A Highly Conserved Region within H2B Is Important for FACT To Act on Nucleosomes Suting Zheng,a J. Brooks Crickard,a Abhinaya Srikanth,a Joseph C. Reesea,b Department of Biochemistry and Molecular Biology, Center for Eukaryotic Gene Regulation,a and Center for RNA Molecular Biology,b Penn State University, University Park, Pennsylvania, USA

T

he genomes of eukaryotes are packaged into chromatin, which is composed of DNA and the four core histones H2A, H2B, H3, and H4 (1, 2). Each histone contains an N-terminal tail that can be decorated with multiple posttranslational modifications. The modification of residues in the tails controls nuclear functions such as gene expression, mitosis, meiosis, mRNA processing, gene silencing, and DNA repair (2–4). The N-terminal tails of histones H3 and H4 are exceptionally well conserved across all eukaryotes, including the sites of posttranslational modifications. The importance of these tails in regulating chromatin transactions has been interrogated by many studies using the genetically tractable yeast model system. In contrast, the amino acid sequence of the H2A and H2B N-terminal tails are relatively divergent between yeasts and metazoans; thus, much less is known about their functions than about those of H3 and H4. While most of the yeast H2B (yH2B) tail shows minimal sequence similarity to its metazoan counterparts, a short basic region (residues 30 to 37) known as the H2B repression (HBR) domain is highly conserved among all eukaryotes (5, 6). The HBR is located adjacent to the ␣-helical structured region in the H2B tail and passes between the gyres of DNA within the structure of the nucleosome. First it was thought to play a role only in the silencing of telomere-proximal genes, but recent analysis of a mutant containing a deletion of the HBR domain (⌬HBR) revealed that it is important also for DNA damage repair and the activation and repression of genes (7, 8). Since the HBR domain residues make contact with DNA, the mutant phenotypes were attributed to weakened histone-DNA interactions. However, the molecular basis for the defects caused by the removal of the HBR domain is not known. Since this region is so well conserved compared to the rest of the H2B tail, it must perform an essential function. Assembly and disassembly of chromatin during transcription require the orchestration of histone-modifying enzymes, ATPdependent remodelers, and histone chaperones (9). The latter class of factors functions by binding the surfaces of histones nor-

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mally buried within the structure of the nucleosome. FACT (facilitates chromatin transactions) was the first histone chaperone found to regulate chromatin during transcription (10–13). First it was thought to function by binding and actively removing H2AH2B dimers to facilitate the passage of RNA polymerase II (RNAPII) (14), but two alternative models have been proposed. Yeast FACT (yFACT), along with the HMG domain protein Nhp6, reorganizes nucleosomes by disrupting DNA-histone contacts. This conclusion was supported by data showing that FACT increased the accessibility of nucleosomal DNA to a greater extent than can be explained by dimer loss and that FACT-Nhp6 remained associated with the altered species (13, 15). In a second model, FACT “shields” DNA binding surfaces on H2A-H2B and displaces DNA from the surface of the dimer (16–18). In both mechanisms, dimer eviction is not obligatory. Recently, insights into the mechanism were partially obtained by solving a high-resolution structure of the middle domain of the Spt16 subunit of FACT from Chaetomium thermophilum bound to the H2A-H2B dimer, which suggests that the binding of a tandem pleckstrin homology-like domain of Spt16 to H2B would disrupt histone-DNA interactions (16). It is still unclear how FACT disassembles nucleosomes, especially in cells. Here we uncovered a function of the HBR domain and the molecular basis for its requirement in essential functions such as transcription and DNA damage resistance. Our results indicate that the HBR domain is important for the histone chaperone

Received 22 April 2013 Returned for modification 14 May 2013 Accepted 1 November 2013 Published ahead of print 18 November 2013 Address correspondence to Joseph C. Reese, [email protected] S.Z. and J.B.C. contributed equally to this study. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/MCB.00478-13

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Histone N-terminal tails play crucial roles in chromatin-related processes. The tails of histones H3 and H4 are highly conserved and well characterized, but much less is known about the functions of the tails of histones H2A and H2B and their sequences are more divergent among eukaryotes. Here we characterized the function of the only highly conserved region in the H2B tail, the H2B repression (HBR) domain. Once thought to play a role only in repression, it also has an uncharacterized function in gene activation and DNA damage responses. We report that deletion of the HBR domain impairs the eviction of nucleosomes at the promoters and open reading frames of genes. A closer examination of the HBR domain mutants revealed that they displayed phenotypes similar to those of histone chaperone complex FACT mutants, including an increase in intragenic transcription and the accumulation of free histones in cells. Biochemical characterization of recombinant nucleosomes indicates that deletion of the HBR domain impairs FACT-dependent removal of H2A-H2B from nucleosomes, suggesting that the HBR domain plays an important role in allowing FACT to disrupt dimer-DNA interactions. We have uncovered a previously unappreciated role for the HBR domain in regulating chromatin structure and have provided insight into how FACT acts on nucleosomes.

