Genome Research

A high-resolution, nucleosome position map of C. elegans reveals a lack of universal sequence-dictated positioning.

Publication Date: 2008 May 13 PMID: 18477713
Authors: Valouev, A. - Ichikawa, J. - Tonthat, T. - Stuart, J. - Ranade, S. - Peckham, H. - Zeng, K. - Malek, J. A. - Costa, G. - McKernan, K. - Sidow, A. - Fire, A. - Johnson, S. M.
Journal: Genome Res

Using the massively parallel technique of Sequencing by Oligonucleotide Ligation and Detection (SOLiD) we have assessed the in vivo positions of more than 44 million putative nucleosome cores in the multicellular genetic model organism Caenorhabditis elegans. These analyses provide a global view of the chromatin architecture of a multicellular animal at extremely high density and resolution. While we observe some degree of reproducible positioning throughout the genome in our mixed stage population of animals, we note that the major chromatin feature in the worm is a diversity of allowed nucleosome positions at the vast majority of individual loci. While absolute positioning of nucleosomes can vary substantially, relative positioning of nucleosomes (in a repeated array structure likely to be maintained at least in part by steric constraints) appears to be a significant property of chromatin structure. The high density of nucleosomal reads enabled a substantial extension of previous analysis describing the usage of individual oligonucleotide sequences along the span of the nucleosome core and linker. We release this dataset, via the UCSC Genome Browser, as a resource for the high-resolution analysis of chromatin conformation and DNA accessibility at individual loci within the C. elegans genome.

post to: CiteULike

A microRNA catalog of the developing chicken embryo identified by a deep sequencing approach.

Publication Date: 2008 May 9 PMID: 18469162
Authors: Glazov, E. A. - Cottee, P. A. - Barris, W. C. - Moore, R. J. - Dalrymple, B. P. - Tizard, M. L.
Journal: Genome Res

MicroRNA (miRNA) and other types of small regulatory RNAs play a crucial role in the regulation of gene expression in eukaryotes. Several distinct classes of small regulatory RNAs have been discovered in recent years. To extend the repertoire of small regulatory RNAs characterized in chickens we used a deep sequencing approach developed by Solexa (now Illumina Inc.). We sequenced three small RNA libraries prepared from different developmental stages of the chicken embryo (days five, seven, and nine) to produce over 9.5 million short sequence reads. We developed a bioinformatics pipeline to distinguish authentic mature miRNA sequences from other classes of small RNAs and short RNA fragments represented in the sequencing data. Using this approach we detected almost all of the previously known chicken miRNAs and their respective miRNA* sequences. In addition we discovered 449 new chicken miRNAs including 88 miRNA candidates. Of these, 430 miRNAs appear to be specific to the avian lineage. Another six new miRNAs had evidence of evolutionary conservation in at least one vertebrate species outside of the bird lineage. The remaining 13 putative miRNAs appear to represent chicken orthologs of known vertebrate miRNAs. We discovered 39 additional putative miRNA candidates originating from miRNA generating intronic sequences known as mirtrons.

post to: CiteULike

Conservation of small RNA pathways in platypus.

Publication Date: 2008 May 7 PMID: 18463306
Authors: Murchison, E. P. - Kheradpour, P. - Sachidanandam, R. - Smith, C. - Hodges, E. - Xuan, Z. - Kellis, M. - Grutzner, F. - Stark, A. - Hannon, G. J.
Journal: Genome Res

Small RNA pathways play evolutionarily conserved roles in gene regulation and defense from parasitic nucleic acids. The character and expression patterns of small RNAs show conservation throughout animal lineages, but specific animal clades also show variations on these recurring themes, including species-specific small RNAs. The monotremes, with only platypus and four species of echidna as extant members, represent the basal branch of the mammalian lineage. Here, we examine the small RNA pathways of monotremes by deep sequencing of six platypus and echidna tissues. We find that highly conserved microRNA species display their signature tissue-specific expression patterns. In addition, we find a large rapidly evolving cluster of microRNAs on platypus chromosome X1, which is unique to monotremes. Platypus and echidna testes contain a robust Piwi-interacting (piRNA) system, which appears to be participating in ongoing transposon defense.

post to: CiteULike

Origin of INSL3-mediated testicular descent in therian mammals.

Publication Date: 2008 May 7 PMID: 18463305
Authors: Park, J. I. - Semyonov, J. - Chang, C. L. - Yi, W. - Warren, W. - Hsu, S. Y.
Journal: Genome Res

Testicular descent is a unique physiological adaptation found in therian mammals allowing optimal spermatogenesis below core body temperature. Recent studies show that INSL3, produced by Leydig cells, and its receptor LGR8 (RXFP2) are essential for mediating the transabdominal phase of testicular descent during early development. However, the origin and genetic basis for this physiological adaptation is not clear. Using syntenic mapping and the functional characterization of contemporary and resurrected relaxin family hormones, we show that derivation of INSL3-mediated testicular descent involved the duplication of an ancestral RLN3-like gene that encodes an indiscriminate ligand for LGR7 (RXFP1) and LGR8. This event was followed by acquisition of the LGR7-selective characteristics by a daughter gene (RLN3) prior to the evolution of the common ancestor of monotremes, marsupials, and placentals. A subsequent mutation of the other daughter gene (INSL3) occurred before the emergence of therian mammals, which then led to the derivation of the reciprocal LGR8-specific characteristics of INSL3. The stepwise evolution of these independent signaling pathways through gene duplication and subsequent divergence is consistent with Darwinian theory of selection and adaptation, and the temporal proximity suggests an association between these genetic events and the concurrent evolution of testicular descent in ancestral therian mammals.

post to: CiteULike

Defensins and the convergent evolution of platypus and reptile venom genes.

Publication Date: 2008 May 7 PMID: 18463304
Authors: Whittington, C. M. - Papenfuss, A. T. - Bansal, P. - Torres, A. M. - Wong, E. S. - Deakin, J. E. - Graves, T. - Alsop, A. - Schatzkamer, K. - Kremitzki, C. - Ponting, C. P. - Temple-Smith, P. - Warren, W. C. - Kuchel, P. W. - Belov, K.
Journal: Genome Res

When the platypus (Ornithorhynchus anatinus) was first discovered, it was thought to be a taxidermist's hoax, as it has a blend of mammalian and reptilian features. It is a most remarkable mammal, not only because it lays eggs but also because it is venomous. Rather than delivering venom through a bite, as do snakes and shrews, male platypuses have venomous spurs on each hind leg. The platypus genome sequence provides a unique opportunity to unravel the evolutionary history of many of these interesting features. While searching the platypus genome for the sequences of antimicrobial defensin genes, we identified three Ornithorhynchus venom defensin-like peptide (OvDLP) genes, which produce the major components of platypus venom. We show that gene duplication and subsequent functional diversification of beta-defensins gave rise to these platypus OvDLPs. The OvDLP genes are located adjacent to the beta-defensins and share similar gene organization and peptide structures. Intriguingly, some species of snakes and lizards also produce venoms containing similar molecules called crotamines and crotamine-like peptides. This led us to trace the evolutionary origins of other components of platypus and reptile venom. Here we show that several venom components have evolved separately in the platypus and reptiles. Convergent evolution has repeatedly selected genes coding for proteins containing specific structural motifs as templates for venom molecules.

post to: CiteULike

Retroposed SNOfall--A mammalian-wide comparison of platypus snoRNAs.

Publication Date: 2008 May 7 PMID: 18463303
Authors: Schmitz, J. - Zemann, A. - Churakov, G. - Kuhl, H. - Grutzner, F. - Reinhardt, R. - Brosius, J.
Journal: Genome Res

Diversification of mammalian species began more than 160 million years ago when the egg-laying monotremes diverged from live bearing mammals. The duck-billed platypus (Ornithorhynchus anatinus) and echidnas are the only potential contemporary witnesses of this period and, thereby, provide a unique insight into mammalian genome evolution. It has become clear that small RNAs are major regulatory agents in eukaryotic cells, and the significant role of non-protein-coding (npc) RNAs in transcription, processing, and translation is now well accepted. Here we show that the platypus genome contains more than 200 small nucleolar (sno) RNAs among hundreds of other diverse npcRNAs. Their comparison among key mammalian groups and other vertebrates enabled us to reconstruct a complete temporal pathway of acquisition and loss of these snoRNAs. In platypus we found cis- and trans-duplication distribution patterns for snoRNAs, which have not been described in any other vertebrates but are known to occur in nematodes. An exciting novelty in platypus is a snoRNA-derived retroposon (termed snoRTE) that facilitates a very effective dispersal of an H/ACA snoRNA via RTE-mediated retroposition. From more than 40,000 detected full-length and truncated genomic copies of this snoRTE, at least 21 are processed into mature snoRNAs. High-copy retroposition via multiple host gene-promoted transcription units is a novel pathway for combining housekeeping function and SINE-like dispersal and reveals a new dimension in the evolution of novel snoRNA function.

post to: CiteULike

Bird-like sex chromosomes of platypus imply recent origin of mammal sex chromosomes.

