The spring-loaded genome: nucleosome redistributions are widespread, transient, and DNA-directed

Abstract

Nucleosome occupancy plays a key role in regulating access to eukaryotic genomes. Although various chromatin regulatory complexes are known to regulate nucleosome occupancy, the role of DNA sequence in this regulation remains unclear, particularly in mammals. To address this problem, we measured nucleosome distribution at high temporal resolution in human cells at hundreds of genes during the reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV). We show that nucleosome redistribution peaks at 24 h post-KSHV reactivation and that the nucleosomal redistributions are widespread and transient. To clarify the role of DNA sequence in these nucleosomal redistributions, we compared the genes with altered nucleosome distribution to a sequence-based computer model and in vitro-assembled nucleosomes. We demonstrate that both the predicted model and the assembled nucleosome distributions are concordant with the majority of nucleosome redistributions at 24 h post-KSHV reactivation. We suggest a model in which loci are held in an unfavorable chromatin architecture and "spring" to a transient intermediate state directed by DNA sequence information. We propose that DNA sequence plays a more considerable role in the regulation of nucleosome positions than was previously appreciated. The surprising findings that nucleosome redistributions are widespread, transient, and DNA-directed shift the current perspective regarding regulation of nucleosome distribution in humans.

Figure 1.

Reactivation of KSHV resulted in widespread, transient nucleosome redistribution. (A) Boxplot of the correlation values for the 472 loci nucleosome distributions between the 0-h time point and the time points following KSHV reactivation. (B) Number of loci with nucleosome distribution changes (r < 0.7) at 6, 12, 24, and 48 h post-reactivation of KSHV. The greatest number of nucleosome distribution changes occurred at 24 h, and the nucleosome redistributions transiently return to the basal state nucleosome distribution (0 h) at 48 h. (C) The correlation values between the nucleosome distributions of the 0-h and post-KSHV reactivation time points for the IL12A, IL1RAP, and IL6R TSSs. (D) The nucleosome distribution from latent (black lines) and reactivated (red lines) post-KSHV time points for three genes: IL12A, IL1RAP, and IL6R loci. The x-axis represents genomic position showing 2 kb centered on a TSS. The y-axis is the log₂ ratio of nucleosomally protected DNA to genomic DNA signal at each probe on the microarray. Gene models from the RefSeq annotations are shown below each gene column.

Figure 2.

Nucleosome redistributions are determined by the underlying DNA sequence. (A) Western blots with the specified antibodies, at various times (in hours) after KSHV reactivation (hpr), of iSLK.219 cells treated with 0.2 µg/mL doxycycline. BRG1 protein levels peaked at 24 h. The RTA immunoblot is included to confirm KSHV reactivation. Beta-actin is included as a loading control. (B) Relative proportions of DNA-directed and DNA-independent nucleosome redistributions at 6, 12, 24, and 48 h post-KSHV reactivation. Approximately two-thirds of the genes with altered nucleosome occupancy at 12 h and 24 h were DNA-directed. (C) Observed and predicted (Gupta et al. 2008) nucleosome distributions. The nucleosome distributions from basal (black lines) KSHV, 24 h post-KSHV reactivation (red lines), and DNA-sequence-based prediction (cyan lines) for the same three genes shown in Figure 1D. The x-axis represents genomic position showing 2 kb centered on a TSS. The y-axis is the log₂ ratio of nucleosomally protected DNA to genomic DNA signal at each probe on the microarray. These plots show strong agreement between the sequence-based model and the reactivated state. Gene models from the RefSeq annotations are shown below each gene column. (D) Measured and assembled nucleosome distributions. The nucleosome distributions from latent (black lines) KSHV, 24 h post-KSHV reactivation (red lines), and assembly (blue lines) for the same three genes shown in Figure 1D. The x-axis represents genomic position showing 2 kb centered on a TSS. The y-axis is the log₂ ratio of nucleosomally protected DNA to genomic DNA signal at each probe on the microarray. These plots show strong agreement between the assembled and the reactivated state. Gene models from the RefSeq annotations are shown below each gene column.

Figure 3.

DNA sequence determined the concerted, widespread, transient redistribution of nucleosomes. (A) Average values for all genes identified as DNA-directed and DNA-independent, calculated by alignment of loci to the TSS for 0 h (black) and 24 h (red). (B) Average values for all genes identified as DNA-directed and DNA-independent, calculated by alignment of loci to the TSS for the predicted (cyan). (C) Average values for all genes identified as DNA-directed and DNA-independent, calculated by alignment of loci to the TSS for the assembly (blue). (D) Average GC (blue) and AT (red) content at each position is represented as a sequence logo below each column.

Figure 4.

Model of chromatin regulation in which nucleosome distributions move from a basal state architecture, to a transient intermediate state, then return to the basal architecture, in response to a common stimulus. The transient intermediate state's architecture is directed by features intrinsic to the underlying DNA sequence (cyan).