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TABLE 1 Strains used in this study Strain

Genotype

Source

PY014

MATa ade2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 hta1-htb1::HIS3; hta2-htb2::LEU2 pMP002(CEN6 TRP1 HTA1 HTB1) Isogenic to PY014, carries pMP031(CEN6 TRP1 HTA1 htb1⌬30-37) Isogenic to PY014, carries pMP032(CEN6 TRP1 HTA1 htb1⌬3-37) MATa his3⌬1 ura3⌬0 leu2⌬0 hht1-hhf1⌬::KanMX hhf2-hht2⌬::NatMX hta1-htb1⌬::HphMX hta2-htb2⌬::NatMX pJH49 [HTA1-HTB1-HHF2-HHT2 LEU2/CEN] MATa his3⌬1 ura3⌬0 leu2⌬0 hht1-hhf1⌬::KanMX hhf2-hht2⌬::NatMX hta1-htb1⌬::HphMX hta2-htb2⌬::NatMX pJH49 [HTA1-htb1(⌬1-32)-HHF2-HHT2 LEU2/CEN] BY4741 with Spt16-TAP::HIS3 BY4741 with Snf6-TAP::HIS3 MATa his4-912␦ lys2-128␦ leu2⌬1 ura3-52 MATa his4-912␦ lys2-128␦ leu2⌬1 ura3-52 spt16-197 PY014 with Spt16-myc::KanMX PY020 with Spt16-myc::KanMX PY021 with Spt16-myc::KanMX PY014 with Asf1-myc::KanMX PY020 with Asf1-myc::KanMX PY021 with Asf1-myc::KanMX

John J. Wyrick

PY020 PY021 MSY1913 MSY1979

FACT to remove H2A-H2B dimers from nucleosomes in vitro, evict nucleosomes at promoters, and restore chromatin structure during transcription elongation. Thus, we have uncovered a function of this highly conserved region of H2B and revealed insights into how FACT acts on nucleosomes. MATERIALS AND METHODS Strains and media. The Saccharomyces cerevisiae strains used in this study are listed in Table 1. Cells were grown in YP medium (1% yeast extract, 2% peptone) with the appropriate carbon source, i.e., 2% dextrose, 3% raffinose, or 2% galactose. The medium was supplemented with 0.05 mg/ml adenine sulfate. For GAL1 induction studies, cells were grown to mid-log phase in YP medium plus 3% raffinose and then galactose was added to 2% for the times indicated in the figures. For testing of hydroxyurea (HU) sensitivity, 3-fold dilutions of log-phase cultures were spotted onto YPAD medium (YP medium supplemented with adenine sulfate and 2% dextrose) or YPAD medium plus 50 mM HU and incubated at 30°C for 48 h. Gene deletion and epitope tagging were carried out by homologous recombination with PCR-generated cassettes (19, 20). RNA isolation, mRNA quantification, and Northern blotting. RNA isolation was carried out as previously described (21). For reverse transcription (RT)-PCR, 10 ␮g of RNA was reverse transcribed and equal amounts of cDNA were analyzed by quantitative PCR (Applied Biosystems) with gene-specific primers. For Northern blotting, 15 ␮g of total RNA was separated on 1.2% formaldehyde-containing agarose gels and transferred to a Hybond-XL membrane (GE Healthcare, Piscataway, NJ) by capillary blotting. After UV cross-linking and prehybridization at 65°C for 4 h, a 32P-labeled probe was added. Signal was detected with a phosphorimager screen and scanned on a Typhoon image documentation system (GE Healthcare, Piscataway, NJ). ChIP. Chromatin immunoprecipitation (ChIP) was performed and analyzed as previously described (22, 23). Yeast cultures were grown to an optical density at 600 nm of 0.7, treated with formaldehyde (1%, vol/vol) for 15 min, and quenched by the addition of glycine to 125 mM. Wholecell extract was prepared by glass bead disruption, and the chromatin was sheared into fragments averaging 200 to 600 bp in size with a Bioruptor (Diagenode). One hundred microliters of whole-cell extract was incubated with 1 to 2 ␮l of antibody overnight. The immunoprecipitated DNA and input DNA were analyzed by real-time PCR. The data are reported as the percent immunoprecipitation (immunoprecipitation signal/input signal) and are the mean and standard deviation of at least three independent experiments.

