CiteULike: Tag genome-wide

Genome-wide transcriptional plasticity underlies cellular adaptation to novel challenge

Shay Stern — 2007-04-24T15:58:25-00:00

Mol Syst Biol, Vol. 3 (24 April 2007)

An optimized split-ubiquitin cDNA-library screening system to identify novel interactors of the human Frizzled 1 receptor

Dietmar Dirnberger — 2008-05-14T11:09:51-00:00

Nucl. Acids Res., Vol. 36, No. 6. (1 April 2008), e37.

The yeast split-ubiquitin system has previously been shown to be suitable to detect protein interactions of membrane proteins and of transcription factors in vivo. Therefore, this technology complements the classical split-transcription factor based yeast two-hybrid system (Y2H). Success or failure of the Y2H depends primarily on the ability to avoid false-negative and false-positive hits that become a limiting factor for the value of the system, especially in large scale proteomic analyses. We provide here a systematic assessment of parameters to help improving the quality of split-ubiquitin cDNA-library screenings. We experimentally defined the optimal 5-fluoroorotic acid (5-FOA) concentration as a key parameter to increase the reproducibility of interactions and, at the same time, to keep non-specific background growth low. Furthermore, we show that the efficacy of the 5-FOA selection is modulated by the plating density of the yeast clones. Moreover, a reporter-specific class of false-positive hits was identified, and a simple phenotypic assay for efficient de-selection was developed. We demonstrate the application of this improved system to identify novel interacting proteins of the human Frizzled 1 receptor. We identified several novel interactors with components of the Wnt-Frizzled signalling pathways and discuss their potential roles as direct mediators of Frizzled receptor signalling. The present work is the first example of a split-ubiquitin interaction screen using an in-situ expressed receptor of the serpentine class, emphasizing the suitability of the described improvements in the screening protocol. 10.1093/nar/gkm1163

Mammalian RNA polymerase II core promoters: insights from genome-wide studies

Albin Sandelin — 2007-05-19T03:15:41-00:00

Nature Reviews Genetics, Vol. 8, No. 6. (08 May 2007), pp. 424-436.

Genome-wide transcription and the implications for genomic organization.

Philipp Kapranov — 2007-05-10T13:57:03-00:00

Nat Rev Genet (8 May 2007)

Recent evidence of genome-wide transcription in several species indicates that the amount of transcription that occurs cannot be entirely accounted for by current sets of genome-wide annotations. Evidence indicates that most of both strands of the human genome might be transcribed, implying extensive overlap of transcriptional units and regulatory elements. These observations suggest that genomic architecture is not colinear, but is instead interleaved and modular, and that the same genomic sequences are multifunctional: that is, used for multiple independently regulated transcripts and as regulatory regions. What are the implications and consequences of such an interleaved genomic architecture in terms of increased information content, transcriptional complexity, evolution and disease states?

Genome-wide expression dynamics of a marine virus and host reveal features of co-evolution

Debbie Lindell — 2007-09-05T18:18:32-00:00

Nature, Vol. 449, No. 7158., pp. 83-86.

Where have all the interactions gone? Estimating the coverage of two-hybrid protein interaction maps

Hailiang Huang — 2007-10-22T04:09:34-00:00

PLoS Computational Biology, Vol. preprint, No. 2007. (1 September 2007), e214.eor.

Yeast two-hybrid screens are an important method for mapping pairwise physical interactions between proteins. The fraction of interactions detected in independent screens can be very small, and an outstanding challenge is to determine the reason for the low overlap. Low overlap can arise from either a high false-positive rate (interaction sets have low overlap because each set is contaminated by a large number of stochastic false positive interactions) or a high false-negative rate (interaction sets have low overlap because each misses many true interactions). We extend capture-recapture theory to provide the first unified model for false-positive and false-negative rates for two hybrid screens. Analysis of yeast, worm and fly data indicates that 25% to 45% of the reported interactions are likely false positives. Membrane proteins have higher false-positive rates on average, and signal transduction proteins have lower rates. The overall false-negative rate ranges from 75% for worm to 90% for fly, which arises from a roughly 50% false-negative rate due to statistical under-sampling and a 55% to 85% false-negative rate due to proteins that appear to be systematically lost from the assays. Finally, statistical model selection conclusively rejects the Erdös-Rényi network model in favor of the power law model for yeast and the truncated power law for worm and fly degree distributions. Much as genome sequencing coverage estimates were essential for planning the human genome sequencing project, the coverage estimates developed here will be valuable for guiding future proteomic screens.

Genome-wide mapping of allele-specific protein-DNA interactions in human cells

Nathaniel Maynard — 2008-03-28T16:35:47-00:00

Nature Methods, Vol. 5, No. 4. (16 March 2008), pp. 307-309.

Genome-wide profiling of DNA methylation reveals a class of normally methylated CpG island promoters.

L Shen — 2007-11-14T08:49:21-00:00

PLoS Genet, Vol. 3, No. 10. (26 October 2007), pp. 2023-2036.

