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The FACT Spt16 ''Peptidase'' Domain Is a Histone H3–H4 Binding Module

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The FACT Spt16 ''Peptidase'' Domain Is a Histone H3–H4 Binding Module The FACT Spt16 ‘‘peptidase’’ domain is a histone H3–H4 binding module Tobias Stuwe, Michael Hothorn*, Erwan Lejeune, Vladimir Rybin, Miriam Bortfeld, Klaus Scheffzek, and Andreas G. Ladurner† European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany Edited by Alan R. Fersht, University of Cambridge, Cambridge, United Kingdom, and approved March 27, 2008 (received for review December 28, 2007) The FACT complex is a conserved cofactor for RNA polymerase II scription and replication by removing one H2A–H2B dimer from elongation through nucleosomes. FACT bears histone chaperone nucleosomes, thus relieving the barrier to polymerase progres- activity and contributes to chromatin integrity. However, the sion. After Pol II passage, FACT may restore the proper molecular mechanisms behind FACT function remain elusive. Here chromatin state (14, 20). we report biochemical, structural, and mutational analyses that There is little mechanistic insight into how FACT may be able identify the peptidase homology domain of the Schizosaccharo- to perform its chaperoning functions. In particular, it is unclear myces pombe FACT large subunit Spt16 (Spt16-N) as a binding how it interacts with histones. To start dissecting the structure module for histones H3 and H4. The 2.1-Å crystal structure of and function of the essential Spt16 subunit of FACT, we sought Spt16-N reveals an aminopeptidase P fold whose enzymatic activ- to identify the molecular functions inherent to this Ͼ100-kDa ity has been lost. Instead, the highly conserved fold directly binds multidomain protein. Structural information on FACT exists for histones H3–H4 through a tight interaction with their globular core the HMG-like module Nhp6A from S. cerevisiae (23), which domains, as well as with their N-terminal tails. Mutations within a binds DNA, and for the middle domain of S. cerevisiae Pob3 (24), conserved surface pocket in Spt16-N or posttranslational modifi- which resembles a double PH-like fold and interacts with cation of the histone H4 tail reduce interaction in vitro, whereas the replication protein A. The highly conserved Spt16 consists of globular domains of H3–H4 and the H3 tail bind distinct Spt16-N three domains: an acidic segment at the C terminus (10) that is surfaces. Our analysis suggests that the N-terminal domain of required for binding histones H2A–H2B in vitro (20) and re- Spt16 may add to the known H2A–H2B chaperone activity of FACT sembles the acidic domains of Nap1, nucleolin, and Asf1 (25–27); by including a H3–H4 tail and H3–H4 core binding function medi- two central domains, one of which interacts with Pob3 (21, 24); ated by the N terminus of Spt16. We suggest that these interactions and an N-terminal region of Ϸ450 residues (Spt16-N) showing may aid FACT-mediated nucleosome reorganization events. homology with aminopeptidases (28). We were intrigued by the presence of a peptidase-like domain within this essential and histone chaperone ͉ histone modifications ͉ protein evolution ͉ conserved histone chaperone [supporting information (SI) Fig. site-directed mutagenesis ͉ transcription S1]. We have characterized biochemical functions of Spt16-N by combining structural approaches with quantitative binding stud- ucleosomes create a natural barrier to RNA polymerase II ies and site-directed mutagenesis. N(Pol II) progression. Transcription of histone-wrapped DNA thus requires factors that promote nucleosome remodel- Results ing, such as the histone chaperone FACT (facilitates chromatin A Catalytically Inactive Enzyme Fold Interacts with Histones H3–H4. transcription), which in human cells purifies as a heterodimer of We expressed and crystallized the N-terminal ‘‘peptidase’’ mod- Spt16 and SSRP1 (1, 2). ule of the S. pombe FACT complex Spt16. The structure of the FACT is a highly conserved complex (2–5). In fungi, SSRP1 domain (residues 1–442, Spt16-N) was solved and refined to is largely encoded by POB3, whereas NHP6 encodes the arche- 2.1-Å resolution (Table S1 and Materials and Methods). Our typal HMG-box of SSRP1. The gene for the Spt16 subunit is structure is highly similar to the recently published structure of essential in Saccharomyces cerevisiae and Schizosaccharomyces the related domain from S. cerevisiae (29), revealing the con- pombe, probably reflecting important chromatin-related func- served pita-bread fold (C-terminal lobe; residues 178–442) of tions in transcription, replication, and DNA repair (4, 6–9). aminopeptidases (30), preceded by a smaller domain (N- Genetic screens identified Spt16 as a factor whose mutation terminal lobe; residues 1–174) (Fig. 1A). Structural homology restores the expression of a Ty1 transposon-silenced reporter searches return a bacterial prolidase and creatinase [Protein gene by promoting its cryptic transcription, known