Genome-wide kinetics of nucleosome turnover determined by metabolic labeling of histones

Abstract

Nucleosome disruption and replacement are crucial activities that maintain epigenomes, but these highly dynamic processes have been difficult to study. Here, we describe a direct method for measuring nucleosome turnover dynamics genome-wide. We found that nucleosome turnover is most rapid over active gene bodies, epigenetic regulatory elements, and replication origins in Drosophila cells. Nucleosomes turn over faster at sites for trithorax-group than polycomb-group protein binding, suggesting that nucleosome turnover differences underlie their opposing activities and challenging models for epigenetic inheritance that rely on stability of histone marks. Our results establish a general strategy for studying nucleosome dynamics and uncover nucleosome turnover differences across the genome that are likely to have functional importance for epigenome maintenance, gene regulation, and control of DNA replication.

Figure 1. CATCH-IT marks sites of histone replacement and reveals kinetics

A) Gene ends analysis of a CATCH-IT experiment from a 3 hour Aha treatment (pulse). All 9820 genes from FlyBase r5.13 with annotated 5′ and 3′ ends were grouped by gene expression quintiles (top 20% to bottom 20% based on GEO#GSM333845), aligned by gene ends, and the log₂-ratios of pulldown DNA/input DNA averaged across genes. B) Same as in (A) but with cells treated with Aha for 3 hours (pulse) followed by a 1.5 hour Met treatment (chase). C) Same as in (A) but for biotin-tagged H3.3 nucleosomes. D) Scatter plot showing the correlation between the pulse and the difference between the pulse and chase, using all 2.1 million probes on the array. E) Same as in (D) but using only genic probes from the highest and lowest gene expression quintiles.

Figure 2. Chromatin landscapes of CATCH-IT, ORC, H3.3, and salt fractions

Chromatin landscapes over a representative euchromatic region from a CATCH-IT time course of 20′, 40′, and 60′ Aha treatment. Also shown are ORC binding, biotinylated H3.3 nucleosomes, and successive 80mM, 600mM and insoluble chromatin salt fractions (17). Genes are shown above chromatin tracks. Those above the line are the top strand and those below the line are on the bottom strand. Right panel shows a magnification of the indicated region of the left panel.

Figure 3. Kinetics of newly synthesized histone incorporation at epigenetic regulatory elements and sites of ORC binding

A) Average CATCH-IT time course signals over GAF and EZ+PSC binding sites. B) Same as in (A) but for a pulse-chase experiment with a 3-hr Aha pulse and 1.5-hr Met chase. C) Average biotinylated H3.3 signals over GAF and EZ+PSC binding sites. D) Average signals from chromatin salt fractions over GAF and EZ+PSC binding sites. E-P) Plots of various chromatin signals aligned at peaks of ORC binding and divided into quintiles by ORC peak score (19).