Genome Research

High-throughput sequence analysis of Ciona intestinalis SL trans-spliced mRNAs: Alternative expression modes and gene function correlates.

Publication Date: 2010 Mar 8 PMID: 20212022
Authors: Matsumoto, J. - Dewar, K. - Wasserscheid, J. - Macmil, S. L. - Wiley, G. B. - Roe, B. A. - Zeller, R. W. - Satou, Y. - Hastings, K. E.
Journal: Genome Res

Pre-mRNA 5' spliced-leader (SL) trans-splicing occurs in some metazoan groups, e.g. tunicate chordates, but not in others, e.g. vertebrates. Functional and evolutionary aspects of SL trans-splicing remain poorly understood and although genome-wide characterization of the trans-spliced mRNA subpopulation would give useful insight, this has not yet been reported for any metazoan. We carried out a high-throughput analysis of the SL trans-spliced mRNA population of the ascidian tunicate Ciona intestinalis by Roche/454 pyrosequencing of SL-PCR-amplified random-primed reverse transcripts of tailbud embryo RNA. We obtained ~250,000 high-quality reads corresponding to 8,790 genes, ~58% of the Ciona total gene number. The great depth of this data revealed new aspects of trans-splicing, including the existence of a significant class of "infrequently trans-spliced" genes, accounting for ~28% of represented genes, that generate largely non-trans-spliced mRNAs, but also produce trans-spliced mRNAs, in part through alternative promoter use. Thus, the conventional qualitative dichotomy of trans-spliced versus non-trans-spliced genes should be supplanted by a more accurate quantitative view recognizing frequently- and infrequently trans-spliced gene categories. Our data include reads representing ~80% of Ciona frequently-trans-spliced genes. Our analysis also revealed significant use of closely-spaced alternative trans-splice acceptor sites which further underscores the mechanistic similarity of cis- and trans-splicing and indicates that the prevalence of +/-3-nt alternative splicing events at tandem acceptor sites, NAGNAG, is driven by spliceosomal mechanisms, and not nonsense-mediated decay, or selection at the protein level. The breadth of gene representation data enabled us to find new correlations between trans-splicing status and gene function, namely the over-representation in the frequently trans-spliced gene class of genes associated with plasma/endomembrane system, Ca2+ homeostasis, and actin cytoskeleton - eukaryotic cell features widely exploited in the metazoa for cell-cell interaction and signaling.

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Analysis of copy number variations among diverse cattle breeds.

Publication Date: 2010 Mar 8 PMID: 20212021
Authors: Liu, G. E. - Hou, Y. - Zhu, B. - Cardone, M. F. - Jiang, L. - Cellamare, A. - Mitra, A. - Alexander, L. J. - Coutinho, L. L. - Dell'aquila, M. E. - Gasbarre, L. C. - Lacalandra, G. - Li, R. W. - Matukumalli, L. K. - Nonneman, D. - Regitano, L. C. - Smith, T. P. - Song, J. - Sonstegard, T. S. - Van Tassell, C. P. - Ventura, M. - Eichler, E. E. - McDaneld, T. G. - Keele, J. W.
Journal: Genome Res

Genomic structural variation is an important and abundant source of genetic and phenotypic variation. Here we describe the first systematic and genome-wide analysis of copy number variations (CNVs) in modern domesticated cattle using array comparative genomic hybridization (array CGH), quantitative PCR (qPCR) and fluorescent in situ hybridization (FISH). The array CGH panel included 90 animals from 11 Bos taurus, 3 Bos indicus and 3 composite breeds for beef, dairy or dual purpose. We identified over 200 candidate CNV regions (CNVRs) in total and 177 within known chromosomes, which harbor or are adjacent to gains or losses. These 177 high-confidence CNVRs cover 28.1 mega bases or ~1.07% of the genome. Over 50% of the CNVRs (89/177) were found in multiple animals or breeds and analysis revealed breed-specific frequency differences and reflected aspects of the known ancestry of these cattle breeds. Selected CNVs were further validated by independent methods using qPCR and FISH. Approximately 67% of the CNVRs (119/177) completely or partially span cattle genes and 61% of the CNVRs (108/177) directly overlap with segmental duplications. The CNVRs span about 400 annotated cattle genes that are significantly enriched for specific biological functions such as immunity, lactation, reproduction and rumination. Multiple gene families, including ULBP, have gone through ruminant lineage-specific gene amplification. We detected and confirmed marked differences in CNV frequencies across diverse breeds, indicating that some cattle CNVs are likely to arise independently in breeds and contribute to breed differences. Our results provide a valuable resource beyond microsatellites and single nucleotide polymorphisms to explore the full dimension of genetic variability for future cattle genomic research.

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The effect of translocation-induced nuclear re-organization on gene expression.