Publication Date: 2008 May 7 PMID: 18463302
Authors: Veyrunes, F. - Waters, P. D. - Miethke, P. - Rens, W. - McMillan, D. - Alsop, A. E. - Grutzner, F. - Deakin, J. E. - Whittington, C. M. - Schatzkamer, K. - Kremitzki, C. L. - Graves, T. - Ferguson-Smith, M. A. - Warren, W. - Marshall Graves, J. A.
Journal: Genome Res

In therian mammals (placentals and marsupials), sex is determined by an XX female: XY male system, in which a gene (SRY) on the Y affects male determination. There is no equivalent in other amniotes, although some taxa (notably birds and snakes) have differentiated sex chromosomes. Birds have a ZW female: ZZ male system with no homology with mammal sex chromosomes, in which dosage of a Z-borne gene (possibly DMRT1) affects male determination. As the most basal mammal group, the egg-laying monotremes are ideal for determining how the therian XY system evolved. The platypus has an extraordinary sex chromosome complex, in which five X and five Y chromosomes pair in a translocation chain of alternating X and Y chromosomes. We used physical mapping to identify genes on the pairing regions between adjacent X and Y chromosomes. Most significantly, comparative mapping shows that, contrary to earlier reports, there is no homology between the platypus and therian X chromosomes. Orthologs of genes in the conserved region of the human X (including SOX3, the gene from which SRY evolved) all map to platypus chromosome 6, which therefore represents the ancestral autosome from which the therian X and Y pair derived. Rather, the platypus X chromosomes have substantial homology with the bird Z chromosome (including DMRT1) and to segments syntenic with this region in the human genome. Thus, platypus sex chromosomes have strong homology with bird, but not to therian sex chromosomes, implying that the therian X and Y chromosomes (and the SRY gene) evolved from an autosomal pair after the divergence of monotremes only 166 million years ago. Therefore, the therian X and Y are more than 145 million years younger than previously thought.

post to: CiteULike

Transcription induces strand specific mutations at the 5' end of human genes.

Publication Date: 2008 May 7 PMID: 18463301
Authors: Polak, P. - Arndt, P. F.
Journal: Genome Res

A regional analysis of nucleotide substitution rates along human genes and their flanking regions allows us to quantify the effect of mutational mechanisms associated with transcription in germline cells. Our analysis reveals three distinct patterns of substitution rates. First, a sharp decline in CpG methylation deamination rate, which is observed in the vicinity of the 5'end of genes. Second, a strand asymmetry in complementary substitution rates, which extends from the 5'end to 1 kbp downstream to the 3'end, associated with transcription coupled repair. Finally, a localized strand asymmetry, an excess of C->T over G->A substitution in the non-template strand confined to the first 1-2 kbp downstream of the 5'end of genes. We hypothesize that higher exposure of the non-template strand near the 5'end of genes leads to a higher cytosine deamination rate. Up to now only the somatic hyper mutation (SHM) pathway has been known to mediate localized and strand specific mutagenic processes associated with transcription in mammalia. The mutational patterns in SHM are induced by cytosine deaminase that just targets single stranded DNA. This DNA conformation is induced by R-loops, which preferentially occur at the 5'ends of genes. We predict that R-loops are extensively formed in the beginning of transcribed regions in germline cells.

post to: CiteULike

The extensive and condition-dependent nature of epistasis among whole-genome-duplicates in yeast.

Publication Date: 2008 May 7 PMID: 18463300
Authors: Musso, G. - Costanzo, M. - Huangfu, M. - Smith, A. M. - Paw, J. - San Luis, B. J. - Boone, C. - Giaever, G. - Nislow, C. - Emili, A. - Zhang, Z.
Journal: Genome Res

Since complete redundancy between extant duplicates (paralogs) is evolutionarily unfavorable, some degree of functional congruency is eventually lost. However, in budding yeast, experimental evidence collected for duplicated metabolic enzymes and in global physical interaction surveys had suggested widespread functional overlap between paralogs. While maintained functional overlap is thought to confer robustness against genetic mutation and facilitate environmental adaptability, it has yet to be determined what properties define paralogs that can compensate for the phenotypic consequence of deleting a sister gene, how extensive this epistasis is, and how adaptable it is towards alternate environmental states. To this end, we have performed a comprehensive experimental analysis of epistasis as indicated by aggravating genetic interactions between paralogs resulting from an ancient Whole-Genome Duplication (WGD) event occurring in the budding yeast S. cerevisiae, and thus were able to compare properties of large numbers of epistatic and non-epistatic paralogs with identical evolutionary times since divergence. We found that over one-third (140) of the 399 examinable WGD paralog pairs were epistatic under standard laboratory conditions, and that additional cases of epistasis became obvious only under media conditions designed to induce cellular stress. Despite a significant increase in within-species sequence co-conservation, analysis of protein interactions revealed that paralogs epistatic under standard laboratory conditions were not more functionally overlapping than those non-epistatic. As experimental condition impacted the functional categorization of paralogs deemed epistatic, and only a fraction of potential stress conditions have been interrogated here, we hypothesize that many epistatic relationships remain unresolved.

post to: CiteULike

DXZ4 chromatin adopts an opposing conformation to that of the surrounding chromosome and acquires a novel inactive X specific role involving CTCF and anti-sense transcripts.

Publication Date: 2008 May 2 PMID: 18456864
Authors: Chadwick, B. P.
Journal: Genome Res

Macrosatellite DNA is composed of large repeat units, arranged in tandem over hundreds of kilobases. The macrosatellite repeat DXZ4, localized at Xq23-24, consists of 50-100 copies of a CpG rich 3kb monomer. Here I report that on the active X chromosome (Xa), DXZ4 is organized into constitutive heterochromatin characterized by a highly organized pattern of H3K9me3. DXZ4 is expressed from both strands and generates an antisense transcript which is processed into small RNAs that directly correlate with H3K9me3 nucleosomes. In contrast, on the inactive X chromosome (Xi) a proportion of DXZ4 is packaged into euchromatin characterized by H3K4me2 and H3K9Ac. The Xi copy of DXZ4 is bound by the chromatin insulator CTCF within a sequence that unidirectionally blocks enhancer-promoter communication. Immediately adjacent to the CTCF binding site is a bi-directional promoter that, like the sequence flanking the CTCF binding region, is completely devoid of CpG methylation on the Xi. As on the Xa, both strands are expressed, but longer antisense transcripts can be detected in addition to the processed small RNAs. The euchromatic organization of DXZ4 on the otherwise heterochromatic Xi, its binding of CTCF and its function as a unidirectional insulator suggests that this macrosatellite has acquired a novel function unique to the process of X chromosome inactivation.

post to: CiteULike

Translation from nonautonomous type IAP retrotransposon is a critical determinant of transposition activity: Implication for retrotransposon-mediated genome evolution.

