The Snf2 Homolog Fun30 Acts as a Homodimeric ATP-dependent Chromatin-remodeling Enzyme

The Saccharomyces cerevisiae Fun30 (Function unknown now 30) protein shares homology with an extended family of Snf2-related ATPases. Here we report the purification of Fun30 principally as a homodimer with a molecular mass of about 250 kDa. Biochemical characterization of this complex reveals that it has ATPase activity stimulated by both DNA and chromatin. Consistent with this, it also binds to both DNA and chromatin. The Fun30 complex also exhibits activity in ATP-dependent chromatin remodeling assays. Interestingly, its activity in histone dimer exchange is high relative to the ability to reposition nucleosomes. Fun30 also possesses a weakly conserved CUE motif suggesting that it may interact specifically with ubiquitinylated proteins. However, in vitro Fun30 was found to have no specificity in its interaction with ubiquitinylated histones. The Saccharomyces cerevisiae Fun30 (Function unknown now 30) protein shares homology with an extended family of Snf2-related ATPases. Here we report the purification of Fun30 principally as a homodimer with a molecular mass of about 250 kDa. Biochemical characterization of this complex reveals that it has ATPase activity stimulated by both DNA and chromatin. Consistent with this, it also binds to both DNA and chromatin. The Fun30 complex also exhibits activity in ATP-dependent chromatin remodeling assays. Interestingly, its activity in histone dimer exchange is high relative to the ability to reposition nucleosomes. Fun30 also possesses a weakly conserved CUE motif suggesting that it may interact specifically with ubiquitinylated proteins. However, in vitro Fun30 was found to have no specificity in its interaction with ubiquitinylated histones. IntroductionThe process of eukaryotic gene regulation is intimately associated with the manipulation of chromatin structure. This is accomplished via a range of strategies that include protein complexes that remodel the structure of chromatin using the energy of ATP hydrolysis (for review, see Ref. 1.Becker P.B. Hörz W. Annu. Rev. Biochem. 2002; 71: 247-273Crossref PubMed Scopus (619) Google Scholar) or covalently modify the core histones by acetylation, methylation, phosphorylation, ubiquitination, sumoylation, and ADP-ribosylation (for review, see Ref. 2.Kouzarides T. Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (7926) Google Scholar). ATP-dependent chromatin remodeling enzymes share a catalytic subunit with homology to the yeast Snf2 protein. The helicase-related motifs within this region are thought to function as a DNA translocating motor (3.Cairns B.R. Nat. Struct. Mol. Biol. 2007; 14: 989-996Crossref PubMed Scopus (194) Google Scholar). Based on the homology within this region, Snf2-related proteins can be assigned to 24 subfamilies, many of which have been broadly conserved during the evolution of eukaryotes (4.Flaus A. Martin D.M. Barton G.J. Owen-Hughes T. Nucleic Acids Res. 2006; 34: 2887-2905Crossref PubMed Scopus (500) Google Scholar). Snf2 family proteins have a diverse range of functions with many, but not all, acting to alter chromatin structure. These ATP-dependent chromatin remodeling enzymes can generate a spectrum of different types of transition in chromatin structure ranging from nucleosome eviction or sliding to the exchange of histone dimers (3.Cairns B.R. Nat. Struct. Mol. Biol. 2007; 14: 989-996Crossref PubMed Scopus (194) Google Scholar).In this report, we investigate the Saccharomyces cerevisiae Snf2 family protein, Fun30 (Function unknown now 30). Fun30 was originally identified as a result of genome sequencing (5.Clark M.W. Zhong W.W. Keng T. Storms R.K. Barton A. Kaback D.B. Bussey H. Yeast. 1992; 8: 133-145Crossref PubMed Scopus (23) Google Scholar) and shares most sequence homology with the Swr1 and Ino80 chromatin remodeling enzymes (4.Flaus A. Martin D.M. Barton G.J. Owen-Hughes T. Nucleic Acids Res. 2006; 34: 2887-2905Crossref PubMed Scopus (500) Google Scholar), both of which are implicated in histone dimer exchange (6.Mizuguchi G. Shen X. Landry J. Wu W.H. Sen S. Wu C. Science. 