Publication Date: 2010 Mar 8 PMID: 20212020
Authors: Harewood, L. - Schutz, F. - Boyle, S. - Perry, P. - Delorenzi, M. - Bickmore, W. A. - Reymond, A.
Journal: Genome Res

Translocations are known to affect the expression of genes at the breakpoints and, in the case of unbalanced translocations, alter the gene copy number. However, a comprehensive understanding of the functional impact of this class of variation is lacking. Here we have studied the effect of balanced chromosomal rearrangements on gene expression by comparing the transcriptomes of cell lines from controls and individuals with the t(11;22)(q23;q11) translocation. The number of differentially expressed transcripts between translocation carrying and control cohorts is significantly higher than that observed between control samples alone, suggesting that balanced rearrangements have a greater effect on gene expression than normal variation. Many of the affected genes are located along the length of the derived chromosome 11. We show that this chromosome is concomitantly altered in its spatial organization, occupying a more central position in the nucleus than its non-rearranged counterpart. Derivative 22-mapping chromosome 22 genes, on the other hand, remain in their usual environment. Our results are consistent with recent studies that experimentally altered nuclear organization and indicated that nuclear position plays a functional role in regulating the expression of some genes in mammalian cells. Our study suggests that chromosomal translocations can result in hitherto unforeseen, large-scale changes in gene expression that are the consequence of alterations in normal chromosome territory positioning. This has consequences for the patterns of gene expression change seen during tumorigenesis associated genome instability and during the karyotype changes that lead to speciation.

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Coexpression network analysis of neural tissue reveals perturbations in developmental processes in schizophrenia.

Publication Date: 2010 Mar 2 PMID: 20197298
Authors: Torkamani, A. - Dean, B. - Schork, N. J. - Thomas, E. A.
Journal: Genome Res

We performed integrated gene coexpression network analysis on two large microarray-based brain gene expression data sets generated from the prefrontal cortex obtained post-mortem from 101 subjects, 47 subjects with schizophrenia and 54 normal control subjects, ranging in age from 19 to 81 years. Twenty-eight modules of coexpressed genes with functional interpretations were detected in both normal subjects and those with schizophrenia. Significant overlap of "case" and "control" module composition was observed, indicating that extensive differences in underlying molecular connectivity are not likely driving pathology in schizophrenia. Modules of coexpressed genes were characterized according to disease association, cell type specificity, and the effects of aging. We find that genes with altered expression in schizophrenia clustered into distinct coexpression networks and that these were associated primarily with neurons. We further identified a robust effect of age on gene expression modules that differentiates normal subjects from those with schizophrenia. In particular, we report that normal age-related decreases in genes related to central nervous system developmental processes, including neurite outgrowth, neuronal differentiation, and dopamine-related cellular signaling, do not occur in subjects with schizophrenia during the aging process. Extrapolating these findings to earlier stages of development supports the concept that schizophrenia pathogenesis begins early in life and is associated with a failure of normal decreases in developmental-related gene expression. These findings provide a novel mechanism for the "developmental" hypothesis of schizophrenia on a molecular level.

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An effective model for natural selection in promoters.

Publication Date: 2010 Mar 1 PMID: 20194951
Authors: Hoffman, M. M. - Birney, E.
Journal: Genome Res

We have produced an evolutionary model for promoters, analogous to the commonly used synonymous/nonsynonymous mutation models for protein-coding sequence. Although our model, called Sunflower, relies on some simple assumptions, it captures enough of the biology of transcription factor action to show clear correlation with other biological features. Sunflower predicts a binding profile of transcription factors to DNA sequence, in which different factors compete for the same potential binding sites. The parametrized model simultaneously estimates a continuous measurement of binding occupancy across the genomic sequence for each factor. We can then introduce a localized mutation, rerun the binding model and record the difference in binding profiles. A single mutation can alter interactions both upstream and downstream of its position due to potential overlapping binding sites, and our statistic captures this domino effect. Over evolutionary time, we observe a clear excess of low-scoring mutations fixed in promoters, consistent with most changes being neutral. However, this is not consistent across all promoters, and some promoters show more rapid divergence. This divergence often occurs in the presence of relatively constant protein coding divergence. Interestingly, different classes of promoters show different sensitivity to mutations, with phosphorylation-related genes having promoters inherently more sensitive to mutations than immune genes. Although there have previously been a number of models attempting to handle transcription factor binding, Sunflower provides a richer biological model, incorporating weak binding sites, and the possibility of competition. The results show the first clear correlations between such a model and evolutionary processes.

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Genome-wide analysis of histone modifications in human pancreatic islets.