The role of CpG island methylation in normal development and cell differentiation is of keen interest, but remains poorly understood. We performed comprehensive DNA methylation profiling of promoter regions in normal peripheral blood by methylated CpG island amplification in combination with microarrays. This technique allowed us to simultaneously determine the methylation status of 6,177 genes, 92% of which include dense CpG islands. Among these 5,549 autosomal genes with dense CpG island promoters, we have identified 4.0% genes that are nearly completely methylated in normal blood, providing another exception to the general rule that CpG island methylation in normal tissue is limited to X inactivation and imprinted genes. We examined seven genes in detail, including ANKRD30A, FLJ40201, INSL6, SOHLH2, FTMT, C12orf12, and DPPA5. Dense promoter CpG island methylation and gene silencing were found in normal tissues studied except testis and sperm. In both tissues, bisulfite cloning and sequencing identified cells carrying unmethylated alleles. Interestingly, hypomethylation of several genes was associated with gene activation in cancer. Furthermore, reactivation of silenced genes could be induced after treatment with a DNA demethylating agent or in a cell line lacking DNMT1 and/or DNMT3b. Sequence analysis identified five motifs significantly enriched in this class of genes, suggesting that cis-regulatory elements may facilitate preferential methylation at these promoter CpG islands. We have identified a group of non-X-linked bona fide promoter CpG islands that are densely methylated in normal somatic tissues, escape methylation in germline cells, and for which DNA methylation is a primary mechanism of tissue-specific gene silencing.

Genome-wide prediction of matrix attachment regions that increase gene expression in mammalian cells

Pierre-Alain Girod — 2007-08-31T15:18:16-00:00

Nat Meth, Vol. 4, No. 9. (2007), pp. 747-753.

Genome-Wide Profiling and Analysis of Arabidopsis siRNAs

Kristin Kasschau — 2007-04-19T14:07:47-00:00

PLoS Biology, Vol. 5, No. 3. (1 March 2007), e57.

Eukaryotes contain a diversified set of small RNA-guided pathways that control genes, repeated sequences, and viruses at the transcriptional and posttranscriptional levels. Genome-wide profiles and analyses of small RNAs, particularly the large class of 24-nucleotide (nt) short interfering RNAs (siRNAs), were done for wild-type Arabidopsis thaliana and silencing pathway mutants with defects in three RNA-dependent RNA polymerase (RDR) and four Dicer-like (DCL) genes. The profiling involved direct analysis using a multiplexed, parallel-sequencing strategy. Small RNA-generating loci, especially those producing predominantly 24-nt siRNAs, were found to be highly correlated with repetitive elements across the genome. These were found to be largely RDR2- and DCL3-dependent, although alternative DCL activities were detected on a widespread level in the absence of DCL3. In contrast, no evidence for RDR2-alternative activities was detected. Analysis of RDR2- and DCL3-dependent small RNA accumulation patterns in and around protein-coding genes revealed that upstream gene regulatory sequences systematically lack siRNA-generating activities. Further, expression profiling suggested that relatively few genes, proximal to abundant 24-nt siRNAs, are regulated directly by RDR2- and DCL3-dependent silencing. We conclude that the widespread accumulation patterns for RDR2- and DCL3-dependent siRNAs throughout the Arabidopsis genome largely reflect mechanisms to silence highly repeated sequences.

Using Phylogeny to Improve Genome-Wide Distant Homology Recognition

Sanne Abeln — 2007-04-12T09:34:57-00:00

PLoS Computational Biology, Vol. 3, No. 1. (1 January 2007), e3.

The gap between the number of known protein sequences and structures continues to widen, particularly as a result of sequencing projects for entire genomes. Recently there have been many attempts to generate structural assignments to all genes on sets of completed genomes using fold-recognition methods. We developed a method that detects false positives made by these genome-wide structural assignment experiments by identifying isolated occurrences. The method was tested using two sets of assignments, generated by SUPERFAMILY and PSI-BLAST, on 150 completed genomes. A phylogeny of these genomes was built and a parsimony algorithm was used to identify isolated occurrences by detecting occurrences that cause a gain at leaf level. Isolated occurrences tend to have high e-values, and in both sets of assignments, a sudden increase in isolated occurrences is observed for e-values >10−8 for SUPERFAMILY and >10−4 for PSI-BLAST. Conditions to predict false positives are based on these results. Independent tests confirm that the predicted false positives are indeed more likely to be incorrectly assigned. Evaluation of the predicted false positives also showed that the accuracy of profile-based fold-recognition methods might depend on secondary structure content and sequence length. We show that false positives generated by fold-recognition methods can be identified by considering structural occurrence patterns on completed genomes; occurrences that are isolated within the phylogeny tend to be less reliable. The method provides a new independent way to examine the quality of fold assignments and may be used to improve the output of any genome-wide fold assignment method.

The road to genome-wide association studies.

Leonid Kruglyak — 2008-02-29T15:46:44-00:00

Nat Rev Genet (19 February 2008)

The recent crop of results from genome-wide association studies might seem like a sudden development. However, this blooming follows a long germination period during which the necessary concepts, resources and techniques were developed and assembled. Here, I look back at how the necessary pieces fell into place, focusing on the less well-chronicled days before the launch of the HapMap project, and speculate about future developments.

