Subunit Composition and Substrate Specificity of a MOF-containing Histone Acetyltransferase Distinct from the Male-specific Lethal (MSL) Complex
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. 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. IntroductionIn eukaryotic cells, chromosomal DNA is packaged with histones and other proteins into chromatin. Alterations in chromatin structure affect the accessibility of chromosomal DNA to enzymes involved in transcription, replication, and repair. Changes in chromatin structure are regulated in at least three different ways: by ATP-dependent remodeling of nucleosomes, by the incorporation of variants of histones H2A and H3 into nucleosomes, and by post-translational modifications of histones (1Clapier C.R. Cairns B.R. Annu. Rev. Biochem. 2009; 78: 273-304Crossref PubMed Scopus (1522) Google Scholar, 2Jin J. Cai Y. Li B. Conaway R.C. Workman J.L. Conaway J.W. Kusch T. Trends Biochem. Sci. 2005; 30: 680-687Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 3Lee K.K. Workman J.L. Nat. Rev. Mol. Cell Biol. 2007; 8: 284-295Crossref PubMed Scopus (770) Google Scholar, 4Rea S. Xouri G. Akhtar A. Oncogene. 2007; 26: 5385-5394Crossref PubMed Scopus (101) Google Scholar, 5Berger S.L. Nature. 2007; 447: 407-412Crossref PubMed Scopus (2126) Google Scholar).Post-translational modifications of histones include acetylation, methylation, phosphorylation, ubiquitination, sumoylation, and ADP-ribosylation (4Rea S. Xouri G. Akhtar A. Oncogene. 2007; 26: 5385-5394Crossref PubMed Scopus (101) Google Scholar, 5Berger S.L. Nature. 2007; 447: 407-412Crossref PubMed Scopus (2126) Google Scholar, 6Bhaumik S.R. Smith E. Shilatifard A. Nat. Struct. Mol. Biol. 2007; 14: 1008-1016Crossref PubMed Scopus (518) Google Scholar). Reversible histone acetylation, controlled by histone acetyltransferases (HATs) 4The abbreviations used are: HAThistone acetyltransferaseHCFhost cell factorMLLmixed lineage in leukemiaMOFmales absent on the firstMSLmale-specific lethalMYSTMoz-Ybf2/Sas3-Sas2-Tip60NSAFnormalized spectral abundance factorNSLnonspecific lethalOGTO-linked N-acetylglucosamine transferaseCBPCREB-binding proteinCREBcAMP-response element-binding proteinHAhemagglutinin. and histone deacetylases, plays an important role in regulation of chromatin structure and function (3Lee K.K. Workman J.L. Nat. Rev. Mol. Cell Biol. 2007; 8: 284-295Crossref PubMed Scopus (770) Google Scholar, 7Kouzarides T. Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (7924) Google Scholar). Based on the nature of their catalytic domains, HATs can be grouped into two distinct families: the GCN5-related N-acetyltransferase (GNAT) family, which includes GCN5 and p300/CBP-associating factor (8Dyda F. Klein D.C. Hickman A.B. Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 81-103Crossref PubMed Scopus (370) Google Scholar), and the Moz-Ybf2/Sas3-Sas2-Tip60 (MYST) family, which is characterized by a highly conserved MYST domain composed of an acetyl-CoA binding motif and a zinc finger (9Pillus L. Curr. Opin. Cell Biol. 2008; 20: 326-333Crossref PubMed Scopus (23) Google Scholar, 10Avvakumov N. Côté J. Oncogene. 2007; 26: 5395-5407Crossref PubMed Scopus (228) Google Scholar). Some MYST family members also have additional structural features such as chromodomains (MOF, Esa1, and Tip60), plant homeodomain-linked zinc fingers (Moz and MORF), and other domains that bind specifically to modified histones or participate in other protein-protein interactions (10Avvakumov N. Côté J. Oncogene. 2007; 26: 5395-5407Crossref PubMed Scopus (228) Google Scholar).Human MOF is an ortholog of the Drosophila MOF HAT. MOF is one of the key components of the dosage compensation or male-specific lethal (MSL) complex. The Drosophila MSL complex is composed of at least five proteins (MSL1, MSL2, MSL3, MLE, and MOF) and two non-coding RNAs (roX1 and roX2). Human cells express an evolutionarily conserved MSL complex that is composed of MOF and at least three additional subunits, including orthologs of MSL1, MSL2, and MSL3. The MOF HAT is believed to be responsible for the majority of histone H4 acetylation at lysine 16 in both Drosophila and human cells (4Rea S. Xouri G. Akhtar A. Oncogene. 