Genomics

Genome-wide Analysis of Hepatic Gene Silencing in Mammalian Cell Hybrids.

Publication Date: 2010 Aug 26 PMID: 20801210
Authors: Bulla, G. A. - Luong, Q. - Shrestha, S. - Reeb, S. - Hickman, S.
Journal: Genomics

Silencing of tissue-specific gene expression in mammalian somatic cell hybrids is a well-documented epigenetic phenomenon which is both profound (involving a large number of genes) and enigmatic. . Our aim was to utilize whole-genome microarray analyses to determine the true extent of gene silencing on a genomic level. By comparing gene expression profiles of hepatoma x fibroblast cell hybrids with those of parental cells, we have identified over 300 liver-enriched genes that are repressed at least 5-fold in the cell hybrids, the majority of which are repressed at least 10-fold. Also, we identify nearly 200 fibroblast-enriched genes that are repressed at least 5-fold. Silenced hepatic genes include several that encode transcription factors and proteins involved in signal transduction pathways.. These data suggest that extensive reprogramming occurs in cell hybrids, leading to a nearly global (although not complete) loss of tissue -specific gene expression.

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A comparative analysis of liver transcriptome suggests divergent liver function among human, mouse and rat.

Publication Date: 2010 Aug 25 PMID: 20800674
Authors: Yu, Y. - Ping, J. - Chen, H. - Jiao, L. - Zheng, S. - Han, Z. G. - Hao, P. - Huang, J.
Journal: Genomics

The human liver plays a vital role in meeting the body's metabolic needs and maintaining homeostasis. To address the molecular mechanisms of liver function, we integrated multiple gene expression datasets from microarray, MPSS, SAGE and EST platforms to generate a transcriptome atlas of the normal human liver. Our results show that 17396 genes are expressed in the human liver. 238 genes were identified as liver enrichment genes, involved in the functions of immune response and metabolic processes, from the MPSS and EST datasets. A comparative analysis of liver transcriptomes was performed in humans, mice and rats with microarray datasets shows that the expression profile of homologous genes remains significantly different between mouse/rat and human, suggesting a functional variance and regulation bias of genes expressed in the livers. The integrated liver transcriptome data should provide a valuable resource for the in-depth understanding of human liver biology and liver disease.

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Genome-wide analysis of expression modes and DNA methylation status at sense-antisense transcript loci in mouse.

Publication Date: 2010 Aug 21 PMID: 20736060
Authors: Watanabe, Y. - Numata, K. - Murata, S. - Osada, Y. - Saito, R. - Nakaoka, H. - Yamamoto, N. - Watanabe, K. - Kato, H. - Abe, K. - Kiyosawa, H.
Journal: Genomics

The functionality of sense-antisense transcripts (SATs), although widespread throughout the mammalian genome, is largely unknown. Here, we analyzed the SATs expression and its associated promoter DNA methylation status by surveying 12 tissues of mice to gain insights into the relationship between expression and DNA methylation of SATs. We have found that sense and antisense expression positively correlate in most tissues. However, in some SATs with tissue-specific expression, the expression level of a transcript from a CpG island-bearing promoter is low when the promoter DNA methylation is present. In these circumstances, the expression level of its opposite strand transcript, especially when it is poly(A)-negative was coincidentally higher. These observations suggest that, albeit the general tendency of sense-antisense simultaneous expression, some antisense transcripts have coordinated expression with its counterpart sense gene promoter methylation. This cross-strand relationship is not a privilege of imprinted genes but seems to occur widely in SATs.

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Chemogenomic profiling of the cellular effects associated with histone H3 acetylation impairment by a quinoline-derived compound.