Estimating the size of the human interactome

Michael Stumpf — 2008-05-13T07:34:25-00:00

Proceedings of the National Academy of Sciences (12 May 2008), 0708078105.

After the completion of the human and other genome projects it emerged that the number of genes in organisms as diverse as fruit flies, nematodes, and humans does not reflect our perception of their relative complexity. Here, we provide reliable evidence that the size of protein interaction networks in different organisms appears to correlate much better with their apparent biological complexity. We develop a stable and powerful, yet simple, statistical procedure to estimate the size of the whole network from subnet data. This approach is then applied to a range of eukaryotic organisms for which extensive protein interaction data have been collected and we estimate the number of interactions in humans to be approx650,000. We find that the human interaction network is one order of magnitude bigger than the Drosophila melanogaster interactome and approx3 times bigger than in Caenorhabditis elegans. 10.1073/pnas.0708078105

A genome-scale computational study of the interplay between transcriptional regulation and metabolism

Tomer Shlomi — 2007-04-17T16:24:45-00:00

Mol Syst Biol, Vol. 3 (17 April 2007)

Genome-wide approaches to studying chromatin modifications

Dustin Schones — 2008-02-18T13:07:54-00:00

Nat Rev Genet, Vol. 9, No. 3. (March 2008), pp. 179-191.

Genome-wide analyses reveal properties of redundant and specific promoter occupancy within the ETS gene family

Peter Hollenhorst — 2007-08-02T13:04:32-00:00

Genes Dev., Vol. 21, No. 15. (1 August 2007), pp. 1882-1894.

The conservation of in vitro DNA-binding properties within families of transcription factors presents a challenge for achieving in vivo specificity. To uncover the mechanisms regulating specificity within the ETS gene family, we have used chromatin immunoprecipitation coupled with genome-wide promoter microarrays to query the occupancy of three ETS proteins in a human T-cell line. Unexpectedly, redundant occupancy was frequently detected, while specific occupancy was less likely. Redundant binding correlated with housekeeping classes of genes, whereas specific binding examples represented more specialized genes. Bioinformatics approaches demonstrated that redundant binding correlated with consensus ETS-binding sequences near transcription start sites. In contrast, specific binding sites diverged dramatically from the consensus and were found further from transcription start sites. One route to specificity was founda highly divergent binding site that facilitates ETS1 and RUNX1 cooperative DNA binding. The specific and redundant DNA-binding modes suggest two distinct roles for members of the ETS transcription factor family. 10.1101/gad.1561707

Genomic analysis of estrogen cascade reveals histone variant H2A.Z associated with breast cancer progression

Sujun Hua — 2008-04-27T23:46:47-00:00

Mol Syst Biol, Vol. 4 (15 April 2008)

Toward simpler and faster genome-wide mutagenesis in mice

Sen Wu — 2007-06-28T22:35:21-00:00

Nature Genetics, Vol. 39, No. 7. (17 June 2007), pp. 922-930.

Worldwide Human Relationships Inferred from Genome-Wide Patterns of Variation

Jun Li — 2008-02-22T08:51:45-00:00

Science, Vol. 319, No. 5866. (22 February 2008), pp. 1100-1104.

Human genetic diversity is shaped by both demographic and biological factors and has fundamental implications for understanding the genetic basis of diseases. We studied 938 unrelated individuals from 51 populations of the Human Genome Diversity Panel at 650,000 common single-nucleotide polymorphism loci. Individual ancestry and population substructure were detectable with very high resolution. The relationship between haplotype heterozygosity and geography was consistent with the hypothesis of a serial founder effect with a single origin in sub-Saharan Africa. In addition, we observed a pattern of ancestral allele frequency distributions that reflects variation in population dynamics among geographic regions. This data set allows the most comprehensive characterization to date of human genetic variation. 10.1126/science.1153717

Dissecting direct reprogramming through integrative genomic analysis

Tarjei Mikkelsen — 2008-05-29T14:02:31-00:00

Nature (28 May 2008)

Genome-wide analysis identifies a general requirement for polarity proteins in endocytic traffic

Zita Balklava — 2007-09-01T06:06:14-00:00

Nature Cell Biology, Vol. 9, No. 9. (19 August 2007), pp. 1066-1073.

A genome-wide analysis in Saccharomyces cerevisiae demonstrates the influence of chromatin modifiers on transcription

Israel Steinfeld — 2007-02-27T01:55:36-00:00

Nature Genetics, Vol. 39, No. 3. (26 February 2007), pp. 303-309.

New models of collaboration in genome-wide association studies: the Genetic Association Information Network

2007-09-05T00:52:38-00:00

Nat Genet, Vol. 39, No. 9. (2007), pp. 1045-1051.

The past, present and future of genome-wide re-annotation.