2004; 303: 343-348Crossref PubMed Scopus (981) Google Scholar, 7.Papamichos-Chronakis M. Krebs J.E. Peterson C.L. Genes Dev. 2006; 20: 2437-2449Crossref PubMed Scopus (160) Google Scholar). Previous studies have shown that yeast fun30 deletions are viable, but temperature sensitive (5.Clark M.W. Zhong W.W. Keng T. Storms R.K. Barton A. Kaback D.B. Bussey H. Yeast. 1992; 8: 133-145Crossref PubMed Scopus (23) Google Scholar), and are resistant to ultraviolet (UV) radiation (8.Barton A.B. Kaback D.B. J. Bacteriol. 1994; 176: 1872-1880Crossref PubMed Google Scholar). The overexpression of Fun30 has been shown to affect chromosome stability, integrity, and segregation (9.Ouspenski I.I. Elledge S.J. Brinkley B.R. Nucleic Acids Res. 1999; 27: 3001-3008Crossref PubMed Scopus (98) Google Scholar). Fun30 has also been shown to be a potential cyclin-dependent kinase (Cdk1)/Cdc28 substrate (10.Ubersax J.A. Woodbury E.L. Quang P.N. Paraz M. Blethrow J.D. Shah K. Shokat K.M. Morgan D.O. Nature. 2003; 425: 859-864Crossref PubMed Scopus (739) Google Scholar). More recently, Fun30 has been found to play a role in gene silencing (11.Neves-Costa A. Will W.R. Vetter A.T. Miller J.R. Varga-Weisz P. Plos One. 2009; 4: e8111Crossref PubMed Scopus (50) Google Scholar).Fun30 is conserved through evolution and its mouse homologue, Etl1 (Enhancer Trap Locus 1), has been identified as being expressed during early development (12.Soininen R. Schoor M. Henseling U. Tepe C. Kisters-Woike B. Rossant J. Gossler A. Mech. Dev. 1992; 39: 111-123Crossref PubMed Scopus (40) Google Scholar). Etl1 is widely expressed but non-essential, although deletion is associated with developmental defects such as skeletal dysplasia, growth retardation, and impaired fertility (13.Schoor M. Schuster-Gossler K. Gossler A. Dev. Dyn. 1993; 197: 227-237Crossref PubMed Scopus (18) Google Scholar, 14.Schoor M. Schuster-Gossler K. Roopenian D. Gossler A. Mech. Dev. 1999; 85: 73-83Crossref PubMed Scopus (32) Google Scholar). The human homolog, SMARCAD1 (previously known as human helicase 1 (hHel1)), has been mapped to the chromosome 4q22–q23 region, which is rich in breakpoints and deletion mutants of genes involved in several human diseases, notably soft tissue leiomyosarcoma, hepatocellular carcinoma, and hematologic malignancies (15.Adra C.N. Donato J.L. Badovinac R. Syed F. Kheraj R. Cai H. Moran C. Kolker M.T. Turner H. Weremowicz S. Shirakawa T. Morton C.C. Schnipper L.E. Drews R. Genomics. 2000; 69: 162-173Crossref PubMed Scopus (34) Google Scholar). It has been recently reported that the binding sites of endogenous SMARCAD1/KIAA1122 are frequently found in the vicinity of transcriptional start sites (16.Okazaki N. Ikeda S. Ohara R. Shimada K. Yanagawa T. Nagase T. Ohara O. Koga H. J. Mol. Biol. 2008; 382: 257-265Crossref PubMed Scopus (22) Google Scholar).To gain insight into the function of Fun30, we have purified it from tagged yeast strains. We obtain Fun30 as a homodimeric complex. This complex displays activity in a range of chromatin remodeling assays. Interestingly, the Fun30 complex displays increased activity in histone dimer exchange assays in comparison to nucleosome sliding. These results suggest that Fun30 function may involve the manipulation of the histone content of nucleosomes.DISCUSSIONThe S. cerevisiae Fun30 protein has been purified and found to exist predominantly as a homodimer. Like other Snf2 family proteins, it is capable of binding nucleosomes, hydrolyzing ATP, and disrupting nucleosomes in an ATP-dependent reaction. Fun30 was found to be especially proficient in catalyzing the exchange of histone dimers between nucleosomes in comparison, for example, to nucleosome sliding. This is consistent with the fact that based on sequence homology Fun30 is most closely related to the Swr1 and Ino80 proteins (4.Flaus A. Martin D.M. Barton G.J. Owen-Hughes T. Nucleic Acids Res. 2006; 34: 2887-2905Crossref PubMed Scopus (500) Google Scholar), which have been reported to have activity in histone exchange (6.Mizuguchi G. Shen X. Landry J. Wu W.H. Sen S. Wu C. Science. 