Publication Date: 2010 Feb 24 PMID: 20181961
Authors: Bhandare, R. - Schug, J. - Le Lay, J. - Fox, A. - Smirnova, O. - Liu, C. - Naji, A. - Kaestner, K. H.
Journal: Genome Res

The global diabetes epidemic poses a major challenge. Epigenetic events contribute to the etiology of diabetes; however, the lack of epigenomic analysis has limited the elucidation of the mechanistic basis for this link. To determine the epigenetic architecture of human pancreatic islets we mapped the genome-wide locations of four histone marks: three associated with gene activation-H3K4me1, H3K4me2, and H3K4me3-and one associated with gene repression, H3K27me3. Interestingly, the promoters of the highly transcribed insulin and glucagon genes are occupied only sparsely by H3K4me2 and H3K4me3. Globally, we identified important relationships between promoter structure, histone modification, and gene expression. We demonstrated co-occurrences of histone modifications including bivalent marks in mature islets. Furthermore, we found a set of promoters that is differentially modified between islets and other cell types. We also use our histone marks to determine which of the known diabetes-associated single-nucleotide polymorphisms are likely to be part of regulatory elements. Our global map of histone marks will serve as an important resource for understanding the epigenetic basis of type 2 diabetes.

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Integrative analysis of the melanoma transcriptome.

Publication Date: 2010 Feb 23 PMID: 20179022
Authors: Berger, M. F. - Levin, J. Z. - Vijayendran, K. - Sivachenko, A. - Adiconis, X. - Maguire, J. - Johnson, L. A. - Robinson, J. - Verhaak, R. G. - Sougnez, C. - Onofrio, R. C. - Ziaugra, L. - Cibulskis, K. - Laine, E. - Barretina, J. - Winckler, W. - Fisher, D. E. - Getz, G. - Meyerson, M. - Jaffe, D. B. - Gabriel, S. B. - Lander, E. S. - Dummer, R. - Gnirke, A. - Nusbaum, C. - Garraway, L. A.
Journal: Genome Res

Global studies of transcript structure and abundance in cancer cells enable the systematic discovery of aberrations that contribute to carcinogenesis, including gene fusions, alternative splice isoforms, and somatic mutations. We developed a systematic approach to characterize the spectrum of cancer-associated mRNA alterations through integration of transcriptomic and structural genomic data, and we applied this approach to generate new insights into melanoma biology. Using paired-end massively parallel sequencing of cDNA (RNA-seq) together with analyses of high-resolution chromosomal copy number data, we identified 11 novel melanoma gene fusions produced by underlying genomic rearrangements, as well as 12 novel readthrough transcripts. We mapped these chimeric transcripts to base-pair resolution and traced them to their genomic origins using matched chromosomal copy number information. We also used these data to discover and validate base-pair mutations that accumulated in these melanomas, revealing a surprisingly high rate of somatic mutation and lending support to the notion that point mutations constitute the major driver of melanoma progression. Taken together, these results may indicate new avenues for target discovery in melanoma, while also providing a template for large-scale transcriptome studies across many tumor types.

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Examination of transcriptional networks reveals an important role for TCFAP2C, SMARCA4, and EOMES in trophoblast stem cell maintenance.

Publication Date: 2010 Mar 8 PMID: 20176728
Authors: Kidder, B. L. - Palmer, S.
Journal: Genome Res

Trophoblast stem cells (TS cells), derived from the trophectoderm (TE) of blastocysts, require transcription factors (TFs) and external signals (FGF4, INHBA/NODAL/TGFB1) for self-renewal. While many reports have focused on TF networks that regulate embryonic stem cell (ES cell) self-renewal and pluripotency, little is know about TF networks that regulate self-renewal in TS cells. To further understand transcriptional networks in TS cells, we used chromatin immunoprecipitation with DNA microarray hybridization (ChIP-chip) analysis to investigate targets of the TFs-TCFAP2C, EOMES, ETS2, and GATA3-and a chromatin remodeling factor, SMARCA4. We then evaluated the transcriptional states of target genes using transcriptome analysis and genome-wide analysis of histone H3 acetylation (AcH3). Our results describe previously unknown transcriptional networks in TS cells, including TF occupancy of genes involved in ES cell self-renewal and pluripotency, co-occupancy of TCFAP2C, SMARCA4, and EOMES at a significant number of genes, and transcriptional regulatory circuitry within the five factors. Moreover, RNAi depletion of Tcfap2c, Smarca4, and Eomes transcripts resulted in a loss of normal colony morphology and down-regulation of TS cell-specific genes, suggesting an important role for TCFAP2C, SMARCA4, and EOMES in TS cell self-renewal. Through genome-wide mapping and global expression analysis of five TF target genes, our data provide a comprehensive analysis of transcriptional networks that regulate TS cell self-renewal.

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Accurate detection and genotyping of SNPs utilizing population sequencing data.