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M. Mitchell Smith Euroscarf Euroscarf Fred Winston Fred Winston This study This study This study This study This study This study

Detection of non-chromatin-bound histones associated with chaperones. Whole-cell extracts of strains containing an epitope-tagged version of Asf1 (Asf1-myc) were prepared by glass bead disruption in E buffer (40 mM HEPES-NaOH [pH 7.5], 0.1% Tween 20, 200 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride [PMSF], 2 ␮g/ml leupeptin, 2 ␮g/ml pepstatin A). Asf1-myc was immunoprecipitated with monoclonal antibody 9E10 and protein A-Sepharose (GE Healthcare, Piscataway, NJ) overnight at 4°C. The beads were washed three times in E buffer, and the immunoprecipitated material was analyzed by Western blotting with H3 and 9E10 antibodies. Purification of FACT, SWI/SNF, and yNap1. FACT was purified from an Spt16-TAP (tandem affinity purification)-tagged strain by the previously published TAP procedure (24). Briefly, 2 liters of yeast culture was collected and lysed by glass bead disruption in E buffer. Cleared lysates were incubated with IgG-Sepharose fast flow (GE Healthcare, Piscataway, NJ), washed in E buffer, and then washed in E buffer containing 500 mM NaCl to reduce the amount of endogenous dimer copurifying with FACT. FACT was eluted by digestion with Tobacco etch virus protease overnight at 4°C and then bound to calmodulin-Sepharose 4B (GE Healthcare, Piscataway, NJ). After washing, FACT was eluted into 50 mM Tris-HCl (pH 8.0)–2.0 mM EGTA–150 mM NaCl–10% glycerol– 0.1% Tween 20. The peak fractions were pooled and dialyzed into 50 mM TrisHCl (pH 8.0)–100 mM NaCl– 0.1% NP-40 –10% glycerol. SWI/SNF was purified from 6 liters of an Snf6-TAP-tagged strain under the same conditions, except that the lysis, wash, and calmodulin elution buffers contained 350 mM NaCl (25). Peak fractions were dialyzed into 10 mM TrisHCl (pH 8.0)–50 mM NaCl– 0.5 mM EDTA– 0.1% NP-40 –10% glycerol. The TAP-tagged proteins were concentrated with microfiltration units (Sartorius Stedim Biotech, Göttingen, Germany). Hexahistidine-tagged yeast Nap1 was overexpressed from pET28 in Escherichia coli BL21(pLysS) and purified on Ni-nitrilotriacetic acid resin. The eluant was passed over a Mono-Q column and eluted with a gradient of 0.1 to 1.0 M KCl. Nap1-containing fractions were pooled and dialyzed into buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM KCl, 1 mM EDTA, and 10% glycerol. Untagged Nhp6 was expressed and purified in E. coli with a plasmid generously provided by Tim Formosa. After cell lysis, Nhp6 was purified by trichloroacetic acid precipitation and SP-Sepharose chromatography as described previously (26). Fractions were analyzed by SDS-PAGE, and peak fractions were pooled, dialyzed against 20 mM Tris-Cl (pH 7.5)–100 mM NaCl–2 mM EDTA–1 mM 2-mercaptoethanol–10% glycerol, and stored at ⫺80°C.