2007; 26: 5385-5394Crossref PubMed Scopus (101) Google Scholar, 11Smith E.R. Pannuti A. Gu W. Steurnagel A. Cook R.G. Allis C.D. Lucchesi J.C. Mol. Cell. Biol. 2000; 20: 312-318Crossref PubMed Scopus (255) Google Scholar, 12Smith E.R. Cayrou C. Huang R. Lane W.S. Côté J. Lucchesi J.C. Mol. Cell. Biol. 2005; 25: 9175-9188Crossref PubMed Scopus (243) Google Scholar, 13Taipale M. Rea S. Richter K. Vilar A. Lichter P. Imhof A. Akhtar A. Mol. Cell. Biol. 2005; 25: 6798-6810Crossref PubMed Scopus (237) Google Scholar).Results of recent studies suggest the existence of additional MOF-containing HAT complexes. Roeder and co-workers (14Dou Y. Milne T.A. Tackett A.J. Smith E.R. Fukuda A. Wysocka J. Allis C.D. Chait B.T. Hess J.L. Roeder R.G. Cell. 2005; 121: 873-885Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar) reported that in addition to the MSL HAT complex, human MOF is stably associated in cells with the mixed lineage in leukemia 1 (MLL1) histone methyltransferase in a multiprotein complex that catalyzes both histone acetylation and methylation and includes the WD repeat protein WDR5 and several other proteins found in mammalian COMPASS-like histone methyltransferases (14Dou Y. Milne T.A. Tackett A.J. Smith E.R. Fukuda A. Wysocka J. Allis C.D. Chait B.T. Hess J.L. Roeder R.G. Cell. 2005; 121: 873-885Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). Also present in their MOF- and MLL1-containing preparations were several TATA box-binding protein-associated factor (TAF) proteins, RING2 and other proteins thought to function in the E2F6 repressive complex, and the forkhead domain-containing MCRS1 protein, which we previously identified as a subunit of the human INO80 chromatin-remodeling complex (15Jin J. Cai Y. Yao T. Gottschalk A.J. Florens L. Swanson S.K. Gutiérrez J.L. Coleman M.K. Workman J.L. Mushegian A. Washburn M.P. Conaway R.C. Conaway J.W. J. Biol. Chem. 2005; 280: 41207-41212Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar).In an independent line of investigation, Akhtar and co-workers (16Mendjan 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. Akhtar A. Mol. Cell. 2006; 21: 811-823Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar) identified a collection of MOF-associated proteins distinct from the subunits of the MSL complex; these proteins included WDR5, the MSL1-like protein NSL1 (KIAA1267, also known as MSLv1 (12Smith E.R. Cayrou C. Huang R. Lane W.S. Côté J. Lucchesi J.C. Mol. Cell. Biol. 2005; 25: 9175-9188Crossref PubMed Scopus (243) Google Scholar, 17Li X. Wu L. Corsa C.A. Kunkel S. Dou Y. Mol. Cell. 2009; 36: 290-301Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar)), plant homeodomain-linked finger-containing proteins PHF20 and PHF20L, O-linked N-acetylglucosamine transferase, isoform 1 (OGT1), host cell factor 1 (HCF1), the human INO80 complex subunit MCRS1, and previously uncharacterized proteins FLJ20436 and FLJ10081, which they designated NSL2 and NSL3, respectively. Although they did not determine whether these MOF-associated proteins were present in one or several discrete protein complexes, they proposed that some or all of them were components of a MOF-containing complex they named the nonspecific lethal (NSL) complex. Importantly, Akhtar and co-workers (16Mendjan 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. Akhtar A. Mol. Cell. 2006; 21: 811-823Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar) did not detect the MLL1 methyltransferase as a MOF-associated protein in their study.In an effort to resolve the discrepancy between these previous studies and to explore further the potential contribution of the human INO80 complex subunit MCRS1 to MOF function, we have carried out a systematic proteomic and biochemical analysis of human MOF-containing complexes. As we describe below, our findings are most consistent with those of Akhtar and co-workers (16Mendjan 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. Akhtar A. Mol. Cell. 2006; 21: 811-823Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar) and establish that MOF is present in at least two discrete multiprotein complexes, the MSL complex and a second complex, which we refer to as the NSL complex in keeping with their nomenclature. In addition, by comparing the substrate specificities of the MSL and NSL complexes, we obtain evidence that MOF HAT activity is differentially regulated by assembly into the MSL complex, where it acetylates nucleosomal histone H4 on lysine 16, and the NSL complex, where it also acetylates nucleosomal histone H4 on lysines 5 and 8.DISCUSSIONIn this report, we have