Publication Date: 2010 Aug 21 PMID: 20732410
Authors: Ruotolo, R. - Tosi, F. - Vernarecci, S. - Ballario, P. - Mai, A. - Filetici, P. - Ottonello, S.
Journal: Genomics

We report the results of a chemogenomic profiling aimed to explore the mode of action of a quinolic analogue of the p300 histone acetyltransferase (HAT) inhibitor anacardic acid, named MC1626. This compound reduced histone H3 acetylation in a dose-dependent manner and the HATs Gcn5 and Rtt109, which specifically target H3 lysines, were the only ones that caused chemical-genetic synthetic sickness with MC1626 when mutated. Deletion of specific Gcn5 (e.g., Ada1) and Rtt109 (e.g., Asf1) multiprotein complex components also enhanced MC1626 sensitivity. In addition to N-terminal H3 lysines, MC1626 inhibits H3-K56 acetylation, a histone modification that, in yeast, is exclusively supported by Rtt109 and indirectly influences DNA integrity. Several DNA repair mutants were found to be sensitive to MC1626. Functional links between histone acetylation impairment by MC1626 and mitochondrion as well as cytoskeleton functionality were also revealed, thus extending the range of non-nuclear processes that are influenced by histone acetylation.

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Bioinformatic analysis of TE-spliced new exons within human, mouse and zebrafish genomes.

Publication Date: 2010 Aug 19 PMID: 20728532
Authors: Kim, D. S. - Huh, J. W. - Kim, Y. H. - Park, S. J. - Kim, H. S. - Chang, K. T.
Journal: Genomics

Recent studies indicate major roles for transposable elements (TEs) in alternative splicing. In this study, we conducted genome-wide alternative splicing analyses focusing on new internal exon birth derived from TEs in human, mouse, and zebrafish genomes. We identified two different exon sets, TE-spliced exons and non-TE-spliced exons. The proportion of TE-spliced exons was nearly twice as high as the proportion of non-TE-spliced exons in the coding sequence (CDS) region. Detailed analysis of various families of TEs in three different species of TE-spliced exons revealed a different pattern in zebrafish. In our analysis, we could identify the functional role of TE insertions in the vertebrate genome affecting mRNA splicing machinery. Their effects can be directly linked to the shift from constitutive to alternative splicing during primate evolution. Our results indicate that TEs have a significant effect on shaping new internal exons in human, mouse, and zebrafish transcriptomes.

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BWtrs: A tool for searching for tandem repeats in DNA sequences based on the Burrows-Wheeler transform.

Publication Date: 2010 Aug 13 PMID: 20709168
Authors: Pokrzywa, R. - Polanski, A.
Journal: Genomics

Genomes of organisms contain a variety of repeated structures of various length and type, interspersed or tandem. Tandem repeats play important role in molecular biology as they are related to genetic backgrounds of inherited diseases, and also they can serve as markers for DNA mapping and DNA fingerprinting. Improving the efficiency of algorithms for searching for tandem repeats in DNA sequences can lead to many useful applications in the area of genomics. We introduce a very efficient, web-based tool for large scale searching for exact tandem repeats in genomes, based on the use of the Burrows-Wheeler Transform. The service is a remarkably efficient and powerful application that allows analyzing complete genomes without any restrictions. The Burrows-Wheeler Tandem Repeat Searcher (BWtrs) is an on-line application that searches for the exact occurrences of tandem repetitions in DNA sequences. The BWtrs service is freely available at: http://bioinfo.polsl.pl/BWtrs. We present examples of the use of our web application and we compare results of our computations with the results obtained by using other existing tools for searches for exact tandem repeats.

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Mutation in erythroid specific transcription factor KLF1 causes Hereditary Spherocytosis in the Nan hemolytic anemia mouse model.

Publication Date: 2010 Aug 5 PMID: 20691777
Authors: Heruth, D. P. - Hawkins, T. - Logsdon, D. P. - Gibson, M. I. - Sokolovsky, I. V. - Nsumu, N. N. - Major, S. L. - Fegley, B. - Woods, G. M. - Lewing, K. B. - Neville, K. A. - Cornetta, K. - Peterson, K. R. - White, R. A.
Journal: Genomics

KLF1 regulates definitive erythropoiesis of red blood cells by facilitating transcription through high affinity binding to CACCC elements within its erythroid specific target genes including those encoding erythrocyte membrane skeleton (EMS) proteins. Deficiencies of EMS proteins in humans lead to the hemolytic anemia Hereditary Spherocytosis (HS) which includes a subpopulation with no known genetic defect. Here we report that a mutation, E339D, in the second zinc finger domain of KLF1 is responsible for HS in the mouse model Nan. The causative nature of this mutation was verified with an allelic test cross between Nan/+ and heterozygous Klf1(+/-) knockout mice. Homology modeling predicted Nan KLF1 binds CACCC elements more tightly, suggesting that Nan KLF1 is a competitive inhibitor of wild-type KLF1. This is the first association of a KLF1 mutation with a disease state in adult mammals and also presents the possibility of being another causative gene for HS in humans.

