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. 2021 Feb 1;10:e65369. doi: 10.7554/eLife.65369

Figure 5. Formation of knobs of highly condensed chromatin is enhanced in G34R and K36 acetyltransferase mutants, correlating with enhanced recruitment of Shugoshin to repressed subtelomeric domains.

(A) Frequency of subtelomeric knob formation observed in H3-WT, H3-G34R, and H3-G34V cells. The number of knobs in a nucleus is shown. Data plots represent mean ± SEM from three independent biological replicates, counting ~200 total nuclei per strain (upper). Example of nuclear knobs observed in WT and H3-G34; scale bar indicates 0.5 µm (bottom). (B) Frequency of subtelomeric knob formation observed in WT, set2Δ, and gcn5Δmst2Δ cells (all 3xH3) with the number of knobs in a nucleus shown. Data plots represent mean ± SEM from three independent biological replicates, counting ~200 total nuclei per strain. (C–F) ChIP analysis of Sgo2-FLAG association with the subtelomeric fah1+ and grt1+ loci in (C) H3-WT, H3-G34R, and H3-G34V cells normalized to act1+. Data were collected and plotted as the mean of six biological replicates ± SEM. * represents a significance of p<0.05 compared with the H3-WT strain. (D) Sgo2-FLAG ChIP in (3xH3) WT, set2Δ, and gcn5Δmst2Δ cells normalized to act1+. Data are the mean of four biological replicates ± SEM. **** represents a significance of p<0.0001 compared with the WT strain. (E–F) Sgo2-FLAG ChIP at the centromeric dh sequences (E) or subtelomeric fah1+ gene (F) in asynchronous or mitotically arrested nda3 mutant cells with sole copy H3-WT or H3-G34R (six replicates), and (G) Sgo2-FLAG ChIP in H3-G34K cells normalized to act1+. Data represents the mean of six biological replicates ± SEM. * represents a significance of p<0.05 compared with the H3-WT strain.

Figure 5.

Figure 5—figure supplement 1. Determining the effect of G34 substitution on antibody recognition of K36ac.

Figure 5—figure supplement 1.

(A) Slot blot analysis to quantitatively assess whether anti-K36 acetyl antibodies equivalently recognize WT, H3-G34R, or H3-G34K peptides bearing H3K36ac modification. Peptides were loaded in twofold serial dilution. Antibodies tested: Abnova PAB31320, Rockland Immunochemicals 600–401-I89, and Thermo Fischer MA5-24672. Ponceau-stained blots were used as the loading control (left).