exploited a MudPIT-based proteomics approach to define the subunit composition of the NSL complex, a MOF-containing HAT complex distinct from the well characterized MSL complex. In addition, we present evidence that the activity and substrate specificity of the MOF HAT is differentially regulated by its assembly into the NSL or MSL complexes. We observe that in contrast to the MSL-associated MOF, which acetylates almost exclusively nucleosomal histone H4 on lysine 16, NSL-associated MOF is capable of catalyzing substantial acetylation of nucleosomal histone H4 on lysines 5, 8, and 16. In this respect, the NSL HAT complex resembles the NuA4 HAT complex, which also specifically acetylates multiple lysines in the H4 N-terminal tail (32Berndsen C.E. Selleck W. McBryant S.J. Hansen J.C. Tan S. Denu J.M. Biochemistry. 2007; 46: 2091-2099Crossref PubMed Scopus (31) Google Scholar, 33Berndsen C.E. Denu J.M. Curr. Opin. Struct. Biol. 2008; 18: 682-689Crossref PubMed Scopus (158) Google Scholar).Prior investigations of the mechanism underlying regulation of MOF HAT activity by subunits of the MSL complex have revealed that association of Drosophila MOF with an MSL1-MSL3 heterodimer leads to strong activation of MOF HAT activity and narrowing of its substrate specificity to just lysine 16 of nucleosomal histone H4 (31Morales V. Straub T. Neumann M.F. Mengus G. Akhtar A. Becker P.B. EMBO J. 2004; 23: 2258-2268Crossref PubMed Scopus (95) Google Scholar). Although it is presently not known how subunits of the NSL complex regulate MOF HAT activity, it was shown previously that the NSL1 protein can bind directly to MOF (16Mendjan 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. Akhtar A. Mol. Cell. 2006; 21: 811-823Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar) and, while our manuscript was in preparation, Dou and co-workers (17Li X. Wu L. Corsa C.A. Kunkel S. Dou Y. Mol. Cell. 2009; 36: 290-301Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) reported that binding of NSL1 to MOF enhances MOF acetylation of histone H4 on lysine 16 and of the DNA binding transcription factor p53. Our definition of an apparently complete set of NSL complex subunits should enable a more thorough analysis of the roles of individual NSL complex subunits in allosteric regulation of MOF HAT activity.It is noteworthy that the NSL complex shares subunits with other chromatin-regulating complexes. The MCRS1 protein is an integral component of both the NSL complex and the INO80 chromatin-remodeling complex. The WDR5 protein is a subunit not only of the NSL complex but also of the MLL/SET1-containing histone H3K4 methyltransferase complexes (34Cho Y.W. Hong T. Hong S. Guo H. Yu H. Kim D. Guszczynski T. Dressler G.R. Copeland T.D. Kalkum M. Ge K. J. Biol. Chem. 2007; 282: 20395-20406Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar) and of the ATAC (ADA2-containing) HAT complex, which includes as catalytic subunits both the GCN5/KAT2 and the ATAC2/KAT14 HATs (35Suganuma T. Gutiérrez J.L. Li B. Florens L. Swanson S.K. Washburn M.P. Abmayr S.M. Workman J.L. Nat. Struct. Mol. Biol. 2008; 15: 364-372Crossref PubMed Scopus (137) Google Scholar, 36Wang Y.L. Faiola F. Xu M. Pan S. Martinez E. J. Biol. Chem. 2008; 283: 33808-33815Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The degree to which the presence of these shared subunits argues for functional links between the NSL, INO80, ATAC, and MLL/SET1-containing histone H3K4 methyltransferase complexes remains to be determined. It has been proposed that WDR5 might serve as a physical link between different chromatin regulatory complexes (14Dou Y. Milne T.A. Tackett A.J. Smith E.R. Fukuda A. Wysocka J. Allis C.D. Chait B.T. Hess J.L. Roeder R.G. Cell. 2005; 121: 873-885Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar, 17Li X. Wu L. Corsa C.A. Kunkel S. Dou Y. Mol. Cell. 2009; 36: 290-301Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar); however, others argue that WDR5 most likely functions as a platform on which different chromatin regulatory complexes assemble independently (27Trievel R.C. Shilatifard A. Nat. Struct. Mol. Biol. 2009; 16: PubMed Scopus Google Scholar). with this we evidence for or assembly of the NSL complex with the INO80 or the MLL/SET1-containing histone H3K4 methyltransferase however, we out the that the NSL complex might with these other chromatin regulatory complexes or to be the