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A comparison between ribo-minus RNA-sequencing and polyA-selected RNA-sequencing.

Publication Date: 2010 Aug 3 PMID: 20688152
Authors: Cui, P. - Lin, Q. - Ding, F. - Xin, C. - Gong, W. - Zhang, L. - Geng, J. - Zhang, B. - Yu, X. - Yang, J. - Hu, S. - Yu, J.
Journal: Genomics

To compare the two RNA-sequencing protocols, ribo-minus RNA-sequencing (rmRNA-seq) and polyA-selected RNA-sequencing (mRNA-seq), we acquired transcriptomic data-52 and 32 million alignable reads of 35 bases in length-from the mouse cerebrum, respectively. We found that a higher proportion, 44% and 25%, of the uniquely alignable rmRNA-seq reads, is in intergenic and intronic regions, respectively, as compared to 23% and 15% from the mRNA-seq dataset. Further analysis made an additional discovery of transcripts of protein-coding genes (such as Histone, Heg1, and Dux), ncRNAs, snoRNAs, snRNAs, and novel ncRNAs as well as repeat elements in rmRNA-seq dataset. This result suggests that rmRNA-seq method should detect more polyA- or bimorphic transcripts. Finally, through comparative analyses of gene expression profiles among multiple datasets, we demonstrated that different RNA sample preparations may result in significant variations in gene expression profiles.

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Development of a versatile, target-oriented tiling microarray assay for measuring allele-specific gene expression.

Publication Date: 2010 Aug 3 PMID: 20688151
Authors: He, H. - Zhang, H. - Wang, X. - Wu, N. - Yang, X. - Chen, R. - Li, Y. - Deng, X. W. - Li, L.
Journal: Genomics

In the study of gene expression, it is often desirable to distinguish transcript pools derived from different alleles present in the same organism. We report here an oligonucleotide tiling microarray designed to specifically target 518 single nucleotide polymorphisms (SNPs) between the two sequenced rice (Oryza sativa) subspecies indica and japonica. The tiling array included all 25-mer probes interrogating each SNP by placing the polymorphic site at all 25 possible positions within the probe. Through hybridization to a titration series in which the japonica- and indica-derived cDNA templates were mixed with altering proportions, a regression model was used to screen for diagnostic probe sets for each SNP. Our result indicates that 284 (55%) SNPs have at least one diagnostic probe pair suitable for distinguishing and quantifying the relative abundance of allele-specific transcripts. As a proof-of-concept, we analyzed allele-specific expression in reciprocal indicaxjaponica F(1) hybrids and detected imbalanced expression at approximately one third of the SNPs. These results were validated by RNA-sequencing and allele-specific real-time PCR experiments. Together, our work demonstrates the utility and advantages of the tiling array method in interrogating large numbers of SNPs for quantifying allele-specific gene expression.

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The probability of nonsense mutation caused by replication-associated mutational pressure is much higher for bacterial genes from lagging than from leading strands.

Publication Date: 2010 Sep PMID: 20600808
Authors: Khrustalev, V. V. - Barkovsky, E. V.
Journal: Genomics

We studied nucleotide usage biases in 4-fold degenerated sites of all the genes from leading and lagging strands of 30 bacterial genomes. The level of guanine in 4-fold degenerated sites (G4f) is significantly lower in genes from lagging strands than in genes from leading strands, probably because of the faster rates of guanine oxidation in single-stranded DNA leading to G to T transversions. The rates of cytosine deamination causing C to T transitions are also higher in lagging strands. We showed that the level of codons able to form stop-codons by the way of G to T transversions and C to T transitions is always higher than the level of codons able to form stop-codons by the way of C to A transversions and G to A transitions. This circumstance can be an explanation of the lower percent of ORFs in lagging strands of bacterial replichores than in leading strands.