2004; 303: 343-348Crossref PubMed Scopus (981) Google Scholar, 7.Papamichos-Chronakis M. Krebs J.E. Peterson C.L. Genes Dev. 2006; 20: 2437-2449Crossref PubMed Scopus (160) Google Scholar). The observation that Fun30 is relatively inefficient in repositioning nucleosomes supports previous work that suggests the mechanisms for dimer exchange and nucleosome sliding are distinct (32.Ferreira H. Somers J. Webster R. Flaus A. Owen-Hughes T. Mol. Cell. Biol. 2007; 27: 4037-4048Crossref PubMed Scopus (102) Google Scholar).The Swr1 complex exhibits specificity in histone exchange directing the incorporation of the histone variant Htz1 (6.Mizuguchi G. Shen X. Landry J. Wu W.H. Sen S. Wu C. Science. 2004; 303: 343-348Crossref PubMed Scopus (981) Google Scholar). It is possible that Fun30 also has specificity in directing exchange of specific histone subtypes; however, in our preliminary studies we obtained no evidence for this. Given that Fun30 contains a weak CUE motif potentially capable of interacting with ubiquitin, one hypothesis we investigated was that Fun30 directs the incorporation or removal of ubiquitinylated histones. However, we could obtain no evidence for specific binding of Fun30 to ubiquitinylated histones (Fig. 7), or the ability to exchange ubiquitinylated histones (data not shown). A caveat to this experiment is that HeLa cells were used as a source of chromatin and the possibility remains that there is specificity for a feature of yeast chromatin we may have missed. It is also worth noting that Fun30 binds DNA better than nucleosome core particles (Fig. 4C) and that this could potentially target the action of Fun30 to accessible regions of the genome.The observation that Fun30 elutes from gel filtration columns in a volume corresponding to a mass of 250 kDa could be interpreted as indicating the presence of a stable dimer. However, as elution volumes were observed to increase progressively when lower concentrations of Fun30 were loaded (data not shown) and TAP- and His-tagged Fun30 preparations interacted with each other in vitro, we favor the existence of a rapid equilibrium between monomeric and dimeric forms in solution. For some other remodeling enzymes, it is clear that the Snf2-related subunit is present as one copy (33.Smith C.L. Horowitz-Scherer R. Flanagan J.F. Woodcock C.L. Peterson C.L. Nat. Struct. Biol. 2003; 10: 141-145Crossref PubMed Scopus (140) Google Scholar). However, in other cases the involvement of a pair of catalytic subunits is an emerging theme (29.Strohner R. Wachsmuth M. Dachauer K. Mazurkiewicz J. Hochstatter J. Rippe K. Längst G. Nat. Struct. Mol. Biol. 2005; 12: 683-690Crossref PubMed Scopus (82) Google Scholar, 34.Racki L.R. Narlikar G.J. Curr. Opin. Genet. Dev. 2008; 18: 137-144Crossref PubMed Scopus (72) Google Scholar). In the case of Fun30, further investigation will be required to confirm that two molecules of Fun30 interact with a single nucleosome, although this is our favored interpretation of the cooperativity observed.The previously reported phenotypes of increased resistance to DNA damage induced by UV (8.Barton A.B. Kaback D.B. J. Bacteriol. 1994; 176: 1872-1880Crossref PubMed Google Scholar) together with our own observation that the deletion of Fun30 results in resistance to UV, ionizing radiation, and resistance to 6-azauracil (data not shown) might indicate an involvement in DNA repair. Interestingly, genome-wide surveys indicate physical and genetic interactions between Tel1 and Rad3, respectively (35.Gavin A.C. Bösche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Höfert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Nature. 2002; 415: 141-147Crossref PubMed Scopus (3974) Google Scholar, 36.Collins S.R. Miller K.M. Maas N.L. Roguev A. Fillingham J. Chu C.S. Schuldiner M. Gebbia M. Recht J. Shales M. Ding H. Xu H. Han J. Ingvarsdottir K. Cheng B. Andrews B. Boone C. Berger S.L. Hieter P. Zhang Z. Brown G.W. Ingles C.J. Emili A. Allis C.D. Toczyski D.P. Weissman J.S. Greenblatt J.F. Krogan N.J. Nature. 