that we have used in their it is that the NSL subunit has been shown to be by the Drosophila and evidence that activity to regulate M. T. Sci. 2009; PubMed Scopus Google Scholar, K. J. 2009; PubMed Scopus Google Scholar). In the it be of to the of between the NSL and complexes in mammalian IntroductionIn eukaryotic cells, chromosomal DNA is packaged with histones and other proteins into chromatin. Alterations in chromatin structure affect the accessibility of chromosomal DNA to enzymes involved in transcription, replication, and repair. Changes in chromatin structure are regulated in at least three different ways: by ATP-dependent remodeling of nucleosomes, by the incorporation of variants of histones H2A and H3 into nucleosomes, and by post-translational modifications of histones (1Clapier C.R. Cairns B.R. Annu. Rev. Biochem. 2009; 78: 273-304Crossref PubMed Scopus (1522) Google Scholar, 2Jin J. Cai Y. Li B. Conaway R.C. Workman J.L. Conaway J.W. Kusch T. Trends Biochem. Sci. 2005; 30: 680-687Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 3Lee K.K. Workman J.L. Nat. Rev. Mol. Cell Biol. 2007; 8: 284-295Crossref PubMed Scopus (770) Google Scholar, 4Rea S. Xouri G. Akhtar A. Oncogene. 2007; 26: 5385-5394Crossref PubMed Scopus (101) Google Scholar, 5Berger S.L. Nature. 2007; 447: 407-412Crossref PubMed Scopus (2126) Google Scholar).Post-translational modifications of histones include acetylation, methylation, phosphorylation, ubiquitination, sumoylation, and ADP-ribosylation (4Rea S. Xouri G. Akhtar A. Oncogene. 2007; 26: 5385-5394Crossref PubMed Scopus (101) Google Scholar, 5Berger S.L. Nature. 2007; 447: 407-412Crossref PubMed Scopus (2126) Google Scholar, 6Bhaumik S.R. Smith E. Shilatifard A. Nat. Struct. Mol. Biol. 2007; 14: 1008-1016Crossref PubMed Scopus (518) Google Scholar). Reversible histone acetylation, controlled by histone acetyltransferases (HATs) 4The abbreviations used are: HAThistone acetyltransferaseHCFhost cell factorMLLmixed lineage in leukemiaMOFmales absent on the firstMSLmale-specific lethalMYSTMoz-Ybf2/Sas3-Sas2-Tip60NSAFnormalized spectral abundance factorNSLnonspecific lethalOGTO-linked N-acetylglucosamine transferaseCBPCREB-binding proteinCREBcAMP-response element-binding proteinHAhemagglutinin. and histone deacetylases, plays an important role in regulation of chromatin structure and function (3Lee K.K. Workman J.L. Nat. Rev. Mol. Cell Biol. 2007; 8: 284-295Crossref PubMed Scopus (770) Google Scholar, 7Kouzarides T. Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (7924) Google Scholar). Based on the nature of their catalytic domains, HATs can be grouped into two distinct families: the GCN5-related N-acetyltransferase (GNAT) family, which includes GCN5 and p300/CBP-associating factor (8Dyda F. Klein D.C. Hickman A.B. Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 81-103Crossref PubMed Scopus (370) Google Scholar), and the Moz-Ybf2/Sas3-Sas2-Tip60 (MYST) family, which is characterized by a highly conserved MYST domain composed of an acetyl-CoA binding motif and a zinc finger (9Pillus L. Curr. Opin. Cell Biol. 2008; 20: 326-333Crossref PubMed Scopus (23) Google Scholar, 10Avvakumov N. Côté J. Oncogene. 2007; 26: 5395-5407Crossref PubMed Scopus (228) Google Scholar). Some MYST family members also have additional structural features such as chromodomains (MOF, Esa1, and Tip60), plant homeodomain-linked zinc fingers (Moz and MORF), and other domains that bind specifically to modified histones or participate in other protein-protein interactions (10Avvakumov N. Côté J. Oncogene. 2007; 26: 5395-5407Crossref PubMed Scopus (228) Google Scholar).Human MOF is an ortholog of the Drosophila MOF HAT. MOF is one of the key components of the dosage compensation or male-specific lethal (MSL) complex. The Drosophila MSL complex is composed of at least five proteins (MSL1, MSL2, MSL3, MLE, and MOF) and two non-coding RNAs (roX1 and roX2). Human cells express an evolutionarily conserved MSL complex that is composed of MOF and at least three additional subunits, including orthologs of MSL1, MSL2, and MSL3. The MOF HAT is believed to be responsible for the majority of histone H4 acetylation at lysine 16 in both Drosophila and human cells (4Rea S. Xouri G. Akhtar A. Oncogene. 