Publication Date: 2010 Mar 5 PMID: 20150320
Authors: Bansal, V. - Harismendy, O. - Tewhey, R. - Murray, S. S. - Schork, N. J. - Topol, E. J. - Frazer, K. A.
Journal: Genome Res

Next-generation sequencing technologies have made it possible to sequence targeted regions of the human genome in hundreds of individuals. Deep sequencing represents a powerful approach for the discovery of the complete spectrum of DNA sequence variants in functionally important genomic intervals. Current methods for single nucleotide polymorphism (SNP) detection are designed to detect SNPs from single individual sequence data sets. Here, we describe a novel method SNIP-Seq (single nucleotide polymorphism identification from population sequence data) that leverages sequence data from a population of individuals to detect SNPs and assign genotypes to individuals. To evaluate our method, we utilized sequence data from a 200-kilobase (kb) region on chromosome 9p21 of the human genome. This region was sequenced in 48 individuals (five sequenced in duplicate) using the Illumina GA platform. Using this data set, we demonstrate that our method is highly accurate for detecting variants and can filter out false SNPs that are attributable to sequencing errors. The concordance of sequencing-based genotype assignments between duplicate samples was 98.8%. The 200-kb region was independently sequenced to a high depth of coverage using two sequence pools containing the 48 individuals. Many of the novel SNPs identified by SNIP-Seq from the individual sequencing were validated by the pooled sequencing data and were subsequently confirmed by Sanger sequencing. We estimate that SNIP-Seq achieves a low false-positive rate of approximately 2%, improving upon the higher false-positive rate for existing methods that do not utilize population sequence data. Collectively, these results suggest that analysis of population sequencing data is a powerful approach for the accurate detection of SNPs and the assignment of genotypes to individual samples.

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Inference of RhoGAP/GTPase regulation using single-cell morphological data from a combinatorial RNAi screen.

Publication Date: 2010 Mar PMID: 20144944
Authors: Nir, O. - Bakal, C. - Perrimon, N. - Berger, B.
Journal: Genome Res

Biological networks are highly complex systems, consisting largely of enzymes that act as molecular switches to activate/inhibit downstream targets via post-translational modification. Computational techniques have been developed to perform signaling network inference using some high-throughput data sources, such as those generated from transcriptional and proteomic studies, but comparable methods have not been developed to use high-content morphological data, which are emerging principally from large-scale RNAi screens, to these ends. Here, we describe a systematic computational framework based on a classification model for identifying genetic interactions using high-dimensional single-cell morphological data from genetic screens, apply it to RhoGAP/GTPase regulation in Drosophila, and evaluate its efficacy. Augmented by knowledge of the basic structure of RhoGAP/GTPase signaling, namely, that GAPs act directly upstream of GTPases, we apply our framework for identifying genetic interactions to predict signaling relationships between these proteins. We find that our method makes mediocre predictions using only RhoGAP single-knockdown morphological data, yet achieves vastly improved accuracy by including original data from a double-knockdown RhoGAP genetic screen, which likely reflects the redundant network structure of RhoGAP/GTPase signaling. We consider other possible methods for inference and show that our primary model outperforms the alternatives. This work demonstrates the fundamental fact that high-throughput morphological data can be used in a systematic, successful fashion to identify genetic interactions and, using additional elementary knowledge of network structure, to infer signaling relations.

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Dynamic changes in the human methylome during differentiation.

Publication Date: 2010 Mar PMID: 20133333
Authors: Laurent, L. - Wong, E. - Li, G. - Huynh, T. - Tsirigos, A. - Ong, C. T. - Low, H. M. - Kin Sung, K. W. - Rigoutsos, I. - Loring, J. - Wei, C. L.
Journal: Genome Res

DNA methylation is a critical epigenetic regulator in mammalian development. Here, we present a whole-genome comparative view of DNA methylation using bisulfite sequencing of three cultured cell types representing progressive stages of differentiation: human embryonic stem cells (hESCs), a fibroblastic differentiated derivative of the hESCs, and neonatal fibroblasts. As a reference, we compared our maps with a methylome map of a fully differentiated adult cell type, mature peripheral blood mononuclear cells (monocytes). We observed many notable common and cell-type-specific features among all cell types. Promoter hypomethylation (both CG and CA) and higher levels of gene body methylation were positively correlated with transcription in all cell types. Exons were more highly methylated than introns, and sharp transitions of methylation occurred at exon-intron boundaries, suggesting a role for differential methylation in transcript splicing. Developmental stage was reflected in both the level of global methylation and extent of non-CpG methylation, with hESC highest, fibroblasts intermediate, and monocytes lowest. Differentiation-associated differential methylation profiles were observed for developmentally regulated genes, including the HOX clusters, other homeobox transcription factors, and pluripotence-associated genes such as POU5F1, TCF3, and KLF4. Our results highlight the value of high-resolution methylation maps, in conjunction with other systems-level analyses, for investigation of previously undetectable developmental regulatory mechanisms.

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Digital transcriptome profiling from attomole-level RNA samples.