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Human-chimpanzee promoter comparisons: property-conserved evolution?

Publication Date: 2010 Sep PMID: 20600807
Authors: Deyneko, I. V. - Kalybaeva, Y. M. - Kel, A. E. - Blocker, H.
Journal: Genomics

Identification of different functional elements and their properties is a fundamental need in biomedical research and phylogenetic comparisons of a growing number of sequenced genomes form a solid basis for this task. Most available phylogenetic approaches are focused on searching for individual sequence alterations, responsible for the observed phenotype, or statistically evaluate observed mutations to infer general trends. However, being applied to close genomes such methods suffer from poor statistics of rare mutations and give only (at its best) coarse results concerning the potential functional importance of the nucleotide differences. However, quantifying the changes in physical properties of DNA allows to see the strength of introduced mutations and hence to classify them for further investigations. In this work we present the comparative sequence analysis of two evolutionarily close species-human and chimpanzee. In contrast to previous studies we evaluate changes in melting enthalpy of DNA rather than count nucleotide mismatches. We find that nucleotide mismatches in promoters were apparently introduced in a correlated manner during the course of evolution, so that, for example, the DNA property "melting enthalpy" was retained. Such property conservation of promoters is significantly different from nucleotide conservation, shows significant positional and functional biases, and seems to represent a novel feature of gene regulation.

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Identification of conserved Drosophila-specific euchromatin-restricted non-coding sequence motifs.

Publication Date: 2010 Sep PMID: 20595017
Authors: Jung, C. H. - Makunin, I. V. - Mattick, J. S.
Journal: Genomics

Non-protein-coding DNA comprises the majority of animal genomes but its functions are largely unknown. We identified over 17,000 different tetranucleotide pairs in the Drosophila melanogaster genome that are over-represented at distances up to 100nt in conserved non-exonic sequences. Those exhibiting the highest information content in surrounding nucleotides were classified into five groups: tRNAs, motifs associated with histone genes, Suppressor-of-Hairy-wing binding sites, and two sets of previously unrecognized motifs (DLM3 and DLM4). There are hundreds to thousands of copies of DLM3 and DLM4, respectively, in the genome, located almost exclusively in non-coding regions. They have similar copy numbers among drosophilids, but are largely absent in other insects. DLM3 is likely a cis-regulatory element, whereas DLM4 sequences are capable of forming a short hairpin structure and are expressed as approximately 80nt RNAs. This work reports the existence of Drosophila genus-specific sequence motifs, and suggests that many more novel functional elements may be discovered in genomes using the general approach outlined herein.

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Chromosomal conservation and sequence diversity of ribosomal RNA genes of two distant Oryza species.

Publication Date: 2010 Sep PMID: 20580815
Authors: Chang, K. D. - Fang, S. A. - Chang, F. C. - Chung, M. C.
Journal: Genomics

Contrary to the chromosomal polymorphism of 45S ribosomal genes (45S rDNA) loci in other Oryza species, each of Oryza australiensis and Oryza brachyantha has only one 45S rDNA locus at the most conserved position of 45S rDNAs in Oryza. O. australiensis and O. brachyantha are known phylogenetically distant and have extremely different genome sizes among diploid Oryza species. This study reveals that the sequences and organizations of intergenic spacer (IGS) for 45S rDNA of both O. australiensis and O. brachyantha are different from other Oryza species. The IGS of O. australiensis contains 13 tandem repeats and only one transcriptional initiation site, while there are four tandem repeats and three transcriptional initiation sites in the IGS of O. brachyantha. Our results suggest different evolution processes of orthologous rDNA loci in the genus Oryza. Here we also demonstrate an efficient strategy to study locus-specific IGS before whole genome sequences data are available.

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Identification of lytic bacteriophage MmP1, assigned to a new member of T7-like phages infecting Morganella morganii.

