Project description:Short-read DNA sequencing technologies provide new tools to answer biological questions. However, high cost and low throughput limit their widespread use, particularly in organisms with smaller genomes such as S. cerevisiae. Although ChIP-Seq in mammalian cell lines is replacing array-based ChIP-chip as the standard for transcription factor binding studies, ChIP-Seq in yeast is still underutilized compared to ChIP-chip. We developed a multiplex barcoding system that allows simultaneous sequencing and analysis of multiple samples using Illumina’s platform. We applied this method to analyze the chromosomal distributions of three yeast DNA binding proteins (Ste12, Cse4 and RNA PolII) and a reference sample (input DNA) in a single experiment and demonstrate its utility for rapid and accurate results at reduced costs. We developed a barcoding ChIP-Seq method for the concurrent analysis of transcription factor binding sites for yeast. Our multiplex strategy generated high quality data that was indistinguishable from data obtained with non-barcoded libraries. None of the barcoded adapters induced differences relative to a non-barcoded adapter when applied to the same DNA sample. We used this method to map the binding sites for Cse4, Ste12 and Pol II throughout the yeast genome and we found 148 binding targets for Cse4, 823 targets for Ste12 and 2508 targets for PolII. Cse4 was strongly bound to all yeast centromeres as expected and the remaining non-centromeric targets correspond to highly expressed genes in rich media, the latter constituting a novel finding. We designed a multiplex short-read DNA sequencing method to perform efficient ChIP-Seq in yeast and other small genome model organisms. This method produces accurate results with higher throughput and reduced cost. Given constant improvements in high-throughput sequencing technologies, increasing multiplexing will be possible to further decrease costs per sample and to accelerate the completion of large consortium projects such as modENCODE.
Project description:Short-read RNA-seq was performed on rRNA-depleted RNA isolated from spores of the budding yeast Saccharomyces cerevisiae that were sorted by mating type.
Project description:We investigated the genome-wide distribution of Okazaki fragments in the commonly used laboratory Saccharomyces cerevisiae strain S288C to study the DNA replication model adopted by the budding yeast. The method based upon lambda exonuclease digestion for purification of RNA-primed replication intermediates was first improved to be suitable for the purification of Okazaki fragments. Then, we used this improved method to purify Okazaki fragments from S288C yeast cells, followed by Illumina sequencing. We found that the expected asymmetric distribution of Okazaki fragments around confirmed replication origins, which was derived from the semi-discontinuous DNA replication model, was not observed on S. cerevisiae chromosomes. Even around two highly efficient replication origins, ARS522 and ARS416, the ratios of Okazaki fragments on both strands were inconsistent with the semi-discontinuous DNA replication model. Our study supported the discontinuous DNA replication model. Besides, we also observed that Okazaki fragments were overpresented in the transcribed regions in S. cerevisiae mitochondrial genome, which indicated the interplay between transcription and DNA replication.
Project description:A network governing DNA integrity was identified in yeast by a global genetic analysis of synthetic fitness or lethality defect (SFL) interactions. Within this network, multiple functional modules or mini-pathways were defined according to their common patterns of global SFL interactions and available protein-protein interaction information. Modules or genes involved in DNA replication, DNA replication checkpoint signaling, and oxidative stress response were identified as the major guardians against lethal spontaneous DNA damage, efficient repair of which requires the functions of the DNA damage checkpoint signaling and multiple DNA repair pathways. This genome-wide genetic interaction network also revealed potential roles of a number of genes and modules in mitotic DNA replication and maintenance of genomic stability. These include DIA2, NPT1, HST3, HST4, and the CSM1/LRS4 module (CSM1m). Likewise, the CTF18 module (CTF18m), previously implicated in sister chromatid cohesion, was found to participate in the DNA replication checkpoint. Keywords: dose response
Project description:GATA transcription factors are highly conserved among eukaryotes and play roles in transcription of genes implicated in cancer progression and hematopoiesis. However, although their consensus binding sites have been well defined in vitro, the in vivo selectivity for recognition by GATA factors remains poorly characterized. Using ChIP-Seq, we identified the Dal80 GATA factor targets in yeast. Our data reveal Dal80 binding to a large set of promoters, sometimes independently of GATA sites, correlating with nitrogen- and/or Dal80-sensitive gene expression. Strikingly, Dal80 was also detected across the body of promoter-bound genes, correlating with high expression. Mechanistic single-gene experiments showed that Dal80 spreading across gene bodies requires active transcription. Consistently, Dal80 co-immunoprecipitated with the initiating and post-initiation forms of RNA Polymerase II. Our work suggests that GATA factors could play dual, synergistic roles during transcription initiation and post-initiation steps, promoting efficient remodeling of the gene expression program in response to environmental changes.