CA Ouzounis — 2005-02-08T17:48:24-00:00

Genome Biol, Vol. 3, No. 2. (2002)

Annotation, the process by which structural or functional information is inferred for genes or proteins, is crucial for obtaining value from genome sequences. We define the process of annotating a previously annotated genome sequence as 're-annotation', and examine the strengths and weaknesses of current manual and automatic genome-wide re-annotation approaches.

Origin of phenotypes: Genes and transcripts.

TR Gingeras — 2007-06-21T09:01:25-00:00

Genome Res, Vol. 17, No. 6. (June 2007), pp. 682-690.

While the concept of a gene has been helpful in defining the relationship of a portion of a genome to a phenotype, this traditional term may not be as useful as it once was. Currently, "gene" has come to refer principally to a genomic region producing a polyadenylated mRNA that encodes a protein. However, the recent emergence of a large collection of unannotated transcripts with apparently little protein coding capacity, collectively called transcripts of unknown function (TUFs), has begun to blur the physical boundaries and genomic organization of genic regions with noncoding transcripts often overlapping protein-coding genes on the same (sense) and opposite strand (antisense). Moreover, they are often located in intergenic regions, making the genic portions of the human genome an interleaved network of both annotated polyadenylated and nonpolyadenylated transcripts, including splice variants with novel 5' ends extending hundreds of kilobases. This complex transcriptional organization and other recently observed features of genomes argue for the reconsideration of the term "gene" and suggests that transcripts may be used to define the operational unit of a genome.

Statistical significance of cis-regulatory modules

Dustin Schones — 2007-01-23T13:59:47-00:00

BMC Bioinformatics, Vol. 8, No. 1. (2007)

BACKGROUND:It is becoming increasingly important for researchers to be able to scan through large genomic regions for transcription factor binding sites or clusters of binding sites forming cis-regulatory modules. Correspondingly, there has been a push to develop algorithms for the rapid detection and assessment of cis-regulatory modules. While various algorithms for this purpose have been introduced, most are not well suited for rapid, large scale scanning.RESULTS:We introduce methods designed for the detection and statistical evaluation of cis-regulatory modules, modeled as either clusters of individual binding sites or as combinations of sites with constrained organization. In order to determine the statistical significance of module sites, we first need a method to determine the statistical significance of single transcription factor binding site matches. We introduce a straightforward method of estimating the statistical significance of single site matches using a database of known promoters to produce data structures that can be used to estimate p-values for binding site matches. We next introduce a technique to calculate the statistical significance of the arrangement of binding sites within a module using a max-gap model. If the module scanned for has defined organizational parameters, the probability of the module is corrected to account for organizational constraints. The statistical significance of single site matches and the architecture of sites within the module can be combined to provide an overall estimation of statistical significance of cis-regulatory module sites.CONCLUSIONS:The methods introduced in this paper allow for the detection and statistical evaluation of single transcription factor binding sites and cis-regulatory modules. The features described are implemented in the Search Tool for Occurrences of Regulatory Motifs (STORM) and MODSTORM software.

Analysis of the vertebrate insulator protein CTCF-binding sites in the human genome.

TH Kim — 2007-07-24T10:04:56-00:00

Cell, Vol. 128, No. 6. (23 March 2007), pp. 1231-1245.

Insulator elements affect gene expression by preventing the spread of heterochromatin and restricting transcriptional enhancers from activation of unrelated promoters. In vertebrates, insulator's function requires association with the CCCTC-binding factor (CTCF), a protein that recognizes long and diverse nucleotide sequences. While insulators are critical in gene regulation, only a few have been reported. Here, we describe 13,804 CTCF-binding sites in potential insulators of the human genome, discovered experimentally in primary human fibroblasts. Most of these sequences are located far from the transcriptional start sites, with their distribution strongly correlated with genes. The majority of them fit to a consensus motif highly conserved and suitable for predicting possible insulators driven by CTCF in other vertebrate genomes. In addition, CTCF localization is largely invariant across different cell types. Our results provide a resource for investigating insulator function and possible other general and evolutionarily conserved activities of CTCF sites.

Integrating physical and genetic maps: from genomes to interaction networks

Andreas Beyer — 2007-08-18T01:43:38-00:00

Nature Reviews Genetics, Vol. 8, No. 9., pp. 699-710.

Large-scale, saturating insertional mutagenesis of the mouse genome.

Alexander Gragerov — 2007-08-29T07:51:34-00:00

Proc Natl Acad Sci U S A (24 August 2007)

We describe the construction of a large-scale, orderly assembly of mutant ES cells, generated with retroviral insertions and having mutational coverage in >90% of mouse genes. We also describe a method for isolating ES cell clones with mutations in specific genes of interest from this library. This approach, which combines saturating random mutagenesis with targeted selection of mutations in the genes of interest, was successfully applied to the gene families of G protein-coupled receptors (GPCRs) and nuclear receptors. Mutant mouse strains in 60 different GPCRs were generated. Applicability of the technique for the GPCR genes, which on average represent fairly small targets for insertional mutagenesis, indicates the general utility of our approach for the rest of the genome. The method also allows for increased scale and automation for the large-scale production of mutant mice, which could substantially expedite the functional characterization of the mouse genome.

