MNase-Sensitive Complexes in Yeast: Nucleosomes and Non-histone Barriers

Abstract

SUMMARY Micrococcal nuclease (MNase) is commonly used to map nucleosomes genome-wide, but nucleosome maps are affected by the degree of digestion. It has been proposed that many yeast promoters are not nucleosome-free but instead occupied by easily digested, unstable, “fragile” nucleosomes. We analyzed the histone content of all MNase-sensitive complexes by MNase-ChIP-seq and sonication-ChIP-seq. We find that yeast promoters are predominantly bound by non-histone protein complexes, with little evidence for fragile nucleosomes. We do detect MNase-sensitive nucleosomes elsewhere in the genome, including at transcription termination sites. However, they have high A/T content, suggesting that MNase sensitivity does not indicate instability, but rather the preference of MNase for A/T-rich DNA, such that A/T-rich nucleosomes are digested faster than G/C-rich nucleosomes. We confirm our observations by analyzing ChIP-exo, chemical mapping, and ATAC-seq data from other laboratories. Thus, histone ChIP-seq experiments are essential to distinguish nucleosomes from other DNA-binding proteins that protect against MNase.

Widespread changes in nucleosome accessibility without changes in nucleosome occupancy during a rapid transcriptional induction

“…While using standard 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). We therefore measured nucleosome occupancy on genes that were regulated during the UPR over a range of MNase concentrations.…”

“…We therefore measured nucleosome occupancy on genes that were regulated during the UPR over a range of MNase concentrations. We included a histone immunoprecipitation step in the protocol, as nucleosome-sized protection might result from nonhistone proteins as opposed to nucleosomes (Mieczkowski et al 2016;Chereji et al 2017). We found numerous examples of locations where nucleosome occupancy was low or undetectable when high MNase concentration (100 U) was used to release nucleosomes, yet occupancy was high at low MNase concentration (1.5 U) (examples in Fig.…”

“…We found previously that increased accessibility to MNase occurs on active genes and therefore were interested in determining how that accessibility changed during an acute response on both active genes and the surrounding regulatory regions. For example, others have shown previously, in disparate organisms, that nucleosomes at gene starts and ends or enhancers can have increased accessibility (Kubik et al 2015;Chereji et al 2016Chereji et al , 2017Iwafuchi-Doi et al 2016). We wished to assess these features during a genome-wide induction.…”

“…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.…”

Unexpected conformational variations of the human centromeric chromatin complex

“…peaks in low-salt fractions may have resulted from disruption of chromatin at the boundaries of CENP-A/B/C due to the action of MNase used in N-ChIP experiments. MNase not only digests away linker DNA but also "nibbles" on free DNA ends and cleaves to a variable extent within nucleosomes (Xi et al 2011;Mieczkowski et al 2016;Chereji et al 2017). MNase also digests RNA, and this might have contributed to the loss of CCAN components by loss of α-satellite RNAs, which are required in cis for full occupancy of CENP-A and CENP-C (McNulty et al 2017).…”