Figure 2. A small predicted helix is the Isw2-recruitment epitope in unscheduled meiotic gene expression (Ume6).

(A) (Top left) Schematic diagram of Ume6 truncation and deletion constructs used to identify the Isw2-recruitment epitope, with the known Sin3-interacting domain depicted as a green square, the DNA binding domain as a dark blue rectangle, and the putative Isw2-recruitment helix as a light blue rectangle. (Bottom left) Modeled helical peptide (by Phyre2) and sequence conservation of the identified Isw2-recruitment motif in Ume6 constructs from other yeasts. Asterisks denote invariant residues. (Right) Nucleosome dyad signal for Ume6 truncation and deletion strains indicates deletion of the region from residues 480 to 507 completely abrogates nucleosome positioning by Isw2 at Ume6 target sites. Vertical dashed gray lines denote wild-type (WT) positions of nucleosomes while vertical dashed pink lines indicate isw2 or ume6-deficient positions of nucleosomes. (B) Genome Browser image showing transcript abundance at three Ume6 target sites for yeast strains lacking Rpd3 with WT Ume6 (gray), Ume6(∆2–479) (blue), and Ume6(∆2–508) (orange). Grossly increased transcription is seen when residues 480–507 are deleted, consistent with expected transcriptional increase associated with loss of Isw2 and Rpd3. Upstream repression sequence (URS) sites are indicated as cyan rectangles. No significant increase in transcription is detected when Ume6 residues 2–479 are deleted. Biological replicates are shown to highlight reproducibility. (C) (Top) Cartoon schematic for ectopic display of the Isw2-recruiting helix (residues 480–507) to the C-terminus of a truncated Ume6 construct lacking Isw2-directed nucleosome positioning. A short SpyTag is appended to the C-terminus of the Ume6 construct and residues 480–507 are fused to the SpyCatcher domain and introduced on a yeast expression vector. (Bottom) Nucleosome dyad signal demonstrating recovery of Isw2-directed nucleosome positions at a subset of Ume6 target genes by the ectopically displayed helical element. Vertical dashed gray lines denote WT positions of nucleosomes while vertical dashed pink lines indicate isw2 or ume6-deficient positions of nucleosomes. URS sites are indicated as cyan rectangles. Individual biological replicates for nucleosome positions after ectopic display of the recruitment helix are provided in Figure 2—figure supplement 4.

Figure 2—figure supplement 1. The unscheduled meiotic gene expression (Ume6) helix between residues 479 and 508 recruits Isw2 to Ume6 targets.

(Left) Schematic representation of the truncation panel initially used to identify the region of Ume6 required for Isw2 recruitment. Green square indicates the part of Ume6 known to recruit SIN3 while the blue rectangle indicates the Ume6 DNA binding domain. (Right) Genome Browser image for a representative locus showing nucleosome dyad signal for the indicated strains. Vertical gray lines denote wild-type (WT) nucleosome positions while vertical pink lines indicate ectopic nucleosome positions associated with truncations beyond 322 N-terminal amino acids. The Isw2-recruitment domain was thus determined to be between residues 322 and 508 in Ume6.

Figure 2—figure supplement 2. The unscheduled meiotic gene expression (Ume6) helix between residues 479 and 508 recruits Isw2 to Ume6 targets.

Meta-analysis of nucleosome dyad signal at the 58 Ume6 target loci associated with cluster 1 in Figure 1B showing loss of residues 479–508 from Ume6 results in ectopic nucleosome positioning while N-terminal deletion of residues 2–479 preserves wild-type (WT) nucleosome positioning at Ume6 sites.

Figure 2—figure supplement 3. The unscheduled meiotic gene expression (Ume6) helix between residues 479 and 508 recruits Isw2 to Ume6 targets.

Genome Browser image demonstrating no loss in Ume6-ChIP signal for relevant Ume6 truncations across all of chromosome XIII. Signal is read-corrected enrichment, relative to genome average.

Figure 2—figure supplement 4. Transcription data support a role of unscheduled meiotic gene expression (Ume6) residues 479–508 for Isw2 recruitment and not Rpd3 activity.

Genome Browser images showing transcript abundance for the three representative Ume6 target loci for indicated Ume6 truncation strains in the presence of functional Rpd3, demonstrating the expected very subtle increase in transcription associated with loss of Isw2 at the INO1 locus. URS sites are indicated as cyan rectangles. Biological replicates for each strain are shown.

Figure 2—figure supplement 5. Transcription data support a role of unscheduled meiotic gene expression (Ume6) residues 479–508 for Isw2 recruitment and not Rpd3 activity.

Volcano plots for genes containing Ume6 motifs showing log2 change in transcription and associated statistical significance for indicated strains. Horizontal dashed line indicates a p value of 0.01. Vertical dashed lines indicate a change in transcription of ±4-fold. Retention of residues 479–508 prevents large-scale increases in transcription associated with deletion of Rpd3 and Isw2 (see blue shaded region).

Figure 2—figure supplement 6. Ectopic display of the unscheduled meiotic gene expression (Ume6) helical element can rescue Isw2 activity at Ume6 targets.

Meta-analysis of nucleosome dyad signal at the 58 Ume6 target loci associated with cluster 1 in Figure 1B showing partial rescue of nucleosome positioning when residues 479–508 are ectopically displayed on the C-terminus of Ume6 ∆2–596 through SpyTag-SpyCatcher pairs. Black arrows indicate wild-type signal gained by ectopic display of the recruitment helix.

Figure 2—figure supplement 7. Ectopic display of the unscheduled meiotic gene expression (Ume6) helical element can rescue Isw2 activity at Ume6 targets.

Genome Browser image showing additional replicates where SpyTag/SpyCatcher-mediated ectopic tethering of Ume6 residues 479–508 recovers wild-type nucleosome positions at Ume6 targets (as in Figure 2C).