Zebrafish promoter microarrays identify actively transcribed embryonic genes
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ABSTRACT:
ORGANISM(S): Danio rerio
PROVIDER: GSE4863 | GEO | 2006/05/22
SECONDARY ACCESSION(S): PRJNA95749
REPOSITORIES: GEO
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Project description:We have designed a zebrafish genomic microarray to identify DNA-protein interactions in the proximal promoter regions of over 11,000 zebrafish genes. Using these microarrays, together with chromatin immunoprecipitation with an antibody directed against tri-methylated lysine 4 of Histone H3, we demonstrate the feasibility of this method in zebrafish. This approach will allow investigators to determine the genomic binding locations of DNA interacting proteins during development and expedite the assembly of the genetic networks that regulate embryogenesis. Genomic array design Microarrays were designed as described below and manufactured by Agilent Technologies (www.agilent.com). Further information on design can be found at http://jura.wi.mit.edu/bioc/gbell/zfish_chip/. Selection of transcription start sites and identification of promoter sequences We interrogated 5 databases: Ensembl, VEGA, Refseq, ZGC full length clones and a database provided by Dr. Leonard Zon (Harvard Medical School, Boston, USA) in order to assemble an extensive list of zebrafish transcripts. The Zon lab database is a hand-curated database of zebrafish genes that have homologues in other species. We included all transcripts that appeared in the manually annotated databases (VEGA, Zon) and in the ZGC full length database. We also identified genes present in any 2 of the 5 databases and included those not already selected. The transcripts were mapped to the zebrafish genome (Zv4, June 2004) obtained from UCSC Bioinformatics (http://genome.ucsc.edu) and the transcription start site (TSS) for each transcript was determined. Transcripts with TSSs within 500bp were clustered into a transcriptional unit (TU) and promoter regions were identified relative to the most upstream TSS. This resulted in the identification of 13,413 TUs and corresponding promoter regions. Each promoter region was extracted and masked for repetitive sequence by RepeatMasker. If the promoter region contained a gap the upstream sequence was also masked. Information on the transcriptional units that were included in the final design can be found at http://jura.wi.mit.edu/bioc/gbell/zfish_chip/. Selection of oligonucleotides 60-mer oligonucleotide probes representing the region between 1.5kb upstream and 0.5kb downstream of the annotated TSS of each transcriptional unit were then designed. Although transcription factors and other DNA binding proteins are known to regulate genes from distances of greater than -1.5kb or + 0.5kb, much information can be gained from regions close to the TSS [45], and the H3K4Me3 mark studied in this paper is found at the most 5â end of a gene, close to the TSS. Selection of 60-mers for the microarrays was essentially as described in [14] using the Zv4 build of the zebrafish genome and a locally customized version of ArrayOligoSelector. 60-mers were chosen so that promoter regions contained approximately one probe every 250bp with a maximum distance between probes for each promoter region set at 600bp. In cases where only one probe could be designed for a particular TU these were not included in the final design. This process yielded 80,839 probes for 11,171 promoter regions We also incorporated several sets of control probes, both positive and negative. On each array there are 1090 probes designed against âgene desertâ regions, which are genomic regions that are unlikely to be bound by transcriptional regulators, and 270 probes designed against Arabidopsis thaliana genes, which are not present in the zebrafish genome (by BLAST). In addition, because our main motivation for making these microarrays is to identify mesodermally-regulated genes we included 7 genes expressed in mesoderm during gastrulation as positive controls (wnt11, flh, vent, msgn1, myod, fgf8, pcdh8). Probes designed against these promoters, which flank from 3-4kb around each TSS, are arrayed 2-4 times on each slide. Since these genes are expressed at gastrula stages to varying degrees, they also serve as a positive controls in this study. Finally there are 2256 controls added by Agilent and a variable number of blank spots. These probes were divided between two microarray slides each with 44,290 features. We refer to these two microarray slides as the âproximal promoter setâ. A proximal promoter set based on these designs as well as an expanded set of 9 slides which contain regions from â9kb to + 3kb relative to the TSS, are available by contacting Agilent (www.agilent.com) or by downloading the design files from http://jura.wi.mit.edu/bioc/gbell/zfish_chip/ for self-manufacture.