Publication Date: 2010 Sep PMID: 20561579
Authors: Zhu, J. - Rao, X. - Tan, Y. - Xiong, K. - Hu, Z. - Chen, Z. - Jin, X. - Li, S. - Chen, Y. - Hu, F.
Journal: Genomics

MmP1 (Morganella morganii phage 1) is a lytic bacteriophage newly isolated from the host bacterium M. morganii. The entire genome was sequenced, and final assembly yielded a 38,234bp linear double-stranded DNA (dsDNA) with a G+C content of 46.5%. In the MmP1 genome, 49 putative genes, 10 putative promoters and 2 predicted sigma-independent terminators were determined through bioinformatic analysis. A striking feature of the MmP1 genome is its high degree of similarity to the T7 group of phages. All of the 49 predicted genes exist on the same DNA strand, and functions were assigned to 35 genes based on the similarity of the homologues deposited in GenBank, which share 30-80% identity to their counterparts in T7-like phages. The analyses of MmP1 using CoreGenes, phylogenetic tree of RNA polymerase and structural proteins have demonstrated that bacteriophage MmP1 should be assigned as a new member of T7-like phages but as a relatively distant member of this family. This is the first report that a T7-like phage adaptively parasitizes in M. morganii, and this will advance our understanding of biodiversity and adaptive evolution of T7-like phages.

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Transcriptional profiling in alopecia areata defines immune and cell cycle control related genes within disease-specific signatures.

Publication Date: 2010 Sep PMID: 20546884
Authors: Subramanya, R. D. - Coda, A. B. - Sinha, A. A.
Journal: Genomics

Alopecia areata (AA) is a non-scarring inflammatory hair loss disease with a complex autoimmune etiopathogenesis that is poorly understood. In order to investigate the pathogenesis of AA at the molecular level, we examined the gene expression profiles in skin samples from lesional (n=10) and non-lesional sites (n=10) of AA patients using Affymetrix Hu95A-v2 arrays. 363 genes were found to be differentially expressed in AA skin compared to non-lesional skin; 97 were up-regulated and 266 were down-regulated. Functional classification of the differentially expressed genes (DEGs) provides evidence for T-cell mediated immune response (CCL5, CXCL10, CD27, ICAM2, ICAM3, IL7R, and CX3CL1), and a possible humoral mechanism (IGHG3, IGHM, and CXCR5) in AA. We also find modulation in gene expression favoring cellular proliferation arrest at various levels (FGF5, FGF18, EREG, and FOXC2) with apoptotic dysregulation (LCK, TNF, TRAF2, and SFN) and decreased expression of hair follicle structural proteins. Further analysis of patients with AAT (<1 year duration, n=4) and AAP (>1 year duration, n=6) of disease revealed 262 DEGs distinctly separating the 2 groups, indicating the existence of gene profiles unique to the initial and later stages of disease.

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Genomic phenotype of non-cultured pulmonary fibroblasts in idiopathic pulmonary fibrosis.

Publication Date: 2010 Sep PMID: 20451601
Authors: Emblom-Callahan, M. C. - Chhina, M. K. - Shlobin, O. A. - Ahmad, S. - Reese, E. S. - Iyer, E. P. - Cox, D. N. - Brenner, R. - Burton, N. A. - Grant, G. M. - Nathan, S. D.
Journal: Genomics

Activated fibroblasts are the central effector cells of the progressive fibrotic process in idiopathic pulmonary fibrosis (IPF). Characterizing the genomic phenotype of isolated fibroblasts is essential to understanding IPF pathogenesis. Comparing the genomic phenotype of non-cultured pulmonary fibroblasts from advanced IPF patients' and normal lungs revealed novel genes, biological processes and concomitant pathways previously unreported in IPF fibroblasts. We demonstrate altered expression in proteasomal constituents, ubiquitination-mediators, Wnt, apoptosis and vitamin metabolic pathways and cell cycle regulators, suggestive of loss of cellular homeostasis. Specifically, FBXO32, CXCL14, BDKRB1 and NMNAT1 were up-regulated, while RARA and CDKN2D were down-regulated. Paradoxically, pro-apoptotic inducers TNFSF10, BAX and CASP6 were also found to be increased. This comprehensive description of altered gene expression in isolated IPF fibroblasts underscores the complex biological processes characteristic of IPF and may provide a foundation for future research into this devastating disease.

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