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. 2022 Mar 28;50(7):3727–3744. doi: 10.1093/nar/gkac175

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© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — Targeted inactivation of Gcn5 in rDNA leads to an increase in H3K9 methylation. (A) H3K9me2 and H3K9me3 levels in 18S rDNA in the wild type (WT) and gcn5Δ strains under glucose-rich conditions (mean + SEM: n = 3). (B) A schematic diagram of Gcn5 domain structure. (top) The tev sequence is inserted to the HAT domain (red segment). (bottom) A diagram showing selective TEV-mediated cleavage of Gcn5 in rDNA. Nucleolus-specific protein Gar2, tagged with TEV protease cleaves Gcn5-tev only in the nucleolus. (C) (top) Immunoblotting of Gcn5-HA (–HA), Gcn5-tev-HA (-tev-HA) and α−Tubulin, in the presence (+) or absence (–) of Gar2-TEVp expression. Gcn5 (–) indicates control data using the wild type with non-tagged Gcn5. (bottom) The bands of Gcn5-HA were quantified and normalized to those of α−Tubulin. (D and E) Bar graph showing the data of ChIP-qPCR experiments for Gcn5-HA enrichment (D) and H3K9me3 levels (E) in 18S rDNA of indicated strains (mean + SEM: n = 3).