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1.
Nucleic Acids Res ; 38(16): e165, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20615900

ABSTRACT

Next-generation sequencing is excellently suited to evaluate the abundance of mRNAs to study gene expression. Here we compare two alternative technologies, cap analysis of gene expression (CAGE) and serial analysis of gene expression (SAGE), for the same RNA samples. Along with quantifying gene expression levels, CAGE can be used to identify tissue-specific transcription start sites, while SAGE monitors 3'-end usage. We used both methods to get more insight into the transcriptional control of myogenesis, studying differential gene expression in differentiated and proliferating C2C12 myoblast cells with statistical evaluation of reproducibility and differential gene expression. Both CAGE and SAGE provided highly reproducible data (Pearson's correlations >0.92 among biological triplicates). With both methods we found around 10,000 genes expressed at levels >2 transcripts per million (approximately 0.3 copies per cell), with an overlap of 86%. We identified 4304 and 3846 genes differentially expressed between proliferating and differentiated C2C12 cells by CAGE and SAGE, respectively, with an overlap of 2144. We identified 196 novel regulatory regions with preferential use in proliferating or differentiated cells. Next-generation sequencing of CAGE and SAGE libraries provides consistent expression levels and can enrich current genome annotations with tissue-specific promoters and alternative 3'-UTR usage.


Subject(s)
Gene Expression Profiling/methods , Myoblasts/metabolism , Sequence Analysis, RNA , 3' Untranslated Regions , Animals , Cell Line , Mice , Models, Biological , Muscle Development/genetics , Oligonucleotide Array Sequence Analysis , Reproducibility of Results , Sequence Alignment , Transcription Initiation Site
2.
Science ; 321(5891): 956-60, 2008 Aug 15.
Article in English | MEDLINE | ID: mdl-18599741

ABSTRACT

The functional complexity of the human transcriptome is not yet fully elucidated. We report a high-throughput sequence of the human transcriptome from a human embryonic kidney and a B cell line. We used shotgun sequencing of transcripts to generate randomly distributed reads. Of these, 50% mapped to unique genomic locations, of which 80% corresponded to known exons. We found that 66% of the polyadenylated transcriptome mapped to known genes and 34% to nonannotated genomic regions. On the basis of known transcripts, RNA-Seq can detect 25% more genes than can microarrays. A global survey of messenger RNA splicing events identified 94,241 splice junctions (4096 of which were previously unidentified) and showed that exon skipping is the most prevalent form of alternative splicing.


Subject(s)
Alternative Splicing , Gene Expression Profiling , Genome, Human , RNA Splice Sites , RNA, Messenger/genetics , Sequence Analysis, RNA , Cell Line , Cell Line, Tumor , Computational Biology , DNA, Complementary , DNA, Intergenic , Exons , Humans , Introns , Oligonucleotide Array Sequence Analysis , RNA Polymerase II/metabolism
3.
Biochem Biophys Res Commun ; 364(4): 1022-5, 2007 Dec 28.
Article in English | MEDLINE | ID: mdl-17971300

ABSTRACT

Little is known about determinants regulating expression of Mannan-binding lectin associated serine protease-2 (MASP-2), the effector component of the lectin pathway of complement activation. Comparative bioinformatic analysis of the MASP2 promoter regions in human, mouse, and rat, revealed conservation of two putative Stat binding sites, termed StatA and StatB. Site directed mutagenesis specific for these sites was performed. Transcription activity was decreased 5-fold when StatB site was mutated in the wildtype reporter gene construct. Gel retardation and competition assays demonstrated that proteins contained in the nuclear extract prepared from HepG2 specifically bound double-stranded StatB oligonucleotides. Supershift analysis revealed Stat3 to be the major specific binding protein. We conclude that Stat3 binding is important for MASP2 promoter activity.


Subject(s)
Gene Expression Regulation/physiology , Mannose-Binding Protein-Associated Serine Proteases/metabolism , STAT3 Transcription Factor/metabolism , Animals , Humans , Mice
4.
Proc Natl Acad Sci U S A ; 103(15): 5682-7, 2006 Apr 11.
Article in English | MEDLINE | ID: mdl-16581909

ABSTRACT

Shared transcription factor binding sites that are conserved in distance and orientation help control the expression of gene products that act together in the same biological context. New bioinformatics approaches allow the rapid characterization of shared promoter structures and can be used to find novel interacting molecules. Here, these principles are demonstrated by using molecules linked to the unique functional unit of the glomerular slit diaphragm. An evolutionarily conserved promoter model was generated by comparative genomics in the proximal promoter regions of the slit diaphragm-associated molecule nephrin. Phylogenetic promoter fingerprints of known elements of the slit diaphragm complex identified the nephrin model in the promoter region of zonula occludens-1 (ZO-1). Genome-wide scans using this promoter model effectively predicted a previously unrecognized slit diaphragm molecule, cadherin-5. Nephrin, ZO-1, and cadherin-5 mRNA showed stringent coexpression across a diverse set of human glomerular diseases. Comparative promoter analysis can identify regulatory pathways at work in tissue homeostasis and disease processes.


