Most technologies applied to genome-wide mapping of chromatin features have serious limitations, and so we have endeavored to develop new strategies that would allow us to better explore nucleosome dynamics in the context of high-throughput readout technologies. One strategy has been to apply epigenomic profiling to the classical salt fractionation method1. Salt competes for interactions between the highly basic histone core and highly acidic DNA, and so salt-solubility measures a nucleosomal physical property, as opposed to surrogate 'marks' such as histone modifications and variants. Chromatin fractions extracted with low salt after micrococcal nuclease (MNase) digestion contain predominantly mononucleosomes and represent classical 'active' chromatin2. We found that profiles of these low-salt-soluble fractions displayed phased nucleosomes downstream of transcriptional start sites, with similar levels of enrichment for active genes regardless of expression level3. Nearly quantitative recovery of chromatin was obtained with 600mM NaCl; however, a small remaining insoluble ('nuclear matrix') fraction was also recovered that is enriched in actively transcribed regions. We found that both low-salt-soluble and insoluble chromatin are rich in sequences that correspond to epigenetic regulatory elements genome-wide. The presence of active chromatin at both extremes of salt solubility suggests that these salt fractions capture protein bound and unbound intermediates in active processes, thus providing a simple, powerful strategy for mapping epigenome dynamics. We also introduced a method for salt-fractionation and ChIP of C. elegans native chromatin4. Genome-wide salt fractionation profiles for both flies and worms used both NimbleGen tiling arrays and Solexa sequencing, and constituted our primary deliverables for the modENCODE consortium, in which I participated as one of eleven Principal Investigators5-8.
To obtain a direct measure of nucleosome dynamics, Roger Deal, a postdoc in the lab now at Emory University, introduced a metabolic labeling strategy, CATCH-IT (for Covalent Attachment of Tags to Capture Histones and Identify Turnover)9. In this method, newly synthesized proteins are labeled with an amino acid analog and derivatized with a biotin moiety, nucleosome core particles are selectively extracted and affinity-purified with streptavidin, and DNA is extracted for genome-wide profiling. Roger successfully obtained genome-wide CATCH-IT profiles for Drosophila cultured cells, and used these data to address the relationship between histone turnover and fundamental processes, including transcriptional initiation and elongation, epigenetic regulation and replication origin activity9.
Sheila Teves, a graduate student in the lab, next applied salt-fractionation and CATCH-IT to study nucleosome turnover during transcription using the classical heat shock system in Drosophila S2 cells. 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 active chromatin, RNA polymerase II (RNAPII), and nucleosome turnover dynamics at single-base-pair resolution10. With heat shock, low-salt-soluble chromatin and stalled RNAPII 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. This suggests that down-regulation of transcription during heat shock involves reduced nucleosome mobility and that this process has evolved to promote heat-shock gene regulation.
Fan Yang, a postdoc in the lab, next applied CATCH-IT to explore nucleosome turnover in mouse cancer lines, which led to a surprising discovery of potential clinical interest. In collaboration with my Hutch colleague Chris Kemp, we aimed to explore the effects of chromosome breaks on nucleosome turnover. To do that, we added sub-lethal doses of doxorubicin (~0.4 µM) or aclarubicin (~0.1 µM), anthracycline DNA intercalators that are among the most commonly used anticancer drugs. We found that these drugs enhance nucleosome turnover around gene promoters and that turnover correlates with gene expression level11. Consistent with a direct action, enhancement of nucleosome turnover around promoters gradually increased with time of exposure to the drugs. Interestingly, enhancement occurred both in wild-type cells and in cells lacking either the p53 tumor suppressor gene or the master regulator of the DNA damage response, ATM, suggesting that anthracycline action on nucleosome dynamics is independent of the DNA damage checkpoint. Our results suggested that anthracycline intercalation promotes nucleosome turnover around promoters by its effect on DNA topology, with possible implications for mechanisms of cell killing during cancer chemotherapy. Shortly after our paper was published11, another group showed that therapeutic doses (~9 µM) of doxorubicin and aclarubicin caused histone eviction around promoters12. We have since proposed that intercalation itself enhances the positive torsion that unwraps nucleosomes and leads to DNA fragility that in therapeutic doses kills cancer cells13. In support of this model, we found that double-strand breaks caused by anthracycline drugs are enriched around active gene promoters14
The idea that torsion caused by anthracycline incorporation increases nucleosome turnover around promoters was in part motivated by Sheila Teves’ follow-up of her Drosophila heat shock study, in which she explored the possibility that DNA torsion underlies nucleosome turnover15. She first introduced a method for high-resolution mapping of underwound DNA, using next-generation sequencing, and then showed that torsion is correlated with gene expression in cells. Accumulation of torsional stress through topoisomerase inhibition resulted in increased Pol II at transcription start sites. Whereas topoisomerase I inhibition resulted in increased nascent RNA transcripts, topoisomerase II inhibition caused little change. Despite the different effects on Pol II elongation, topoisomerase inhibition resulted in increased nucleosome turnover and salt solubility within gene bodies, thus suggesting that the elongation-independent effects of torsional stress on nucleosome dynamics contributes to the destabilization of nucleosomes.