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Catch me if you can a
abc
Maria Hondele & Andreas G Ladurner a
Department of Physiological Chemistry; Butenandt Institute and LMU Biomedical Center, Faculty of Medicine; Ludwig Maximilians University of Munich; Munich, Germany b
Munich Cluster for Systems Neurology (SyNergy); Munich, Germany
c
Center for Integrated Protein Science Munich (CIPSM); Munich, Germany Published online: 05 Dec 2013.
To cite this article: Maria Hondele & Andreas G Ladurner (2013) Catch me if you can, Nucleus, 4:6, 443-449, DOI: 10.4161/nucl.27235 To link to this article: http://dx.doi.org/10.4161/nucl.27235
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Nucleus 4:6, 443–449; November/December 2013; © 2013 Landes Bioscience
Catch me if you can Maria Hondele1 and Andreas G Ladurner1,2,3,* Department of Physiological Chemistry; Butenandt Institute and LMU Biomedical Center, Faculty of Medicine; Ludwig Maximilians University of Munich; Munich, Germany; 2Munich Cluster for Systems Neurology (SyNergy); Munich, Germany; 3Center for Integrated Protein Science Munich (CIPSM); Munich, Germany
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Keywords: histone chaperone, transcription through chromatin, chromatin, nucleosome (reorganization), thermodynamics / kinetic of nucleosome (un)folding, structure / crystallography *Correspondence to: Andreas G Ladurner; Email: [email protected] Submitted: 10/10/2013 Revised: 11/12/2013 Accepted: 11/17/2013 http://dx.doi.org/10.4161/nucl.27235 Extra View to: Hondele M, Stuwe T, Hassler M, Halbach F, Bowman A, Zhang ET, Nijmeijer B, Kotthoff C, Rybin V, Amlacher S, et al. Structural basis of histone H2A-H2B recognition by the essential chaperone FACT. Nature 2013; 499:1114; PMID:23698368; http://dx.doi.org/10.1038/ nature12242
ucleosomes confer a barrier to processes that require access to the eukaryotic genome such as transcription, DNA replication and repair. A variety of ATP-dependent nucleosome remodeling machines and ATP-independent histone chaperones facilitate nucleosome dynamics by depositing or evicting histones and unwrapping the DNA. It is clear that remodeling machines can use the energy from ATP to actively destabilize, translocate or disassemble nucleosomes. But how do ATP-independent histone chaperones, which “merely” bind histones, contribute to this process? Using our recent structural analysis of the conserved and essential eukaryotic histone chaperone FACT in complex with histones H2AH2B as an example, we suggest that FACT capitalizes on transiently exposed surfaces of the nucleosome. By binding these surfaces, FACT stabilizes thermodynamically unfavorable intermediates of the intrinsically dynamic nucleosome particle. This makes the nucleosome permissive to DNA and RNA polymerases, providing temporary access, passage, and read-out.
Introduction The eukaryotic FACT complex is an essential and highly conserved eukaryotic histone chaperone. It assists the progression of DNA and RNA polymerases, for example, by facilitating transcriptional initiation and elongation, DNA replication and repair of the chromatinized DNA template.1-6 Further, it promotes
the genome-wide integrity of chromatin structure, including the suppression of cryptic transcription.5 Genetic and biochemical assays have shown that direct binding of the histone heterodimer H2A-H2B is crucial for FACT’s chaperone function.2,7-9 However, an absence of any structural insight into how FACT exactly recognizes nucleosome components meant that the mechanism of how FACT facilitates access to the DNA template remained somewhat unclear: Does it partially disassemble the nucleosome by removing one or both H2A-H2B dimers, as has been observed?1-6 Or does it simply “slacken” the nucleosome particle by detaching DNA from the histone octamer core?5,8 The advantages of the latter mechanism would be that leaving the histone octamer intact could help to preserve chromatin integrity and the regulatory role of stable histone marks. Further, it is likely that the un- and re-wrapping of DNA from the histones happens on a faster time-scale than the partial or complete dis- and re-assembly of the nucleosome core particle. In an effort to investigate some of these biologically vital functions, we recently solved the structure of the H2A-H2B chaperone domain of FACT in complex with the histone heterodimer.2,7-10 The structure together with biochemical and in vivo data give much support to the hypothesis that FACT stabilizes a nucleosome state where the DNA is partially peeled off the histone octamer. In this model, FACT would stabilize partly unfolded nucleosome intermediates in
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How the histone chaperone FACT capitalizes on nucleosome breathing
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How does FACT Mediate its Vital Engagement with Histones H2A-H2B? FACT is composed of two subunits: Spt16 and Pob3 in yeast, or Spt16 and SSRP1 in humans.1,4 In our recent analysis, which includes the 2.35 Å crystal structure of a complex between the C. thermophilum yeast Spt16M domain with the globular core domains of histone H2A (13–106) and H2B (24–122) (Fig. 1A), we show that the Spt16M module of FACT’s Spt16 subunit establishes the evolutionarily conserved histone H2AH2B binding and chaperoning function.10 Spt16M adopts a tandem PHL (pleckstrin homology-like) fold,11 with a novel α-helical feature at the C-terminus, which we termed the “U-turn.” This extension engages in a strong hydrophobic interaction (K D ~400 nM) with the N-terminal α1 helix of histone H2B. Yeast cells carrying mutations in U-turn residues were unable to grow, therefore the U-turn motif represents a protein surface that is essential to FACT’s function and cell viability. We also found that two other surfaces of Spt16 contribute to H2A-H2B binding electrostatically and stabilize the complex kinetically. First, a conserved acidic patch on the second PHL domain of Spt16M interacts with the unstructured basic N-terminal tail of histone H2B. And second, the unstructured acidic Spt16C domain forms a high-affinity electrostatic interaction with the histone H2A-H2B dimer, confirming earlier data showing that the C-terminal end of human Spt16 is required for H2A-H2B binding in vitro.2,7 Quite unexpectedly, the structure revealed that the tandem PHL module is structurally and functionally related to the histone H3-H4 chaperones Rtt10612-14 and Pob3M15 (RMSD ~3 Å), which is itself another component of FACT. In light of this, it is not surprising that Spt16M also binds histones H3-H4. The region of Spt16M that mediates this interaction remains to be defined, but it must lie outside of the U-turn, which is a feature unique to Spt16M, since a mutant that
abrogates H2A-H2B binding is still able to interact with histones H3-H4.10 As is the case with the chaperone Rtt106, Spt16M interacts with the αN helix of histone H3, although the extent to which it may prefer H3 to be acetylated at the residue Lys 56 remains to be clearly established. Our data thus also show that tandem PHL modules establish an evolutionary conserved family of histone H3-H4 binding proteins. It is possible that further PHL proteins with H3-H4 chaperone function remain to be biochemically identified, considering that it can be difficult to computationally predict the pleckstrin homology fold from the amino acid sequence.
Spt16M Makes Multiple Interactions with the Histone Octamer The high-resolution snapshot of the Spt16M−H2A-H2B complex serves as a structural platform for determining the mechanism(s) by which FACT couples the recognition of hydrophobic and electrostatic features of H2A-H2B to productive nucleosome reorganization. Combining structure and biochemistry, we show that Spt16M interacts with three proximal surface patches of the histone octamer that organize the first 30 bp of nucleosomal DNA (Fig. 1B)16 : the H2B N-terminal tail, the H2B α1 helix and the H3 αN helix. Further, we envision the acidic Spt16C domain interacting with exposed, positively charged histone surfaces.