Publication Date: 2008 May 2 PMID: 18456863
Authors: Saito, E. S. - Keng, V. W. - Takeda, J. - Horie, K.
Journal: Genome Res

Retrotransposons constitute a major component of the genome and their proliferation significantly impacts genome evolution. Retrotransposons can propagate autonomously or nonautonomously. Nonautonomous type transposition occurs through trans-complementation by autonomous type retrotransposons. While autonomous type retrotransposons have been studied extensively, the translation products from nonautonomous type retrotransposons are not well characterized. In a previous study, we isolated both autonomous and nonautonomous type intracisternal A particle (IAP) elements from the mouse genome and established a tissue culture assay to examine trans-complementation of nonautonomous type IAP element. Using this system in the present study, we determined an active role for the translation product from nonautonomous type IAP element. Point mutations that either eliminated or truncated the IAP protein were introduced and their effects on trans-complementation were examined. Trans-complementation efficiency correlated with the expression of nonautonomous type IAP protein. The effect of nonautonomous type IAP protein was observed only when it was provided in cis, suggesting an interaction of nonautonomous type IAP protein and its transcript immediately after transcription. Interaction of autonomous and nonautonomous type IAP proteins was demonstrated by immunostaining and coimmunoprecipitation assay. Based on these findings, we propose a model in which nonautonomous type IAP protein associates with its transcript, recruits autonomous type IAP protein, and promotes the assembly of transposition competent IAP particle. The active role of the nonautonomous type IAP protein revealed in this study may provide a new insight into retrotransposon proliferation within the genome.

post to: CiteULike

A systematic analysis of intronic sequences downstream of 5' splice sites reveals a widespread role for U-rich motifs and TIA1/TIAL1 proteins in alternative splicing regulation.

Publication Date: 2008 May 2 PMID: 18456862
Authors: Aznarez, I. - Barash, Y. - Shai, O. - He, D. - Zielenski, J. - Tsui, L. C. - Parkinson, J. - Frey, B. - Rommens, J. M. - Blencowe, B.
Journal: Genome Res

To identify human intronic sequences associated with 5' splice site recognition, we performed a systematic search for motifs enriched in introns downstream of both constitutive and alternative cassette exons. Significant enrichment was observed for U-rich motifs within 100 nucleotides downstream of 5' splice sites of both classes of exons, with the highest enrichment between positions +6 and +30. Exons adjacent to U-rich intronic motifs contain lower frequencies of exonic splicing enhancers and higher frequencies of exonic splicing silencers, compared to exons not followed by U-rich intronic motifs. These findings motivated us to explore the possibility of a widespread role for U-rich motifs in promoting exon inclusion. Since cytotoxic granule-associated RNA binding protein (TIA1) and TIA1-like 1 (TIAL1; also known as "TIAR") were previously shown in vitro to bind to U-rich motifs downstream of 5' splice sites, and to facilitate 5' splice site recognition in vitro and in vivo, we investigated whether these factors function more generally in the regulation of splicing of exons followed by U-rich intronic motifs. Simultaneous knockdown of TIA1 and TIAL1 resulted in increased skipping of 36/41 (87.8%) of alternatively spliced exons associated with U-rich motifs, but did not affect 32/33 (97%) alternatively spliced exons that are not associated with U-rich motifs. The increase in exon skipping correlated with the proximity of the first U-rich motif and the overall "U-richness" of the adjacent intronic region. The majority of the alternative splicing events regulated by TIA1/TIAL1 are conserved in mouse and the corresponding genes are associated with diverse cellular functions. Based on our results, we estimate that ~ 15% of alternative cassette exons are regulated by TIA1/TIAL1 via U-rich intronic elements.

post to: CiteULike

Two strategies for gene regulation by promoter nucleosomes.

Publication Date: 2008 Apr 30 PMID: 18448704
Authors: Tirosh, I. - Barkai, N.
Journal: Genome Res

Chromatin structure is central for the regulation of gene expression, but its genome-wide organization is only beginning to be understood. Here, we examine the connection between patterns of nucleosome occupancy and the capacity to modulate gene expression upon changing conditions, i.e. transcriptional plasticity. By analyzing a genome-wide data of nucleosome positioning in yeast, we find that the presence of nucleosomes close to the transcription start site is associated with high transcriptional plasticity, while nucleosomes at more distant upstream positions are negatively correlated with transcriptional plasticity. Based on this, we identify two typical promoter structures associated with low or high plasticity, respectively. The first class is characterized by a relatively large nucleosome free region close to the start site coupled with well-positioned nucleosomes further upstream, whereas the second class displays a more evenly distributed and dynamic nucleosome positioning, with high occupancy close to the start site. The two classes are further distinguished by multiple promoter features, including histone turnover, binding sites location, H2A.Z occupancy, expression noise and expression diversity. Analysis of nucleosome positioning in human promoters reproduce the main observations. Our results suggest two distinct strategies for gene regulation by chromatin, which are selectively employed by different genes.

post to: CiteULike

Evolutionary dynamics of segmental duplications from human Y-chromosomal euchromatin/heterochromatin transition regions.

Publication Date: 2008 Apr 29 PMID: 18445620
Authors: Kirsch, S. - Munch, C. - Jiang, Z. - Cheng, Z. - Chen, L. - Batz, C. - Eichler, E. E. - Schempp, W.
Journal: Genome Res

Human chromosomal regions enriched in segmental duplications are subject to extensive genomic reorganization. Such regions are particularly informative for illuminating the evolutionary history of a given chromosome. We have analyzed 866-kb of Y-chromosomal non-palindromic segmental duplications delineating four euchromatin/heterochromatin transitions regions (Yp11.2/Yp11.1, Yq11.1/Yq11.21, Yq11.23/Yq12, and Yq12/PAR2). Several computational methods were applied to decipher the segmental duplication architecture and identify the ancestral origin of the 41 different duplicons. Combining computational and comparative FISH analysis, we reconstruct the evolutionary history of these regions. Our analysis indicates a continuous process of transposition of duplicated sequences onto the evolving higher primate Y chromosome providing unique insights into the development of species-specific Y-chromosomal and autosomal duplicons Phylogenetic sequence comparisons show that duplicons of the human Yp11.2/Yp11.1 region were already present in the macaque-human ancestor as multiple paralogs located predominantly in subtelomeric regions. In contrast, duplicons from the Yq11.1/Yq11.21, Yq11.23/Yq12 and Yq12/PAR2 regions show no evidence of duplication in rhesus macaque, but map to the pericentromeric regions in the chimpanzee and human. This suggests an evolutionary shift in the direction of duplicative transposition events from subtelomeric in Old World monkeys to pericentromeric in the human/ape lineage. Extensive chromosomal relocation of autosomal-duplicated sequences from euchromatin/heterochromatin transition regions to interstitial regions as demonstrated on the pygmy chimpanzee Y chromosome support a model where substantial reorganization and amplification of duplicated sequences may contribute to speciation.

post to: CiteULike

Identification of active transcriptional regulatory modules by the functional assay of DNA from nucleosome-free regions.

Publication Date: 2008 Apr 25 PMID: 18441229
Authors: Yaragatti, M. - Basilico, C. - Dailey, L.
Journal: Genome Res

The identification of transcriptional regulatory modules within mammalian genomes is a prerequisite to understanding the mechanisms controlling regulated gene expression. While high-throughput microarray- and sequencing-based approaches have been used to map the genomic locations of sites of nuclease hypersensitivity or target DNA sequences bound by specific protein factors, the identification of regulatory elements using functional assays, which would provide important complementary data, has been relatively rare. Here we present a method that permits the functional identification of active transcriptional regulatory modules using a simple procedure for the isolation and analysis of DNA derived from nucleosome-free regions (NFRs), the 2% of the cellular genome that contains these elements. The more than 100 new active regulatory DNAs identified in this manner from F9 cells correspond to both promoter-proximal and distal elements, and display several features predicted for endogenous transcriptional regulators, including localization within DNase-accessible chromatin and CpG islands, and proximity to expressed genes. Furthermore, comparison with published ChIP-seq data of ES-cell chromatin shows that the functional elements we identified correspond with genomic regions enriched for H3K4me3, a histone modification associated with active transcriptional regulatory elements, and that the correspondence of H3K4me3 with our promoter-distal elements is largely ES-cell specific. The majority of the distal elements exhibit enhancer activity. Importantly, these functional DNA fragments are an average 149 bp in length, greatly facilitating future applications to identify transcription factor binding sites mediating their activity. Thus, this approach provides a tool for the high-resolution identification of the functional components of active promoters and enhancers.

post to: CiteULike

Direct targets of the TRP63 transcription factor revealed by a combination of gene expression profiling and reverse engineering.