2007; 446: 806-810Crossref PubMed Scopus (718) Google Scholar). Mutations to the histone variant Htz1 also have repair phenotypes, and Fun30 exhibits synthetic lethality with Htz1 and several components of the Swr1 complex, which directs its incorporation (Vps71, Vps72, and Arp6) (37.Krogan N.J. Keogh M.C. Datta N. Sawa C. Ryan O.W. Ding H. Haw R.A. Pootoolal J. Tong A. Canadien V. Richards D.P. Wu X. Emili A. Hughes T.R. Buratowski S. Greenblatt J.F. Mol. Cell. 2003; 12: 1565-1576Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). Thus, despite the lack of specificity for H2AZ in vitro, Fun30 functionally interacts with Htz1 in some way. The recent observation that Htz1 is targeted to nucleosomes adjacent to nucleosome-free regions resulted in speculation that the action of Swr1 might be targeted to regions of exposed DNA (38.Hartley P.D. Madhani H.D. Cell. 2009; 137: 445-458Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Nonspecific histone dimer exchange especially with S-phase could potentially contribute to this process. Fun30 has the potential to accelerate this process and based on its in vitro DNA binding properties could be targeted to nucleosome-free regions. Interactions between Fun30 and proteins involved in transcription (Taf13, Rpo21, Rpc40, and Rpc34) (36.Collins S.R. Miller K.M. Maas N.L. Roguev A. Fillingham J. Chu C.S. Schuldiner M. Gebbia M. Recht J. Shales M. Ding H. Xu H. Han J. Ingvarsdottir K. Cheng B. Andrews B. Boone C. Berger S.L. Hieter P. Zhang Z. Brown G.W. Ingles C.J. Emili A. Allis C.D. Toczyski D.P. Weissman J.S. Greenblatt J.F. Krogan N.J. Nature. 2007; 446: 806-810Crossref PubMed Scopus (718) Google Scholar), cell cycle progression (Orc2 and Orc5, (39.Suter B. Tong A. Chang M. Yu L. Brown G.W. Boone C. Rine J. Genetics. 2004; 167: 579-591Crossref PubMed Scopus (93) Google Scholar), Cks1 (36.Collins S.R. Miller K.M. Maas N.L. Roguev A. Fillingham J. Chu C.S. Schuldiner M. Gebbia M. Recht J. Shales M. Ding H. Xu H. Han J. Ingvarsdottir K. Cheng B. Andrews B. Boone C. Berger S.L. Hieter P. Zhang Z. Brown G.W. Ingles C.J. Emili A. Allis C.D. Toczyski D.P. Weissman J.S. Greenblatt J.F. Krogan N.J. Nature. 2007; 446: 806-810Crossref PubMed Scopus (718) Google Scholar), Clb2, and Cdc28 (10.Ubersax J.A. Woodbury E.L. Quang P.N. Paraz M. Blethrow J.D. Shah K. Shokat K.M. Morgan D.O. Nature. 2003; 425: 859-864Crossref PubMed Scopus (739) Google Scholar) have also been identified. Although it is intriguing that Fun30 is linked to the process of DNA replication, repair, and transcription, which all involve histone exchange, further studies are required to reveal the nature of this association. IntroductionThe process of eukaryotic gene regulation is intimately associated with the manipulation of chromatin structure. This is accomplished via a range of strategies that include protein complexes that remodel the structure of chromatin using the energy of ATP hydrolysis (for review, see Ref. 1.Becker P.B. Hörz W. Annu. Rev. Biochem. 2002; 71: 247-273Crossref PubMed Scopus (619) Google Scholar) or covalently modify the core histones by acetylation, methylation, phosphorylation, ubiquitination, sumoylation, and ADP-ribosylation (for review, see Ref. 2.Kouzarides T. Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (7926) Google Scholar). ATP-dependent chromatin remodeling enzymes share a catalytic subunit with homology to the yeast Snf2 protein. The helicase-related motifs within this region are thought to function as a DNA translocating motor (3.Cairns B.R. Nat. Struct. Mol. Biol. 2007; 14: 989-996Crossref PubMed Scopus (194) Google Scholar). Based on the homology within this region, Snf2-related proteins can be assigned to 24 subfamilies, many of which have been broadly conserved during the evolution of eukaryotes (4.Flaus A. Martin D.M. Barton G.J. Owen-Hughes T. Nucleic Acids Res. 2006; 34: 2887-2905Crossref PubMed Scopus (500) Google Scholar). Snf2 family proteins have a diverse range of functions with many, but not all, acting to alter chromatin structure. These ATP-dependent chromatin remodeling enzymes can generate a spectrum of different types of transition in chromatin structure ranging from nucleosome eviction or sliding to the exchange of histone dimers (3.Cairns B.R. Nat. Struct. Mol. Biol. 2007; 14: 989-996Crossref PubMed Scopus (194) Google Scholar).In this report, we investigate the Saccharomyces cerevisiae Snf2 family protein, Fun30 (Function unknown now 30). Fun30 was originally identified as a result of genome sequencing (5.Clark M.W. Zhong W.W. Keng T. Storms R.K. Barton A. Kaback D.B. Bussey H. Yeast. 