Heat shock reduces stalled RNA polymerase II and nucleosome turnover genome-wide

Abstract

Heat shock rapidly induces expression of a subset of genes while globally repressing transcription, making it an attractive system to study alterations in the chromatin landscape that accompany changes in gene regulation. We characterized these changes in Drosophila cells by profiling classical low-salt-soluble chromatin, RNA polymerase II (Pol II), and nucleosome turnover dynamics at single-base-pair resolution. With heat shock, low-salt-soluble chromatin and stalled Pol II levels were found to decrease within gene bodies, but no overall changes were detected at transcriptional start sites. Strikingly, nucleosome turnover decreased genome-wide within gene bodies upon heat shock in a pattern similar to that observed with inhibition of Pol II elongation, especially at genes involved in the heat-shock response. Relatively high levels of nucleosome turnover were also observed throughout the bodies of genes with paused Pol II. These observations suggest that down-regulation of transcription during heat shock involves reduced nucleosome mobility and that this process has evolved to promote heat-shock gene regulation. Our ability to precisely map both nucleosomal and subnucleosomal particles directly from low-saltsoluble chromatin extracts to assay changes in the chromatin landscape provides a simple general strategy for epigenome characterization.

Partially Assembled Nucleosome Structures at Atomic Detail

“…For example, a 2011 study (31) used nuclease protection assays to identify a peculiar pattern of lengths of protected DNA fragments in active genes of Drosophila (right column of Table 2). In addition to the expected peak at~147 bp, corresponding to the intact nucleosome, three additional, lower intensity satellite peaks were observed (31). The oscillatory profile of DNA protection for fragments shorter than 147 bp was in contrast to the flat profiles that characterize inactive genes.…”

“…Further, because one of our goals here is to demonstrate utility of our atomistic models for interpretation of both in vivo and in vitro experiments (28,31,32), we focus on just three key PANS: the disome, the tetrasome, and the hexasome (Table 1). These subnucleosomal structures have been deemed important in vivo, and are already well characterized in vitro, both thermodynamically and by low-resolution methods such as Förster resonance energy transfer (FRET) or small-angle x-ray scattering (SAXS).…”

A Quantitative Proteomic Analysis of In Vitro Assembled Chromatin

“…After another washing step in EXNonidet P-40 (10 mM Hepes pH 7.6, 1.5 mM MgCl 2 , 0.5 mM EGTA, 10% (v/v) Glycerol, 0.05% Nonidet P-40) beads were resuspended in a total volume of 240 l containing 45 l DREX and an ATP regenerating system (3 mM ATP, 30 mM creatine phosphate, 10 g creatine kinase/ml, 3 mM MgCl 2 , and 1 mM DTT). To study the influence of protein acetylation, 1 The abbreviations used are: SWATH, sequential window acquisition of all theoretical fragment ions; Aa, amino acid; Ac, acetylation; CAF, chromatin assembly factor; CE, collision energy; CHRAC, Chromatin Accessibility Complex; DDA, data-dependent acquisition; DIA, data-independent acquisition; Dre4, Dras3-Roughened-ecdysoneless region 4; DREX, Drosophila Embryonic Extract; FDR, false discovery rate; Gnf1, Germ line transcription factor 1; GO, Gene Ontology; H1/H2A/H2B/H3/H4, histone 1/2A/2B/3/4; HDAC, histone deacetylase; HMG, High mobility group protein; Hsp, Heat shock protein; iBAQ, intensity based absolute quantitation; Irbp, Inverted repeat-binding protein; ISWI, Imitation SWI; LFQ, label-free quantitation; MBT complex, Myb-MuvB/DREAM transcriptional regulatory complex; MNase, Micrococcal nuclease; MCM, Minichromosome Maintenance; MRM-HR, multiple reaction monitoring; Nurf, Nucleosome remodeling factor; PCNA, Proliferating cell nuclear antigen; PTM, post-translational modification; Rfc38, Replication factor C 38kD subunit; RpA, Replication protein A; SSRP, structure specific recognition protein; Tcp1, T-complex protein 1; TSA, Trichostatin A; XIC, extracted ion chromatogram.…”

“…DNA replication, transcription and repair continuously disturb the conformation of chromatin, which results in a relatively high rate of histone turnover (1) and poses a constant threat to the maintenance of epigenetic information (2,3). Therefore, chromatin assembly has to be controlled thoroughly to ensure a proper chromatin structure.…”

“…In this study, we used immobilized linear DNA to rapidly isolate in vitro assembled chromatin at different time points and determined its protein composition in a time resolved manner using sequential window acquisition of all theoretical fragment ions (SWATH) 1 -MS-based label-free protein quantitation. A comparison with the proteomic investigation of chromatin assembled in vivo (13) reveals an almost 80% overlap with the orthologue proteins assembled in vitro.…”