2010-07-01 | E-GEOD-4863 | biostudies-arrayexpress
Project description:We have designed a zebrafish genomic microarray to identify DNA-protein interactions in the proximal promoter regions of over 11,000 zebrafish genes. Using these microarrays, together with chromatin immunoprecipitation with an antibody directed against tri-methylated lysine 4 of Histone H3, we demonstrate the feasibility of this method in zebrafish. This approach will allow investigators to determine the genomic binding locations of DNA interacting proteins during development and expedite the assembly of the genetic networks that regulate embryogenesis.
| S-EPMC1779600 | biostudies-literature
Project description:The Paf1 complex in yeast has been reported to influence a multitude of steps in gene expression through interactions with RNA polymerase II (Pol II) and chromatin-modifying complexes; however, it is unclear which of these many activities are primary functions of Paf1 and are conserved in metazoans. We have identified and characterized the Drosophila homologs of three subunits of the yeast Paf1 complex and found striking differences between the yeast and Drosophila Paf1 complexes. We demonstrate that although Drosophila Paf1, Rtf1, and Cdc73 colocalize broadly with actively transcribing, phosphorylated Pol II, and all are recruited to activated heat shock genes with similar kinetics; Rtf1 does not appear to be a stable part of the Drosophila Paf1 complex. RNA interference (RNAi)-mediated depletion of Paf1 or Rtf1 leads to defects in induction of Hsp70 RNA, but tandem RNAi-chromatin immunoprecipitation assays show that loss of neither Paf1 nor Rtf1 alters the density or distribution of phosphorylated Pol II on the active Hsp70 gene. However, depletion of Paf1 reduces trimethylation of histone H3 at lysine 4 in the Hsp70 promoter region and significantly decreases the recruitment of chromatin-associated factors Spt6 and FACT, suggesting that Paf1 may manifest its effects on transcription through modulating chromatin structure.
| S-EPMC1317635 | biostudies-literature
Project description:Background:Animal mitochondrial genomes typically encode 37 genes: 13 proteins, 22 tRNAs and two rRNAs. However, many species represent exceptions to that rule. Bivalvia along with Nematoda and Platyhelminthes are often suspected to fully or partially lack the ATP synthase subunit 8 (atp8) gene. This raises the question as to whether they are really lacking this gene or is this maybe an annotation problem? Among bivalves, Mytilus edulis has been inferred to lack an ATP8 gene since the characterization of its mitochondrial genome in 1992. Even though recent bioinformatic analyses suggested that atp8 is present in Mytilus spp., due to high divergence in predicted amino acid sequences, the existence of a functional atp8 gene in this group remains controversial. Results:Here we demonstrate that M. edulis mitochondrial open reading frames suggested to be atp8 (in male and female mtDNAs) are actively translated proteins. We also provide evidence that both proteins are an integral part of the ATP synthase complex based on in-gel detection of ATP synthase activity and two-dimensional Blue-Native and SDS polyacrylamide electrophoresis. Conclusion:Many organisms (e.g., Bivalvia along with Nematoda and Platyhelminthes) are considered to be lacking certain mitochondrial genes often only based on poor similarity between protein coding gene sequences in genetically closed species. In some situations, this may lead to the inference that the ATP8 gene is absent, when it is in fact present, but highly divergent. This shows how important complementary role protein-based approaches, such as those in the present study, can provide to bioinformatic, genomic studies (i.e., ability to confirm the presence of a gene).
| S-EPMC5995098 | biostudies-literature
Project description:While it is widely acknowledged that the ubiquitin-proteasome system plays an important role in transcription, little is known concerning the mechanistic basis, in particular the spatial organization of proteasome-dependent proteolysis at the transcription site. Here, we show that proteasomal activity and tetraubiquitinated proteins concentrate to nucleoplasmic microenvironments in the euchromatin. Such proteolytic domains are immobile and distinctly positioned in relation to transcriptional processes. Analysis of gene arrays and early genes in Caenorhabditis elegans embryos reveals that proteasomes and proteasomal activity are distantly located relative to transcriptionally active genes. In contrast, transcriptional inhibition generally induces local overlap of proteolytic microdomains with components of the transcription machinery and degradation of RNA polymerase II. The results establish that spatial organization of proteasomal activity differs with respect to distinct phases of the transcription cycle in at least some genes, and thus might contribute to the plasticity of gene expression in response to environmental stimuli.