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JR1397 JR1709 FY120 FY348 JR1256 JR1260 JR1261 JR1477 JR1478 JR1479

John J. Wyrick John J. Wyrick M. Mitchell Smith

HBR Mediates FACT Activity

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buffer containing 2 mM CaCl2. Calmodulin-Sepharose 4B (GE Healthcare, Piscataway, NJ) was then added for another 20 min of incubation to capture the FACT-dimer complexes. After the beads were washed three times in binding buffer, proteins were eluted by the addition of SDS loading buffer. The amount of H2A-H2B dimer bound to the beads was detected by Coomassie blue staining of SDS-PAGE gels. Fluorescence-based high-throughput (HI-FI) assays were conducted with 384-well microtiter plates, and the data were analyzed as described previously (30). Recombinant yeast H2AQ114C was purified and the conjugated to Alexa Fluor 488-maleimide by incubation overnight at 4°C in unfolding buffer. The protein was then refolded with either wild-type or ⌬HBR mutant H2B and purified on Biorex70 as described above. The binding conditions were as follows: 10 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM MgCl2, 5% glycerol, 1 mM tris(2-carboxyethyl)phosphine, and 100 ng/␮l BSA. Increasing amounts of FACT were titrated into 1 nM dimer, and fluorescence was detected with a Typhoon Scanner (GE Healthcare, Piscataway, NJ). The data were processed with ImageQuant 5.2. The background, minus FACT, was subtracted from the values. The point of saturation was set to 1.0, and the relative fluorescence was calculated for the remaining values. The data were exported into Excel, and each determination was fitted to a logarithmic curve. Kd values were determined for each curve and averaged from the six replicates.

RESULTS

The HBR domain is important for transcription activation. Gene expression profiling of H2B mutants suggests that the N-terminal domain is principally required for the repression of a subset of genes and that the HBR domain comprising residues 30 to 37 is necessary and sufficient for this function (7). To confirm this, we measured the uninduced level of GAL1 transcription in strains lacking most of the H2B tail (H2B ⌬1-32), the HBR domain specifically (H2B ⌬30-37), or the entire H2B N-terminal domain (H2B ⌬3-37). The results show that any mutant lacking the HBR domain displayed ⬃2- to 3-fold derepression of GAL1 when grown in raffinose (Fig. 1A), consistent with its known role in repressing uninduced transcription. The importance of the HBR domain in gene activation is less clear; thus, we analyzed the expression of GAL1 by inducing it with galactose. GAL1 was induced up to 1,200-fold within 90 min in wild-type and H2B ⌬1-32 mutant cells (Fig. 1B). However, further deletion of amino acids in the HBR domain (3-37⌬) or deletion of the HBR domain specifically (30-37⌬) reduced GAL1 transcription to 40 or 60% of that in wild-type levels (Fig. 1B). To determine the step in transcription activation impaired by the HBR domain deletion, we monitored preinitiation complex (PIC) formation by measuring TATA binding protein (TBP) and RNAPII levels at GAL1 with the ChIP assay. Deletion of the HBR domain strongly reduced the recruitment of TBP and RNAPII to the GAL1 promoter (Fig. 1C and D). Thus, the HBR domain is required for the full activation of genes and deleting it impairs PIC formation. The HBR domain promotes nucleosome disassembly. An essential step in transcription initiation is the remodeling and eviction of promoter nucleosomes, which is necessary for the general transcription machinery to access DNA (9). We evaluated nucleosome eviction at GAL1 in wild-type and H2B mutant cells by monitoring H2A and H3 cross-linking in the repressed (dextrose) and activated (galactose) states. Nucleosomes were evicted from the GAL1 promoter in wild-type cells, as indicated by the 4- to 5-fold reduction in H2A and H3 cross-linking (Fig. 2A and B). Strikingly, the HBR domain mutants displayed a significant nucleosome eviction defect; H2A and H3 densities were 2- to 3- and 4- to

