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. 2022 Mar 1;36(5-6):313–330. doi: 10.1101/gad.349039.121

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© 2022 Barry et al.; Published by Cold Spring Harbor Laboratory Press

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Figure 4. — Rap1 forms a complex with and modulates the histone acetyltransferase (HAT) activity of Tip60/Epc1. (A) Schematic illustration of mouse Epc1 and Rap1 protein domains and deletion mutant constructs used for co-IP assays. (B) Co-IP of Flag-Epc1 deletion mutants and Myc-Rap1 in cotransfected HEK293T cells. Western blot analysis for input and IP sample was performed with the indicated antibody. (C) Co-IP of the Flag-Epc1 EPcA domain and Myc-Rap1. (D) Co-IP of Flag-Rap1 deletion mutants and Myc-Epc1. (E) Co-IP of Flag-Epc1, Myc-Tip60, and HA-Rap1. (F) Histone acetyltransferase (HAT) activity assays performed using co-IPs from E on core histones. Data are the counts per minute (CPM) mean ± standard deviation of n = 2 technical replicates; two-way ANOVA, and Tukey's multiple comparison test. (*) P-value < 0.0001. (G) HAT activity assay using affinity-purified human EPC1/TIP60 and RAP1 (increasing amounts) on short oligonucleosome chromatin. Bovine serum albumin (BSA) was a negative control. Data are CPM mean ± standard deviation of n = 3 technical replicates; two-way ANOVA, and Tukey's multiple comparison test. (*) P-value < 0.01.