| S-EPMC3113580 | biostudies-literature
Project description:Histone H3 trimethylation at lysine 36 (H3K36me3) is an important histone mark involved in both transcription elongation and DNA mismatch repair (MMR). It is known that H3K36me3 recruits the mismatch-recognition protein MutS? to replicating chromatin via its physical interaction with MutS?'s PWWP domain, but the exact role of H3K36me3 in transcription is undefined. Using ChIP combined with whole-genome DNA sequencing analysis, we demonstrate here that H3K36me3, together with MutS?, is involved in protecting against mutation, preferentially in actively transcribed genomic regions. We found that H3K36me3 and MutS? are much more co-enriched in exons and actively transcribed regions than in introns and nontranscribed regions. The H3K36me3-MutS? co-enrichment correlated with a much lower mutation frequency in exons and actively transcribed regions than in introns and nontranscribed regions. Correspondingly, depleting H3K36me3 or disrupting the H3K36me3-MutS? interaction elevated the spontaneous mutation frequency in actively transcribed genes, but it had little influence on the mutation frequency in nontranscribed or transcriptionally inactive regions. Similarly, H2O2-induced mutations, which mainly cause base oxidations, preferentially occurred in actively transcribed genes in MMR-deficient cells. The data presented here suggest that H3K36me3-mediated MMR preferentially safeguards actively transcribed genes not only during replication by efficiently correcting mispairs in early replicating chromatin but also during transcription by directly or indirectly removing DNA lesions associated with a persistently open chromatin structure.
| S-EPMC5961058 | biostudies-literature
Project description:In mammalian cells, the expression level of the cyclin B1 gene plays a critical role in the progression through mitosis. Here we demonstrate that the transcriptional activity of the human cyclin B1 promoter, as well as the rate of gene transcription, is high during mitosis. Indeed, the cyclin B1 promoter maintains an open chromatin configuration at the mitotic stage. Consistent with this, we show that the cyclin B1 promoter is occupied and bound to NF-Y during mitosis in vivo. Our results provide the first example of RNA polymerase II-dependent transcription during mitosis in mammalian cells.
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Project description:The human genome must be packaged and organized in a functional manner for the regulation of DNA replication and transcription. The nuclear scaffold/matrix, consisting of structural and functional nuclear proteins, remains after extraction of nuclei and anchors loops of DNA. In the search for cis-elements functioning as chromatin domain boundaries, we identified 453 nuclear scaffold attachment sites purified by lithium-3,5-iodosalicylate extraction of HeLa nuclei across 30 Mb of the human genome studied by the ENCODE pilot project. The scaffold attachment sites mapped predominately near expressed genes and localized near transcription start sites and the ends of genes but not to boundary elements. In addition, these regions were enriched for RNA polymerase II and transcription factor binding sites and were located in early replicating regions of the genome. We believe these sites correspond to genome-interactions mediated by transcription factors and transcriptional machinery immobilized on a nuclear substructure.
| S-EPMC3056778 | biostudies-literature
Project description:In the mammalian genome, each histone family contains multiple replication-dependent paralogs, which are found in clusters where their transcription is thought to be coupled to the cell cycle. Here, we wanted to interrogate the transcriptional regulation of these paralogs during retinal development and aging. We employed deep sequencing, quantitative PCR, in situ hybridization (ISH), and microarray analysis, which revealed that replication-dependent histone genes were not only transcribed in progenitor cells but also in differentiating neurons. Specifically, by ISH analysis we found that different histone genes were actively transcribed in a subset of neurons between postnatal day 7 and 14. Interestingly, within a histone family, not all paralogs were transcribed at the same level during retinal development. For example, expression of Hist1h1b was higher embryonically, while that of Hist1h1c was higher postnatally. Finally, expression of replication-dependent histone genes was also observed in the aging retina. Moreover, transcription of replication-dependent histones was independent of rapamycin-mediated mTOR pathway inactivation. Overall, our data suggest the existence of variant nucleosomes produced by the differential expression of the replication-dependent histone genes across retinal development. Also, the expression of a subset of replication-dependent histone isotypes in senescent neurons warrants re-examining these genes as "replication-dependent." Thus, our findings underscore the importance of understanding the transcriptional regulation of replication-dependent histone genes in the maintenance and functioning of neurons.
| S-EPMC4614993 | biostudies-literature
Project description:Active protein translation can be assessed and measured using ribosome profiling sequencing strategies. Existing approaches make use of sequence fragment length or frame occupancy to differentiate between active translation and background noise, however they do not consider additional characteristics inherent to the technology which limits their overall accuracy. Here, we present an analytical tool that models the overall tri-nucleotide periodicity of ribosomal occupancy using a classifier based on spectral coherence. Our software, SPECtre, examines the relationship of normalized ribosome profiling read coverage over a rolling series of windows along a transcript against an idealized reference signal. A comparison of SPECtre against current methods on existing and new data shows a marked improvement in accuracy for detecting active translation and exhibits overall high sensitivity at a low false discovery rate. Classification of actively translated transcripts in ribosome profiling data derived from human neuroblastoma SH-SY5Y cells, and data previously published derived from mouse embryonic stem cells and zebrafish embryos.
2016-07-01 | E-GEOD-75947 | biostudies-arrayexpress