Publication Date: 2008 Apr 25 PMID: 18441228
Authors: Della Gatta, G. - Bansal, M. - Ambesi-Impiombato, A. - Antonini, D. - Missero, C. - di Bernardo, D.
Journal: Genome Res

Genome-wide identification of bona-fide targets of transcription factors in mammalian cells is still a challenge. We present a novel integrated computational and experimental approach to identify direct targets of a transcription factor. This consists of measuring time-course (dynamic) gene expression profiles upon perturbation of the transcription factor under study, and in applying a novel "reverse-engineering" algorithm (TSNI) to rank genes according to their probability of being direct targets. Using primary keratinocytes as a model system, we identified novel transcriptional target genes of TRP63, a crucial regulator of skin development. TSNI-predicted TRP63 target genes were validated by Trp63 knockdown and by ChIP-chip to identify TRP63-bound regions in vivo. Our study revealed that short sampling times, in the order of minutes, are needed to capture the dynamics of gene expression in mammalian cells. We show that TRP63 transiently regulates a subset of its direct targets, thus highlighting the importance of considering temporal dynamics when identifying transcriptional targets. Using this approach, we uncovered a previously unsuspected transient regulation of the AP-1 complex by TRP63 through direct regulation of a subset of AP-1 components. The integrated experimental and computational approach described here is readily applicable to other transcription factors in mammalian systems and is complementary to genome-wide identification of transcription-factor binding sites.

post to: CiteULike

Detection and characterization of silencers and enhancer-blockers in the greater CFTR locus.

Publication Date: 2008 Apr 24 PMID: 18436892
Authors: Petrykowska, H. - Vockley, C. - Elnitski, L.
Journal: Genome Res

Silencers and enhancer-blockers (EBs) are cis-acting, negative regulatory elements (NREs) that control interactions between promoters and enhancers. Although relatively uncharacterized in terms of biological mechanisms, these elements are likely to be abundant in the genome. We developed an experimental strategy to identify silencers and EBs using transient transfection assays. A known insulator and EB from the chicken beta-globin locus, cHS4, served as a control element for these assays. We examined forty-seven sequences from a 1.8 Mb region of human chromosome 7 for silencer and EB activities. The majority of functional elements displayed directional and promoter-specific activities. A limited number of sequences acted in a dual manner, as both silencers and EBs. We examined genomic data, epigenetic modifications and sequence motifs within these regions. Strong silencer elements contained a novel CT-rich motif, often in multiple copies. Deletion of the motif from three regions caused a measurable loss of silencing ability in these sequences. Moreover, five duplicate occurrences of this motif were identified in the cHS4 insulator. These motifs provided an explanation for an uncharacterized silencing activity we measured in the insulator element. Overall, we identified 15 novel NREs, which contribute new insights into the prevalence and composition of sequences that negatively regulate gene expression.

post to: CiteULike

Comparative proteogenomics: Combining mass spectrometry and comparative genomics to analyze multiple genomes.

Publication Date: 2008 Apr 21 PMID: 18426904
Authors: Gupta, N. - Benhamida, J. - Bhargava, V. - Goodman, D. - Kain, E. - Kerman, I. - Nguyen, N. - Ollikainen, N. - Rodriguez, J. - Wang, J. - Lipton, M. S. - Romine, M. - Bafna, V. - Smith, R. D. - Pevzner, P. A.
Journal: Genome Res

Recent proliferation of low-cost DNA sequencing techniques will soon lead to an explosive growth in the number of sequenced genomes and will turn manual annotations into a luxury. Mass spectrometry recently emerged as a valuable technique for proteogenomic annotations that improves on the state-of-the-art in predicting genes and other features. However, previous proteogenomic approaches were limited to a single genome and did not take advantage of analyzing mass spectrometry data from multiple genomes at once. We show that such comparative proteogenomics approach (like comparative genomics) allows one to address the problems that remained beyond the reach of the traditional "single proteome" approach in mass-spectrometry. In particular, we show how comparative proteogenomics addresses the notoriously difficult problem of "one-hit-wonders" in proteomics, improves on the existing gene prediction tools in genomics and allows identification of rare post-translational modifications. We therefore argue that complementing DNA sequencing projects by comparative proteogenomics projects can be a viable approach to improve both genomic and proteomic annotations.

post to: CiteULike

Identification of the source of ancient remains through genomic sequencing.

Publication Date: 2008 Apr 21 PMID: 18426903
Authors: Blow, M. J. - Zhang, T. - Woyke, T. - Spelle, C. F. - Krivoshapkin, A. - Yang, D. - Derevianko, A. - Rubin, E. M.
Journal: Genome Res

Studies of ancient DNA have been hindered by the preciousness of remains, the small quantities of undamaged DNA accessible, and the limitations associated with conventional PCR amplification. In these studies, we developed and applied a genome-wide adapter-mediated emulsion PCR amplification protocol to ancient mammalian samples between 45,000 and 69,000 years old. Using GS20 and Solexa sequencing technologies, we examined over one hundred megabases of DNA from amplified extracts, revealing unbiased sequence coverage with substantial amounts of non-redundant nuclear sequences from the sample sources and negligible levels of human contamination. We consistently recorded over five hundred-fold increases such that nanogram quantities of starting material could be amplified to microgram quantities. Application of our protocol to a 50,000 year old uncharacterized bone sample that was unsuccessful in mitochondrial PCR provided sufficient nuclear sequences for comparison with extant mammals and subsequent phylogenetic classification of the remains. The combined use of emulsion PCR amplification and high throughput sequencing allows for the generation of large quantities DNA sequence data from ancient remains. Using such techniques, even small amounts of ancient remains with low levels of endogenous DNA preservation may yield substantial quantities of nuclear DNA, enabling novel applications of ancient DNA genomics to the investigation of extinct phyla.

post to: CiteULike

Genetic-linkage mapping of complex hereditary disorders to a whole-genome molecular-interaction network.

Publication Date: 2008 Apr 16 PMID: 18417725
Authors: Iossifov, I. - Zheng, T. - Baron, M. - Gilliam, T. C. - Rzhetsky, A.
Journal: Genome Res

Common hereditary neurodevelopmental disorders such as autism, bipolar disorder, and schizophrenia are, most likely, both genetically multifactorial and heterogeneous. Because of these characteristics traditional methods for genetic analysis fail when applied to such diseases. To address the problem we propose a novel probabilistic framework that combines the standard genetic linkage formalism with whole-genome molecular-interaction data to predict pathways or networks of interacting genes that contribute to common heritable disorders. We apply the model to three large genotype-phenotype datasets, and identify a small number of highly-significant candidate genes for autism (24), bipolar disorder (21) and schizophrenia (25), and predict a number of gene targets likely to be shared among the disorders.

post to: CiteULike

Rapid comparative genomic analysis for clinical microbiology: The Francisella tularensis paradigm.

Publication Date: 2008 May PMID: 18407970
Authors: La Scola, B. - Elkarkouri, K. - Li, W. - Wahab, T. - Fournous, G. - Rolain, J. M. - Biswas, S. - Drancourt, M. - Robert, C. - Audic, S. - Lofdahl, S. - Raoult, D.
Journal: Genome Res

It is critical to avoid delays in detecting strain manipulations, such as the addition/deletion of a gene or modification of genes for increased virulence or antibiotic resistance, using genome analysis during an epidemic outbreak or a bioterrorist attack. Our objective was to evaluate the efficiency of genome analysis in such an emergency context by using contigs produced by pyrosequencing without time-consuming finishing processes and comparing them to available genomes for the same species. For this purpose, we analyzed a clinical isolate of Francisella tularensis subspecies holarctica (strain URFT1), a potential biological weapon, and compared the data obtained with available genomic sequences of other strains. The technique provided 1,800,530 bp of assembled sequences, resulting in 480 contigs. We found by comparative analysis with other strains that all the gaps but one in the genome sequence were caused by repeats. No new genes were found, but a deletion was detected that included three putative genes and part of a fourth gene. The set of 35 candidate LVS virulence attenuation genes was identified, as well as a DNA gyrase mutation associated with quinolone resistance. Selection for variable sequences in URFT1 allowed the design of a strain-specific, highly effective typing system that was applied to 74 strains and six clinical specimens. The analysis presented herein may be completed within approximately 6 wk, a duration compatible with that required by an urgent context. In the bioterrorism context, it allows the rapid detection of strain manipulation, including intentionally added virulence genes and genes that support antibiotic resistance.

post to: CiteULike

Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis.