Publication Date: 2010 Mar 4 PMID: 20133332
Authors: Ozsolak, F. - Goren, A. - Gymrek, M. - Guttman, M. - Regev, A. - Bernstein, B. E. - Milos, P. M.
Journal: Genome Res

Accurate profiling of minute quantities of RNA in a global manner can enable key advances in many scientific and clinical disciplines. Here, we present low-quantity RNA sequencing (LQ-RNAseq), a high-throughput sequencing-based technique allowing whole transcriptome surveys from subnanogram RNA quantities in an amplification/ligation-free manner. LQ-RNAseq involves first-strand cDNA synthesis from RNA templates, followed by 3' polyA tailing of the single-stranded cDNA products and direct single molecule sequencing. We applied LQ-RNAseq to profile S. cerevisiae polyA+ transcripts, demonstrate the reproducibility of the approach across different sample preparations and independent instrument runs, and establish the absolute quantitative power of this method through comparisons with other reported transcript profiling techniques and through utilization of RNA spike-in experiments. We demonstrate the practical application of this approach to define the transcriptional landscape of mouse embryonic and induced pluripotent stem cells, observing transcriptional differences, including over 100 genes exhibiting differential expression between these otherwise very similar stem cell populations. This amplification-independent technology, which utilizes small quantities of nucleic acid and provides quantitative measurements of cellular transcripts, enables global gene expression measurements from minute amounts of materials and offers broad utility in both basic research and translational biology for characterization of rare cells.

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Positioned and G/C-capped poly(dA:dT) tracts associate with the centers of nucleosome-free regions in yeast promoters.

Publication Date: 2010 Mar 5 PMID: 20133331
Authors: Wu, R. - Li, H.
Journal: Genome Res

Eukaryotic transcriptional regulation is mediated by the organization of nucleosomes in promoter regions. Most Saccharomyces cerevisiae promoters have a highly stereotyped chromatin organization, where nucleosome-free regions (NFR) are flanked by well-ordered nucleosomes. We have found that yeast promoters fall into two classes differing in NFR sharpness, and that this distinction follows a known transcriptional dichotomy in yeast genes. A class of yeast promoters having well-defined NFRs are characterized by positioned patterns of poly(dA:dT) tracts with several novel features. First, poly(dA:dT) tracts are localized in a strand-dependent manner, with poly(dA) tracts lying proximal to transcriptional start sites and poly(dT) tracts lying distal, and collectively define a symmetry axis that is coincident with NFR centers. Second, poly(dA:dT) tracts are preferentially "capped" by G:C residues on the terminus proximal to the symmetry axis. Both signature features co-vary with fine positional variations between NFRs, establishing a closely knit relationship between poly(dA:dT) tracts, their capping patterns, and the central coordinates of NFRs. We found that these features are unique to promoters with well-defined NFRs, and that these promoters display significant difference between in vitro and in vivo nucleosome occupancy patterns. These observations are consistent with a model in which localized and G:C-capped poly(dA:dT) tracts initiate or facilitate the formation of NFRs at their center, possibly with chromatin remodeling and transcriptional machines involved.

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ATRX interacts with H3.3 in maintaining telomere structural integrity in pluripotent embryonic stem cells.

Publication Date: 2010 Mar PMID: 20110566
Authors: Wong, L. H. - McGhie, J. D. - Sim, M. - Anderson, M. A. - Ahn, S. - Hannan, R. D. - George, A. J. - Morgan, K. A. - Mann, J. R. - Choo, K. H.
Journal: Genome Res

ATRX (alpha thalassemia/mental retardation syndrome X-linked) belongs to the SWI2/SNF2 family of chromatin remodeling proteins. Besides the ATPase/helicase domain at its C terminus, it contains a PHD-like zinc finger at the N terminus. Mutations in the ATRX gene are associated with X-linked mental retardation (XLMR) often accompanied by alpha thalassemia (ATRX syndrome). Although ATRX has been postulated to be a transcriptional regulator, its precise roles remain undefined. We demonstrate ATRX localization at the telomeres in interphase mouse embryonic stem (ES) cells in synchrony with the incorporation of H3.3 during telomere replication at S phase. Moreover, we found that chromobox homolog 5 (CBX5) (also known as heterochromatin protein 1 alpha, or HP1 alpha) is also present at the telomeres in ES cells. We show by coimmunoprecipitation that this localization is dependent on the association of ATRX with histone H3.3, and that mutating the K4 residue of H3.3 significantly diminishes ATRX and H3.3 interaction. RNAi-knockdown of ATRX induces a telomere-dysfunction phenotype and significantly reduces CBX5 enrichment at the telomeres. These findings suggest a novel function of ATRX, working in conjunction with H3.3 and CBX5, as a key regulator of ES-cell telomere chromatin.

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Widespread and tissue specific age-related DNA methylation changes in mice.

