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Series GSE90458

Query DataSets for GSE90458

Status

Public on Nov 01, 2021

Title

Nucleosomes inhibit intragenic binding of Rap1p and subsequent cryptic transcription [MNase-seq]

Organism

Saccharomyces cerevisiae

Experiment type

Genome binding/occupancy profiling by high throughput sequencing

Summary

Nucleosomes are the primary unit of chromatin structure and inhibit key intracellular interactions which regulate how eukaryotic genetic information is read, including transcription factor (TF) binding to DNA. In theory, the sterics of nucleosome-DNA interactions could inhibit TF binding to all nucleosome-bound DNA. In reality, however, nucleosome inhibition of TF binding is variable both across a genome and even within a single nucleosome. Prior studies have demonstrated that nucleosome occupancy, nucleosome positioning, TF binding site (TFBS) affinity & TFBS location can each influence TF targeting in vivo, however, this work has been largely correlative and no study has dissected collective from individual contributions of these important variables. Therefore, to address this deficiency, we have mapped genome-wide changes in chromatin structure and TF binding following experimental nucleosome depletion. Heuristic modeling of results confirmed that chromatin specification of TF targeting is a multivariate, context-specific, process, with most informative features originating from additive interactions between cis and trans variables. Additionally, we found compelling evidence that nucleosomes serve to inhibit intra-genic TF binding, as evidenced by an increasing number of sub-optimal TFBS occupied following nucleosome depletion. The majority of these conditional loci were located within open reading frames and parallel transcriptome analysis revealed their occurrence was linked to generation of intragenic cryptic transcripts. Together results support the idea that chromatin structure has evolved to coordinate specification of regulatory factor targeting & gene expression. Findings have functional relevance to emerging datasets on genetic and epigenetic variation in human disease states.

Overall design

H4 shutoff (UKY403) and isogenic control (UKY412) strains of S. cerevisiae were obtained from the Grunstein lab. Cells were maintained on agar plates containing YPG media (1% yeast extract, 2% peptone, 2% galactose). For time-course experiments, cells were initially inoculated in 5ml YPG liquid media and cultured at 30C. Logarithmically growing cells were then transferred and diluted into larger 1-1.5L YPG cultures for growth overnight at 30C while shaking in containers with 4x equivalent free-air volume. The following morning, mid-log phase cells (OD600 ~0.6-1.2) were collected by centrifugation, washed 2x within 5 minutes with ~250-500ml YP dextrose (YPD), and re-suspended in equivalent volume of YPD media for 0, 2, and 4 hours of continued growth at 30C. After each time-point samples were taken for MNase-seq, ChIP-seq, and RNA-seq studies. RNA-seq samples were immediately flash-frozen in an ethanol-dry ice bath, whereas samples for ChIP and MNase were first cross-linked at room temperature for 30 min with 1% weight/volume formaldehyde prior to flash freezing.

Contributor(s)

Buck MJ, Bard J, Rizzo J

Citation missing

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Submission date

Nov 23, 2016

Last update date

Nov 01, 2021

Contact name

Michael Buck

E-mail(s)

mjbuck@buffalo.edu