2007; 26: 5385-5394Crossref PubMed Scopus (101) Google Scholar, 11Smith E.R. Pannuti A. Gu W. Steurnagel A. Cook R.G. Allis C.D. Lucchesi J.C. Mol. Cell. Biol. 2000; 20: 312-318Crossref PubMed Scopus (255) Google Scholar, 12Smith E.R. Cayrou C. Huang R. Lane W.S. Côté J. Lucchesi J.C. Mol. Cell. Biol. 2005; 25: 9175-9188Crossref PubMed Scopus (243) Google Scholar, 13Taipale M. Rea S. Richter K. Vilar A. Lichter P. Imhof A. Akhtar A. Mol. Cell. Biol. 2005; 25: 6798-6810Crossref PubMed Scopus (237) Google Scholar).Results of recent studies suggest the existence of additional MOF-containing HAT complexes. Roeder and co-workers (14Dou Y. Milne T.A. Tackett A.J. Smith E.R. Fukuda A. Wysocka J. Allis C.D. Chait B.T. Hess J.L. Roeder R.G. Cell. 2005; 121: 873-885Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar) reported that in addition to the MSL HAT complex, human MOF is stably associated in cells with the mixed lineage in leukemia 1 (MLL1) histone methyltransferase in a multiprotein complex that catalyzes both histone acetylation and methylation and includes the WD repeat protein WDR5 and several other proteins found in mammalian COMPASS-like histone methyltransferases (14Dou Y. Milne T.A. Tackett A.J. Smith E.R. Fukuda A. Wysocka J. Allis C.D. Chait B.T. Hess J.L. Roeder R.G. Cell. 2005; 121: 873-885Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). Also present in their MOF- and MLL1-containing preparations were several TATA box-binding protein-associated factor (TAF) proteins, RING2 and other proteins thought to function in the E2F6 repressive complex, and the forkhead domain-containing MCRS1 protein, which we previously identified as a subunit of the human INO80 chromatin-remodeling complex (15Jin J. Cai Y. Yao T. Gottschalk A.J. Florens L. Swanson S.K. Gutiérrez J.L. Coleman M.K. Workman J.L. Mushegian A. Washburn M.P. Conaway R.C. Conaway J.W. J. Biol. Chem. 2005; 280: 41207-41212Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar).In an independent line of investigation, Akhtar and co-workers (16Mendjan 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. Akhtar A. Mol. Cell. 2006; 21: 811-823Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar) identified a collection of MOF-associated proteins distinct from the subunits of the MSL complex; these proteins included WDR5, the MSL1-like protein NSL1 (KIAA1267, also known as MSLv1 (12Smith E.R. Cayrou C. Huang R. Lane W.S. Côté J. Lucchesi J.C. Mol. Cell. Biol. 2005; 25: 9175-9188Crossref PubMed Scopus (243) Google Scholar, 17Li X. Wu L. Corsa C.A. Kunkel S. Dou Y. Mol. Cell. 2009; 36: 290-301Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar)), plant homeodomain-linked finger-containing proteins PHF20 and PHF20L, O-linked N-acetylglucosamine transferase, isoform 1 (OGT1), host cell factor 1 (HCF1), the human INO80 complex subunit MCRS1, and previously uncharacterized proteins FLJ20436 and FLJ10081, which they designated NSL2 and NSL3, respectively. Although they did not determine whether these MOF-associated proteins were present in one or several discrete protein complexes, they proposed that some or all of them were components of a MOF-containing complex they named the nonspecific lethal (NSL) complex. Importantly, Akhtar and co-workers (16Mendjan 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. Akhtar A. Mol. Cell. 2006; 21: 811-823Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar) did not detect the MLL1 methyltransferase as a MOF-associated protein in their study.In an effort to resolve the discrepancy between these previous studies and to explore further the potential contribution of the human INO80 complex subunit MCRS1 to MOF function, we have carried out a systematic proteomic and biochemical analysis of human MOF-containing complexes. As we describe below, our findings are most consistent with those of Akhtar and co-workers (16Mendjan 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. Akhtar A. Mol. Cell. 2006; 21: 811-823Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar) and establish that MOF is present in at least two discrete multiprotein complexes, the MSL complex and a second complex, which we refer to as the NSL complex in keeping with their nomenclature. In addition, by comparing the substrate specificities of the MSL and NSL complexes, we obtain evidence that MOF HAT activity is differentially regulated by assembly into the MSL complex, where it acetylates nucleosomal histone H4 on lysine 16, and the NSL complex, where it also acetylates nucleosomal histone H4 on lysines 5 and 8.