Dataset Information

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.

SUBMITTER: Cai Y

PROVIDER: S-EPMC2836030 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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

Cai Yong Y Jin Jingji J Swanson Selene K SK Cole Michael D MD Choi Seung Hyuk SH Florens Laurence L Washburn Michael P MP Conaway Joan W JW Conaway Ronald C RC

The Journal of biological chemistry 20091214 7

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 t ...[more]

PMID: 20018852

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Project description:In this study we focus on the MYST-type acetyltransferase MOF (males-absent-on-the-first, KAT8), which is the effector enzyme of two transcription regulator complexes in Dro-sophila. As part of the male-specific-lethal dosage compensation complex (MSL-DCC, or DCC for short), the enzyme is targeted to transcribed genes on the X chromosome, where it acetylates H4K16 to unfold the chromatin fiber to boost transcription (15-17). In the context of the NSL complex (16) MOF has been reported to acetylate different combina-tions of histone H4 lysines 5, 8, 12 and 16 at promoters in different cells, illustrating the potential of complex subunits to modulate substrate specificity (18-21). Defining the substrate selectivity of KATs in vivo poses several challenges (1). The intrin-sic substrate selectivity of enzymes may be modulated by its molecular environment, be it the complex assembly or the chromatin context. Redundant activities of different en-zymes, indirect effects and the action of lysine deacetylases (KDACs) may occlude the consequences of loss-of-function manipulation. Furthermore, the specificity of the anti-bodies that are commonly used to detect distinct acetylated lysines in histones may be compromised due to low-level off-target effects, interference of modifications next to acetylated lysines, and the tendency of Kac-antibodies to react with relaxed selectivity with oligo-acetylated histone tails (22-25). A more reliable strategy involves the detection of modified histone peptides by mass spectrometry, which unequivocally detects individ-ual modifications and defined combinations of modifications and has a much better dy-namic range than traditional Western blotting (26-30). In a complementary approach one may determine the intrinsic substrate selectivity of defined recombinant enzymes and KAT complexes in biochemical assays (31). We re-cently reconstituted a recombinant MOF-containing core DCC (here termed ‘4-MSL’, since it contains the subunits MOF, MSL1, MSL2 and MSL3 (32)) and now report on a study of its substrate selectivity on recombinant nucleosome arrays. Pilot experiments using traditional Western blotting of diagnostic antibodies suggested that, in addition to the expected H4K16 acetylation, H4K12 was modified. Since this finding disagrees with the prevailing view that the DCC selectively acetylates H4K16, we applied the targeted mass spectrometry described in (26) to our in vitro reactions, focusing on acetylation of the H4 N-terminus. We compared the acetylation reaction of the 4-MSL complex with another MYST enzyme, dTip60 (KAT5), in the context of a trimeric complex, akin to the yeast piccolo NuA4 complex, which is thought to have a more relaxed lysine selectivity on the H4 N-terminus (33-35). We found that during short reaction times, the 4-MSL complex acetylated H4K16 with excellent selectivity, while the dTip60piccolo complex preferentially modified H4K12. To our surprise, we also observed that during longer incubations the 4-MSL complex progres-sively acetylated K12, K8 and K5, leading to a complex mixture of oligo-acetylated H4. Mathematical modeling suggests that MOF recognizes and acetylates H4K16 with high selectivity, but remains bound to the substrate and continues to acetylate more N-terminal H4 lysines in a processive manner. Because the MSL-DCC in vivo contains long, non-coding roX RNA (15,36-38), we ex-plored whether RNA affected the specificity of the reaction. We observed that the addi-tion of RNA to the HAT reaction improves the specificity of the reaction and suppresses the generation of oligo-acetylated forms, possibly by reducing the dwell time of the en-zyme on the nucleosome substrate. The comparison of MOF and dTip60 activities in the context of recombinant HAT com-plexes revealed significant differences in activity and specificity. Our study describes a powerful experimental approach and conceptual framework for the analysis of physiolog-ically relevant components that modulate intrinsic activities.

Dataset Information

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

Publications

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

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