Subject(s)
Membrane Proteins/genetics , Podocytes/physiology , Promoter Regions, Genetic , Transcription Factors/genetics , Animals , Antigens, CD , Cadherins/genetics , Conserved Sequence , Evolution, Molecular , Gene Expression Regulation , Humans , Intercellular Junctions/genetics , Mice , Models, Genetic , Phosphoproteins/genetics , Rats , Vertebrates , Zonula Occludens-1 Protein
5.
Biochemistry ; 45(12): 3863-74, 2006 Mar 28.
Article in English | MEDLINE | ID: mdl-16548514

ABSTRACT

Matrix metalloproteinase-20 (MMP-20, enamelysin) has a highly restricted pattern of expression. In healthy tissues, MMP-20 is observed in the enamel organ and pulp organ of developing teeth and is present only as an activated enzyme. To identify other tissues that may express MMP-20, we performed a systematic mouse tissue expression screen. Among the non-tooth tissues assayed, MMP-20 transcripts were identified only in minute quantities within the large intestine. The murine Mmp20 promoter was cloned, sequenced, and assessed for potential tooth-specific regulatory elements. In silico analysis identified four promoter modules that were common to Mmp20 and at least two of three coregulated predominantly tooth-specific genes that encode ameloblastin, amelogenin, and enamelin. We asked if the highly restricted MMP-20 expression pattern was associated with a broad substrate specificity that might preclude its expression in other tissues. An iterative mixture-based random doedecamer peptide library screen with Edman sequencing of MMP-20 cleavage products revealed that, among MMPs previously screened, MMP-20 had unique substrate preferences. These preferences indicate that MMP-20 has a deep and wide catalytic pocket that can accommodate substrates with large aromatic residues in the P1' position. On the basis of matrices derived from the peptide library data, we identified and then confirmed that type V collagen is an MMP-20 substrate. Since type V collagen is not present in dental enamel but is an otherwise widely distributed collagen, and since only active MMP-20 has been observed in teeth, our data suggest that control of MMP-20 activity is primarily regulated by transcriptional means.


Subject(s)
Collagen Type V/metabolism , Matrix Metalloproteinases/metabolism , Tooth/enzymology , Animals , Animals, Newborn , Base Sequence , Blotting, Southern , Catalytic Domain , Chromatography, High Pressure Liquid , Cloning, Molecular , DNA Primers , Electrophoresis, Polyacrylamide Gel , Hydrolysis , Matrix Metalloproteinase 20 , Matrix Metalloproteinases/chemistry , Matrix Metalloproteinases/genetics , Matrix Metalloproteinases/isolation & purification , Mice , Promoter Regions, Genetic , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction
6.
Genome Res ; 12(2): 349-54, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11827955

ABSTRACT

Scaffold/matrix attachment regions (S/MARs) are essential regulatory DNA elements of eukaryotic cells. They are major determinants of locus control of gene expression and can shield gene expression from position effects. Experimental detection of S/MARs requires substantial effort and is not suitable for large-scale screening of genomic sequences. In silico prediction of S/MARs can provide a crucial first selection step to reduce the number of candidates. We used experimentally defined S/MAR sequences as the training set and generated a library of new S/MAR-associated, AT-rich patterns described as weight matrices. A new tool called SMARTest was developed that identifies potential S/MARs by performing a density analysis based on the S/MAR matrix library (http://www.genomatix.de/cgi-bin/smartest_pd/smartest.pl). S/MAR predictions were evaluated by using six genomic sequences from animal and plant for which S/MARs and non-S/MARs were experimentally mapped. SMARTest reached a sensitivity of 38% and a specificity of 68%. In contrast to previous algorithms, the SMARTest approach does not depend on the sequence context and is suitable to analyze long genomic sequences up to the size of whole chromosomes. To demonstrate the feasibility of large-scale S/MAR prediction, we analyzed the recently published chromosome 22 sequence and found 1198 S/MAR candidates.


Subject(s)
Computational Biology/methods , DNA/genetics , Nuclear Matrix/genetics , Algorithms , Animals , Binding Sites/genetics , Chickens , DNA/metabolism , DNA, Plant/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Databases, Genetic , Humans , Mice , Nuclear Matrix/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oryza/genetics , Software
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