title: Biological chromodynamics: a general method for measuring protein occupancy across the genome by calibrating ChIP-seq.
abstract: Sequencing DNA fragments associated with proteins following in vivo cross-linking with formaldehyde (known as ChIP-seq) has been used extensively to describe the distribution of proteins across genomes. It is not widely appreciated that this method merely estimates a protein's distribution and cannot reveal changes in occupancy between samples. To do this, we tagged with the same epitope orthologous proteins in Saccharomyces cerevisiae and Candida glabrata, whose sequences have diverged to a degree that most DNA fragments longer than 50 bp are unique to just one species. By mixing defined numbers of C. glabrata cells (the calibration genome) with S. cerevisiae samples (the experimental genomes) prior to chromatin fragmentation and immunoprecipitation, it is possible to derive a quantitative measure of occupancy (the occupancy ratio - OR) that enables a comparison of occupancies not only within but also between genomes. We demonstrate for the first time that this 'internal standard' calibration method satisfies the sine qua non for quantifying ChIP-seq profiles, namely linearity over a wide range. Crucially, by employing functional tagged proteins, our calibration process describes a method that distinguishes genuine association within ChIP-seq profiles from background noise. Our method is applicable to any protein, not merely highly conserved ones, and obviates the need for the time consuming, expensive, and technically demanding quantification of ChIP using qPCR, which can only be performed on individual loci. As we demonstrate for the first time in this paper, calibrated ChIP-seq represents a major step towards documenting the quantitative distributions of proteins along chromosomes in different cell states, which we term biological chromodynamics.
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK Department of Molecular Biology and Biotechnology, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Bin
authors.initials: B
authors.lastname: Hu
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
authors.forename: Naomi
authors.initials: N
authors.lastname: Petela
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
authors.forename: Alexander
authors.initials: A
authors.lastname: Kurze
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
authors.forename: Kok-Lung
authors.initials: KL
authors.lastname: Chan
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
authors.forename: Christophe
authors.initials: C
authors.lastname: Chapard
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK kim.nasmyth@bioch.ox.ac.uk.
authors.forename: Kim
authors.initials: K
authors.lastname: Nasmyth
mesh_terms: D002138:Calibration; D041221:Candida glabrata / Q000235:genetics; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000032:analysis; D047369:Chromatin Immunoprecipitation / Q000379:methods* / Q000592:standards; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis; D004268:DNA-Binding Proteins / Q000032:analysis*; D005656:Fungal Proteins / Q000032:analysis; D016681:Genome, Fungal; D059014:High-Throughput Nucleotide Sequencing; D050505:Mutant Proteins / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017422:Sequence Analysis, DNA; D000097722:Cohesins
chemical_list: D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D050505:Mutant Proteins; D000097722:Cohesins