Electrostatic Interactions Drive Rapid Complex Formation Many protein–protein interactions are known to be driven by initial electrostatic interactions (which are not necessarily part of the main interaction interface) that then help successive, stable hydrophobic interfaces to form, since the attractive forces of electrostatic interactions have greater reach in solution and promote faster complex assembly.17 The Spt16M–H2A-H2B interactions we have described perfectly fit this model: the highly charged basic H2B N-terminal tail and the unstructured acidic Spt16C domain could mediate the first electrostatic interactions between chaperone and histone, facilitating the
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successive (hydrophobic) binding of the Spt16M U-turn to H2B α1. In fact, the solvent-accessible N-terminal tail of H2B exits from the nucleosome between the gyres of DNA (Fig. 1B) and is quite accessible. It is therefore possible that it would mediate some of the first interactions of FACT with the nucleosome (ref. 7 and our data), and indeed we observe that while its contribution at thermodynamic equilibrium is negligible, it has an essential role in kinetically stabilizing the complex between chaperone and histones. We also see that the unstructured acidic Spt16C domain strongly interacts with H2A-H2B (K D ~30 nM) and might also make initial contacts. However, the other two interaction sites, the H2B α1 and H3 αN helices, are DNA-covered in the folded, canonical structure of the nucleosome core particle (Fig. 1B).16 So how should the chaperone FACT get access?
FACT Captures “Breathing” Nucleosomes The nucleosome is not a static particle, but rather a dynamic complex: the first ~30 basepairs of DNA constantly and progressively unwrap and rewrap from the histone octamer,18 a process that has been termed “nucleosome breathing.” We suggest that FACT can capitalize on these intrinsic nucleosome dynamics to gradually invade the nucleosome particle and develop stronger interactions with the two DNA-covered Spt16M binding patches on the histone octamer (Fig. 2A). Shielding of the histones’ DNA-interaction sites and prevention of unproductive DNA-histone interactions are a characteristic feature of histone chaperones (reviewed in 19, 20). The H3 αN helix stabilizes the first 10 bp of DNA at the nucleosomal superhelix location 6.5. This stretch of DNA is detached about 20–60% of the time,18 and the H3 αN helix is therefore quite accessible. The detachment probability decreases to 10% at a position 27 bp into the nucleosome (superhelical location 4.5), where the hydrophobic patch on H2B encompassing the region α1/L1/ α2 forms the second-strongest histoneDNA contact. This would be expected to greatly decrease the probability of diffusion-mediated interactions with Spt16M.
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order to catalyze the passage of nucleic acid polymerases on the chromatinized DNA template.
However, if the chaperone domain is already captured by the H2B tail or by the H3 αN helix, Spt16M could afford to just “sit and wait” until the H2B α1 site becomes accessible. In summary, we showed that Spt16M interacts with the two major DNA-histone octamer contacts that coordinate the outermost ~30 base pairs of DNA. Therefore, we and others7-9 suggest that FACT could passively stabilize a state of the dynamic nucleosome complex where this stretch of DNA is displaced, capturing an alternative nucleosome arrangement that occurs upon spontaneous nucleosome breathing. In support of this model, there is good evidence to show that this region of nucleosomal DNA becomes hypersensitive to enzymatic and chemical degradation in the presence of FACT.8 The resulting nucleosome particle would be reorganized to make DNA more accessible, without removing a H2A-H2B dimer or disassembling the nucleosome core particle. This is further supported by the fact that the structure of the Spt16M–H2A-H2B complex suggests no direct interference of the interactions between H2A-H2B and H3-H4 by Spt16M. The mechanism we propose is ATP-independent and clearly distinct from the nucleosome remodeling machinery. Rather, we suggest that FACT could function as a “wedge” that passively capitalizes on the spontaneous movements
of the DNA in order to creep into the nucleosome.
Where Does the DNA Go when Displaced by Spt16? If FACT is able to peel off some of the DNA from the histone proteins within the canonical nucleosome core particle, then is the (lost) binding energy between DNA and histones somehow compensated for? The Spt16M module displays distinct positively charged surface patches on the PHL-1 domain that could neutralize the negative charges of the dissociated DNA (see Supplementary Figure 2 of ref. 10). Indeed, both the tandem PHL domain Spt16M or the isolated PHL-1 domain bind DNA in EMSA assays, just like the structurally homologous Pob3M and Rtt106 domains,14 while the PHL-2 domain does not bind DNA (Hondele M and Laudurner AG, unpublished data). In the superposition of the Spt16M–H2AH2B complex onto the nucleosome structure (Fig. 2A), the basic patches on PHL-1 would be in a good position to catch, neutralize and stabilize the displaced DNA, thereby compensating for some of the broken histone-DNA binding energy. In fungal Spt16 sequences, an unstructured loop in PHL-1 is enriched for positively charged residues. A homologous stretch is absent or less distinctive in higher eukaryotes
(Fig. 3). However, this loss could be evolutionarily compensated for by the DNAbinding HMG box of SSRP1, which is not present in the yeast Pob3 homolog. The chaperone FACT could thus easily capture DNA surfaces normally involved in histone interactions, helping to prevent unproductive protein-DNA interactions.
FACT Breaks Strong Octamer-DNA Contacts to Allow Polymerase Passage The enzyme RNA polymerase II by itself cannot generate enough force to transcribe through nucleosomes in vitro, since breaking DNA-histone contacts poses a strong energetic barrier (see ref. 21 for review). Yet if contacts between the H2A-H2B dimer and nucleosomal DNA are already broken, this sufficiently lowers the energetic barrier to permit polymerase passage.22 Recently, Hsieh et al.9 showed that FACT does exactly this: it helps to partially uncoil DNA from H2A-H2B and its presence is sufficient to facilitate transcription through the nucleosome. Since chemically crosslinking the histone octamer core does not inhibit FACT action, nor transcription, the authors assume that FACT does not evict the H2A-H2B dimer or introduce major changes in the overall structure of the histone octamer, such as the interface between H2A-H2B
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Figure 1. Crystal structure of the Spt16M- H2A-H2B complex and overview of three surfaces of the histone octamer recognizes by Spt16M. (A) The 2.35 Å crystal structure of the trimeric complex between Ch. thermophilum Spt16M and the globular cores (g) of histone H2A and histone H2B reveals the interaction is mediated by Spt16’s U-turn motif (red) and the α1 helix of H2B (dark blue). Spt16M PHL-1 (light orange), Spt16M PHL-2 (dark orange); H2A (light green), N-termini (N), C-termini (C). (B) Close-up view of the histone octamer surfaces recognized by Spt16M (sphere representation of the amino acid side chains): The H2B N-terminal tail (light pink) extrudes between the gyres of DNA and might serve as an initial, solvent-accessible point of attachment for Spt16 to the nucleosome. The αN helix of H3 (blue) and the α1 helix of H2B (dark pink) are DNA-covered in the structure of the canonical nucleosome but become accessible upon DNA breathing.
and H3-H4. The authors propose that by shielding or “breaking” DNA-histone contacts the chaperone FACT lowers the thermodynamic barrier sufficiently to facilitate polymerase passage. Our structure of the Spt16M–H2A-H2B complex shows that a nucleosome-engaged Spt16M
module would fulfill these mechanistic criteria. On a related note, we speculate that the torsional forces generated by RNA Polymerase II could further help to lift DNA off the histone octamer core and thereby facilitate access of Spt16M to the H2B α1 helix.