The Binding Sites for the Chromatin Insulator Protein CTCF Map to DNA Methylation-Free Domains Genome-Wide

Rituparna Mukhopadhyay — 2007-01-30T15:50:16-00:00

Genome Res., Vol. 14, No. 8. (1 August 2004), pp. 1594-1602.

All known vertebrate chromatin insulators interact with the highly conserved, multivalent 11-zinc finger nuclear factor CTCF to demarcate expression domains by blocking enhancer or silencer signals in a position-dependent manner. Recent observations document that the properties of CTCF include reading and propagating the epigenetic state of the differentially methylated H19 imprinting control region. To assess whether these findings may reflect a universal role for CTCF targets, we identified more than 200 new CTCF target sites by generating DNA microarrays of clones derived from chromatin-immunopurified (ChIP) DNA followed by ChIP-on-chip hybridization analysis. Target sites include not only known loci involved in multiple cellular functions, such as metabolism, neurogenesis, growth, apoptosis, and signalling, but potentially also heterochromatic sequences. Using a novel insulator trapping assay, we also show that the majority of these targets manifest insulator functions with a continuous distribution of stringency. As these targets are generally DNA methylation-free as determined by antibodies against 5-methylcytidine and a methyl-binding protein (MBD2), a CTCF-based network correlates with genome-wide epigenetic states. 10.1101/gr.2408304

Genome-Wide Analysis of Protein-DNA Interactions.

TH Kim — 2007-06-23T23:16:02-00:00

Annu Rev Genomics Hum Genet, Vol. 7 (2006), pp. 81-102.

The human genome is predominantly composed of nonprotein-coding sequences whose function remains largely undefined. A significant portion of the noncoding DNA is believed to serve as transcriptional regulatory elements that control gene expression in specific cell types at appropriate developmental stages. Identifying these regulatory sequences and determining the mechanisms by which they act present a great challenge in the postgenomic era. Previous investigations using genetic, molecular, and biochemical approaches have uncovered a large number of proteins involved in regulating transcription. Knowledge of the genomic locations of DNA binding for these proteins in the nucleus should define the identity and nature of the transcriptional regulatory sequences and reveal the gene regulatory networks in cells. Chromatin immunoprecipitation (ChIP) is a common method for detecting interactions between a protein and a DNA sequence in vivo. In recent years, this method has been combined with DNA microarrays and other high-throughput technologies to enable genome-wide identification of DNA-binding sites for various nuclear proteins. Here, we review recent advances in ChIP-based methods for genome-wide detection of protein-DNA interactions, and discuss their significance in enhancing our knowledge of the gene regulatory networks and epigenetic mechanisms in cells.

Meiotic crossover hotspots contained in haplotype block boundaries of the mouse genome.

Liisa Kauppi — 2007-08-12T17:51:34-00:00

Proc Natl Acad Sci U S A (9 August 2007)

Fertility requires successful chromosome segregation in meiosis, which in most sexual organisms depends on the formation of appropriately placed crossovers. The nonrandom genome-wide distributions of meiotic recombination events have been examined at the molecular level experimentally in yeast and by inference from linkage disequilibrium patterns in humans. Thus far, no method has existed for pinpointing sites of crossing-over on a genome-wide scale in an experimentally tractable animal whose genome size and complexity models that of humans. Here, we present a genomic approach to identify mouse crossover hotspots, based on targeting haplotype block boundaries. This represents a previously undescribed method potentially applicable to large-scale mouse hotspot identification. Using this method, we have successfully predicted the location of two previously uncharacterized crossover hotspots in male mice. As increasing amounts of single-nucleotide polymorphism data emerge, this approach will be useful for investigating the recombination landscape of the mouse genome.

Distinguishing protein-coding and noncoding genes in the human genome

Michele Clamp — 2007-11-26T23:07:17-00:00

Proceedings of the National Academy of Sciences (26 November 2007), 0709013104.

Although the Human Genome Project was completed 4 years ago, the catalog of human protein-coding genes remains a matter of controversy. Current catalogs list a total of approx24,500 putative protein-coding genes. It is broadly suspected that a large fraction of these entries are functionally meaningless ORFs present by chance in RNA transcripts, because they show no evidence of evolutionary conservation with mouse or dog. However, there is currently no scientific justification for excluding ORFs simply because they fail to show evolutionary conservation: the alternative hypothesis is that most of these ORFs are actually valid human genes that reflect gene innovation in the primate lineage or gene loss in the other lineages. Here, we reject this hypothesis by carefully analyzing the nonconserved ORFsspecifically, their properties in other primates. We show that the vast majority of these ORFs are random occurrences. The analysis yields, as a by-product, a major revision of the current human catalogs, cutting the number of protein-coding genes to approx20,500. Specifically, it suggests that nonconserved ORFs should be added to the human gene catalog only if there is clear evidence of an encoded protein. It also provides a principled methodology for evaluating future proposed additions to the human gene catalog. Finally, the results indicate that there has been relatively little true innovation in mammalian protein-coding genes. 10.1073/pnas.0709013104

Prediction of Gene Expression in Embryonic Structures of Drosophila melanogaster

Anastasia Samsonova — 2007-07-24T18:58:17-00:00

PLoS Computational Biology, Vol. 3, No. 7. (1 July 2007), e144.

