Figure 4

GCN5 acetylates ISWI at K753. (A) Alignment of Drosophila ISWI sequences spanning aa 740–769 with the corresponding residues from Xenopus ISWI (xISWI), human hSNF2H, hSNF2L and with the 30 N-terminal amino acids of histone H3. A consensus of highly conserved residues is given. The arrowhead points to K753 indISWI. (B) Peptides corresponding to the histone H3 N-terminal tail (aa 1–19, left panel) and to aa 740–759 of ISWI (right panel) were acetylated in vitro by GCN5, p300 or MOF as in Figure 2. Peptide acetylation was determined from incorporation of [³H]-acetate and by subtracting the values for autoacetylation of enzymes (from reactions lacking substrate) and the values for non-specific adsorption of label to the peptides (from reactions lacking enzyme) from the experimental values. (C) MALDI analysis of the in vitro acetylated ISWI peptide (aa 740–759). The acetylation reaction was stopped by addition of 10% acetic acid, and the reaction products were analyzed by MALDI-TOF. The upper spectrum represents the unmodified peptide (M-H⁺ = 2580.29 amu) from a control reaction lacking acetyl-CoA. In the lower spectrum the arrow points to a peak representing the product of the acetylation reaction (M-H⁺ = 2622.32 amu). A Deltamass (Dm) of 42.01 amu indicates mono-acetylation. A second peak of very low intensity (asterisk) suggests minor di-acetylation (M-H⁺ = 2664.33). (D) Nanospray sequencing of ISWI peptide (aa 743–759) after in vitro acetylation and removal of the C-terminal cysteine by V8 protease. Upper spectrum: sequencing of the unmodified peptide with a (M-3H⁺) value of 702.67 confirms the sequence IYYFRKTVGYKVPKNTE with unmodified K748, K753 and K756. The spectrum is enlarged over the region ₇₅₀VGYKV₇₅₄ in order to observe the y7 ion (M-H⁺ = 815.46) corresponding to the unacetylated K753. Lower spectrum: sequencing of the peptide with a (M-3H⁺) value of 716.73 reveals the sequence IYYFRKTVGYK_AcVPKNTE, with only K753 being acetylated, (mass of the y7 ion M-H⁺ = 857.47, which differs in mass from the corresponding unmodified y7 ion in the upper spectrum by 42.012 amu). Detailed analysis of K748 and K756 revealed no acetylation at these sites (data notshown). (E) Recombinant ISWI and derivatives bearing the indicated point mutations were acetylated by GCN5 and acetylated proteins were visualized by autoradiography (upper panel) and Coomassie staining (lower panel). (F) Recombinant wildtype (WT) and mutant ISWI proteins were incubated with [g-³²P]-ATP in the absence (control), or presence of 110 ng of free or nucleosomal DNA (Nuc) as indicated. ATP hydrolysis was detected by thin-layer chromatography and quantified by PhosphoImager analysis. ATPase activity is indicated as percentage of ATP hydrolysed.