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Size fractionated NET-Seq reveals a conserved architecture around yeast genes

ABSTRACT: Genomes from yeast to human are subject to pervasive transcription. A single round of pervasive transcription is sufficient to alter local chromatin conformation, nucleosome dynamics and gene expression, but is hard to distinguish from background signals. Size fractionated native elongating transcript sequencing (sfNET-Seq) was developed to precisely map nascent transcripts independent of expression levels. RNAPII-associated nascent transcripts are fractionation into different size ranges before library construction. When anchored to the transcription start sites (TSS) of annotated genes, the combined pattern of the output metagenes gives the expected reference pattern. Bioinformatic pattern matching to the reference pattern identified 9542 transcription units in Saccharomyces cerevisiae, of which 47% are coding and 53% are non-coding. 3113 (33%) are unannotated non-coding transcription units. Anchoring all transcription units to the TSS or polyadenylation site (PAS) of annotated genes reveals distinctive architectures of linked pairs of divergent transcripts approximately 200nt apart. The Reb1 transcription factor is enriched 30nt downstream of the PAS only when an upstream (TSS -60nt with respect to PAS) non-coding transcription unit co-occurs with a downstream (TSS+150nt) coding transcription unit and acts to limit levels of upstream antisense transcripts. The potential for extensive transcriptional interference is evident from low abundance unannotated transcription units with variable TSS (median -240nt) initiating within a 500nt window upstream of, and transcribing over, the promoters of protein coding genes. This study confirms a highly interleaved yeast genome with different types of transcription units altering the chromatin landscape in distinctive ways, with the potential to exert extensive regulatory control.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE246153 | GEO | 2024/02/06

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.

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Size fractionated NET-Seq reveals a conserved architecture around yeast genes

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