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FACT could Destabilize the Histone Octamer Core The superposition model also identifies a steric clash between a surface bulge of Spt16M’s PHL-2 and the H3-H4 dimer of the other nucleosome half-disc, which
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Figure 2. The FACT chaperone domain may compete with DNA for binding to histones H2A-H2B. The structure of the Spt16M–H2A-H2B complex (Spt16M in light green, U-turn in blue, H2A in yellow, H2B in red) was superimposed onto the structure of the nucleosome core particle (histones in gray, DNA in orange, dyad marked with φ) by aligning histones H2A-H2B. Intrinsic dynamics of the nucleosome, DNA “breathing” (A) and nucleosome “gaping” (B), are indicated by arrows. The path of the DNA putatively displaced during breathing is sketched in dotted lines (A).
locates below the H2B α1 helix bound by the Spt16M U-turn (Fig. 2B). This steric clash could be resolved by flexibility in the Spt16M-H2A-H2B interface, by nucleosome “gaping,” an oyster shell-like movement of two nucleosome half-discs around a H3-H3′ interface hinge,23 or by moving the H2A-H2B dimer away from the octamer. In physiological salt, contacts between H2A-H2B and H3-H4 are weak and the octamer is mainly stabilized by the presence of DNA. Therefore, although Spt16M does not actively disturb contacts between the H2A-H2B dimer and the H3-H4 tetramer, Spt16Minduced displacement of DNA and steric clashes with the other nucleosome halfdisc might be sufficient to release the H2A-H2B dimer from the nucleosome, as observed in vitro.2,8
Spt16M Likely Chaperones Canonical, Variant, and Modified H2A-H2B Alike The read-out of chromatin is regulated by post-translational modifications of histone proteins. These modifications can recruit or stabilize the chromatin binding of “reader” proteins or selectively block the binding of others. Several of these modifications have been well characterized for histone H3, but H2B in contrast only shows a few modifications that may be functional. Some of these localize directly to the interface with Spt16M and might therefore regulate the interaction between histone and chaperone.24,25 For example, phosphorylation of H2B Ser33 on the H2B α1 helix was found
to promote transcriptional activation in D. melanogaster.24 In the Spt16M-H2AH2B crystal structure, this residue is hydrogen bonded to a chloride ion, which is further coordinated by Spt16M Asn916 and H2B Ile36. The negatively charged phosphate group of Ser33 might well substitute the chloride, and thus we suggest that H2B Ser33 phosphorylation could stabilize the FACT–histone complex. In contrast, our structure suggests that Spt16M binding to histones might not be altered by H2A or H2B ubiquitination. During transcription elongation, FACT shows functional interaction with histone ubiquitin modifications: ubiquitination of H2A (at K119 in mammals) blocks FACT recruitment and inhibits transcription elongation.26 In contrast, FACT stimulates the ubiquitination of H2B K123 in yeast (K120 in mammals) and is in return retained at actively transcribed ORFs. Together with the PAF complex, FACT and H2B monoubiquitination (H2Bub1) cooperate to promote transcription elongation27 and preserve chromatin structure.28 Neither H2A K119 nor H2B K123 are visible in the structure of the complex. However, both ubiquitination-sites locate to the C-terminus of the histone proteins, opposite to where Spt16M binds H2B α1. Thus, the modifications will probably not directly block FACT binding and direct recognition of ubiquitin is also unlikely, since the H2A and H2B modifications have opposite effects. Since H2Bub, but not H2Aub, interferes with chromatin compaction and maintains an open and accessible chromatin fiber,29,30 ubiquitination may instead promote a chromatin
state that is more accessible (H2Bub) or inaccessible (H2Aub) to the binding of the chaperone and other factors. The interactions we observed between Spt16M and the H2A-H2B are mediated by H2B residues that are conserved in all other isoforms of this histone heterodimer, such as complexes formed between H2B and H2A variants. In fact, most sequence variance for H2A variants localizes to the C-terminus of the histone protein, which is far from the interaction site on the H2B α1 helix. Similarly, only very few and rare histone variants of H2B are known, and none of these sequence variations localize to the interfaces with Spt16M. Thus, in summary, FACT may engage with canonical and variant or modified H2AH2B heterodimers alike, acting as a very general H2A-H2B chaperone.
The Hydrophobic Patch of H2B has so far Escaped Functional Analysis Thus far, large-scale genetic screens using scanning alanine mutagenesis of all histone residues have found no requirement of the hydrophobic H2B α1 patch, which interacts with the Spt16M U-turn, for several tested biological functions (e.g., transcription, replication).31,32 This is somewhat surprising, but the lack of obvious phenotype might be explained by the rather hydrophobic character of the alanine residue used as replacement; single point mutants of this type would be predicted to conserve the overall hydrophobic properties of this FACT-interacting surface. In the case of Spt16M, for example,
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Figure 3. Alignment of selected Spt16M PHL-1 residues of different species. Secondary structure elements are marked above the sequence. Fungal Pob3 does not contain a HMG box, but the SSRP1 protein of higher eukaryotes does; the order of sequences is the same as for the PHL-1 sequence alignment.
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Holo-FACT acts as a Scaffold to Integrate Nuclear Processes We also reported the structure of FACT’s heterodimerization domain. Like Spt16M, Pob3M or Rtt106, the Spt16D and Pob3N complex is composed of PHL modules. However, the Spt16D-Pob3N heterodimer does not bind histones, rather our preliminary biochemical analysis showed that this region connects FACT to replicative DNA polymerases. Since Pob3M also interacts with RPA, the single-stranded DNA binding protein,15 this suggests that coupling of the chaperone FACT to the replication machinery might be important, promoting nucleosome (dis-) assembly during DNA replication. Together with the published structures of Spt16N33,34 and Pob3M,15 the structures of all globular domains of FACT have now been solved. Apart from Spt16N, which adopts the canonical “pita-bread” fold of amino-peptidases, all domains are composed of pleckstrin homologylike modules. It is possible that these histone-binding modules may have originated from domain fold amplification during evolution. All published structures are from yeast proteins, but since the sequences and secondary structures References 1. Orphanides G, Wu WH, Lane WS, Hampsey M, Reinberg D. The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins. Nature 1999; 400:284-8; PMID:10421373; http://dx.doi.org/10.1038/22350 2. Belotserkovskaya R, Oh S, Bondarenko VA, Orphanides G, Studitsky VM, Reinberg D. FACT facilitates transcription-dependent nucleosome alteration. Science 2003; 301:1090-3; PMID:12934006; http://dx.doi.org/10.1126/science.1085703 3. Mason PB, Struhl K. The FACT complex travels with elongating RNA polymerase II and is important for the fidelity of transcriptional initiation in vivo. Mol Cell Biol 2003; 23:8323-33; PMID:14585989; http:// dx.doi.org/10.1128/MCB.23.22.8323-8333.2003
of FACT subunits are very conserved, we can assume that the observed features also apply to the proteins from human and other species. In fact, the human Spt16M module readily binds H2A-H2B.10 We believe that the modular nature of the FACT complex may allow the chaperone to effect multiple interactions with distinct histones, DNA and a variety of other nuclear factors. The linkers connecting the globular FACT domains are predicted to be (at least) partially α-helical, but we believe that they do not rigidly connect the globular modules. Rather, each domain may act as a more or less “independent” unit that binds histones or other nuclear factors. To summarize, only one globular domain of FACT, Spt16M, binds histones H2A-H2B. However, three out of the four globular domains—Spt16N, Pob3M, and Spt16M—interact with H3-H4 at least in vitro. Spt16M seems to be the strongest binder and primarily recognizes a peptide spanning H3 residues (46–65).10 Further, unstructured domains such as the highly acidic Spt16C region, promote further histone interaction through vital electrostatic interactions. We suggest that the tethering of all these domains together might have a recruiting function, to “keep things in place” as a scaffold, thus increasing the processivity of many distinct nuclear processes involving the dynamic reorganization of the chromatin template.