Publication Date: 2010 Mar PMID: 20107151
Authors: Maegawa, S. - Hinkal, G. - Kim, H. S. - Shen, L. - Zhang, L. - Zhang, J. - Zhang, N. - Liang, S. - Donehower, L. A. - Issa, J. P.
Journal: Genome Res

Aberrant methylation of promoter CpG islands in cancer is associated with silencing of tumor-suppressor genes, and age-dependent hypermethylation in normal appearing mucosa may be a risk factor for human colon cancer. It is not known whether this age-related DNA methylation phenomenon is specific to human tissues. We performed comprehensive DNA methylation profiling of promoter regions in aging mouse intestine using methylated CpG island amplification in combination with microarray analysis. By comparing C57BL/6 mice at 3-mo-old versus 35-mo-old for 3627 detectable autosomal genes, we found 774 (21%) that showed increased methylation and 466 (13%) that showed decreased methylation. We used pyrosequencing to quantitatively validate the microarray data and confirmed linear age-related methylation changes for all 12 genomic regions examined. We then examined 11 changed genomic loci for age-related methylation in other tissues. Of these, three of 11 showed similar changes in lung, seven of 11 changed in liver, and six of 11 changed in spleen, though to a lower degree than the changes seen in colon. There was partial conservation between age-related hypermethylation in human and mouse intestines, and Polycomb targets in embryonic stem cells were enriched among the hypermethylated genes. Our findings demonstrate a surprisingly high rate of hyper- and hypomethylation as a function of age in normal mouse small intestine tissues and a strong tissue-specificity to the process. We conclude that epigenetic deregulation is a common feature of aging in mammals.

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Impact of replication timing on non-CpG and CpG substitution rates in mammalian genomes.

Publication Date: 2010 Feb 18 PMID: 20103589
Authors: Chen, C. L. - Rappailles, A. - Duquenne, L. - Huvet, M. - Guilbaud, G. - Farinelli, L. - Audit, B. - d'Aubenton-Carafa, Y. - Arneodo, A. - Hyrien, O. - Thermes, C.
Journal: Genome Res

Neutral nucleotide substitutions occur at varying rates along genomes, and it remains a major issue to unravel the mechanisms that cause these variations and to analyze their evolutionary consequences. Here, we study the role of replication in the neutral substitution pattern. We obtained a high-resolution replication timing profile of the whole human genome by massively parallel sequencing of nascent BrdU-labeled replicating DNA. These data were compared to the neutral substitution rates along the human genome, obtained by aligning human and chimpanzee genomes using macaque and orangutan as outgroups. All substitution rates increase monotonously with replication timing even after controlling for local or regional nucleotide composition, crossover rate, distance to telomeres, and chromatin compaction. The increase in non-CpG substitution rates might result from several mechanisms including the increase in mutation-prone activities or the decrease in efficiency of DNA repair during the S phase. In contrast, the rate of C --> T transitions in CpG dinucleotides increases in later-replicating regions due to increasing DNA methylation level that reflects a negative correlation between timing and gene expression. Similar results are observed in the mouse, which indicates that replication timing is a main factor affecting nucleotide substitution dynamics at non-CpG sites and constitutes a major neutral process driving mammalian genome evolution.

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Population differentiation as a test for selective sweeps.

Publication Date: 2010 Mar PMID: 20086244
Authors: Chen, H. - Patterson, N. - Reich, D.
Journal: Genome Res

Selective sweeps can increase genetic differentiation among populations and cause allele frequency spectra to depart from the expectation under neutrality. We present a likelihood method for detecting selective sweeps that involves jointly modeling the multilocus allele frequency differentiation between two populations. We use Brownian motion to model genetic drift under neutrality, and a deterministic model to approximate the effect of a selective sweep on single nucleotide polymorphisms (SNPs) in the vicinity. We test the method with extensive simulated data, and demonstrate that in some scenarios the method provides higher power than previously reported approaches to detect selective sweeps, and can provide surprisingly good localization of the position of a selected allele. A strength of our technique is that it uses allele frequency differentiation between populations, which is much more robust to ascertainment bias in SNP discovery than methods based on the allele frequency spectrum. We apply this method to compare continentally diverse populations, as well as Northern and Southern Europeans. Our analysis identifies a list of loci as candidate targets of selection, including well-known selected loci and new regions that have not been highlighted by previous scans for selection.

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Nitrogen depletion in the fission yeast Schizosaccharomyces pombe causes nucleosome loss in both promoters and coding regions of activated genes.