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Reconstitution of yeast nucleosomes. Recombinant yeast histones were expressed in E. coli and purified by ion-exchange chromatography as described previously (27, 28). Individual histones were denatured and refolded into H2A-H2B dimers and H3-H4 tetramers by dialysis. The dimers and tetramers were bound individually to Biorex-70 resin for 4 h at 4°C, and the resin was washed with 10 mM Tris-HCl (pH 8.0)– 0.5 mM EDTA–500 mM NaCl for H2A-H2B and the same buffer containing 750 mM NaCl for H3-H4. The H2A-H2B dimers and H3-H4 tetramers were eluted with 1 or 2 M NaCl, respectively, and glycerol was added to 50%; this was followed by storage at ⫺20°C. DNA containing the 601 nucleosome-positioning sequence was generated by PCR amplification and gel purified prior to reconstitution. To assemble nucleosomes, purified dimers and tetramers were incubated with yNap1 on ice for 30 min in buffer containing 10 mM HEPES-NaOH (pH 7.5), 50 mM NaCl, 0.5 mM EGTA, 100 ng/␮l bovine serum albumin (BSA), and 10% glycerol; this was followed by the addition of template DNA and incubation for 4 h at 30°C. Nucleosome assembly was analyzed on a 4% native acrylamide gel. Nucleosome mobilization assay. Swi/Snf was preincubated with 50 fmol of radiolabeled recombinant nucleosomes for 20 min in 10 mM Tris-HCl (pH 8.0)–100 mM NaCl–5 mM MgCl2–1 mM dithiothreitol (DTT)–100 ng/␮l BSA–10% glycerol; this was followed by the addition of 1 mM ATP for 1 h at 30°C. Swi/Snf was competed off the nucleosome by the addition of 100 ng/␮l salmon sperm DNA prior to electrophoresis on 4% native acrylamide gels (29). Images were captured on a phosphorimager screen and analyzed on a Typhoon scanner (GE Healthcare, Piscataway, NJ). FACT chaperone activity. Recombinant yeast histones were assembled onto a biotinylated 601 nucleosome-positioning sequence and then immobilized on M-280 streptavidin Dynabeads for 1 h at room temperature in buffer containing 20 mM HEPES-KOH (pH 7.5), 70 mM KCl, 0.1% NP-40, 250 ng/␮l BSA, and 10% glycerol. Nucleosomes were washed twice with 20 mM HEPES-KOH (pH 7.5)–70 mM KCl-5 mM MgCl2– 0.5 mM EGTA–1 mM DTT– 0.02% NP-40. Immobilized nucleosomes (75 ng) were incubated with FACT for 1 h at room temperature in buffer containing 20 mM HEPES-NaOH (pH 7.5), 200 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.5 mM EGTA, 0.02% NP-40, and 100 ng/␮l BSA. The beads were then collected, and the supernatants were removed. The amounts of histone H2B and H3 retained on the beads and released into the supernatant were analyzed by Western blotting (Active motif 39237 and Abcam 1791). Nucleosome and H2A-H2B dimer association. Whole-cell extract from a Spt16 TAP-tagged strain was incubated with IgG-Sepharose 4B (GE Healthcare, Piscataway, NJ) overnight at 4°C. The IgG beads were washed three times in binding buffer (20 mM Tris [pH 7.4], 200 mM NaCl, 1 mM MgCl2, 0.1% Triton X-100, 10% glycerol, 0.5 mM DTT, 0.5 mM PMSF) and once in binding buffer containing 500 mM NaCl to remove the endogenous H2A-H2B dimer that copurifies with FACT. The beads were then returned to binding buffer containing 200 mM NaCl. Beads containing approximately 500 ng FACT were incubated with 500 ng wild-type or ⌬HBR mutant nucleosomes for 1 h at room temperature. The beads were washed three times in binding buffer (200 mM NaCl) and boiled in SDS loading buffer. Nucleosomes associated with FACT were analyzed by Western blotting with H3 antibody (Abcam 1791). Binding of FACT-Nhp6 to recombinant nucleosomes was performed as described previously (26). Briefly, Spt16-Pob3 purified from yeast was mixed with recombinant Nhp6 in an equimolar ratio, and the complex was bound to radiolabeled nucleosome core particles (NCPs) by incubation in binding buffer (10 mM HEPES [pH 7.5], 120 mM NaCl, 0.2 mM 2-mercaptoethanol, 0.1 ␮g/␮l BSA, 12% sucrose, 2% glycerol) for 10 min at 30°C. The products were then separated by 4% native PAGE in 0.5⫻ Tris-borate-EDTA for 2 h at room temperature. Gels were then dried, exposed to a phosphorimager screen, and scanned. FACT-H2A-H2B dimer pulldown was performed by incubating 500 ng purified FACT and 500 ng of recombinant yeast wild-type or ⌬HBR mutant dimer in solution for 20 min at room temperature in binding

Zheng et al.

mutant cells was measured by RT-PCR. Cultures were grown to log phase in medium containing 3% raffinose. The amount of GAL1 mRNA was normalized to that of scR1, a polymerase III-transcribed RNA. (B) GAL1 induction in wild-type and H2B mutant cells was measured after shifting the cells from 3% raffinose to 2% galactose for the times indicated. ChIP analysis of RNAP II (C) and TBP (D) recruitment to the GAL1 promoter upon activation. IP, immunoprecipitation.