Understanding how sets of genes are coordinately regulated in space and time to generate the diversity of cell types that characterise complex metazoans is a major challenge in modern biology. The use of high-throughput approaches, such as large-scale in situ hybridisation and genome-wide expression profiling via DNA microarrays, is beginning to provide insights into the complexities of development. However, in many organisms the collection and annotation of comprehensive in situ localisation data is a difficult and time-consuming task. Here, we present a widely applicable computational approach, integrating developmental time-course microarray data with annotated in situ hybridisation studies, that facilitates the de novo prediction of tissue-specific expression for genes that have no in vivo gene expression localisation data available. Using a classification approach, trained with data from microarray and in situ hybridisation studies of gene expression during Drosophila embryonic development, we made a set of predictions on the tissue-specific expression of Drosophila genes that have not been systematically characterised by in situ hybridisation experiments. The reliability of our predictions is confirmed by literature-derived annotations in FlyBase, by overrepresentation of Gene Ontology biological process annotations, and, in a selected set, by detailed gene-specific studies from the literature. Our novel organism-independent method will be of considerable utility in enriching the annotation of gene function and expression in complex multicellular organisms.

Quantitative analysis of cell nucleus organisation.

C Shiels — 2007-08-08T13:23:15-00:00

PLoS Comput Biol, Vol. 3, No. 7. (27 July 2007)

There are almost 1,300 entries for higher eukaryotes in the Nuclear Protein Database. The proteins' subcellular distribution patterns within interphase nuclei can be complex, ranging from diffuse to punctate or microspeckled, yet they all work together in a coordinated and controlled manner within the three-dimensional confines of the nuclear volume. In this review we describe recent advances in the use of quantitative methods to understand nuclear spatial organisation and discuss some of the practical applications resulting from this work.

Toward a universal microarray: prediction of gene expression through nearest-neighbor probe sequence identification.

Thomas E Royce — 2007-08-11T10:09:31-00:00

Nucleic Acids Res (7 August 2007)

A generic DNA microarray design applicable to any species would greatly benefit comparative genomics. We have addressed the feasibility of such a design by leveraging the great feature densities and relatively unbiased nature of genomic tiling microarrays. Specifically, we first divided each Homo sapiens Refseq-derived gene's spliced nucleotide sequence into all of its possible contiguous 25 nt subsequences. For each of these 25 nt subsequences, we searched a recent human transcript mapping experiment's probe design for the 25 nt probe sequence having the fewest mismatches with the subsequence, but that did not match the subsequence exactly. Signal intensities measured with each gene's nearest-neighbor features were subsequently averaged to predict their gene expression levels in each of the experiment's thirty-three hybridizations. We examined the fidelity of this approach in terms of both sensitivity and specificity for detecting actively transcribed genes, for transcriptional consistency between exons of the same gene, and for reproducibility between tiling array designs. Taken together, our results provide proof-of-principle for probing nucleic acid targets with off-target, nearest-neighbor features.

High-resolution profiling of histone methylations in the human genome.

A Barski — 2007-07-03T10:08:18-00:00

Cell, Vol. 129, No. 4. (18 May 2007), pp. 823-837.

Histone modifications are implicated in influencing gene expression. We have generated high-resolution maps for the genome-wide distribution of 20 histone lysine and arginine methylations as well as histone variant H2A.Z, RNA polymerase II, and the insulator binding protein CTCF across the human genome using the Solexa 1G sequencing technology. Typical patterns of histone methylations exhibited at promoters, insulators, enhancers, and transcribed regions are identified. The monomethylations of H3K27, H3K9, H4K20, H3K79, and H2BK5 are all linked to gene activation, whereas trimethylations of H3K27, H3K9, and H3K79 are linked to repression. H2A.Z associates with functional regulatory elements, and CTCF marks boundaries of histone methylation domains. Chromosome banding patterns are correlated with unique patterns of histone modifications. Chromosome breakpoints detected in T cell cancers frequently reside in chromatin regions associated with H3K4 methylations. Our data provide new insights into the function of histone methylation and chromatin organization in genome function.

A high-resolution map of active promoters in the human genome

Tae Kim — 2005-07-01T19:24:29-00:00

Nature (29 June 2005)

Direct isolation and identification of promoters in the human genome.

TH Kim — 2007-07-24T13:58:29-00:00

Genome Res, Vol. 15, No. 6. (June 2005), pp. 830-839.

