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. 2016 Dec 8;100(1):91–104. doi: 10.1016/j.ajhg.2016.11.011

Figure 4.

Figure 4

Functional Characterization of BRPF1 Variants In Vitro

(A) Interaction of BRPF1 and the variants with KAT6A, ING5, and MEAF6. KAT6A was produced in HEK293 cells as a FLAG-tagged fusion protein along with HA-tagged BRPF1 (or the p.Pro370Ser variant), ING5, and MEAF6 as indicated. Soluble protein extracts were prepared for affinity purification on anti-FLAG agarose, and bound proteins were eluted with the FLAG peptide for immunoblotting with anti-FLAG and -HA antibodies.

(B) Histone acetylation assays. HeLa oligonucleosomes were used as substrates for acetylation by proteins affinity purified in (A). Acetylation of histone H3 was detected with antibodies recognizing histone H3 and its acetylated forms as indicated.

(C and D) Same as (A) except that different BRPF1 variants were analyzed. Unexpectedly, the variant p.Trp315Leufs∗26 still showed strong interaction with MEAF6; whether this is due to the extra 26 residues introduced after the reading frameshift remains unclear.

(E) Histone acetylation assays. Proteins affinity-purified in (D) were used for the enzymatic assays as in (B).