Subunit composition and substrate specificity of a MOF-containing histone acetyltransferase distinct from the male-specific lethal (MSL) complex

Abstract

Human MOF (MYST1), a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs), is the human ortholog of the Drosophila males absent on the first (MOF) protein. MOF is the catalytic subunit of the male-specific lethal (MSL) HAT complex, which plays a key role in dosage compensation in the fly and is responsible for a large fraction of histone H4 lysine 16 (H4K16) acetylation in vivo. MOF was recently reported to be a component of a second HAT complex, designated the non-specific lethal (NSL) complex (Mendjan, S., Taipale, M., Kind, J., Holz, H., Gebhardt, P., Schelder, M., Vermeulen, M., Buscaino, A., Duncan, K., Mueller, J., Wilm, M., Stunnenberg, H. G., Saumweber, H., and Akhtar, A. (2006) Mol. Cell 21, 811-823). Here we report an analysis of the subunit composition and substrate specificity of the NSL complex. Proteomic analyses of complexes purified through multiple candidate subunits reveal that NSL is composed of nine subunits. Two of its subunits, WD repeat domain 5 (WDR5) and host cell factor 1 (HCF1), are shared with members of the MLL/SET family of histone H3 lysine 4 (H3K4) methyltransferase complexes, and a third subunit, MCRS1, is shared with the human INO80 chromatin-remodeling complex. In addition, we show that assembly of the MOF HAT into MSL or NSL complexes controls its substrate specificity. Although MSL-associated MOF acetylates nucleosomal histone H4 almost exclusively on lysine 16, NSL-associated MOF exhibits a relaxed specificity and also acetylates nucleosomal histone H4 on lysines 5 and 8.

FIGURE 1.

Subunit compositions of MOF-containing complexes. A, MudPIT analyses of MOF-containing complexes. Aliquots of anti-FLAG (Fl) agarose eluates prepared from cell lines expressing the indicated proteins or from control HEK293FRT or Hela cells were subjected to MudPIT. The intensity of the coloring corresponds to the NSAF for each protein and is a rough reflection of the relative abundance of the protein in the samples. (NSAF)_k = NSAF calculated for an individual protein k; SpC = spectral count; L = protein length in amino acids; and i = all proteins detected in the MudPIT runs. PTIP, Pax transactivation domain-interacting protein; h, human. B, Western blot analysis of MOF-containing complexes. Anti-FLAG agarose eluates from FLAG-INO80E- (as a negative control), FLAG-MCRS1-, FLAG-MSL3L1-, and FLAG-NSL3-expressing HEK293FRT cells were subjected to SDS-PAGE in 4–20% gradient gels, and proteins were identified by Western blotting with the indicated antibodies. C, representative silver-stained gels of proteins associated with MSL and NSL complexes. The positions and relative molecular masses in kDa of protein size standards are indicated at the left of the gels.

FIGURE 2.

Both MSL and NSL complexes acetylate nucleosomal histone H4. A, aliquots of anti-FLAG (Fl) agarose eluates from cells expressing the indicated proteins were subjected to SDS-PAGE in 4–20% gradient gels, and MOF was identified by Western blotting with anti-MOF antibody. h, human. B, HAT activities associated with MOF-containing complexes. Anti-FLAG agarose eluates from equivalent numbers of FLAG-Ies2- (as a negative control), FLAG-MSL3L1-, FLAG-NSL3-, and FLAG-MCRS1-expressing cells were prepared. As determined by Western blotting (panel A), the relative concentrations of the MOF in MSL and NSL complexes were similar in anti-FLAG agarose eluates prepared from the different cell lines and used in HAT assays. HAT assays were performed by mixing HeLa long oligonucleosomes, [³H]acetyl CoA, and 1.0 μl (lanes 2, 3, 5, and 7) or 2.0 μl (lanes 4, 6, and 8) of the indicated anti-FLAG agarose eluates. Proteins were visualized by Coomassie Blue R250 staining (lower gel) and acetylated histones were visualized by autoradiography (top gel).

FIGURE 3.

NSL-associated MOF has a broader substrate specificity than MSL-associated MOF. A, acetylation of Hela long oligonucleosomes by MSL- and NSL-associated MOF. HAT assays were performed with HeLa long oligonucleosomes, [³H]AcCoA, and 1 μl (lane 2 and lane 4) or 2 μl (lane 3 and lane 5) of the indicated anti-FLAG (Fl) agarose eluates, and acetylated hisones were visualized by autoradiography. The MSL and NSL complexes used in these experiments contained approximately equal concentrations of MOF (Fig. 2A). B, acetylation of free recombinant histones by MSL- and NSL-associated MOF. HAT assays were performed with core histones, unlabeled acetyl CoA, and 0.6 μl (lanes 2 and 5), 1.2 μl (lanes 3 and 6), or 2.4 μl (lanes 4 and 7) of the indicated anti-FLAG agarose eluates, and modified residues of histone H4 were detected by Western blotting with acetylation-specific antibodies. C, acetylation of recombinant polynucleosomes by MSL- and NSL-associated MOF. HAT assays were performed with reconstituted polynucleosomes, unlabeled acetyl CoA, and 0.6 μl (lanes 2 and 5), 1.2 μl (lanes 3 and 6), or 2.4 μl (lanes 4 and 7) of the indicated anti-FLAG agarose eluates, and modified histone H4 residues were detected by Western blotting with acetylation-specific antibodies.

FIGURE 4.

HAT activities of free MOF and MSL- and NSL-associated MOF. Anti-FLAG (Fl) agarose eluates from cells stably expressing FLAG-MOF (a mixture of MSL and NSL complexes), FLAG-PHF20 (NSL complex), or free MOF, expressed in and purified from insect cells, were assayed for HAT activity using recombinant polynucleosomes as substrate. Reactions contained the indicated relative molar amounts of free and MSL- and/or NSL-associated MOF. Acetylated histone H4 residues were detected by Western blotting.