Tandem PHD fingers of MORF/MOZ acetyltransferases display selectivity for acetylated histone H3 and are required for the association with chromatin

Abstract

MORF [MOZ (monocytic leukemia zinc-finger protein)-related factor] and MOZ are catalytic subunits of histone acetyltransferase (HAT) complexes essential in hematopoiesis, neurogenesis, skeletogenesis and other developmental programs and implicated in human leukemias. The canonical HAT domain of MORF/MOZ is preceded by a tandem of plant homeodomain (PHD) fingers whose biological roles and requirements for MORF/MOZ activity are unknown. Here, we demonstrate that the tandem PHD1/2 fingers of MORF recognize the N-terminal tail of histone H3. Acetylation of Lys9 (H3K9ac) or Lys14 (H3K14ac) enhances binding of MORF PHD1/2 to unmodified H3 peptides twofold to threefold. The selectivity for acetylated H3 tail is conserved in the double PHD1/2 fingers of MOZ. This interaction requires the intact N-terminus of histone H3 and is inhibited by trimethylation of Lys4. Biochemical analysis using NMR, fluorescence spectroscopy and mutagenesis identified key amino acids of MORF PHD1/2 necessary for the interaction with histones. Fluorescence microscopy and immunoprecipitation experiments reveal that both PHD fingers are required for binding to H3K14ac in vivo and localization to chromatin. The HAT assays indicate that the interaction with H3K14ac may promote enzymatic activity in trans. Together, our data suggest that the PHD1/2 fingers play a role in MOZ/MORF HATs association with the chromatic regions enriched in acetylated marks.

Fig. 1

The MORF PHD1/2 fingers bind histone H3. (a) Architecture of MORF. The tandem PHD1/2 fingers are colored yellow. (b) Superimposed ¹H,¹⁵N HSQC spectra of 0.1 mM MORF PHD1/2, collected as indicated H3 peptides were separately titrated in. The spectra are color-coded according to the protein-peptide ratio (inset). (c) Alignment of the PHD1/2 sequences: absolutely, moderately and weakly conserved residues are colored brown, yellow and green, respectively. Each tenth residue of MORF PHD1/2 is marked by a dot and labeled.

Fig. 2

Selectivity of the MORF PHD1/2 fingers. (a) Binding affinities of the MORF PHD1/2 fingers for indicated peptides were measured by tryptophan fluorescence ^(a) and NMR ^(b). NB- no binding. (b) Representative binding curves used to determine K_ds of the PHD1/2-H3K9ac and PHD1/2-H3K9me3 interactions. (c, e) Superimposed ¹H,¹⁵N HSQC spectra of MORF PHD1/2, collected as indicated H3 peptides were added stepwise. (d) Western blot of bound MORF PHD1/2 (beads) following in-solution pulldowns with the indicated peptides. Control shows detection of unbound protein following incubation with peptide-bead conjugate.

Fig. 3

The selectivity for acetylated H3 is conserved. (a) Superimposed ¹H,¹⁵N HSQC spectra of MOZ PHD1/2, recorded during addition of the indicated H3 peptides. The spectra are color-coded according to the protein-peptide ratio. (b) Binding affinities of the MOZ PHD1/2 fingers for indicated H3 peptides were measured by tryptophan fluorescence. (c) Western blot of bound MOZ PHD1/2 (beads) following in-solution pulldowns with the indicated peptides. Control shows detection of unbound protein following incubation with peptide-bead conjugate.

Fig. 4

Identification of the histone H3K9ac binding site of the MORF PHD1/2 fingers. (a) Histograms show normalized ¹H,¹⁵N chemical shift changes in backbone amides of ¹⁵N-labeled MORF PHD1/2 upon addition of short and long H3K9ac peptides at a protein:peptide ratio of 1:5. The most perturbed residues are labeled. (b) Mutations of the binding site residues abolish interaction of the MORF PHD1/2 fingers with H3K9ac. Superimposed ¹H,¹⁵N HSQC spectra of MORF PHD1/2 mutants, collected as H3K9ac peptide was titrated in. (c) Binding affinity of D285A MORF PHD1/2 was estimated by NMR.

Fig. 5

The PHD1/2 fingers of MORF are required for the interaction with histone H3 in cells. (a) IPs of the Flag-MORF [N-terminal region of MORF (N-MORF, amino acids 1–352) and N-MORFΔPHD1/2 (amino acids 1–215), in which the PHD1/2 fingers are deleted] from HEK293 cells and immunoblotting with indicated antibodies. (b) IPs of the Flag-N-MORF from HEK293 cells +/− TSA and immunoblotting with indicated antibodies. (c) HEK293 cells were transfected with indicated GFP-MORF constructs, and subcellular localization of the GFP signal was detected by live green fluorescence microscopy. (d, e) HAT assays with indicated substrates and MOZ-BRPF1-ING5-hEaf6 complex purified from co-transfected cells were quantified by liquid scintillation (d) and gel fluorography (e).

Fig. 6

Crosstalk between the PHD fingers present in the MORF/MOZ, BRPF1 and ING5 subunits of the MOZ/MORF HAT complexes. The ING5 PHD finger recognizes H3K4me3, an epigenetic mark that inhibits binding of the PHD fingers of either MORF/MOZ or BRPF1.