MNase-seq Mapping of Nucleosome Positioning and Occupancy at Inducible Promoters in Saccharomyces cerevisiae Following Galactose Induction

Two conditions (glucose-repressed, galactose-induced 30 min), biological triplicate (n=3). Three MNase concentrations per sample (light, medium, heavy digestion) to build an occupancy titration = 18 digestions. Each sample yields a mononucleosome gel-purified pool. Time-course aliquots (0, 15, 30, 60 min post-galactose) for a subset. Cultures grown to mid-log (OD600 0.5-0.7); harvest synchronized by rapid formaldehyde crosslinking. Samples randomized across the digestion plate.

BSL-1 for S. cerevisiae. Formaldehyde is a carcinogen and respiratory sensitizer — handle and quench in a fume hood, wear nitrile gloves and goggles, collect formaldehyde waste separately. 2-mercaptoethanol is toxic and malodorous (fume hood). Phenol-chloroform (if used) is corrosive/toxic — halogenated organic waste. Ethidium-free gel stains preferred; if EtBr used, follow institutional disposal. Standard lab PPE and yeast biological waste handling.

MNase titration itself is the key internal control: light vs heavy digestion distinguishes stable from labile nucleosomes (occupancy that disappears at low MNase = fragile). Naked-DNA MNase control (deproteinized genomic DNA digested in parallel) maps MNase sequence-cleavage bias for correction. Glucose-repressed samples are the negative/baseline condition for GAL induction. Housekeeping promoters (ACT1/PGK1) serve as no-change internal references. Input genomic DNA for normalization.

Clear mononucleosome band (~150 bp) at the chosen MNase level. Genome-wide well-positioned +1 nucleosome arrays downstream of NDRs; characteristic phasing pattern. At GAL1-10/GAL7: NDR widening >100 bp and >50% occupancy loss of -1/+1 nucleosomes by 30 min galactose. ACT1/PGK1 unchanged. Fragment-length distribution peaking at ~147 bp; fraction in nucleosomes high.

• S. cerevisiae strain BY4741, grown in YP + 2% raffinose, induced with 2% galactose
• 37% formaldehyde (crosslinking to 1% final); 2.5 M glycine (quench)
• Zymolyase 100T (spheroplasting, 0.5 mg/mL) + sorbitol buffer (1 M sorbitol, 50 mM Tris pH 7.5, 10 mM 2-mercaptoethanol)
• Micrococcal nuclease (NEB M0247), titrated (e.g., 5, 20, 60 U)
• MNase digestion buffer: 1 M sorbitol, 50 mM NaCl, 10 mM Tris pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.075% NP-40, spermidine
• 0.5 M EDTA/EGTA stop; SDS/Proteinase K for reversal; RNase A
• Agarose for gel-purification of the ~150 bp mononucleosome band; QIAquick gel extraction
• NEBNext Ultra II library kit, AMPure XP beads

To generate base-pair-resolution nucleosome occupancy maps across the S. cerevisiae genome by digesting crosslinked chromatin with a titration of micrococcal nuclease and sequencing the protected mononucleosomal DNA (~147 bp). The MNase titration distinguishes well-positioned, stable nucleosomes from fragile/labile ones and from non-nucleosomal protections, enabling accurate occupancy quantification at inducible promoters.

1. Grow 50 mL BY4741 in YP-raffinose to OD600 0.6; split, add 2% galactose to induced set; incubate 30 min (or time course).
2. Crosslink 1% formaldehyde 15 min RT; quench with 125 mM glycine 5 min.
3. Pellet, wash; spheroplast with Zymolyase in sorbitol buffer 30°C 20-30 min until ~80% spheroplasts (phase-dark by microscopy).
4. Gently pellet spheroplasts; resuspend in MNase digestion buffer; split into three tubes for light/medium/heavy MNase.
5. Digest 37°C 20 min; stop with EDTA/EGTA on ice.
6. Reverse crosslinks: add SDS to 1% + Proteinase K, 65°C overnight; treat with RNase A 37°C 1 h.
7. Purify DNA (phenol-chloroform or column); run on 2% agarose; excise the ~150 bp mononucleosome band; gel-extract.
8. Confirm mononucleosome enrichment on TapeStation; choose the digestion giving ~80% mononucleosome.
9. Prepare PE libraries (NEBNext Ultra II, 8-10 cycles); sequence PE 50 to ~30-50M reads for dense yeast coverage.

Independent variables: carbon source/induction state (glucose vs galactose) and MNase concentration (titration). Dependent variables: nucleosome occupancy (normalized fragment midpoint density), dyad positioning, and NDR width at target promoters. Controlled variables: growth phase (OD600 0.6), crosslinking time, spheroplasting extent, digestion temperature/time, and fragment-size selection (mononucleosome band).

We hypothesize that galactose induction will trigger eviction of the -1 and +1 nucleosomes flanking the nucleosome-depleted region (NDR) of GAL1-10 and GAL7 promoters within 30 minutes, widening the NDR by >100 bp and reducing local nucleosome occupancy by >50%, while constitutively expressed housekeeping promoters (e.g., ACT1, PGK1) show no significant repositioning.

Trim and align PE reads to sacCer3 with Bowtie2; keep properly paired fragments 120-180 bp (mononucleosome). Compute fragment midpoints/dyads; generate occupancy and dyad-density tracks with DANPOS3 (which models occupancy, fuzziness, and position shifts) or nucleR. Correct for MNase bias using the naked-DNA digestion track. Quantify NDR width and per-nucleosome occupancy at GAL/control promoters; metaplots aligned to TSS with deepTools.

No mononucleosome band: incomplete spheroplasting — verify >80% spheroplasts before digestion and ensure fresh Zymolyase. Over-digestion (sub-147 bp smear, naked DNA): lower MNase units or shorten to 10 min. Under-digestion (oligonucleosome ladder): raise MNase or extend digestion. Weak GAL eviction signal: confirm galactose induction by RT-qPCR of GAL1 mRNA and ensure raffinose (not glucose) pre-growth to avoid catabolite repression carryover. MNase bias artifacts at AT-rich regions: always subtract/normalize against the naked-DNA digestion control.

DANPOS3 compares glucose vs galactose, reporting position-shift and occupancy-change p-values per nucleosome (Poisson/negative-binomial test); BH FDR q<0.05. Target-promoter occupancy change tested across triplicates with a paired two-sided t-test (alpha 0.05) versus baseline, Bonferroni-corrected over the assayed loci. Power: triplicate MNase-seq at ~40M PE reads detects >40% occupancy changes at well-positioned nucleosomes with >0.8 power.