The histone H4 acetyltransferase MOF uses a C2HC zinc finger for substrate recognition

Abstract

Site-specific acetylation of histone H4 by MOF is central to establishing the hyperactive male X chromosome in Drosophila. MOF belongs to the MYST family of histone acetyltransferases (HATs) characterized by an unusual C2HC-type zinc finger close to their HAT domains. The function of these rare zinc fingers is unknown. We found that this domain is essential for HAT activity, in addition to the established catalytic domain. MOF uses its zinc finger to contact the globular part of the nucleosome as well as the histone H4 N-terminal tail substrate. Point mutations that leave the zinc-finger structure intact nevertheless abolish its interaction with the nucleosome. Our data document a novel role of the C2HC-type finger in nucleosome binding and HAT activity.

Fig. 1. Structural features of MOF. (A) Schematic representation of the known domains of MOF. CD, chromodomain; Zn, zinc finger-containing domain; HAT, histone acetyltransferase domain. Point mutations in the CD and HAT domains are indicated. (B) Alignment of the conserved sequences of the MYST family members containing the C₂HC zinc finger. (C) Summary of point mutations in the zinc-finger region generated and analyzed in this study.

Fig. 2. Mutations in the zinc finger impair HAT activity of MOF. (A) Point mutants in the chromodomain (Y416D, W426G), HAT domain (G691E) and zinc finger (C577G) were tested for acetylation of Drosophila histones. (B) The HAT activity of MOF (black bars) but not HAT1 (gray bars) is sensitive to the zinc chelator 1,10-phenanthroline. HAT assays contained Drosophila histones, 3H acetyl CoA and 100 ng of either MOF (lanes 2–5) or HAT1 (lanes 6–9) in the presence of 5, 10 or 15 mM 1,10-phenanthroline (lanes 3–5 and 7–9), respectively. Lane 1, control reaction without enzyme.

Fig. 3. The C₂HC zinc finger is involved in acetyltransferase activity and chromatin binding of MOF. (A) Only mutations in or adjacent to the zinc finger affect HAT activity. See Figure 1C for an overview of the mutations. HAT assays were performed as described in Figure 2A. (B) The zinc finger, but not the chromodomain, is involved in nucleosome binding. Drosophila embryo extracts were used to assemble chromatin on linear DNA immobilized on paramagmetic beads. Three hundred nanograms of either chromatinized (N) or free DNA template (D) were used for each binding reaction with 100 ng of each MOF derivative. Stable binding was analyzed by western blot analysis. I, 20% of total input protein. Position of the molecular weight markers (in kDa) is indicated. An arrow indicates the position of full-length MOF. (C) RNA EMSA with either 150 ng (odd lanes) or 300 ng (even lanes) of MOF derivatives as indicated. Lane 1, free RNA probe.

Fig. 3. The C₂HC zinc finger is involved in acetyltransferase activity and chromatin binding of MOF. (A) Only mutations in or adjacent to the zinc finger affect HAT activity. See Figure 1C for an overview of the mutations. HAT assays were performed as described in Figure 2A. (B) The zinc finger, but not the chromodomain, is involved in nucleosome binding. Drosophila embryo extracts were used to assemble chromatin on linear DNA immobilized on paramagmetic beads. Three hundred nanograms of either chromatinized (N) or free DNA template (D) were used for each binding reaction with 100 ng of each MOF derivative. Stable binding was analyzed by western blot analysis. I, 20% of total input protein. Position of the molecular weight markers (in kDa) is indicated. An arrow indicates the position of full-length MOF. (C) RNA EMSA with either 150 ng (odd lanes) or 300 ng (even lanes) of MOF derivatives as indicated. Lane 1, free RNA probe.

Fig. 3. The C₂HC zinc finger is involved in acetyltransferase activity and chromatin binding of MOF. (A) Only mutations in or adjacent to the zinc finger affect HAT activity. See Figure 1C for an overview of the mutations. HAT assays were performed as described in Figure 2A. (B) The zinc finger, but not the chromodomain, is involved in nucleosome binding. Drosophila embryo extracts were used to assemble chromatin on linear DNA immobilized on paramagmetic beads. Three hundred nanograms of either chromatinized (N) or free DNA template (D) were used for each binding reaction with 100 ng of each MOF derivative. Stable binding was analyzed by western blot analysis. I, 20% of total input protein. Position of the molecular weight markers (in kDa) is indicated. An arrow indicates the position of full-length MOF. (C) RNA EMSA with either 150 ng (odd lanes) or 300 ng (even lanes) of MOF derivatives as indicated. Lane 1, free RNA probe.

Fig. 4. Substrate recognition by MOF requires the histone H4 tail. (A) Interaction assay. Chromatin was assembled from recombinant Xenopus histones using the NAP1 histone chaperone. The histone mixes that were used to reconstitute chromatin are indicated above the lanes. Histones from which the N-terminus has been deleted (i.e. the globular domains) are indicated with the prefix ‘g’. D, free DNA; I, 20% input protein. Bound proteins were resolved by SDS–PAGE and detected by western blot analysis. (B) HAT activity in the absence (–) or presence (+) of MOF using different recombinant histone substrates, as indicated. (C) Tail-dependence of the interaction of the Y580G derivative. Binding of wild-type MOF and Y580G derivative to nucleosomes either lacking all histones tails (lanes 3 and 4) or displaying just the H4 N-terminus (lanes 1 and 2) was analyzed as in (A).

Fig. 4. Substrate recognition by MOF requires the histone H4 tail. (A) Interaction assay. Chromatin was assembled from recombinant Xenopus histones using the NAP1 histone chaperone. The histone mixes that were used to reconstitute chromatin are indicated above the lanes. Histones from which the N-terminus has been deleted (i.e. the globular domains) are indicated with the prefix ‘g’. D, free DNA; I, 20% input protein. Bound proteins were resolved by SDS–PAGE and detected by western blot analysis. (B) HAT activity in the absence (–) or presence (+) of MOF using different recombinant histone substrates, as indicated. (C) Tail-dependence of the interaction of the Y580G derivative. Binding of wild-type MOF and Y580G derivative to nucleosomes either lacking all histones tails (lanes 3 and 4) or displaying just the H4 N-terminus (lanes 1 and 2) was analyzed as in (A).