as a suppres- Data Bank ID codes 1CHM and 1PV9 (31, 32); DALI (55) sor of Ty1 phenotype, [SptϪ] (4, 7, 10, 11). Several POB3 alleles genes display [SptϪ] phenotypes (12), indicating that the main- tenance of correct chromatin structure involves Pol II cofactors Author contributions: T.S. and M.H. contributed equally to this work; T.S., M.H., E.L., and such as FACT (13). Indeed, FACT acts as a coactivator of A.G.L. designed research; T.S., M.H., and M.B. purified proteins and carried out biochemical assays, T.S. and M.H. crystallized the proteins and collected diffraction data, M.H. phased transcriptional initiation and elongation (14), and many Spt16 and refined the structures, and V.R. conducted ultracentrifugation assay; T.S., M.B., and alleles display genetic interactions with basal transcription fac- A.G.L. contributed new reagents/analytic tools; T.S., M.H., E.L., V.R., K.S., and A.G.L. tors. Furthermore, FACT subunits biochemically interact with analyzed data; and T.S., M.H., E.L., K.S., and A.G.L. wrote the paper. the Pol II elongation complex Paf1 (15), bind the coding region The authors declare no conflict of interest. of transcribed Pol II genes, and are recruited to inducible genes This article is a PNAS Direct Submission. upon activation (16–19). Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, FACT’s biological roles in transcription (and replication) may www.pdb.org (PDB ID codes 3CB5 and 3CB6). stem from its histone chaperone activity (20, 21). Histone *Present address: The Salk Institute, Plant Biology Laboratory, 10010 North Torrey Pines chaperones stimulate reactions involving the transfer of histones Road, La Jolla, CA 92037. (22), thereby mediating chromatin reorganization. At the mo- †To whom correspondence should be addressed. E-mail: [email protected]. lecular level, FACT binds nucleosomes and destabilizes inter- This article contains supporting information online at www.pnas.org/cgi/content/full/ actions between H2A–H2B dimers and (H3–H4)2 tetramers 0712293105/DCSupplemental. (20). Mechanistically this suggests that FACT may help tran- © 2008 by The National Academy of Sciences of the USA 8884–8889 ͉ PNAS ͉ July 1, 2008 ͉ vol. 105 ͉ no. 26 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712293105 Downloaded by guest on October 2, 2021 the conserved Spt16-N domain may bind histones using GST A pull-down assays between Spt16-N and recombinant core his- tones H2A–H2B or H3–H4, as well as native Drosophila octam- ers. Surprisingly, the Spt16-N peptidase module directly interacts with both recombinant and native histones H3–H4 (Fig. 2 A and B). Stable H3–H4 association occurs even under high ionic N N-terminal lobe strength, in contrast to histones H2A–H2B, which do not bind N Spt16-N specifically (Fig. 2A). Isothermal titration calorimetry 90º (ITC) assays confirm binding of recombinant H3–H4 to Spt16-N under physiological salt concentrations (Fig. S3A). To test whether the interacting proteins form a stable complex, we measured the interaction between the proteins by comigration on size-exclusion chromatography. Spt16-N, together with re- folded histone H2A–H2B dimers, shows no shift in its elution volume (Fig. S2C). Thus, H2A–H2B do not bind Spt16-N under these conditions, consistent with Spt16 requiring its acidic C terminus for H2A–H2B interaction (20). In contrast, we observe C-terminal lobe comigration of Spt16-N with H3–H4, as indicated by the shift to C C higher molecular masses of Spt16-N when bound to histones H3–H4 (Fig. 2C). There is an equilibrium between H3–H4 B dimers and tetramers that depends on pH and protein and salt concentration (33). Based on column calibration using molecular mass markers, the two shifted peaks correspond approximately to a dimer of H3–H4 bound to one Spt16-N domain, as well as two Spt16-N domains bound to a H3–H4 tetramer. To resolve the stoichiometry of the complex extrapolated from our size-exclusion chromatography, we have performed analyt- 90º ical ultracentrifugation. The analysis shows that Spt16-N forms BIOCHEMISTRY a 1:1 complex with H3–H4 dimers and, to a lesser extent, a peak that fits the molecular masses of two Spt16-N modules with four H3–H4 histones (Fig. 3A). Taken together, our structural and biochemical data reveal that Spt16-N uses an ancient enzymatic fold to mediate molecular interactions with histones H3–H4 in vitro. Spt16-N Interacts with the Globular Domains of Histone H3 and H4. To determine whether Spt16-N binds histones H3–H4 through their globular core domains or through their N-terminal tails, we used 0% 100% gel-filtration and GST pull-down assays to measure the binding Sequence conservation of Spt16-N to the globular domains of tailless H3 and H4 Fig. 1. Structure of the aminopeptidase domain of Spt16. (A) View of the complexes (Fig. 3B and Fig. S3B). The assays reveal that Spt16-N N-terminal domain (residues 1–442) of S. pombe FACT subunit Spt16 in two makes direct interactions with the globular domains of histone orientations.
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