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

Abstract

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.

Keywords: fragile nucleosomes; micrococcal nuclease bias; micrococcal nuclease-sensitive nucleosomes; non-histone barrier complex; yeast chromatin.

Figure 1. MNase-Sensitive Complexes at Yeast Promoters Do Not Contain Histones

(A) MNase-sensitive complexes at promoters contain very little H4 or H2B. Average DNA fragment center distributions are shown for all genes relative to the dyad of the +1 nucleosome for five levels of MNase digestion (inputs) for YDC439 (HA-tagged H4) and YDC443 (HA-tagged H2B) and for IPs from the most- and least-digested samples. Note that the inputs show a peak between the +1 and −1 nucleosomes that decreases with increasing MNase concentration, whereas there is no such peak in the IPs. (B) 2D occupancies for the HA–H2B input samples (left in A; see Figure S1A for equivalent plots for HA–H4). The peak located between the +1 and −1 nucleosomes observed in the inputs in A is due to proteins that protect relatively short DNA fragments from MNase. (C) 2D occupancies of the mildly digested H2B and H4 IP samples shown in (B) and Figure S1A. (D) Average occupancy plots for H2B and H4 for the same strains obtained by sonication-ChIP-seq (no MNase was used); two biological replicate experiments are shown for each strain. The corresponding 2D occupancy heatmaps are shown in Figure S1D. See also Figure S1.

Figure 2. Transcription Termination Sites Are Occupied by MNase-Sensitive Nucleosomes

(A) Average nucleosome dyad distribution for all genes indicates weak nucleosome phasing relative to the TTS (data for 200U MNase). (B) The weak phasing at the TTS is mostly due to neighboring TSSs. Heatmap of the dyad distribution at the TTS with convergent genes (top) separated from tandem genes (bottom) and sorted according to the distance between the genes. (C) Average dyad distributions for convergent genes (blue line) and tandem genes (red line) show that nucleosome phasing due to the TTS is very poor. (D) MNase-sensitive nucleosomes occupy the TTS. In the MNase-ChIP-seq data, average dyad distribution plots for input samples show that the complexes covering the TTS are digested relatively rapidly; similar plots for the IPs show that the complexes contain both H4 and H2B. (E) 2D occupancy heatmaps for the H2B input data in (D). (F) 2D occupancy heatmaps for the H2B and H4 IPs from the least-digested samples in (D). (G) Sonication-ChIP-seq data show weak depletion of H2B and H4 at the TTS (two biological replicate experiments are shown) consistent with moderately high nucleosome occupancy at the TTS. Panels (D)–(G) contain the TTSs from all convergent genes plus tandem genes that are >1 kb from a downstream TSS. See also Figure S2.

Figure 3. A/T-Rich Nucleosomes Identified by Chemical Mapping Are MNase Sensitive

(A) Heatmap analysis of the 5,000 most A/T-rich nucleosomes identified by chemical mapping (Henikoff et al., 2014) aligned on the nucleosome dyad and sorted according to A/T content. (B) Genomic distribution of A/T-rich nucleosomes identified in A. Note that an A/T-rich nucleosome can be counted more than once; e.g., a −1 nucleosome can also be the +1 nucleosome on a neighboring divergent gene. (C) The rate of MNase digestion depends on the A/T content of a nucleosome. MNase-ChIP-seq input occupancy (HA–H2B) for the 5,000 chemically mapped nucleosomes was aligned and sorted as in (B). (D) MNase-sensitive A/T-rich complexes contain H4 and H2B. Average occupancy plots for the 5,000 nucleosomes are shown, with inputs and IPs for the least digested samples. (E) Sonication-ChIP-seq: H4 and H2B are only mildly depleted from A/T-rich sites; two biological replicate experiments are shown for each strain. See also Figure S3.