Publication Date: 2008 May PMID: 18403782
Authors: Stinear, T. P. - Seemann, T. - Harrison, P. F. - Jenkin, G. A. - Davies, J. K. - Johnson, P. D. - Abdellah, Z. - Arrowsmith, C. - Chillingworth, T. - Churcher, C. - Clarke, K. - Cronin, A. - Davis, P. - Goodhead, I. - Holroyd, N. - Jagels, K. - Lord, A. - Moule, S. - Mungall, K. - Norbertczak, H. - Quail, M. A. - Rabbinowitsch, E. - Walker, D. - White, B. - Whitehead, S. - Small, P. L. - Brosch, R. - Ramakrishnan, L. - Fischbach, M. A. - Parkhill, J. - Cole, S. T.
Journal: Genome Res

Mycobacterium marinum, a ubiquitous pathogen of fish and amphibia, is a near relative of Mycobacterium tuberculosis, the etiologic agent of tuberculosis in humans. The genome of the M strain of M. marinum comprises a 6,636,827-bp circular chromosome with 5424 CDS, 10 prophages, and a 23-kb mercury-resistance plasmid. Prominent features are the very large number of genes (57) encoding polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) and the most extensive repertoire yet reported of the mycobacteria-restricted PE and PPE proteins, and related-ESX secretion systems. Some of the NRPS genes comprise a novel family and seem to have been acquired horizontally. M. marinum is used widely as a model organism to study M. tuberculosis pathogenesis, and genome comparisons confirmed the close genetic relationship between these two species, as they share 3000 orthologs with an average amino acid identity of 85%. Comparisons with the more distantly related Mycobacterium avium subspecies paratuberculosis and Mycobacterium smegmatis reveal how an ancestral generalist mycobacterium evolved into M. tuberculosis and M. marinum. M. tuberculosis has undergone genome downsizing and extensive lateral gene transfer to become a specialized pathogen of humans and other primates without retaining an environmental niche. M. marinum has maintained a large genome so as to retain the capacity for environmental survival while becoming a broad host range pathogen that produces disease strikingly similar to M. tuberculosis. The work described herein provides a foundation for using M. marinum to better understand the determinants of pathogenesis of tuberculosis.

post to: CiteULike

Large-scale analysis of gene clustering in bacteria.

Publication Date: 2008 May 8 PMID: 18390694
Authors: Yang, Q. - Sze, S. H.
Journal: Genome Res

An important strategy to study operons and their evolution is to investigate clustering of related genes across multiple bacterial genomes. Although existing algorithms are available that can identify gene clusters across two or more genomes, very few algorithms are efficient enough to study gene clusters across hundreds of genomes. We observe that a querying strategy can be used to analyze gene clusters across a large number of genomes and develop an efficient algorithm to identify all related clusters on a genome from a given query cluster. We use this algorithm to study gene clustering in 400 bacterial genomes by starting from a well-characterized list of operons in Escherichia coli K12 and perform comparative analysis of operon occurrences, gene orientations, and rearrangements both within and across clusters. We show that important biological insights can be obtained by comparing results across these categories. A software program implementing the algorithm (GCQuery) and supplementary data containing detailed results are available at http://faculty.cs.tamu.edu/shsze/gcquery.

post to: CiteULike

Nonrecurrent MECP2 duplications mediated by genomic architecture-driven DNA breaks and break-induced replication repair.

Publication Date: 2008 May 7 PMID: 18385275
Authors: Bauters, M. - Van Esch, H. - Friez, M. J. - Boespflug-Tanguy, O. - Zenker, M. - Vianna-Morgante, A. M. - Rosenberg, C. - Ignatius, J. - Raynaud, M. - Hollanders, K. - Govaerts, K. - Vandenreijt, K. - Niel, F. - Blanc, P. - Stevenson, R. E. - Fryns, J. P. - Marynen, P. - Schwartz, C. E. - Froyen, G.
Journal: Genome Res

Recurrent submicroscopic genomic copy number changes are the result of nonallelic homologous recombination (NAHR). Nonrecurrent aberrations, however, can result from different nonexclusive recombination-repair mechanisms. We previously described small microduplications at Xq28 containing MECP2 in four male patients with a severe neurological phenotype. Here, we report on the fine-mapping and breakpoint analysis of 16 unique microduplications. The size of the overlapping copy number changes varies between 0.3 and 2.3 Mb, and FISH analysis on three patients demonstrated a tandem orientation. Although eight of the 32 breakpoint regions coincide with low-copy repeats, none of the duplications are the result of NAHR. Bioinformatics analysis of the breakpoint regions demonstrated a 2.5-fold higher frequency of Alu interspersed repeats as compared with control regions, as well as a very high GC content (53%). Unexpectedly, we obtained the junction in only one patient by long-range PCR, which revealed nonhomologous end joining as the mechanism. Breakpoint analysis in two other patients by inverse PCR and subsequent array comparative genomic hybridization analysis demonstrated the presence of a second duplicated region more telomeric at Xq28, of which one copy was inserted in between the duplicated MECP2 regions. These data suggest a two-step mechanism in which part of Xq28 is first inserted near the MECP2 locus, followed by breakage-induced replication with strand invasion of the normal sister chromatid. Our results indicate that the mechanism by which copy number changes occur in regions with a complex genomic architecture can yield complex rearrangements.

post to: CiteULike

New binary polymorphisms reshape and increase resolution of the human Y chromosomal haplogroup tree.

Publication Date: 2008 May PMID: 18385274
Authors: Karafet, T. M. - Mendez, F. L. - Meilerman, M. B. - Underhill, P. A. - Zegura, S. L. - Hammer, M. F.
Journal: Genome Res

Markers on the non-recombining portion of the human Y chromosome continue to have applications in many fields including evolutionary biology, forensics, medical genetics, and genealogical reconstruction. In 2002, the Y Chromosome Consortium published a single parsimony tree showing the relationships among 153 haplogroups based on 243 binary markers and devised a standardized nomenclature system to name lineages nested within this tree. Here we present an extensively revised Y chromosome tree containing 311 distinct haplogroups, including two new major haplogroups (S and T), and incorporating approximately 600 binary markers. We describe major changes in the topology of the parsimony tree and provide names for new and rearranged lineages within the tree following the rules presented by the Y Chromosome Consortium in 2002. Several changes in the tree topology have important implications for studies of human ancestry. We also present demography-independent age estimates for 11 of the major clades in the new Y chromosome tree.

post to: CiteULike

Extensive variation between inbred mouse strains due to endogenous L1 retrotransposition.

Publication Date: 2008 May 7 PMID: 18381897
Authors: Akagi, K. - Li, J. - Stephens, R. M. - Volfovsky, N. - Symer, D. E.
Journal: Genome Res

Numerous inbred mouse strains comprise models for human diseases and diversity, but the molecular differences between them are mostly unknown. Several mammalian genomes have been assembled, providing a framework for identifying structural variations. To identify variants between inbred mouse strains at a single nucleotide resolution, we aligned 26 million individual sequence traces from four laboratory mouse strains to the C57BL/6J reference genome. We discovered and analyzed over 10,000 intermediate-length genomic variants (from 100 nucleotides to 10 kilobases), distinguishing these strains from the C57BL/6J reference. Approximately 85% of such variants are due to recent mobilization of endogenous retrotransposons, predominantly L1 elements, greatly exceeding that reported in humans. Many genes' structures and expression are altered directly by polymorphic L1 retrotransposons, including Drosha (also called Rnasen), Parp8, Scn1a, Arhgap15, and others, including novel genes. L1 polymorphisms are distributed nonrandomly across the genome, as they are excluded significantly from the X chromosome and from genes associated with the cell cycle, but are enriched in receptor genes. Thus, recent endogenous L1 retrotransposition has diversified genomic structures and transcripts extensively, distinguishing mouse lineages and driving a major portion of natural genetic variation.

post to: CiteULike

SNP-specific array-based allele-specific expression analysis.

Publication Date: 2008 May PMID: 18369178
Authors: Bjornsson, H. T. - Albert, T. J. - Ladd-Acosta, C. M. - Green, R. D. - Rongione, M. A. - Middle, C. M. - Irizarry, R. A. - Broman, K. W. - Feinberg, A. P.
Journal: Genome Res

We have developed an optimized array-based approach for customizable allele-specific gene expression (ASE) analysis. The central features of the approach are the ability to select SNPs at will for detection, and the absence of need to PCR amplify the target. A surprisingly long probe length (39-49 nt) was needed for allelic discrimination. Reconstitution experiments demonstrate linearity of ASE over a broad range. Using this approach, we have discovered at least two novel imprinted genes, NLRP2, which encodes a member of the inflammasome, and OSBPL1A, which encodes a presumed oxysterol-binding protein, were both preferentially expressed from the maternal allele. In contrast, ERAP2, which encodes an aminopeptidase, did not show preferential parent-of-origin expression, but rather, cis-acting nonimprinted differential allelic control. The approach is scalable to the whole genome and can be used for discovery of functional epigenetic modifications in patient samples.

post to: CiteULike

Power and SNP tagging in whole mitochondrial genome association studies.