1992; 8: 133-145Crossref PubMed Scopus (23) Google Scholar) and shares most sequence homology with the Swr1 and Ino80 chromatin remodeling enzymes (4.Flaus A. Martin D.M. Barton G.J. Owen-Hughes T. Nucleic Acids Res. 2006; 34: 2887-2905Crossref PubMed Scopus (500) Google Scholar), both of which are implicated in histone dimer exchange (6.Mizuguchi G. Shen X. Landry J. Wu W.H. Sen S. Wu C. Science. 2004; 303: 343-348Crossref PubMed Scopus (981) Google Scholar, 7.Papamichos-Chronakis M. Krebs J.E. Peterson C.L. Genes Dev. 2006; 20: 2437-2449Crossref PubMed Scopus (160) Google Scholar). Previous studies have shown that yeast fun30 deletions are viable, but temperature sensitive (5.Clark M.W. Zhong W.W. Keng T. Storms R.K. Barton A. Kaback D.B. Bussey H. Yeast. 1992; 8: 133-145Crossref PubMed Scopus (23) Google Scholar), and are resistant to ultraviolet (UV) radiation (8.Barton A.B. Kaback D.B. J. Bacteriol. 1994; 176: 1872-1880Crossref PubMed Google Scholar). The overexpression of Fun30 has been shown to affect chromosome stability, integrity, and segregation (9.Ouspenski I.I. Elledge S.J. Brinkley B.R. Nucleic Acids Res. 1999; 27: 3001-3008Crossref PubMed Scopus (98) Google Scholar). Fun30 has also been shown to be a potential cyclin-dependent kinase (Cdk1)/Cdc28 substrate (10.Ubersax J.A. Woodbury E.L. Quang P.N. Paraz M. Blethrow J.D. Shah K. Shokat K.M. Morgan D.O. Nature. 2003; 425: 859-864Crossref PubMed Scopus (739) Google Scholar). More recently, Fun30 has been found to play a role in gene silencing (11.Neves-Costa A. Will W.R. Vetter A.T. Miller J.R. Varga-Weisz P. Plos One. 2009; 4: e8111Crossref PubMed Scopus (50) Google Scholar).Fun30 is conserved through evolution and its mouse homologue, Etl1 (Enhancer Trap Locus 1), has been identified as being expressed during early development (12.Soininen R. Schoor M. Henseling U. Tepe C. Kisters-Woike B. Rossant J. Gossler A. Mech. Dev. 1992; 39: 111-123Crossref PubMed Scopus (40) Google Scholar). Etl1 is widely expressed but non-essential, although deletion is associated with developmental defects such as skeletal dysplasia, growth retardation, and impaired fertility (13.Schoor M. Schuster-Gossler K. Gossler A. Dev. Dyn. 1993; 197: 227-237Crossref PubMed Scopus (18) Google Scholar, 14.Schoor M. Schuster-Gossler K. Roopenian D. Gossler A. Mech. Dev. 1999; 85: 73-83Crossref PubMed Scopus (32) Google Scholar). The human homolog, SMARCAD1 (previously known as human helicase 1 (hHel1)), has been mapped to the chromosome 4q22–q23 region, which is rich in breakpoints and deletion mutants of genes involved in several human diseases, notably soft tissue leiomyosarcoma, hepatocellular carcinoma, and hematologic malignancies (15.Adra C.N. Donato J.L. Badovinac R. Syed F. Kheraj R. Cai H. Moran C. Kolker M.T. Turner H. Weremowicz S. Shirakawa T. Morton C.C. Schnipper L.E. Drews R. Genomics. 2000; 69: 162-173Crossref PubMed Scopus (34) Google Scholar). It has been recently reported that the binding sites of endogenous SMARCAD1/KIAA1122 are frequently found in the vicinity of transcriptional start sites (16.Okazaki N. Ikeda S. Ohara R. Shimada K. Yanagawa T. Nagase T. Ohara O. Koga H. J. Mol. Biol. 2008; 382: 257-265Crossref PubMed Scopus (22) Google Scholar).To gain insight into the function of Fun30, we have purified it from tagged yeast strains. We obtain Fun30 as a homodimeric complex. This complex displays activity in a range of chromatin remodeling assays. Interestingly, the Fun30 complex displays increased activity in histone dimer exchange assays in comparison to nucleosome sliding. These results suggest that Fun30 function may involve the manipulation of the histone content of nucleosomes.

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