Transcriptional regulatory elements play essential roles in gene expression during animal development and cellular response to environmental signals, but our knowledge of these regions in the human genome is limited despite the availability of the complete genome sequence. Promoters mark the start of every transcript and are an important class of regulatory elements. A large, complex protein structure known as the pre-initiation complex (PIC) is assembled on all active promoters, and the presence of these proteins distinguishes promoters from other sequences in the genome. Using components of the PIC as tags, we isolated promoters directly from human cells as protein-DNA complexes and identified the resulting DNA sequences using genomic tiling microarrays. Our experiments in four human cell lines uncovered 252 PIC-binding sites in 44 semirandomly selected human genomic regions comprising 1% (30 megabase pairs) of the human genome. Nearly 72% of the identified fragments overlap or immediately flank 5' ends of known cDNA sequences, while the remainder is found in other genomic regions that likely harbor putative promoters of unannotated transcripts. Indeed, molecular analysis of the RNA isolated from one cell line uncovered transcripts initiated from over half of the putative promoter fragments, and transient transfection assays revealed promoter activity for a significant proportion of fragments when they were fused to a luciferase reporter gene. These results demonstrate the specificity of a genome-wide analysis method for mapping transcriptional regulatory elements and also indicate that a small, yet significant number of human genes remains to be discovered.

An integrated resource for genome-wide identification and analysis of human tissue-specific differentially methylated regions (tDMRs)

Vardhman Rakyan — 2008-06-25T10:34:37-00:00

Genome Res. (24 June 2008), gr.077479.108.

We report a novel resource (methylation profiles of DNA, mPod) for human genome-wide tissue-specific DNA methylation profiles. mPod consists of three fully integrated parts, genome-wide DNA methylation reference profiles of 13 normal somatic tissues, placenta, sperm and an immortalized cell line, a visualization tool that has been integrated with the Ensembl genome browser and a new algorithm for the analysis of immunoprecipitation-based DNA methylation profiles. We demonstrate the utility of our resource by identifying the first comprehensive genome-wide set of tissue-specific differentially methylated regions (tDMRs) that may play a role in cellular identity and the regulation of tissue-specific genome function. We also discuss the implications of our findings with respect to the regulatory potential of regions with varied CpG density, gene expression, transcription factor motifs, gene ontology and correlation with other epigenetic marks such as histone modifications. 10.1101/gr.077479.108

Genome-wide maps of chromatin state in pluripotent and lineage-committed cells

Tarjei Mikkelsen — 2007-07-01T23:06:35-00:00

Nature (01 July 2007)

Systematic discovery of regulatory motifs in conserved regions of the human genome, including thousands of CTCF insulator sites.

X Xie — 2007-05-11T08:32:24-00:00

Proc Natl Acad Sci U S A, Vol. 104, No. 17. (24 April 2007), pp. 7145-7150.

Conserved noncoding elements (CNEs) constitute the majority of sequences under purifying selection in the human genome, yet their function remains largely unknown. Experimental evidence suggests that many of these elements play regulatory roles, but little is known about regulatory motifs contained within them. Here we describe a systematic approach to discover and characterize regulatory motifs within mammalian CNEs by searching for long motifs (12-22 nt) with significant enrichment in CNEs and studying their biochemical and genomic properties. Our analysis identifies 233 long motifs (LMs), matching a total of approximately 60,000 conserved instances across the human genome. These motifs include 16 previously known regulatory elements, such as the histone 3'-UTR motif and the neuron-restrictive silencer element, as well as striking examples of novel functional elements. The most highly enriched motif (LM1) corresponds to the X-box motif known from yeast and nematode. We show that it is bound by the RFX1 protein and identify thousands of conserved motif instances, suggesting a broad role for the RFX family in gene regulation. A second group of motifs (LM2*) does not match any previously known motif. We demonstrate by biochemical and computational methods that it defines a binding site for the CTCF protein, which is involved in insulator function to limit the spread of gene activation. We identify nearly 15,000 conserved sites that likely serve as insulators, and we show that nearby genes separated by predicted CTCF sites show markedly reduced correlation in gene expression. These sites may thus partition the human genome into domains of expression.

Genomic surveys by methylation-sensitive SNP analysis identify sequence-dependent allele-specific DNA methylation.

Kristi Kerkel — 2008-06-25T09:22:52-00:00

Nature genetics (22 June 2008)

Allele-specific DNA methylation (ASM) is a hallmark of imprinted genes, but ASM in the larger nonimprinted fraction of the genome is less well characterized. Using methylation-sensitive SNP analysis (MSNP), we surveyed the human genome at 50K and 250K resolution, identifying ASM as recurrent genotype call conversions from heterozygosity to homozygosity when genomic DNAs were predigested with the methylation-sensitive restriction enzyme HpaII. Using independent assays, we confirmed ASM at 16 SNP-tagged loci distributed across various chromosomes. At 12 of these loci (75%), the ASM tracked strongly with the sequence of adjacent SNPs. Further analysis showed allele-specific mRNA expression at two loci from this methylation-based screen-the vanin and CYP2A6-CYP2A7 gene clusters-both implicated in traits of medical importance. This recurrent phenomenon of sequence-dependent ASM has practical implications for mapping and interpreting associations of noncoding SNPs and haplotypes with human phenotypes.

Transcript annotation in FANTOM3: mouse gene catalog based on physical cDNAs.