Publication Date: 2008 May 7 PMID: 18356315
Authors: McRae, A. F. - Byrne, E. M. - Zhao, Z. Z. - Montgomery, G. W. - Visscher, P. M.
Journal: Genome Res

The application of genetic association studies to detect mitochondrial variants responsible for phenotypic variation has recently been demonstrated. However, the only power estimates currently available are based on the use of mitochondrial haplogroups, which can only tag a small fraction of the common variation in the mitochondrial genome. Here, power estimates are derived for a SNP-based study design for both disease (case-control) and quantitative trait mapping studies. Power is estimated using simulations based on a collection of publicly available mitochondrial sequences of European origin. The power when testing all common mitochondrial SNPs is shown to be equivalent to that when testing only tagging SNPs, despite the relatively high ratio of tagging SNPs to total SNPs resulting from the tagging of all SNPs with a minor allele frequency greater than 1%. The sample size requirements of mitochondrial genome association studies are compared with that of nuclear whole-genome studies. Remarkably, the trade off between the number of tests being performed and the proportion of phenotypic variance explained for a fixed effect size results in approximately equal sample sizes required for both study types, although the per individual cost for the mitochondrial association study is much less. To test the representation of the sequences used in the power simulations, a sample of 3839 individuals from 1037 Australian families was genotyped for 69 tagging SNPs. The strong concordance in allele frequencies and linkage disequilibrium between the European sequences and the Australian sample indicates that the results presented here are transferable across populations of European descent.

post to: CiteULike

Genome-wide analysis of microsatellite polymorphism in chicken circumventing the ascertainment bias.

Publication Date: 2008 May 2 PMID: 18356314
Authors: Brandstrom, M. - Ellegren, H.
Journal: Genome Res

Studies of microsatellites evolution based on marker data almost inherently suffer from an ascertainment bias because there is selection for the most mutable and polymorphic loci during marker development. To circumvent this bias we took advantage of whole-genome shotgun sequence data from three unrelated chicken individuals that, when aligned to the genome reference sequence, give sequence information on two chromosomes from about one-fourth (375,000) of all microsatellite loci containing di- through pentanucleotide repeat motifs in the chicken genome. Polymorphism is seen at loci with as few as five repeat units, and the proportion of dimorphic loci then increases to 50% for sequences with approximately 10 repeat units, to reach a maximum of 75%-80% for sequences with 15 or more repeat units. For any given repeat length, polymorphism increases with decreasing GC content of repeat motifs for dinucleotides, nonhairpin-forming trinucleotides, and tetranucleotides. For trinucleotide repeats which are likely to form hairpin structures, polymorphism increases with increasing GC content, indicating that the relative stability of hairpins affects the rate of replication slippage. For any given repeat length, polymorphism is significantly lower for imperfect compared to perfect repeats and repeat interruptions occur in >15% of loci. However, interruptions are not randomly distributed within repeat arrays but are preferentially located toward the ends. There is negative correlation between microsatellite abundance and single nucleotide polymorphism (SNP) density, providing large-scale genomic support for the hypothesis that equilibrium microsatellite distributions are governed by a balance between rate of replication slippage and rate of point mutation.

post to: CiteULike

Velvet: Algorithms for de novo short read assembly using de Bruijn graphs.

Publication Date: 2008 May PMID: 18349386
Authors: Zerbino, D. R. - Birney, E.
Journal: Genome Res

We have developed a new set of algorithms, collectively called "Velvet," to manipulate de Bruijn graphs for genomic sequence assembly. A de Bruijn graph is a compact representation based on short words (k-mers) that is ideal for high coverage, very short read (25-50 bp) data sets. Applying Velvet to very short reads and paired-ends information only, one can produce contigs of significant length, up to 50-kb N50 length in simulations of prokaryotic data and 3-kb N50 on simulated mammalian BACs. When applied to real Solexa data sets without read pairs, Velvet generated contigs of approximately 8 kb in a prokaryote and 2 kb in a mammalian BAC, in close agreement with our simulated results without read-pair information. Velvet represents a new approach to assembly that can leverage very short reads in combination with read pairs to produce useful assemblies.

post to: CiteULike

A gene expression network model of type 2 diabetes links cell cycle regulation in islets with diabetes susceptibility.

Publication Date: 2008 May PMID: 18347327
Authors: Keller, M. P. - Choi, Y. - Wang, P. - Belt Davis, D. - Rabaglia, M. E. - Oler, A. T. - Stapleton, D. S. - Argmann, C. - Schueler, K. L. - Edwards, S. - Steinberg, H. A. - Chaibub Neto, E. - Kleinhanz, R. - Turner, S. - Hellerstein, M. K. - Schadt, E. E. - Yandell, B. S. - Kendziorski, C. - Attie, A. D.
Journal: Genome Res

Insulin resistance is necessary but not sufficient for the development of type 2 diabetes. Diabetes results when pancreatic beta-cells fail to compensate for insulin resistance by increasing insulin production through an expansion of beta-cell mass or increased insulin secretion. Communication between insulin target tissues and beta-cells may initiate this compensatory response. Correlated changes in gene expression between tissues can provide evidence for such intercellular communication. We profiled gene expression in six tissues of mice from an obesity-induced diabetes-resistant and a diabetes-susceptible strain before and after the onset of diabetes. We studied the correlation structure of mRNA abundance and identified 105 co-expression gene modules. We provide an interactive gene network model showing the correlation structure between the expression modules within and among the six tissues. This resource also provides a searchable database of gene expression profiles for all genes in six tissues in lean and obese diabetes-resistant and diabetes-susceptible mice, at 4 and 10 wk of age. A cell cycle regulatory module in islets predicts diabetes susceptibility. The module predicts islet replication; we found a strong correlation between (2)H(2)O incorporation into islet DNA in vivo and the expression pattern of the cell cycle module. This pattern is highly correlated with that of several individual genes in insulin target tissues, including Igf2, which has been shown to promote beta-cell proliferation, suggesting that these genes may provide a link between insulin resistance and beta-cell proliferation.

post to: CiteULike

De novo search for non-coding RNA genes in the AT-rich genome of Dictyostelium discoideum: Performance of Markov-dependent genome feature scoring.

Publication Date: 2008 May 7 PMID: 18347326
Authors: Larsson, P. - Hinas, A. - Ardell, D. H. - Kirsebom, L. A. - Virtanen, A. - Soderbom, F.
Journal: Genome Res

Genome data are increasingly important in the computational identification of novel regulatory non-coding RNAs (ncRNAs). However, most ncRNA gene-finders are either specialized to well-characterized ncRNA gene families or require comparisons of closely related genomes. We developed a method for de novo screening for ncRNA genes with a nucleotide composition that stands out against the background genome based on a partial sum process. We compared the performance when assuming independent and first-order Markov-dependent nucleotides, respectively, and used Karlin-Altschul and Karlin-Dembo statistics to evaluate the significance of hits. We hypothesized that a first-order Markov-dependent process might have better power to detect ncRNA genes since nearest-neighbor models have been shown to be successful in predicting RNA structures. A model based on a first-order partial sum process (analyzing overlapping dinucleotides) had better sensitivity and specificity than a zeroth-order model when applied to the AT-rich genome of the amoeba Dictyostelium discoideum. In this genome, we detected 94% of previously known ncRNA genes (at this sensitivity, the false positive rate was estimated to be 25% in a simulated background). The predictions were further refined by clustering candidate genes according to sequence similarity and/or searching for an ncRNA-associated upstream element. We experimentally verified six out of 10 tested ncRNA gene predictions. We conclude that higher-order models, in combination with other information, are useful for identification of novel ncRNA gene families in single-genome analysis of D. discoideum. Our generalizable approach extends the range of genomic data that can be searched for novel ncRNA genes using well-grounded statistical methods.

post to: CiteULike

Genomic evolution of the placenta using co-option and duplication and divergence.

