| 1. |
- Birney, Ewan, et al.
(författare)
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Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project
- 2007
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 447:7146, s. 799-816
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Tidskriftsartikel (refereegranskat)abstract
- We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.
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| 2. |
- Rada-Iglesias, Alvaro, et al.
(författare)
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Butyrate mediates decrease of histone acetylation centered on transcription start sites and down-regulation of associated genes
- 2007
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Ingår i: Genome Research. - : Cold Spring Harbor Laboratory. - 1088-9051 .- 1549-5469. ; 17:6, s. 708-719
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Tidskriftsartikel (refereegranskat)abstract
- Butyrate is a histone deacetylase inhibitor (HDACi) with anti-neoplastic properties, which theoretically reactivates epigenetically silenced genes by increasing global histone acetylation. However, recent studies indicate that a similar number or even more genes are down-regulated than up-regulated by this drug. We treated hepatocarcinoma HepG2 cells with butyrate and characterized the levels of acetylation at DNA-bound histones H3 and H4 by ChIP-chip along the ENCODE regions. In contrast to the global increases of histone acetylation, many genomic regions close to transcription start sites were deacetylated after butyrate exposure. In order to validate these findings, we found that both butyrate and trichostatin A treatment resulted in histone deacetylation at selected regions, while nucleosome loss or changes in histone H3 lysine 4 trimethylation (H3K4me3) did not occur in such locations. Furthermore, similar histone deacetylation events were observed when colon adenocarcinoma HT-29 cells were treated with butyrate. In addition, genes with deacetylated promoters were down-regulated by butyrate, and this was mediated at the transcriptional level by affecting RNA polymerase II (POLR2A) initiation/elongation. Finally, the global increase in acetylated histones was preferentially localized to the nuclear periphery, indicating that it might not be associated to euchromatin. Our results are significant for the evaluation of HDACi as anti-tumourogenic drugs, suggesting that previous models of action might need to be revised, and provides an explanation for the frequently observed repression of many genes during HDACi treatment.
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| 3. |
- Gustafsdottir, Sigrun M., et al.
(författare)
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In vitro analysis of DNA-protein interactions by proximity ligation
- 2007
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 104:9, s. 3067-3072
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Tidskriftsartikel (refereegranskat)abstract
- Protein-binding DNA sequence elements encode a variety of regulated functions of genomes. Information about such elements is currently in a state of rapid growth, but improved methods are required to characterize the sequence specificity of DNA-binding proteins. We have established an in vitro method for specific and sensitive solution-phase analysis of interactions between proteins and nucleic acids in nuclear extracts, based on the proximity ligation assay. The reagent consumption is very low, and the excellent sensitivity of the assay enables analysis of as few as 1-10 cells. We show that our results are highly reproducible, quantitative, and in good agreement with both EMSA and predictions obtained by using a motif finding software. This assay can be a valuable tool to characterize in-depth the sequence specificity of DNA-binding proteins and to evaluate effects of polymorphisms in known transcription factor binding sites.
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| 4. |
- Rada-Iglesias, Alvaro, et al.
(författare)
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Binding sites for metabolic disease related transcription factors inferred at base pair resolution by chromatin immunoprecipitation and genomic microarrays
- 2005
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Ingår i: Human Molecular Genetics. - : Oxford University Press (OUP). - 0964-6906 .- 1460-2083. ; 14:22, s. 3435-3447
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Tidskriftsartikel (refereegranskat)abstract
- We present a detailed in vivo characterization of hepatocyte transcriptional regulation in HepG2 cells, using chromatin immunoprecipitation and detection on PCR fragment-based genomic tiling path arrays covering the encyclopedia of DNA element (ENCODE) regions. Our data suggest that HNF-4α and HNF-3β, which were commonly bound to distal regulatory elements, may cooperate in the regulation of a large fraction of the liver transcriptome and that both HNF-4α and USF1 may promote H3 acetylation to many of their targets. Importantly, bioinformatic analysis of the sequences bound by each transcription factor (TF) shows an over-representation of motifs highly similar to the in vitro established consensus sequences. On the basis of these data, we have inferred tentative binding sites at base pair resolution. Some of these sites have been previously found by in vitro analysis and some were verified in vitro in this study. Our data suggests that a similar approach could be used for the in vivo characterization of all predicted/uncharacterized TF and that the analysis could be scaled to the whole genome.
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| 5. |
- Replogle, Kirstin, et al.