Where Are We Now? Our ultimate goal is to understand how the holo-FACT complex reorganizes the nucleosome, and therefore to analyze 4.
Wittmeyer J, Joss L, Formosa T. Spt16 and Pob3 of Saccharomyces cerevisiae form an essential, abundant heterodimer that is nuclear, chromatin-associated, and copurifies with DNA polymerase alpha. Biochemistry 1999; 38:8961-71; PMID:10413469; http://dx.doi.org/10.1021/bi982851d 5. Kaplan CD, Laprade L, Winston F. Transcription elongation factors repress transcription initiation from cryptic sites. Science 2003; 301:10969; PMID:12934008; http://dx.doi.org/10.1126/ science.1087374 6. Lejeune E, Bortfeld M, White SA, Pidoux AL, Ekwall K, Allshire RC, Ladurner AG. The chromatinremodeling factor FACT contributes to centromeric heterochromatin independently of RNAi. Curr Biol 2007; 17:1219-24; PMID:17614284; http://dx.doi. org/10.1016/j.cub.2007.06.028
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how the individual modules of the multidomain chaperone interact together with the various features of the nucleosome core particle to successfully reorganize DNAhistone contacts. To gain a full picture of this highly dynamic process, methods such as cryo-electron microscopy, crosslinkingmass spectrometry and single-molecule in vitro and in vivo FRET will probably be most useful to capture “snapshots” of the dynamic complex of the full-length chaperone bound to the nucleosome, at different stages of nucleosome reorganization. Further work is also necessary at the cellular level in order to reveal the temporal aspects of how FACT promotes nucleosome dynamics. In particular, it will be interesting to study its interactions with the DNA and RNA polymerase machineries and how FACT integrates these diverse functions. This will lead us to an understanding of how this apparently passive, “non-aggressive” protein compelx has sufficient energy to facilitate the progression of DNA and RNA polymerases, and at the same time helps to ensure that chromatin structure is established properly and remains as intact as possible. In short: faithful to its function as a genuine chaperone. Disclosure of Potential Conflicts of Interest
No potential conflict of interest was disclosed. Acknowledgments
We are grateful to Corey Laverty and Markus Hassler for comments on the manuscript. This work was supported by the LMU Munich and the Boehringer Ingelheim Fonds (M.H.). 7. Winkler DD, Muthurajan UM, Hieb AR, Luger K. Histone chaperone FACT coordinates nucleosome interaction through multiple synergistic binding events. J Biol Chem 2011; 286:41883-92; PMID:21969370; http://dx.doi.org/10.1074/jbc. M111.301465 8. Xin H, Takahata S, Blanksma M, McCullough L, Stillman DJ, Formosa T. yFACT induces global accessibility of nucleosomal DNA without H2AH2B displacement. Mol Cell 2009; 35:365-76; PMID:19683499; http://dx.doi.org/10.1016/j. molcel.2009.06.024 9. Hsieh FK, Kulaeva OI, Patel SS, Dyer PN, Luger K, Reinberg D, Studitsky VM. Histone chaperone FACT action during transcription through chromatin by RNA polymerase II. Proc Natl Acad Sci U S A 2013; 110:7654-9; PMID:23610384; http://dx.doi. org/10.1073/pnas.1222198110
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we found that mutation of four hydrophobic U-turn residues to alanine is not sufficient to disrupt the complex with H2A-H2B. However, mutation to serine residues clearly is. Hydrophobic-tocharged H2A mutants in the H2A α1 helix might thus be necessary for functional analysis of histone mutants.
19. Elsässer SJ, D’Arcy S. Towards a mechanism for histone chaperones. Biochim Biophys Acta 2012; 1819:211–221. 20. Hondele M, Ladurner AG. The chaperone-histone partnership: for the greater good of histone traffic and chromatin plasticity. Curr Opin Struct Biol 2011; 21:698-708; PMID:22054910; http://dx.doi. org/10.1016/j.sbi.2011.10.003 21. Kulaeva OI, Hsieh FK, Chang HW, Luse DS, Studitsky VM. Mechanism of transcription through a nucleosome by RNA polymerase II. Biochim Biophys Acta 2013; 1829:76–83. 22. Kulaeva OI, Gaykalova DA, Pestov NA, Golovastov VV, Vassylyev DG, Artsimovitch I, Studitsky VM. Mechanism of chromatin remodeling and recovery during passage of RNA polymerase II. Nat Struct Mol Biol 2009; 16:1272-8; PMID:19935686; http:// dx.doi.org/10.1038/nsmb.1689 23. Mozziconacci J, Victor JM. Nucleosome gaping supports a functional structure for the 30nm chromatin fiber. J Struct Biol 2003; 143:72-6; PMID:12892727; http://dx.doi.org/10.1016/S1047-8477(03)00102-3 24. Maile T, Kwoczynski S, Katzenberger RJ, Wassarman DA, Sauer F. TAF1 activates transcription by phosphorylation of serine 33 in histone H2B. Science 2004; 304:1010-4; PMID:15143281; http://dx.doi. org/10.1126/science.1095001 25. Mahajan K, Fang B, Koomen JM, Mahajan NP. H2B Tyr37 phosphorylation suppresses expression of replication-dependent core histone genes. Nat Struct Mol Biol 2012; 19:930-7; PMID:22885324; http:// dx.doi.org/10.1038/nsmb.2356 26. Zhou W, Zhu P, Wang J, Pascual G, Ohgi KA, Lozach J, Glass CK, Rosenfeld MG. Histone H2A monoubiquitination represses transcription by inhibiting RNA polymerase II transcriptional elongation. Mol Cell 2008; 29:69-80; PMID:18206970; http:// dx.doi.org/10.1016/j.molcel.2007.11.002 27. Pavri R, Zhu B, Li G, Trojer P, Mandal S, Shilatifard A, Reinberg D. Histone H2B monoubiquitination functions cooperatively with FACT to regulate elongation by RNA polymerase II. Cell 2006; 125:70317; PMID:16713563; http://dx.doi.org/10.1016/j. cell.2006.04.029 28. Fleming AB, Kao CF, Hillyer C, Pikaart M, Osley MA. H2B ubiquitylation plays a role in nucleosome dynamics during transcription elongation. Mol Cell 2008; 31:57-66; PMID:18614047; http://dx.doi. org/10.1016/j.molcel.2008.04.025