Publication Date: 2010 Mar PMID: 20086243
Authors: Kristell, C. - Orzechowski Westholm, J. - Olsson, I. - Ronne, H. - Komorowski, J. - Bjerling, P.
Journal: Genome Res

Gene transcription is associated with local changes in chromatin, both in nucleosome positions and in chemical modifications of the histones. Chromatin dynamics has mostly been studied on a single-gene basis. Those genome-wide studies that have been made primarily investigated steady-state transcription. However, three studies of genome-wide changes in chromatin during the transcriptional response to heat shock in the budding yeast Saccharomyces cerevisiae revealed nucleosome eviction in promoter regions but only minor effects in coding regions. Here, we describe the short-term response to nitrogen starvation in the fission yeast Schizosaccharomyces pombe. Nitrogen depletion leads to a fast induction of a large number of genes in S. pombe and is thus suitable for genome-wide studies of chromatin dynamics during gene regulation. After 20 min of nitrogen removal, 118 transcripts were up-regulated. The distribution of regulated genes throughout the genome was not random; many up-regulated genes were found in clusters, while large parts of the genome were devoid of up-regulated genes. Surprisingly, this up-regulation was associated with nucleosome eviction of equal magnitudes in the promoters and in the coding regions. The nucleosome loss was not limited to induction by nitrogen depletion but also occurred during cadmium treatment. Furthermore, the lower nucleosome density persisted for at least 60 min after induction. Two highly induced genes, urg1(+) and urg2(+), displayed a substantial nucleosome loss, with only 20% of the nucleosomes being left in the coding region. We conclude that nucleosome loss during transcriptional activation is not necessarily limited to promoter regions.

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Copy number abnormalities in sporadic canine colorectal cancers.

Publication Date: 2010 Mar PMID: 20086242
Authors: Tang, J. - Le, S. - Sun, L. - Yan, X. - Zhang, M. - Macleod, J. - Leroy, B. - Northrup, N. - Ellis, A. - Yeatman, T. J. - Liang, Y. - Zwick, M. E. - Zhao, S.
Journal: Genome Res

Human colorectal cancer (CRC) is one of the better-understood systems for studying the genetics of cancer initiation and progression. To develop a cross-species comparison strategy for identifying CRC causative gene or genomic alterations, we performed array comparative genomic hybridization (aCGH) to investigate copy number abnormalities (CNAs), one of the most prominent lesion types reported for human CRCs, in 10 spontaneously occurring canine CRCs. The results revealed for the first time a strong degree of genetic homology between sporadic canine and human CRCs. First, we saw that between 5% and 22% of the canine genome was amplified/deleted in these tumors, and that, reminiscent of human CRCs, the total altered sequences directly correlated to the tumor's progression stage, origin, and likely microsatellite instability status. Second, when mapping the identified CNAs onto syntenic regions of the human genome, we noted that the canine orthologs of genes participating in known human CRC pathways were recurrently disrupted, indicating that these pathways might be altered in the canine CRCs as well. Last, we observed a significant overlapping of CNAs between human and canine tumors, and tumors from the two species were clustered according to the tumor subtypes but not the species. Significantly, compared with the shared CNAs, we found that species-specific (especially human-specific) CNAs localize to evolutionarily unstable regions that harbor more segmental duplications and interspecies genomic rearrangement breakpoints. These findings indicate that CNAs recurrent between human and dog CRCs may have a higher probability of being cancer-causative, compared with CNAs found in one species only.

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Genome-wide discovery of human heart enhancers.

Publication Date: 2010 Mar PMID: 20075146
Authors: Narlikar, L. - Sakabe, N. J. - Blanski, A. A. - Arimura, F. E. - Westlund, J. M. - Nobrega, M. A. - Ovcharenko, I.
Journal: Genome Res

The various organogenic programs deployed during embryonic development rely on the precise expression of a multitude of genes in time and space. Identifying the cis-regulatory elements responsible for this tightly orchestrated regulation of gene expression is an essential step in understanding the genetic pathways involved in development. We describe a strategy to systematically identify tissue-specific cis-regulatory elements that share combinations of sequence motifs. Using heart development as an experimental framework, we employed a combination of Gibbs sampling and linear regression to build a classifier that identifies heart enhancers based on the presence and/or absence of various sequence features, including known and putative transcription factor (TF) binding specificities. In distinguishing heart enhancers from a large pool of random noncoding sequences, the performance of our classifier is vastly superior to four commonly used methods, with an accuracy reaching 92% in cross-validation. Furthermore, most of the binding specificities learned by our method resemble the specificities of TFs widely recognized as key players in heart development and differentiation, such as SRF, MEF2, ETS1, SMAD, and GATA. Using our classifier as a predictor, a genome-wide scan identified over 40,000 novel human heart enhancers. Although the classifier used no gene expression information, these novel enhancers are strongly associated with genes expressed in the heart. Finally, in vivo tests of our predictions in mouse and zebrafish achieved a validation rate of 62%, significantly higher than what is expected by chance. These results support the existence of underlying cis-regulatory codes dictating tissue-specific transcription in mammalian genomes and validate our enhancer classifier strategy as a method to uncover these regulatory codes.