N Maeda — 2008-03-08T23:22:28-00:00

PLoS Genet, Vol. 2, No. 4. (April 2006)

The international FANTOM consortium aims to produce a comprehensive picture of the mammalian transcriptome, based upon an extensive cDNA collection and functional annotation of full-length enriched cDNAs. The previous dataset, FANTOM2, comprised 60,770 full-length enriched cDNAs. Functional annotation revealed that this cDNA dataset contained only about half of the estimated number of mouse protein-coding genes, indicating that a number of cDNAs still remained to be collected and identified. To pursue the complete gene catalog that covers all predicted mouse genes, cloning and sequencing of full-length enriched cDNAs has been continued since FANTOM2. In FANTOM3, 42,031 newly isolated cDNAs were subjected to functional annotation, and the annotation of 4,347 FANTOM2 cDNAs was updated. To accomplish accurate functional annotation, we improved our automated annotation pipeline by introducing new coding sequence prediction programs and developed a Web-based annotation interface for simplifying the annotation procedures to reduce manual annotation errors. Automated coding sequence and function prediction was followed with manual curation and review by expert curators. A total of 102,801 full-length enriched mouse cDNAs were annotated. Out of 102,801 transcripts, 56,722 were functionally annotated as protein coding (including partial or truncated transcripts), providing to our knowledge the greatest current coverage of the mouse proteome by full-length cDNAs. The total number of distinct non-protein-coding transcripts increased to 34,030. The FANTOM3 annotation system, consisting of automated computational prediction, manual curation, and final expert curation, facilitated the comprehensive characterization of the mouse transcriptome, and could be applied to the transcriptomes of other species.

Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs

Y Okazaki — 2008-03-08T23:19:20-00:00

Nature, Vol. 420 (December 2002), pp. 563-573.

Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 `transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.

Integrative annotation of 21,037 human genes validated by full-length cDNA clones.

T Imanishi — 2005-11-18T12:04:44-00:00

PLoS Biol, Vol. 2, No. 6. (June 2004)

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology.

Identification and Classification of Conserved RNA Secondary Structures in the Human Genome.

Jakob Skou Pedersen — 2006-04-24T15:27:06-00:00

PLoS Comput Biol, Vol. 2, No. 4. (21 April 2006)

The discoveries of microRNAs and riboswitches, among others, have shown functional RNAs to be biologically more important and genomically more prevalent than previously anticipated. We have developed a general comparative genomics method based on phylogenetic stochastic context-free grammars for identifying functional RNAs encoded in the human genome and used it to survey an eight-way genome-wide alignment of the human, chimpanzee, mouse, rat, dog, chicken, zebra-fish, and puffer-fish genomes for deeply conserved functional RNAs. At a loose threshold for acceptance, this search resulted in a set of 48,479 candidate RNA structures. This screen finds a large number of known functional RNAs, including 195 miRNAs, 62 histone 3'UTR stem loops, and various types of known genetic recoding elements. Among the highest-scoring new predictions are 169 new miRNA candidates, as well as new candidate selenocysteine insertion sites, RNA editing hairpins, RNAs involved in transcript auto regulation, and many folds that form singletons or small functional RNA families of completely unknown function. While the rate of false positives in the overall set is difficult to estimate and is likely to be substantial, the results nevertheless provide evidence for many new human functional RNAs and present specific predictions to facilitate their further characterization.

Local RNA base pairing probabilities in large sequences

Stephan Bernhart — 2006-02-27T16:11:45-00:00

Bioinformatics, Vol. 22, No. 5. (1 March 2006), pp. 614-615.

Prediction of locally stable RNA secondary structures for genome-wide surveys.

IL Hofacker — 2005-03-09T21:14:48-00:00

Bioinformatics, Vol. 20, No. 2. (22 January 2004), pp. 186-190.

MOTIVATION: Recently novel classes of functional RNAs, most prominently the miRNAs have been discovered, strongly suggesting that further types of functional RNAs are still hidden in the recently completed genomic DNA sequences. Only few techniques are known, however, to survey genomes for such RNA genes. When sufficiently similar sequences are not available for comparative approaches the only known remedy is to search directly for structural features. RESULTS: We present here efficient algorithms for computing locally stable RNA structures at genome-wide scales. Both the minimum energy structure and the complete matrix of base pairing probabilities can be computed in theta(N x L2) time and theta(N + L2) memory in terms of the length N of the genome and the size L of the largest secondary structure motifs of interest. In practice, the 100 Mb of the complete genome of Caenorhabditis elegans can be folded within about half a day on a modern PC with a search depth of L = 100. This is sufficient example for a survey for miRNAs. AVAILABILITY: The software described in this contribution will be available for download at http://www.tbi.univie.ac.at/~ivo/RNA/ as part of the Vienna RNA Package.

Genome-wide association studies for complex traits: consensus, uncertainty and challenges

Mark Mccarthy — 2008-04-18T17:25:58-00:00

Nature Reviews Genetics, Vol. 9, No. 5., pp. 356-369.