29. Fierz B, Chatterjee C, McGinty RK, Bar-Dagan M, Raleigh DP, Muir TW. Histone H2B ubiquitylation disrupts local and higher-order chromatin compaction. Nat Chem Biol 2011; 7:113-9; PMID:21196936; http://dx.doi.org/10.1038/nchembio.501 30. Chandrasekharan MB, Huang F, Sun ZW. Ubiquitination of histone H2B regulates chromatin dynamics by enhancing nucleosome stability. Proc Natl Acad Sci U S A 2009; 106:16686-91; PMID:19805358; http://dx.doi.org/10.1073/ pnas.0907862106 31. Matsubara K, Sano N, Umehara T, Horikoshi M. Global analysis of functional surfaces of core histones with comprehensive point mutants. Genes Cells 2007; 12:13-33; PMID:17212652; http://dx.doi. org/10.1111/j.1365-2443.2007.01031.x 32. Nakanishi S, Sanderson BW, Delventhal KM, Bradford WD, Staehling-Hampton K, Shilatifard A. A comprehensive library of histone mutants identifies nucleosomal residues required for H3K4 methylation. Nat Struct Mol Biol 2008; 15:8818; PMID:18622391; http://dx.doi.org/10.1038/ nsmb.1454 33. VanDemark AP, Xin H, McCullough L, Rawlins R, Bentley S, Heroux A, Stillman DJ, Hill CP, Formosa T. Structural and functional analysis of the Spt16p N-terminal domain reveals overlapping roles of yFACT subunits. J Biol Chem 2008; 283:5058-68; PMID:18089575; http://dx.doi.org/10.1074/jbc. M708682200 34. Stuwe T, Hothorn M, Lejeune E, Rybin V, Bortfeld M, Scheffzek K, Ladurner AG. The FACT Spt16 “peptidase” domain is a histone H3-H4 binding module. Proc Natl Acad Sci U S A 2008; 105:88849; PMID:18579787; http://dx.doi.org/10.1073/ pnas.0712293105
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10. Hondele M, Stuwe T, Hassler M, Halbach F, Bowman A, Zhang ET, Nijmeijer B, Kotthoff C, Rybin V, Amlacher S, et al. Structural basis of histone H2AH2B recognition by the essential chaperone FACT. Nature 2013; 499:111-4; PMID:23698368; http:// dx.doi.org/10.1038/nature12242 11. Kemble DJ, Whitby FG, Robinson H, McCullough LL, Formosa T, Hill CP. Structure of the Spt16 middle domain reveals functional features of the histone chaperone FACT. J Biol Chem 2013; 288:10188-94; PMID:23417676; http://dx.doi.org/10.1074/jbc. C113.451369 12. Su D, Hu Q, Li Q, Thompson JR, Cui G, Fazly A, Davies BA, Botuyan MV, Zhang Z, Mer G. Structural basis for recognition of H3K56-acetylated histone H3-H4 by the chaperone Rtt106. Nature 2012; 483:104-7; PMID:22307274; http://dx.doi. org/10.1038/nature10861 13. Zunder RM, Antczak AJ, Berger JM, Rine J. Two surfaces on the histone chaperone Rtt106 mediate histone binding, replication, and silencing. Proc Natl Acad Sci U S A 2012; 109:E144-53; PMID:22198837; http://dx.doi.org/10.1073/pnas.1119095109 14. Liu Y, Huang H, Zhou BO, Wang SS, Hu Y, Li X, Liu J, Zang J, Niu L, Wu J, et al. Structural analysis of Rtt106p reveals a DNA binding role required for heterochromatin silencing. J Biol Chem 2010; 285:425162; PMID:20007951; http://dx.doi.org/10.1074/jbc. M109.055996 15. VanDemark AP, Blanksma M, Ferris E, Heroux A, Hill CP, Formosa T. The structure of the yFACT Pob3-M domain, its interaction with the DNA replication factor RPA, and a potential role in nucleosome deposition. Mol Cell 2006; 22:36374; PMID:16678108; http://dx.doi.org/10.1016/j. molcel.2006.03.025 16. Luger K, Mäder AW, Richmond RK, Sargent DF, Richmond TJ. Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 1997; 389:251-60; PMID:9305837; http://dx.doi. org/10.1038/38444 17. Schreiber G, Fersht AR. Rapid, electrostatically assisted association of proteins. Nat Struct Biol 1996; 3:427-31; PMID:8612072; http://dx.doi. org/10.1038/nsb0596-427 18. Koopmans WJA, Buning R, Schmidt T, van Noort J. spFRET using alternating excitation and FCS reveals progressive DNA unwrapping in nucleosomes. Biophys J 2009; 97:195-204; PMID:19580757; http://dx.doi.org/10.1016/j.bpj.2009.04.030
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Catch me if you can a
abc
Maria Hondele & Andreas G Ladurner a
Department of Physiological Chemistry; Butenandt Institute and LMU Biomedical Center, Faculty of Medicine; Ludwig Maximilians University of Munich; Munich, Germany b
Munich Cluster for Systems Neurology (SyNergy); Munich, Germany
c
Center for Integrated Protein Science Munich (CIPSM); Munich, Germany Published online: 05 Dec 2013.
To cite this article: Maria Hondele & Andreas G Ladurner (2013) Catch me if you can, Nucleus, 4:6, 443-449, DOI: 10.4161/nucl.27235 To link to this article: http://dx.doi.org/10.4161/nucl.27235
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Nucleus 4:6, 443–449; November/December 2013; © 2013 Landes Bioscience
Catch me if you can Maria Hondele1 and Andreas G Ladurner1,2,3,* Department of Physiological Chemistry; Butenandt Institute and LMU Biomedical Center, Faculty of Medicine; Ludwig Maximilians University of Munich; Munich, Germany; 2Munich Cluster for Systems Neurology (SyNergy); Munich, Germany; 3Center for Integrated Protein Science Munich (CIPSM); Munich, Germany
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Keywords: histone chaperone, transcription through chromatin, chromatin, nucleosome (reorganization), thermodynamics / kinetic of nucleosome (un)folding, structure / crystallography *Correspondence to: Andreas G Ladurner; Email: [email protected] Submitted: 10/10/2013 Revised: 11/12/2013 Accepted: 11/17/2013 http://dx.doi.org/10.4161/nucl.27235 Extra View to: Hondele M, Stuwe T, Hassler M, Halbach F, Bowman A, Zhang ET, Nijmeijer B, Kotthoff C, Rybin V, Amlacher S, et al. Structural basis of histone H2A-H2B recognition by the essential chaperone FACT. Nature 2013; 499:1114; PMID:23698368; http://dx.doi.org/10.1038/ nature12242
ucleosomes confer a barrier to processes that require access to the eukaryotic genome such as transcription, DNA replication and repair. A variety of ATP-dependent nucleosome remodeling machines and ATP-independent histone chaperones facilitate nucleosome dynamics by depositing or evicting histones and unwrapping the DNA. It is clear that remodeling machines can use the energy from ATP to actively destabilize, translocate or disassemble nucleosomes. But how do ATP-independent histone chaperones, which “merely” bind histones, contribute to this process? Using our recent structural analysis of the conserved and essential eukaryotic histone chaperone FACT in complex with histones H2AH2B as an example, we suggest that FACT capitalizes on transiently exposed surfaces of the nucleosome. By binding these surfaces, FACT stabilizes thermodynamically unfavorable intermediates of the intrinsically dynamic nucleosome particle. This makes the nucleosome permissive to DNA and RNA polymerases, providing temporary access, passage, and read-out.
