrotus franciscanus. As expected, single-nucleotide substitutions and small indels occur freely at many positions within the regulatory modules of these two species, as they do outside the regulatory modules. However, large indels (>20 bp) are statistically almost absent within the regulatory modules, although they are common in flanking intergenic or intronic sequence. The result helps to explain the patterns of evolutionary sequence divergence characteristic of cis-regulatory DNA. 10.1073/pnas.0505291102

Functional Analysis of Chicken Sox2 Enhancers Highlights an Array of Diverse Regulatory Elements that Are Conserved in Mammals

Masanori Uchikawa — 2007-10-15T11:47:00-00:00

Developmental Cell, Vol. 4, No. 4. (April 2003), pp. 509-519.

Sox2 expression marks neural and sensory primordia at various stages of development. A 50 kb genomic region of chicken Sox2 was isolated and scanned for enhancer activity utilizing embryo electroporation, resulting in identification of a battery of enhancers. Although Sox2 expression in the early embryonic CNS appears uniform, it is actually pieced together by five separate enhancers with distinct spatio-temporal specificities, including the one activated by the neural induction signals emanating from Hensen's node. Enhancers for Sox2 expression in the lens and nasal/otic placodes and in the neural crest were also determined. These functionally identified Sox2 enhancers exactly correspond to the extragenic sequence blocks conspicuously conserved between chicken and mammals, which are not discernible by sequence comparison among mammals.

Combinatorial epigenetics, "junk DNA", and the evolution of complex organisms.

E Zuckerkandl — 2007-04-20T12:07:30-00:00

Gene, Vol. 390, No. 1-2. (1 April 2007), pp. 232-242.

At certain evolutionary junctures, two or more mutations participating in the build-up of a new complex function may be required to become available simultaneously in the same individuals. How could this happen in higher organisms whose populations are small compared to those of microbes, and in which chances of combined nearly simultaneous highly specific favorable mutations are correspondingly low? The question can in principle be answered for regulatory evolution, one of the basic processes of evolutionary change. A combined resetting of transcription rates in several genes could occur in the same individual. It is proposed that, in eukaryotes, changes in epigenetic trends and epigenetically transforming encounters between alternative chromatin structures could arise frequently enough so as to render probable particular conjunctions of changed transcription rates. Such conjunctions could involve mutational changes with low specificity requirements in gene-associated regions of non-protein-coding sequences. The effects of such mutations, notably when they determine the use of histone variants and covalent modifications of histones, can be among those that migrate along chromatin. Changes in chromatin structure are often cellularly inheritable over at least a limited number of generations of cells, and of individuals when the germ line is involved. SINEs and LINEs, which have been considered "junk DNA", are among the repeat sequences that would appear liable to have teleregulatory effects on the function of a nearby promoter, through changes in their numbers and distribution. There may also be present preexisting unstably inheritable epigenetic trends leading to cellular variegation, trends endemic in a cell population based on DNA sequences previously established in the neighborhood. Either way, epigenetically conditioned teleregulatory trends may display only limited penetrance. The imposition at a distance of new chromatin structures with regulatory impact can occur in cis as well as in trans, and is examined as intrachromosomally spreading teleregulation and interchromosomal "gene kissing". The chances for two or more particular epigenetically determined regulatory trends to occur together in a cell are increased thanks to the proposed low specificity requirements for most of the pertinent sequence changes in intergenic and intronic DNA or in the distribution of middle repetitive sequences that have teleregulatory impact. Inheritable epigenetic changes ("epimutations") with effects at a distance would then perdure over the number of generations required for "assimilation" of the several regulatory novelties through the occurrence and selection, gene by gene, of specific classical mutations. These mutations would have effects similar to the epigenetic effects, yet would provide stability and penetrance. The described epigenetic/genetic partnership may well at times have opened the way toward certain complex new functions. Thus, the presence of "junk DNA", through co-determining the (higher or lower) order and the variants of chromatin structure with regulatory effects at a distance, might make an important contribution to the evolution of complex organisms.

Scanning Human Gene Deserts for Long-Range Enhancers

Marcelo Nobrega — 2006-08-04T00:01:53-00:00

Science, Vol. 302, No. 5644. (17 October 2003), 413.

10.1126/science.1088328

Megabase deletions of gene deserts result in viable mice.