(författare)
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The Songbird Neurogenomics (SoNG) Initiative: Community-based tools and strategies for study of brain gene function and evolution
- 2008
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 9:131
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Tidskriftsartikel (refereegranskat)abstract
- Background: Songbirds hold great promise for biomedical, environmental and evolutionary research. A complete draft sequence of the zebra finch genome is imminent, yet a need remains for application of genomic resources within a research community traditionally focused on ethology and neurobiological methods. In response, we developed a core set of genomic tools and a novel collaborative strategy to probe gene expression in diverse songbird species and natural contexts. Results: We end-sequenced cDNAs from zebra finch brain and incorporated additional sequences from community sources into a database of 86,784 high quality reads. These assembled into 31,658 non-redundant contigs and singletons, which we annotated via BLAST search of chicken and human databases. The results are publicly available in the ESTIMA: Songbird database. We produced a spotted cDNA microarray with 20,160 addresses representing 17,214 non-redundant products of an estimated 11,500-15,000 genes, validating it by analysis of immediate-early gene (zenk) gene activation following song exposure and by demonstrating effective cross hybridization to genomic DNAs of other songbird species in the Passerida Parvorder. Our assembly was also used in the design of the "Lund-zfa" Affymetrix array representing similar to 22,000 non-redundant sequences. When the two arrays were hybridized to cDNAs from the same set of male and female zebra finch brain samples, both arrays detected a common set of regulated transcripts with a Pearson correlation coefficient of 0.895. To stimulate use of these resources by the songbird research community and to maintain consistent technical standards, we devised a "Community Collaboration" mechanism whereby individual birdsong researchers develop experiments and provide tissues, but a single individual in the community is responsible for all RNA extractions, labelling and microarray hybridizations. Conclusion: Immediately, these results set the foundation for a coordinated set of 25 planned experiments by 16 research groups probing fundamental links between genome, brain, evolution and behavior in songbirds. Energetic application of genomic resources to research using songbirds should help illuminate how complex neural and behavioral traits emerge and evolve.
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| 6. |
- Santos-Neto, Alvaro J., et al.
(författare)
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Capillary Column Switching Restricted-Access Media-Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry System for Simultaneous and Direct Analysis of Drugs in Biofluids
- 2007
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 79:16, s. 6359-6367
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Tidskriftsartikel (refereegranskat)abstract
- Capillary online restricted-access media-liquid chromatography-electrospray ionization-tandem mass spectrometry (RAM-LC-ESI-MS/MS) for direct analysis of drugs and metabolites spiked in biological fluids was developed. Using a column switching setup it was possible to perform effective sample preparation and analysis of raw biological fluids (plasma and urine) without matrix effects in the electrospray mass spectrometric detection step. The peak focusing efficiency of the extraction column was more effective in backflush compared to foreflush mode. The system was able to concentrate diminished samples of polar drugs and their metabolites reaching quantifiable results as low as 1 ng/mL utilizing a sample volume of only 333 nL of biofluids. New column hardware was developed to circumvent clogging problems experienced with plasma injections. The glass fiber filter frit, which is commonly used, was replaced with a short piece of 20 μm i.d. fused silica capillary. The extraction columns were able to handle up to 60 injections and showed a high loading capacity, making the saturation of the MS detector the limiting factor on the linear dynamic range. The simultaneous separation and detection of 10 drugs and metabolites was obtained in 8 min of analysis, including the online sample preparation and enrichment step.
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| 7. |
- Santos-Neto, Alvaro J., et al.
(författare)
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Simultaneous analysis of five antidepressant drugs using direct injection of biofluids in a capillary restricted-access media-liquid chromatography-tandem mass spectrometry system
- 2008
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1189:1-2, s. 514-522
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Tidskriftsartikel (refereegranskat)abstract
- Direct analysis, with minimal sample pretreatment, of antidepressant drugs, fluoxetine, imipramine, desipramine, amitriptyline, and nortriptyline in biofluids was developed with a total run time of 8 min. The setup consists of two HPLC pumps, injection valve, capillary RAM-ADS-C18 pre-column and a capillary analytical C 18 column connected by means of a six-port valve in backflush mode. Detection was performed with ESI-MS/MS and only 1 mu m of sample was injected. Validation was adequately carried out using FLU-d(5) as internal standard. Calibration curves were constructed under a linear range of 1-250 ng mL(-1) in plasma, being the limit of quantification (LOQ), determined as 1 ng mL(-1), for all the analytes. With the described approach it was possible to reach a quantified mass sensitivity of 0.3 pg for each analyte (equivalent to 1.1-1.3 fmol), translating to a lower sample consumption (in the order of 103 less sample than using conventional methods).
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| 8. |
- Serra, Elias, et al.
(författare)
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Immobilization of lipase in ordered mesoporous materials : Effect of textural and structural parameters
- 2008
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Ingår i: Microporous and Mesoporous Materials. - : Elsevier BV. - 1387-1811 .- 1873-3093. ; 114:03-jan, s. 201-213
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Tidskriftsartikel (refereegranskat)abstract
- A systematic study dealing with the influence of several parameters oil the immobilization of lipase in ordered mesoporous materials (OMM) is presented here. In a first step, a series of OMM have been synthesized trying to cover the most relevant structures. The aim is to get variation in the key properties susceptible of influencing their behavior as lipase supports, such as the structure (cubic or hexagonal), the nature of the pores (channel-like or cage-like), the connectivity of the porous network and the pore size. Also, by following the co-condensation technique, 5-10%-methylated analogues of the pure-silica materials have been prepared. All the samples have been fully characterized with XRD, TEM (including 3D reconstruction), SEM, TGA and N-2 isotherms, and the incorporation of the organic function has been demonstrated by Si-29 NMR. All of them have been tested as supports in the immobilization of Candida antarctica Lipase B (CaLB) and the leaching of the enzyme in aqueous media evaluated. With such a systematic approach, valuable information on the influence of the textural properties and the nature of the porous network oil the yields of immobilization and enzyme desorption have been stated. Very interestingly, leaching of the enzyme can be diminished until it practically disappears without being covalently bonded to the wall, which places the ordered mesoporous materials at the starting point of a new scenario in enzyme immobilization on preexisting supports.
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