Publication Date: 2008 May PMID: 18340042
Authors: Knox, K. - Baker, J. C.
Journal: Genome Res

The invention of the placenta facilitated the evolution of mammals. How the placenta evolved from the simple structure observed in birds and reptiles into the complex organ that sustains human life is one of the great mysteries of evolution. By using a timecourse microarray analysis including the entire lifetime of the placenta, we uncover molecular and genomic changes that underlie placentation and find that two distinct evolutionary mechanisms were utilized during placental evolution in mice and human. Ancient genes involved in growth and metabolism were co-opted for use during early embryogenesis, likely enabling the accelerated development of extraembryonic tissues. Recently duplicated genes are utilized at later stages of placentation to meet the metabolic needs of a diverse range of pregnancy physiologies. Together, these mechanisms served to develop the specialized placenta, a novel structure that led to expansion of the eutherian mammal, including humankind.

post to: CiteULike

Genome-wide analysis of Fis binding in Escherichia coli indicates a causative role for A-/AT-tracts.

Publication Date: 2008 May 7 PMID: 18340041
Authors: Cho, B. K. - Knight, E. M. - Barrett, C. L. - Palsson, B. O.
Journal: Genome Res

We determined the genome-wide distribution of the nucleoid-associated protein Fis in Escherichia coli using chromatin immunoprecipitation coupled with high-resolution whole genome-tiling microarrays. We identified 894 Fis-associated regions across the E. coli genome. A significant number of these binding sites were found within open reading frames (33%) and between divergently transcribed transcripts (5%). Analysis indicates that A-tracts and AT-tracts are an important signal for preferred Fis-binding sites, and that A(6)-tracts in particular constitute a high-affinity signal that dictates Fis phasing in stretches of DNA containing multiple and variably spaced A-tracts and AT-tracts. Furthermore, we find evidence for an average of two Fis-binding regions per supercoiling domain in the chromosome of exponentially growing cells. Transcriptome analysis shows that approximately 21% of genes are affected by the deletion of fis; however, the changes in magnitude are small. To address the differential Fis bindings under growth environment perturbation, ChIP-chip analysis was performed using cells grown under aerobic and anaerobic growth conditions. Interestingly, the Fis-binding regions are almost identical in aerobic and anaerobic growth conditions-indicating that the E. coli genome topology mediated by Fis is superficially identical in the two conditions. These novel results provide new insight into how Fis modulates DNA topology at a genome scale and thus advance our understanding of the architectural bases of the E. coli nucleoid.

post to: CiteULike

Multiple waves of recent DNA transposon activity in the bat, Myotis lucifugus.

Publication Date: 2008 May PMID: 18340040
Authors: Ray, D. A. - Feschotte, C. - Pagan, H. J. - Smith, J. D. - Pritham, E. J. - Arensburger, P. - Atkinson, P. W. - Craig, N. L.
Journal: Genome Res

DNA transposons, or class 2 transposable elements, have successfully propagated in a wide variety of genomes. However, it is widely believed that DNA transposon activity has ceased in mammalian genomes for at least the last 40 million years. We recently reported evidence for the relatively recent activity of hAT and Helitron elements, two distinct groups of DNA transposons, in the lineage of the vespertilionid bat Myotis lucifugus. Here, we describe seven additional families that have also been recently active in the bat lineage. Early vespertilionid genome evolution was dominated by the activity of Helitrons, mariner-like and Tc2-like elements. This was followed by the colonization of Tc1-like elements, and by a more recent explosion of hAT-like elements. Finally, and most recently, piggyBac-like elements have amplified within the Myotis genome and our results indicate that one of these families is probably still expanding in natural populations. Together, these data suggest that there has been tremendous recent activity of various DNA transposons in the bat lineage that far exceeds those previously reported for any mammalian lineage. The diverse and recent populations of DNA transposons in genus Myotis will provide an unprecedented opportunity to study the impact of this class of elements on mammalian genome evolution and to better understand what makes some species more susceptible to invasion by genomic parasites than others.

post to: CiteULike

ALLPATHS: De novo assembly of whole-genome shotgun microreads.

Publication Date: 2008 May PMID: 18340039
Authors: Butler, J. - Maccallum, I. - Kleber, M. - Shlyakhter, I. A. - Belmonte, M. K. - Lander, E. S. - Nusbaum, C. - Jaffe, D. B.
Journal: Genome Res

New DNA sequencing technologies deliver data at dramatically lower costs but demand new analytical methods to take full advantage of the very short reads that they produce. We provide an initial, theoretical solution to the challenge of de novo assembly from whole-genome shotgun "microreads." For 11 genomes of sizes up to 39 Mb, we generated high-quality assemblies from 80x coverage by paired 30-base simulated reads modeled after real Illumina-Solexa reads. The bacterial genomes of Campylobacter jejuni and Escherichia coli assemble optimally, yielding single perfect contigs, and larger genomes yield assemblies that are highly connected and accurate. Assemblies are presented in a graph form that retains intrinsic ambiguities such as those arising from polymorphism, thereby providing information that has been absent from previous genome assemblies. For both C. jejuni and E. coli, this assembly graph is a single edge encompassing the entire genome. Larger genomes produce more complicated graphs, but the vast majority of the bases in their assemblies are present in long edges that are nearly always perfect. We describe a general method for genome assembly that can be applied to all types of DNA sequence data, not only short read data, but also conventional sequence reads.

post to: CiteULike

De novo bacterial genome sequencing: Millions of very short reads assembled on a desktop computer.

Publication Date: 2008 May PMID: 18332092
Authors: Hernandez, D. - Francois, P. - Farinelli, L. - Osteras, M. - Schrenzel, J.
Journal: Genome Res

Novel high-throughput DNA sequencing technologies allow researchers to characterize a bacterial genome during a single experiment and at a moderate cost. However, the increase in sequencing throughput that is allowed by using such platforms is obtained at the expense of individual sequence read length, which must be assembled into longer contigs to be exploitable. This study focuses on the Illumina sequencing platform that produces millions of very short sequences that are 35 bases in length. We propose a de novo assembler software that is dedicated to process such data. Based on a classical overlap graph representation and on the detection of potentially spurious reads, our software generates a set of accurate contigs of several kilobases that cover most of the bacterial genome. The assembly results were validated by comparing data sets that were obtained experimentally for Staphylococcus aureus strain MW2 and Helicobacter acinonychis strain Sheeba with that of their published genomes acquired by conventional sequencing of 1.5- to 3.0-kb fragments. We also provide indications that the broad coverage achieved by high-throughput sequencing might allow for the detection of clonal polymorphisms in the set of DNA molecules being sequenced.

post to: CiteULike

Detecting polymorphic regions in Arabidopsis thaliana with resequencing microarrays.

Publication Date: 2008 May 7 PMID: 18323538
Authors: Zeller, G. - Clark, R. M. - Schneeberger, K. - Bohlen, A. - Weigel, D. - Ratsch, G.
Journal: Genome Res

Whole-genome oligonucleotide resequencing arrays have allowed the comprehensive discovery of single nucleotide polymorphisms (SNPs) in eukaryotic genomes of moderate to large size. With this technology, the detection rate for isolated SNPs is typically high. However, it is greatly reduced when other polymorphisms are located near a SNP as multiple mismatches inhibit hybridization to arrayed oligonucleotides. Contiguous tracts of suppressed hybridization therefore typify polymorphic regions (PRs) such as clusters of SNPs or deletions. We developed a machine learning method, designated margin-based prediction of polymorphic regions (mPPR), to predict PRs from resequencing array data. Conceptually similar to hidden Markov models, the method is trained with discriminative learning techniques related to support vector machines, and accurately identifies even very short polymorphic tracts (<10 bp). We applied this method to resequencing array data previously generated for the euchromatic genomes of 20 strains (accessions) of the best-characterized plant, Arabidopsis thaliana. Nonredundantly, 27% of the genome was included within the boundaries of PRs predicted at high specificity ( approximately 97%). The resulting data set provides a fine-scale view of polymorphic sequences in A. thaliana; patterns of polymorphism not apparent in SNP data were readily detected, especially for noncoding regions. Our predictions provide a valuable resource for evolutionary genetic and functional studies in A. thaliana, and our method is applicable to similar data sets in other species. More broadly, our computational approach can be applied to other segmentation tasks related to the analysis of genomic variation.

post to: CiteULike

Comprehensive high-throughput arrays for relative methylation (CHARM).