Introduction The eukaryotic FACT complex is an essential and highly conserved eukaryotic histone chaperone. It assists the progression of DNA and RNA polymerases, for example, by facilitating transcriptional initiation and elongation, DNA replication and repair of the chromatinized DNA template.1-6 Further, it promotes
the genome-wide integrity of chromatin structure, including the suppression of cryptic transcription.5 Genetic and biochemical assays have shown that direct binding of the histone heterodimer H2A-H2B is crucial for FACT’s chaperone function.2,7-9 However, an absence of any structural insight into how FACT exactly recognizes nucleosome components meant that the mechanism of how FACT facilitates access to the DNA template remained somewhat unclear: Does it partially disassemble the nucleosome by removing one or both H2A-H2B dimers, as has been observed?1-6 Or does it simply “slacken” the nucleosome particle by detaching DNA from the histone octamer core?5,8 The advantages of the latter mechanism would be that leaving the histone octamer intact could help to preserve chromatin integrity and the regulatory role of stable histone marks. Further, it is likely that the un- and re-wrapping of DNA from the histones happens on a faster time-scale than the partial or complete dis- and re-assembly of the nucleosome core particle. In an effort to investigate some of these biologically vital functions, we recently solved the structure of the H2A-H2B chaperone domain of FACT in complex with the histone heterodimer.2,7-10 The structure together with biochemical and in vivo data give much support to the hypothesis that FACT stabilizes a nucleosome state where the DNA is partially peeled off the histone octamer. In this model, FACT would stabilize partly unfolded nucleosome intermediates in
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How the histone chaperone FACT capitalizes on nucleosome breathing
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How does FACT Mediate its Vital Engagement with Histones H2A-H2B? FACT is composed of two subunits: Spt16 and Pob3 in yeast, or Spt16 and SSRP1 in humans.1,4 In our recent analysis, which includes the 2.35 Å crystal structure of a complex between the C. thermophilum yeast Spt16M domain with the globular core domains of histone H2A (13–106) and H2B (24–122) (Fig. 1A), we show that the Spt16M module of FACT’s Spt16 subunit establishes the evolutionarily conserved histone H2AH2B binding and chaperoning function.10 Spt16M adopts a tandem PHL (pleckstrin homology-like) fold,11 with a novel α-helical feature at the C-terminus, which we termed the “U-turn.” This extension engages in a strong hydrophobic interaction (K D ~400 nM) with the N-terminal α1 helix of histone H2B. Yeast cells carrying mutations in U-turn residues were unable to grow, therefore the U-turn motif represents a protein surface that is essential to FACT’s function and cell viability. We also found that two other surfaces of Spt16 contribute to H2A-H2B binding electrostatically and stabilize the complex kinetically. First, a conserved acidic patch on the second PHL domain of Spt16M interacts with the unstructured basic N-terminal tail of histone H2B. And second, the unstructured acidic Spt16C domain forms a high-affinity electrostatic interaction with the histone H2A-H2B dimer, confirming earlier data showing that the C-terminal end of human Spt16 is required for H2A-H2B binding in vitro.2,7 Quite unexpectedly, the structure revealed that the tandem PHL module is structurally and functionally related to the histone H3-H4 chaperones Rtt10612-14 and Pob3M15 (RMSD ~3 Å), which is itself another component of FACT. In light of this, it is not surprising that Spt16M also binds histones H3-H4. The region of Spt16M that mediates this interaction remains to be defined, but it must lie outside of the U-turn, which is a feature unique to Spt16M, since a mutant that
abrogates H2A-H2B binding is still able to interact with histones H3-H4.10 As is the case with the chaperone Rtt106, Spt16M interacts with the αN helix of histone H3, although the extent to which it may prefer H3 to be acetylated at the residue Lys 56 remains to be clearly established. Our data thus also show that tandem PHL modules establish an evolutionary conserved family of histone H3-H4 binding proteins. It is possible that further PHL proteins with H3-H4 chaperone function remain to be biochemically identified, considering that it can be difficult to computationally predict the pleckstrin homology fold from the amino acid sequence.
Spt16M Makes Multiple Interactions with the Histone Octamer The high-resolution snapshot of the Spt16M−H2A-H2B complex serves as a structural platform for determining the mechanism(s) by which FACT couples the recognition of hydrophobic and electrostatic features of H2A-H2B to productive nucleosome reorganization. Combining structure and biochemistry, we show that Spt16M interacts with three proximal surface patches of the histone octamer that organize the first 30 bp of nucleosomal DNA (Fig. 1B)16 : the H2B N-terminal tail, the H2B α1 helix and the H3 αN helix. Further, we envision the acidic Spt16C domain interacting with exposed, positively charged histone surfaces.
Electrostatic Interactions Drive Rapid Complex Formation Many protein–protein interactions are known to be driven by initial electrostatic interactions (which are not necessarily part of the main interaction interface) that then help successive, stable hydrophobic interfaces to form, since the attractive forces of electrostatic interactions have greater reach in solution and promote faster complex assembly.17 The Spt16M–H2A-H2B interactions we have described perfectly fit this model: the highly charged basic H2B N-terminal tail and the unstructured acidic Spt16C domain could mediate the first electrostatic interactions between chaperone and histone, facilitating the
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successive (hydrophobic) binding of the Spt16M U-turn to H2B α1. In fact, the solvent-accessible N-terminal tail of H2B exits from the nucleosome between the gyres of DNA (Fig. 1B) and is quite accessible. It is therefore possible that it would mediate some of the first interactions of FACT with the nucleosome (ref. 7 and our data), and indeed we observe that while its contribution at thermodynamic equilibrium is negligible, it has an essential role in kinetically stabilizing the complex between chaperone and histones. We also see that the unstructured acidic Spt16C domain strongly interacts with H2A-H2B (K D ~30 nM) and might also make initial contacts. However, the other two interaction sites, the H2B α1 and H3 αN helices, are DNA-covered in the folded, canonical structure of the nucleosome core particle (Fig. 1B).16 So how should the chaperone FACT get access?
FACT Captures “Breathing” Nucleosomes The nucleosome is not a static particle, but rather a dynamic complex: the first ~30 basepairs of DNA constantly and progressively unwrap and rewrap from the histone octamer,18 a process that has been termed “nucleosome breathing.” We suggest that FACT can capitalize on these intrinsic nucleosome dynamics to gradually invade the nucleosome particle and develop stronger interactions with the two DNA-covered Spt16M binding patches on the histone octamer (Fig. 2A). Shielding of the histones’ DNA-interaction sites and prevention of unproductive DNA-histone interactions are a characteristic feature of histone chaperones (reviewed in 19, 20). The H3 αN helix stabilizes the first 10 bp of DNA at the nucleosomal superhelix location 6.5. This stretch of DNA is detached about 20–60% of the time,18 and the H3 αN helix is therefore quite accessible. The detachment probability decreases to 10% at a position 27 bp into the nucleosome (superhelical location 4.5), where the hydrophobic patch on H2B encompassing the region α1/L1/ α2 forms the second-strongest histoneDNA contact. This would be expected to greatly decrease the probability of diffusion-mediated interactions with Spt16M.
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order to catalyze the passage of nucleic acid polymerases on the chromatinized DNA template.
However, if the chaperone domain is already captured by the H2B tail or by the H3 αN helix, Spt16M could afford to just “sit and wait” until the H2B α1 site becomes accessible. In summary, we showed that Spt16M interacts with the two major DNA-histone octamer contacts that coordinate the outermost ~30 base pairs of DNA. Therefore, we and others7-9 suggest that FACT could passively stabilize a state of the dynamic nucleosome complex where this stretch of DNA is displaced, capturing an alternative nucleosome arrangement that occurs upon spontaneous nucleosome breathing. In support of this model, there is good evidence to show that this region of nucleosomal DNA becomes hypersensitive to enzymatic and chemical degradation in the presence of FACT.8 The resulting nucleosome particle would be reorganized to make DNA more accessible, without removing a H2A-H2B dimer or disassembling the nucleosome core particle. This is further supported by the fact that the structure of the Spt16M–H2A-H2B complex suggests no direct interference of the interactions between H2A-H2B and H3-H4 by Spt16M. The mechanism we propose is ATP-independent and clearly distinct from the nucleosome remodeling machinery. Rather, we suggest that FACT could function as a “wedge” that passively capitalizes on the spontaneous movements
of the DNA in order to creep into the nucleosome.
