Abstract
Activation of transcription requires alteration of chromatin by complexes that increase the accessibility of nucleosomal DNA. Removing nucleosomes from regulatory sequences has been proposed to play a significant role in activation. We tested whether changes in nucleosome occupancy occurred on the set of genes that is activated by the unfolded protein response (UPR). We observed no decrease in occupancy on most promoters, gene bodies, and enhancers. Instead, there was an increase in the accessibility of nucleosomes, as measured by micrococcal nuclease (MNase) digestion and ATAC-seq (assay for transposase-accessible chromatin [ATAC] using sequencing), that did not result from removal of the nucleosome. Thus, changes in nucleosome accessibility predominate over changes in nucleosome occupancy during rapid transcriptional induction during the UPR.
Highlights
Many sequence-specific DNA-binding regulatory factors and components of the general transcription machinery are inhibited by nucleosome formation (Kornberg 1974)
To identify time points at which we expected nucleosomes to change during the unfolded protein response (UPR), we looked at UPR initiation in S2 cells by measuring splicing of the key UPR transcription factors (TFs) XPB-1 (Supplemental Fig. S1A, B) and using RNA sequencing (RNA-seq) to examine genome-wide changes in regulation (Fig. 1A; Supplemental Fig. S1C)
While using standard micrococcal nuclease (MNase)-seq protocols, which have been applied effectively in organisms such as Saccharomyces cerevisiae, we noted variations in measuring nucleosome occupancy from experiment to experiment that confounded interpretation. We attributed this to the fact that nucleosomes are variably released by different MNase concentrations, changes that are likely to be influenced by H1 association, histone variants, and compaction (Iwafuchi-Doi et al 2016; Mieczkowski et al 2016; Chereji et al 2017)
Summary
Many sequence-specific DNA-binding regulatory factors and components of the general transcription machinery are inhibited by nucleosome formation (Kornberg 1974). The use of several MNase concentrations allowed us to capture nucleosomes that are preferentially released at both high and low levels of MNase, providing a more comprehensive occupancy map than is likely to be obtained at a single MNase amount (Iwafuchi-Doi et al 2016; Mieczkowski et al 2016; Chereji et al 2017) These experiments, further supported by experiments performed using ATACseq (assay for transposase-accessible chromatin [ATAC] using sequencing), allowed us to determine whether a nucleosome was accessible or inaccessible in addition to its genomic location. In promoters and enhancers, predominant changes during the UPR occurred in nucleosome accessibility, not in nucleosome occupancy
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