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Evolutionary constraint facilitates interpretation of genetic variation in resequenced human genomes.

Publication Date: 2010 Mar PMID: 20067941
Authors: Goode, D. L. - Cooper, G. M. - Schmutz, J. - Dickson, M. - Gonzales, E. - Tsai, M. - Karra, K. - Davydov, E. - Batzoglou, S. - Myers, R. M. - Sidow, A.
Journal: Genome Res

Here, we demonstrate how comparative sequence analysis facilitates genome-wide base-pair-level interpretation of individual genetic variation and address two questions of importance for human personal genomics: first, whether an individual's functional variation comes mostly from noncoding or coding polymorphisms; and, second, whether population-specific or globally-present polymorphisms contribute more to functional variation in any given individual. Neither has been definitively answered by analyses of existing variation data because of a focus on coding polymorphisms, ascertainment biases in favor of common variation, and a lack of base-pair-level resolution for identifying functional variants. We resequenced 575 amplicons within 432 individuals at genomic sites enriched for evolutionary constraint and also analyzed variation within three published human genomes. We find that single-site measures of evolutionary constraint derived from mammalian multiple sequence alignments are strongly predictive of reductions in modern-day genetic diversity across a range of annotation categories and across the allele frequency spectrum from rare (<1%) to high frequency (>10% minor allele frequency). Furthermore, we show that putatively functional variation in an individual genome is dominated by polymorphisms that do not change protein sequence and that originate from our shared ancestral population and commonly segregate in human populations. These observations show that common, noncoding alleles contribute substantially to human phenotypes and that constraint-based analyses will be of value to identify phenotypically relevant variants in individual genomes.

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Population genetic inference from genomic sequence variation.

Publication Date: 2010 Mar PMID: 20067940
Authors: Pool, J. E. - Hellmann, I. - Jensen, J. D. - Nielsen, R.
Journal: Genome Res

Population genetics has evolved from a theory-driven field with little empirical data into a data-driven discipline in which genome-scale data sets test the limits of available models and computational analysis methods. In humans and a few model organisms, analyses of whole-genome sequence polymorphism data are currently under way. And in light of the falling costs of next-generation sequencing technologies, such studies will soon become common in many other organisms as well. Here, we assess the challenges to analyzing whole-genome sequence polymorphism data, and we discuss the potential of these data to yield new insights concerning population history and the genomic prevalence of natural selection.

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Genome-wide evidence for selection acting on single amino acid repeats.

Publication Date: 2010 Feb 11 PMID: 20056893
Authors: Haerty, W. - Golding, G. B.
Journal: Genome Res

Low complexity and homopolymer sequences within coding regions are known to evolve rapidly. While their expansion may be deleterious, there is increasing evidence for a functional role associated with these amino acid sequences. Homopolymer sequences are thought to evolve mostly through replication slippage and, therefore, they may be expected to be longer in regions with relaxed selective constraint. Within the coding sequences of eukaryotes, alternatively spliced exons are known to evolve under relaxed constraints in comparison to those exons that are constitutively spliced because they are not included in all of the mature mRNA of a gene. This relaxed exposure to selection leads to faster rates of evolution for alternatively spliced exons in comparison to constitutively spliced exons. Here, we have tested the effect of splicing on the structure (composition, length) of homopolymer sequences in relation to the splicing pattern in which they are found. We observed a significant relationship between alternative splicing and homopolymer sequences with alternatively spliced genes being enriched in number and length of homopolymer sequences. We also observed lower codon diversity and longer homocodons, suggesting a balance between slippage and point mutations linked to the constraints imposed by selection.

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Human genetic variation recognizes functional elements in noncoding sequence.

Publication Date: 2010 Mar PMID: 20032171
Authors: Lomelin, D. - Jorgenson, E. - Risch, N.
Journal: Genome Res

Noncoding DNA, particularly intronic DNA, harbors important functional elements that affect gene expression and RNA splicing. Yet, it is unclear which specific noncoding sites are essential for gene function and regulation. To identify functional elements in noncoding DNA, we characterized genetic variation within introns using ethnically diverse human polymorphism data from three public databases-PMT, NIEHS, and SeattleSNPs. We demonstrate that positions within introns corresponding to known functional elements involved in pre-mRNA splicing, including the branch site, splice sites, and polypyrimidine tract show reduced levels of genetic variation. Additionally, we observed regions of reduced genetic variation that are candidates for distance-dependent localization sites of functional elements, possibly intronic splicing enhancers (ISEs). Using several bioinformatics approaches, we provide additional evidence that supports our hypotheses that these regions correspond to ISEs. We conclude that studies of genetic variation can successfully discriminate and identify functional elements in noncoding regions. As more noncoding sequence data become available, the methods employed here can be utilized to identify additional functional elements in the human genome and provide possible explanations for phenotypic associations.

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