Where Does the DNA Go when Displaced by Spt16? If FACT is able to peel off some of the DNA from the histone proteins within the canonical nucleosome core particle, then is the (lost) binding energy between DNA and histones somehow compensated for? The Spt16M module displays distinct positively charged surface patches on the PHL-1 domain that could neutralize the negative charges of the dissociated DNA (see Supplementary Figure 2 of ref. 10). Indeed, both the tandem PHL domain Spt16M or the isolated PHL-1 domain bind DNA in EMSA assays, just like the structurally homologous Pob3M and Rtt106 domains,14 while the PHL-2 domain does not bind DNA (Hondele M and Laudurner AG, unpublished data). In the superposition of the Spt16M–H2AH2B complex onto the nucleosome structure (Fig. 2A), the basic patches on PHL-1 would be in a good position to catch, neutralize and stabilize the displaced DNA, thereby compensating for some of the broken histone-DNA binding energy. In fungal Spt16 sequences, an unstructured loop in PHL-1 is enriched for positively charged residues. A homologous stretch is absent or less distinctive in higher eukaryotes
(Fig. 3). However, this loss could be evolutionarily compensated for by the DNAbinding HMG box of SSRP1, which is not present in the yeast Pob3 homolog. The chaperone FACT could thus easily capture DNA surfaces normally involved in histone interactions, helping to prevent unproductive protein-DNA interactions.
FACT Breaks Strong Octamer-DNA Contacts to Allow Polymerase Passage The enzyme RNA polymerase II by itself cannot generate enough force to transcribe through nucleosomes in vitro, since breaking DNA-histone contacts poses a strong energetic barrier (see ref. 21 for review). Yet if contacts between the H2A-H2B dimer and nucleosomal DNA are already broken, this sufficiently lowers the energetic barrier to permit polymerase passage.22 Recently, Hsieh et al.9 showed that FACT does exactly this: it helps to partially uncoil DNA from H2A-H2B and its presence is sufficient to facilitate transcription through the nucleosome. Since chemically crosslinking the histone octamer core does not inhibit FACT action, nor transcription, the authors assume that FACT does not evict the H2A-H2B dimer or introduce major changes in the overall structure of the histone octamer, such as the interface between H2A-H2B
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Figure 1. Crystal structure of the Spt16M- H2A-H2B complex and overview of three surfaces of the histone octamer recognizes by Spt16M. (A) The 2.35 Å crystal structure of the trimeric complex between Ch. thermophilum Spt16M and the globular cores (g) of histone H2A and histone H2B reveals the interaction is mediated by Spt16’s U-turn motif (red) and the α1 helix of H2B (dark blue). Spt16M PHL-1 (light orange), Spt16M PHL-2 (dark orange); H2A (light green), N-termini (N), C-termini (C). (B) Close-up view of the histone octamer surfaces recognized by Spt16M (sphere representation of the amino acid side chains): The H2B N-terminal tail (light pink) extrudes between the gyres of DNA and might serve as an initial, solvent-accessible point of attachment for Spt16 to the nucleosome. The αN helix of H3 (blue) and the α1 helix of H2B (dark pink) are DNA-covered in the structure of the canonical nucleosome but become accessible upon DNA breathing.
and H3-H4. The authors propose that by shielding or “breaking” DNA-histone contacts the chaperone FACT lowers the thermodynamic barrier sufficiently to facilitate polymerase passage. Our structure of the Spt16M–H2A-H2B complex shows that a nucleosome-engaged Spt16M
module would fulfill these mechanistic criteria. On a related note, we speculate that the torsional forces generated by RNA Polymerase II could further help to lift DNA off the histone octamer core and thereby facilitate access of Spt16M to the H2B α1 helix.
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FACT could Destabilize the Histone Octamer Core The superposition model also identifies a steric clash between a surface bulge of Spt16M’s PHL-2 and the H3-H4 dimer of the other nucleosome half-disc, which
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Figure 2. The FACT chaperone domain may compete with DNA for binding to histones H2A-H2B. The structure of the Spt16M–H2A-H2B complex (Spt16M in light green, U-turn in blue, H2A in yellow, H2B in red) was superimposed onto the structure of the nucleosome core particle (histones in gray, DNA in orange, dyad marked with φ) by aligning histones H2A-H2B. Intrinsic dynamics of the nucleosome, DNA “breathing” (A) and nucleosome “gaping” (B), are indicated by arrows. The path of the DNA putatively displaced during breathing is sketched in dotted lines (A).
locates below the H2B α1 helix bound by the Spt16M U-turn (Fig. 2B). This steric clash could be resolved by flexibility in the Spt16M-H2A-H2B interface, by nucleosome “gaping,” an oyster shell-like movement of two nucleosome half-discs around a H3-H3′ interface hinge,23 or by moving the H2A-H2B dimer away from the octamer. In physiological salt, contacts between H2A-H2B and H3-H4 are weak and the octamer is mainly stabilized by the presence of DNA. Therefore, although Spt16M does not actively disturb contacts between the H2A-H2B dimer and the H3-H4 tetramer, Spt16Minduced displacement of DNA and steric clashes with the other nucleosome halfdisc might be sufficient to release the H2A-H2B dimer from the nucleosome, as observed in vitro.2,8
Spt16M Likely Chaperones Canonical, Variant, and Modified H2A-H2B Alike The read-out of chromatin is regulated by post-translational modifications of histone proteins. These modifications can recruit or stabilize the chromatin binding of “reader” proteins or selectively block the binding of others. Several of these modifications have been well characterized for histone H3, but H2B in contrast only shows a few modifications that may be functional. Some of these localize directly to the interface with Spt16M and might therefore regulate the interaction between histone and chaperone.24,25 For example, phosphorylation of H2B Ser33 on the H2B α1 helix was found
to promote transcriptional activation in D. melanogaster.24 In the Spt16M-H2AH2B crystal structure, this residue is hydrogen bonded to a chloride ion, which is further coordinated by Spt16M Asn916 and H2B Ile36. The negatively charged phosphate group of Ser33 might well substitute the chloride, and thus we suggest that H2B Ser33 phosphorylation could stabilize the FACT–histone complex. In contrast, our structure suggests that Spt16M binding to histones might not be altered by H2A or H2B ubiquitination. During transcription elongation, FACT shows functional interaction with histone ubiquitin modifications: ubiquitination of H2A (at K119 in mammals) blocks FACT recruitment and inhibits transcription elongation.26 In contrast, FACT stimulates the ubiquitination of H2B K123 in yeast (K120 in mammals) and is in return retained at actively transcribed ORFs. Together with the PAF complex, FACT and H2B monoubiquitination (H2Bub1) cooperate to promote transcription elongation27 and preserve chromatin structure.28 Neither H2A K119 nor H2B K123 are visible in the structure of the complex. However, both ubiquitination-sites locate to the C-terminus of the histone proteins, opposite to where Spt16M binds H2B α1. Thus, the modifications will probably not directly block FACT binding and direct recognition of ubiquitin is also unlikely, since the H2A and H2B modifications have opposite effects. Since H2Bub, but not H2Aub, interferes with chromatin compaction and maintains an open and accessible chromatin fiber,29,30 ubiquitination may instead promote a chromatin
state that is more accessible (H2Bub) or inaccessible (H2Aub) to the binding of the chaperone and other factors. The interactions we observed between Spt16M and the H2A-H2B are mediated by H2B residues that are conserved in all other isoforms of this histone heterodimer, such as complexes formed between H2B and H2A variants. In fact, most sequence variance for H2A variants localizes to the C-terminus of the histone protein, which is far from the interaction site on the H2B α1 helix. Similarly, only very few and rare histone variants of H2B are known, and none of these sequence variations localize to the interfaces with Spt16M. Thus, in summary, FACT may engage with canonical and variant or modified H2AH2B heterodimers alike, acting as a very general H2A-H2B chaperone.