MA Nóbrega — 2006-04-05T14:49:51-00:00

Nature, Vol. 431, No. 7011. (21 October 2004), pp. 988-993.

The functional importance of the roughly 98% of mammalian genomes not corresponding to protein coding sequences remains largely undetermined. Here we show that some large-scale deletions of the non-coding DNA referred to as gene deserts can be well tolerated by an organism. We deleted two large non-coding intervals, 1,511 kilobases and 845 kilobases in length, from the mouse genome. Viable mice homozygous for the deletions were generated and were indistinguishable from wild-type littermates with regard to morphology, reproductive fitness, growth, longevity and a variety of parameters assaying general homeostasis. Further detailed analysis of the expression of multiple genes bracketing the deletions revealed only minor expression differences in homozygous deletion and wild-type mice. Together, the two deleted segments harbour 1,243 non-coding sequences conserved between humans and rodents (more than 100 base pairs, 70% identity). Some of the deleted sequences might encode for functions unidentified in our screen; nonetheless, these studies further support the existence of potentially 'disposable DNA' in the genomes of mammals.

Discovering DNA regulatory elements with bacteria

Martha Bulyk — 2005-08-04T22:52:11-00:00

Nature Biotechnology, Vol. 23, No. 8., pp. 942-944.

DNA microarray technologies for measuring protein-DNA interactions

Martha Bulyk — 2006-08-08T10:45:47-00:00

Current Opinion in Biotechnology, Vol. 17, No. 4. (August 2006), pp. 422-430.

DNA-binding proteins have key roles in many cellular processes, including transcriptional regulation and replication. Microarray-based technologies permit the high-throughput identification of binding sites and enable the functional roles of these binding proteins to be elucidated. In particular, microarray readout either of chromatin immunoprecipitated DNA-bound proteins (ChIP-chip) or of DNA adenine methyltransferase fusion proteins (DamID) enables the identification of in vivo genomic target sites of proteins. A complementary approach to analyse the in vitro binding of proteins directly to double-stranded DNA microarrays (protein binding microarrays; PBMs), permits rapid characterization of their DNA binding site sequence specificities. Recent advances in DNA microarray synthesis technologies have facilitated the definition of DNA-binding sites at much higher resolution and coverage, and advances in these and emerging technologies will further increase the efficiencies of these exciting new approaches.

A supervised hidden Markov model framework for efficiently segmenting tiling array data in transcriptional and ChIP-chip experiments: systematically incorporating validated biological knowledge.

Jiang Du — 2006-10-23T15:32:54-00:00

Bioinformatics (12 October 2006)

MOTIVATION: Large-scale tiling array experiments are becoming increasingly common in genomics. In particular, the ENCODE project requires the consistent segmentation of many different tiling array data sets into "active regions" (e.g. finding transfrags from transcriptional data and putative binding sites from ChIP-chip experiments). Previously, such segmentation was done in an unsupervised fashion mainly based on characteristics of the signal distribution in the tiling array data itself. Here we propose a supervised framework for doing this. It has the advantage of explicitly incorporating validated biological knowledge into the model and allowing for formal training and testing. Methodology: In particular, we use a hidden Markov model (HMM) framework, which is capable of explicitly modeling the dependency between neighboring probes and whose extended version (the generalized HMM) also allows explicit description of state duration density. We introduce a formal definition of the tiling-array analysis problem, and explain how we can use this to describe sampling small genomic regions for experimental validation to build up a gold-standard set for training and testing. We then describe various ideal and practical sampling strategies (e.g. maximizing signal entropy within a selected region versus using gene annotation or known promoters as positives for transcription or ChIP-chip data, respectively). RESULTS: For the practical sampling and training strategies, we show how the size and noise in the validated training data affects the performance of an HMM applied to the ENCODE transcriptional and ChIP-chip experiments. In particular, we show that the HMM framework is able to efficiently process tiling array data as well as or better than previous approaches. For the idealized sampling strategies, we show how we can assess their performance in a simulation framework and how a maximum entropy approach, which samples sub-regions with very different signal intensities, gives the maximally performing gold-standard. This latter result has strong implications for the optimum way medium-scale validation experiments should be carried out to verify the results of the genome-scale tiling array experiments. SUPPLEMENTARY INFORMATION: The supplementary materials are available at http://tiling.gersteinlab.org/hmm/.