Figure 4. tRNA Genes Are Occupied by TFIIIB-TFIIIC Complexes, which Are Less Accessible to MNase Than Nucleosomes

(A) tRNA genes are occupied by non-histone protein complexes that are digested more slowly than the flanking nucleosomes. Average DNA fragment center distribution plots from MNase-ChIP-seq are for all tRNA genes aligned on the tRNA gene start site. The IPs show that H4 and H2B are absent. (B) 2D occupancy heatmaps for the H2B input data in (A). (C) 2D occupancy heatmaps for the H2B and H4 IPs from extensively digested samples in (A). tRNA genes are bound by non-histone proteins with smaller footprints than a nucleosome. (D) Sonication-ChIP-seq confirms the very low levels of H2B and H4 at tRNA genes. (E) tRNA genes are occupied by a TFIIIB-TFIIIC complex: MNase-ChIP-seq for Brf1 (a TFIIIB sub-unit) and Tfc1 (a TFIIIC sub-unit). MNase first liberates the complete complex, which has a footprint similar to that of a nucleosome, and then cuts between TFIIIB, which binds upstream of the start site, and TFIIIC, which binds to the tRNA gene itself (Nagarajavel et al., 2013). See also Figure S4.

Figure 5. The GAL1-GAL10 Regulatory Region Is Bound by an MNase-Sensitive Complex that Does Not Contain Histones

(A) The upstream activating region in the divergent GAL1-GAL10 gene promoter (UASg) is bound by an MNase-sensitive complex with a smaller footprint than a nucleosome (120–130 bp). (B–D) MNase-ChIP-seq (B), Sonication-ChIP-seq (C), and chemical mapping (D) show that this MNase-sensitive complex does not contain H2B or H4.

Figure 6. MNase-Sensitive Nucleosomes and MNase-Sensitive Non-histone Complexes Have Distinct Nucleotide Compositions

(A) Two types of MNase-sensitive site can be distinguished by chemical cleavage (Henikoff et al., 2014). MNase-sensitive sites were identified by analysis of relative rates of MNase digestion. The histogram shows the average chemical cleavage density within these MNase-sensitive regions. The bimodal distribution separates the non-nucleosomal sites (low chemical cleavage density due to absence of H4) from nucleosomal sites (high cleavage density). (B) Heatmap of chemical cleavage at MNase-sensitive sites. The horizontal white line indicates the separation of the two peaks in (A). (C) Heatmaps showing an MNase digestion series for the MNase-sensitive complexes aligned and sorted as in (B). (D and E) MNase-ChIP-seq (D) and sonication-ChIP-seq (E) confirm that there are two types of MNase-sensitive complex: non-histone complexes (top) and nucleosomes (bottom). (F) Heatmap of the A/T content of MNase-sensitive regions aligned and sorted as in (B). MNase-sensitive nucleosomes are A/T rich, whereas MNase-sensitive non-histone complexes have much lower A/T content. (G) MNase-sensitive non-histone-DNA complexes are hypersensitive to DNase I (Hesselberth et al., 2009), but MNase-sensitive nucleosomes are not. The non-nucleosomal MNase-sensitive regions are DNase I hypersensitive sites and are bound by transcription factors such as TBP (Paul et al., 2015; Zentner and Henikoff, 2013; Zentner et al., 2015), Abf1 (Paul et al., 2015; Zentner and Henikoff, 2013; Zentner et al., 2015), and Reb1 (Paul et al., 2015; Zentner and Henikoff, 2013; Zentner et al., 2015). See also Figure S5.

Figure 7. The Extent of MNase Digestion Affects the Nucleotide Composition of Mono-nucleosomal DNA

(A) Probability distribution function for the G/C content of the mono-nucleosomes obtained in an MNase titration (50 to 400U). The G/C content of mono-nucleosomes increases during digestion as G/C-rich nucleosomes are released from oligo-nucleosomes and A/T-rich nucleosomes are destroyed. (B) Cumulative distribution function for nucleosomal G/C content.