The Hydrophobic Patch of H2B has so far Escaped Functional Analysis Thus far, large-scale genetic screens using scanning alanine mutagenesis of all histone residues have found no requirement of the hydrophobic H2B α1 patch, which interacts with the Spt16M U-turn, for several tested biological functions (e.g., transcription, replication).31,32 This is somewhat surprising, but the lack of obvious phenotype might be explained by the rather hydrophobic character of the alanine residue used as replacement; single point mutants of this type would be predicted to conserve the overall hydrophobic properties of this FACT-interacting surface. In the case of Spt16M, for example,
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Figure 3. Alignment of selected Spt16M PHL-1 residues of different species. Secondary structure elements are marked above the sequence. Fungal Pob3 does not contain a HMG box, but the SSRP1 protein of higher eukaryotes does; the order of sequences is the same as for the PHL-1 sequence alignment.
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Holo-FACT acts as a Scaffold to Integrate Nuclear Processes We also reported the structure of FACT’s heterodimerization domain. Like Spt16M, Pob3M or Rtt106, the Spt16D and Pob3N complex is composed of PHL modules. However, the Spt16D-Pob3N heterodimer does not bind histones, rather our preliminary biochemical analysis showed that this region connects FACT to replicative DNA polymerases. Since Pob3M also interacts with RPA, the single-stranded DNA binding protein,15 this suggests that coupling of the chaperone FACT to the replication machinery might be important, promoting nucleosome (dis-) assembly during DNA replication. Together with the published structures of Spt16N33,34 and Pob3M,15 the structures of all globular domains of FACT have now been solved. Apart from Spt16N, which adopts the canonical “pita-bread” fold of amino-peptidases, all domains are composed of pleckstrin homologylike modules. It is possible that these histone-binding modules may have originated from domain fold amplification during evolution. All published structures are from yeast proteins, but since the sequences and secondary structures References 1. Orphanides G, Wu WH, Lane WS, Hampsey M, Reinberg D. The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins. Nature 1999; 400:284-8; PMID:10421373; http://dx.doi.org/10.1038/22350 2. Belotserkovskaya R, Oh S, Bondarenko VA, Orphanides G, Studitsky VM, Reinberg D. FACT facilitates transcription-dependent nucleosome alteration. Science 2003; 301:1090-3; PMID:12934006; http://dx.doi.org/10.1126/science.1085703 3. Mason PB, Struhl K. The FACT complex travels with elongating RNA polymerase II and is important for the fidelity of transcriptional initiation in vivo. Mol Cell Biol 2003; 23:8323-33; PMID:14585989; http:// dx.doi.org/10.1128/MCB.23.22.8323-8333.2003
of FACT subunits are very conserved, we can assume that the observed features also apply to the proteins from human and other species. In fact, the human Spt16M module readily binds H2A-H2B.10 We believe that the modular nature of the FACT complex may allow the chaperone to effect multiple interactions with distinct histones, DNA and a variety of other nuclear factors. The linkers connecting the globular FACT domains are predicted to be (at least) partially α-helical, but we believe that they do not rigidly connect the globular modules. Rather, each domain may act as a more or less “independent” unit that binds histones or other nuclear factors. To summarize, only one globular domain of FACT, Spt16M, binds histones H2A-H2B. However, three out of the four globular domains—Spt16N, Pob3M, and Spt16M—interact with H3-H4 at least in vitro. Spt16M seems to be the strongest binder and primarily recognizes a peptide spanning H3 residues (46–65).10 Further, unstructured domains such as the highly acidic Spt16C region, promote further histone interaction through vital electrostatic interactions. We suggest that the tethering of all these domains together might have a recruiting function, to “keep things in place” as a scaffold, thus increasing the processivity of many distinct nuclear processes involving the dynamic reorganization of the chromatin template.
Where Are We Now? Our ultimate goal is to understand how the holo-FACT complex reorganizes the nucleosome, and therefore to analyze 4.
Wittmeyer J, Joss L, Formosa T. Spt16 and Pob3 of Saccharomyces cerevisiae form an essential, abundant heterodimer that is nuclear, chromatin-associated, and copurifies with DNA polymerase alpha. Biochemistry 1999; 38:8961-71; PMID:10413469; http://dx.doi.org/10.1021/bi982851d 5. Kaplan CD, Laprade L, Winston F. Transcription elongation factors repress transcription initiation from cryptic sites. Science 2003; 301:10969; PMID:12934008; http://dx.doi.org/10.1126/ science.1087374 6. Lejeune E, Bortfeld M, White SA, Pidoux AL, Ekwall K, Allshire RC, Ladurner AG. The chromatinremodeling factor FACT contributes to centromeric heterochromatin independently of RNAi. Curr Biol 2007; 17:1219-24; PMID:17614284; http://dx.doi. org/10.1016/j.cub.2007.06.028
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how the individual modules of the multidomain chaperone interact together with the various features of the nucleosome core particle to successfully reorganize DNAhistone contacts. To gain a full picture of this highly dynamic process, methods such as cryo-electron microscopy, crosslinkingmass spectrometry and single-molecule in vitro and in vivo FRET will probably be most useful to capture “snapshots” of the dynamic complex of the full-length chaperone bound to the nucleosome, at different stages of nucleosome reorganization. Further work is also necessary at the cellular level in order to reveal the temporal aspects of how FACT promotes nucleosome dynamics. In particular, it will be interesting to study its interactions with the DNA and RNA polymerase machineries and how FACT integrates these diverse functions. This will lead us to an understanding of how this apparently passive, “non-aggressive” protein compelx has sufficient energy to facilitate the progression of DNA and RNA polymerases, and at the same time helps to ensure that chromatin structure is established properly and remains as intact as possible. In short: faithful to its function as a genuine chaperone. Disclosure of Potential Conflicts of Interest
No potential conflict of interest was disclosed. Acknowledgments
We are grateful to Corey Laverty and Markus Hassler for comments on the manuscript. This work was supported by the LMU Munich and the Boehringer Ingelheim Fonds (M.H.). 7. Winkler DD, Muthurajan UM, Hieb AR, Luger K. Histone chaperone FACT coordinates nucleosome interaction through multiple synergistic binding events. J Biol Chem 2011; 286:41883-92; PMID:21969370; http://dx.doi.org/10.1074/jbc. M111.301465 8. Xin H, Takahata S, Blanksma M, McCullough L, Stillman DJ, Formosa T. yFACT induces global accessibility of nucleosomal DNA without H2AH2B displacement. Mol Cell 2009; 35:365-76; PMID:19683499; http://dx.doi.org/10.1016/j. molcel.2009.06.024 9. Hsieh FK, Kulaeva OI, Patel SS, Dyer PN, Luger K, Reinberg D, Studitsky VM. Histone chaperone FACT action during transcription through chromatin by RNA polymerase II. Proc Natl Acad Sci U S A 2013; 110:7654-9; PMID:23610384; http://dx.doi. org/10.1073/pnas.1222198110
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we found that mutation of four hydrophobic U-turn residues to alanine is not sufficient to disrupt the complex with H2A-H2B. However, mutation to serine residues clearly is. Hydrophobic-tocharged H2A mutants in the H2A α1 helix might thus be necessary for functional analysis of histone mutants.
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