Publication Date: 2008 May PMID: 18316654
Authors: Irizarry, R. A. - Ladd-Acosta, C. - Carvalho, B. - Wu, H. - Brandenburg, S. A. - Jeddeloh, J. A. - Wen, B. - Feinberg, A. P.
Journal: Genome Res

This study was originally conceived to test in a rigorous way the specificity of three major approaches to high-throughput array-based DNA methylation analysis: (1) MeDIP, or methylated DNA immunoprecipitation, an example of antibody-mediated methyl-specific fractionation; (2) HELP, or HpaII tiny fragment enrichment by ligation-mediated PCR, an example of differential amplification of methylated DNA; and (3) fractionation by McrBC, an enzyme that cuts most methylated DNA. These results were validated using 1466 Illumina methylation probes on the GoldenGate methylation assay and further resolved discrepancies among the methods through quantitative methylation pyrosequencing analysis. While all three methods provide useful information, there were significant limitations to each, specifically bias toward CpG islands in MeDIP, relatively incomplete coverage in HELP, and location imprecision in McrBC. However, we found that with an original array design strategy using tiling arrays and statistical procedures that average information from neighboring genomic locations, much improved specificity and sensitivity could be achieved, e.g., approximately 100% sensitivity at 90% specificity with McrBC. We term this approach "comprehensive high-throughput arrays for relative methylation" (CHARM). While this approach was applied to McrBC analysis, the array design and computational algorithms are fractionation method-independent and make this a simple, general, relatively inexpensive tool suitable for genome-wide analysis, and in which individual samples can be assayed reliably at very high density, allowing locus-level genome-wide epigenetic discrimination of individuals, not just groups of samples. Furthermore, unlike the other approaches, CHARM is highly quantitative, a substantial advantage in application to the study of human disease.

post to: CiteULike

Copy number variation at the 7q11.23 segmental duplications is a susceptibility factor for the Williams-Beuren syndrome deletion.

Publication Date: 2008 May PMID: 18292220
Authors: Cusco, I. - Corominas, R. - Bayes, M. - Flores, R. - Rivera-Brugues, N. - Campuzano, V. - Perez-Jurado, L. A.
Journal: Genome Res

Large copy number variants (CNVs) have been recently found as structural polymorphisms of the human genome of still unknown biological significance. CNVs are significantly enriched in regions with segmental duplications or low-copy repeats (LCRs). Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder caused by a heterozygous deletion of contiguous genes at 7q11.23 mediated by nonallelic homologous recombination (NAHR) between large flanking LCRs and facilitated by a structural variant of the region, a approximately 2-Mb paracentric inversion present in 20%-25% of WBS-transmitting progenitors. We now report that eight out of 180 (4.44%) WBS-transmitting progenitors are carriers of a CNV, displaying a chromosome with large deletion of LCRs. The prevalence of this CNV among control individuals and non-transmitting progenitors is much lower (1%, n = 600), thus indicating that it is a predisposing factor for the WBS deletion (odds ratio 4.6-fold, P = 0.002). LCR duplications were found in 2.22% of WBS-transmitting progenitors but also in 1.16% of controls, which implies a non-statistically significant increase in WBS-transmitting progenitors. We have characterized the organization and breakpoints of these CNVs, encompassing approximately 100-300 kb of genomic DNA and containing several pseudogenes but no functional genes. Additional structural variants of the region have also been defined, all generated by NAHR between different blocks of segmental duplications. Our data further illustrate the highly dynamic structure of regions rich in segmental duplications, such as the WBS locus, and indicate that large CNVs can act as susceptibility alleles for disease-associated genomic rearrangements in the progeny.

post to: CiteULike

Scanning the human genome at kilobase resolution.

Publication Date: 2008 May PMID: 18292219
Authors: Chen, J. - Kim, Y. C. - Jung, Y. C. - Xuan, Z. - Dworkin, G. - Zhang, Y. - Zhang, M. Q. - Wang, S. M.
Journal: Genome Res

Normal genome variation and pathogenic genome alteration frequently affect small regions in the genome. Identifying those genomic changes remains a technical challenge. We report here the development of the DGS (Ditag Genome Scanning) technique for high-resolution analysis of genome structure. The basic features of DGS include (1) use of high-frequent restriction enzymes to fractionate the genome into small fragments; (2) collection of two tags from two ends of a given DNA fragment to form a ditag to represent the fragment; (3) application of the 454 sequencing system to reach a comprehensive ditag sequence collection; (4) determination of the genome origin of ditags by mapping to reference ditags from known genome sequences; (5) use of ditag sequences directly as the sense and antisense PCR primers to amplify the original DNA fragment. To study the relationship between ditags and genome structure, we performed a computational study by using the human genome reference sequences as a model, and analyzed the ditags experimentally collected from the well-characterized normal human DNA GM15510 and the leukemic human DNA of Kasumi-1 cells. Our studies show that DGS provides a kilobase resolution for studying genome structure with high specificity and high genome coverage. DGS can be applied to validate genome assembly, to compare genome similarity and variation in normal populations, and to identify genomic abnormality including insertion, inversion, deletion, translocation, and amplification in pathological genomes such as cancer genomes.

post to: CiteULike

Genome-wide mapping and characterization of hypomethylated sites in human tissues and breast cancer cell lines.

Publication Date: 2008 May PMID: 18256232
Authors: Shann, Y. J. - Cheng, C. - Chiao, C. H. - Chen, D. T. - Li, P. H. - Hsu, M. T.
Journal: Genome Res

We have developed a method for mapping unmethylated sites in the human genome based on the resistance of TspRI-digested ends to ExoIII nuclease degradation. Digestion with TspRI and methylation-sensitive restriction endonuclease HpaII, followed by ExoIII and single-strand DNA nuclease allowed removal of DNA fragments containing unmethylated HpaII sites. We then used array comparative genomic hybridization (CGH) to map the sequences depleted by these procedures in human genomes derived from five human tissues, a primary breast tumor, and two breast tumor cell lines. Analysis of methylation patterns of the normal tissue genomes indicates that the hypomethylated sites are enriched in the 5' end of widely expressed genes, including promoter, first exon, and first intron. In contrast, genomes of the MCF-7 and MDA-MB-231 cell lines show extensive hypomethylation in the intragenic and intergenic regions whereas the primary tumor exhibits a pattern between those of the normal tissue and the cell lines. A striking characteristic of tumor cell lines is the presence of megabase-sized hypomethylated zones. These hypomethylated zones are associated with large genes, fragile sites, evolutionary breakpoints, chromosomal rearrangement breakpoints, tumor suppressor genes, and with regions containing tissue-specific gene clusters or with gene-poor regions containing novel tissue-specific genes. Correlation with microarray analysis shows that genes with a hypomethylated sequence 2 kb up- or downstream of the transcription start site are highly expressed, whereas genes with extensive intragenic and 3' untranslated region (UTR) hypomethylation are silenced. The method described herein can be used for large-scale screening of changes in the methylation pattern in the genome of interest.

post to: CiteULike

Quality scores and SNP detection in sequencing-by-synthesis systems.

Publication Date: 2008 May PMID: 18212088
Authors: Brockman, W. - Alvarez, P. - Young, S. - Garber, M. - Giannoukos, G. - Lee, W. L. - Russ, C. - Lander, E. S. - Nusbaum, C. - Jaffe, D. B.
Journal: Genome Res

Promising new sequencing technologies, based on sequencing-by-synthesis (SBS), are starting to deliver large amounts of DNA sequence at very low cost. Polymorphism detection is a key application. We describe general methods for improved quality scores and accurate automated polymorphism detection, and apply them to data from the Roche (454) Genome Sequencer 20. We assess our methods using known-truth data sets, which is critical to the validity of the assessments. We developed informative, base-by-base error predictors for this sequencer and used a variant of the phred binning algorithm to combine them into a single empirically derived quality score. These quality scores are more useful than those produced by the system software: They both better predict actual error rates and identify many more high-quality bases. We developed a SNP detection method, with variants for low coverage, high coverage, and PCR amplicon applications, and evaluated it on known-truth data sets. We demonstrate good specificity in single reads, and excellent specificity (no false positives in 215 kb of genome) in high-coverage data.

post to: CiteULike