5-fold higher than those in wild-type cells, respectively (Fig. 2A and B). Similar to what was observed at the promoter of GAL1, the HBR domain mutants displayed higher nucleosome density within the open reading frame (ORF) (Fig. 2C). The nucleosome eviction defect was also observed at two constitutively activated genes, PMA1 and PYK1. The cross-linking of H3 was approximately 2-fold higher over the promoter (Fig. 2D). The increased nucleosome density at PMA1 and PYK1 also correlated with reduced mRNA levels and RNAPII recruitment at these loci (data not shown). These results indicate that the HBR domain is important for the disassembly of nucleosomes at promoters, which is essential for PIC assembly and transcription activation. We next monitored the kinetics of nucleosome eviction after shifting cells from the noninducing (raffinose) to the activated (galactose) condition. Activation of transcription led to a dramatic decrease in H2A and H3 cross-linking over the promoter in wild-type cells, but strikingly, this was significantly reduced in the ⌬HBR mutants (Fig. 2E and F). The nucleosome density in the mutants was 2- to 3-fold higher than that in the wild-type cells by 90 min. The absolute levels of H2A cross-linking in the mutants were similar to those in wild-type cells during the early stages of activation (15 and 30 min). However, the nucleosome occupancy was lower in raffinose (t ⫽ 0 min), so the rate of removal (change) was less than that observed in wild-type cells even at the earlier time points. These findings suggest that HBR is important for the removal of promoter nucleosomes during activation. HBR deletion does not affect the activity or recruitment of SWI/SNF. ATP-dependent chromatin remodeling enzymes use energy derived from ATP hydrolysis to disrupt or alter the association of histones with DNA (31, 32). Previous studies have shown that the SWI/SNF complex is recruited to GAL1 and is required for nucleosome eviction during gene activation (33–35); thus, it is possible that the HBR domain is required for SWI/SNF recruit-

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ment or activity. To test this, we examined the recruitment of SWI/SNF by monitoring the cross-linking of the catalytic subunit of the complex, Swi2, to the promoter of GAL1. In the wild-type cells, the activation of GAL1 correlated with an ⬃10-fold recruitment of SWI/SNF, and importantly, the level of Swi2 cross-linking was similar in the H2B tail mutants (Fig. 3A). Thus, the nucleosome eviction defect in the ⌬HBR mutants is not caused by reduced SWI/SNF recruitment. The recruitment of chromatin remodeling complexes to promoters is the first step in the disassembly process. It is possible that SWI/SNF is recruited but is unable to recognize or remodel the ⌬HBR mutant nucleosomes. Removal of a short basic patch from the H4 tail impaired nucleosome sliding by ISWI chromatin remodelers without affecting nucleosome binding (36, 37). Therefore, substrate binding and catalytic activities of ATP-dependent chromatin remodelers can be uncoupled by histone mutations. We examined if the HBR domain is required for SWI/SNF to remodel chromatin by using an in vitro sliding assay. Recombinant wild-type and ⌬HBR mutant (⌬30-37) mononucleosomes were prepared by assembling recombinant yeast histones onto a DNA fragment containing the 601 nucleosome-positioning sequence (Fig. 3B). Interestingly, ⌬HBR mutant nucleosomes show greater mobility than the wild-type version, suggesting that removal of the HBR domain affects the canonical structure of the nucleosome. Alternatively, deletion of the six basic amino acids in the HBR domain caused the nucleosomes to migrate faster. The nature of this change is not clear and requires further investigation. We then examined the remodeling of the wild-type and mutant nucleosomes and found that SWI/SNF slid the ⌬HBR mutant and wild-type nucleosomes equally well (Fig. 3C and D). Collectively, our results suggest that the nucleosome eviction defect in the ⌬HBR mutant is not caused by a defect in SWI/SNF recruitment or activity.

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FIG 1 The HBR domain is important for GAL1 expression. (A) The uninduced level of GAL1 mRNA in wild-type and H2B ⌬1-32, H2B ⌬30-37, and H2B ⌬3-37

HBR Mediates FACT Activity

The HBR mutant displays signature FACT mutant phenotypes. ATP-dependent remodelers collaborate with histone chaperones to remove nucleosomes from promoters (9, 38, 39). The ⌬HBR mutation may impact the ability of chaperones to assist in the removal of nucleosomes from promoters. The FACT complex, consisting of Spt16/Pob3, is required for nucleosome eviction at promoters during transcription (12, 13). Histone eviction and PIC formation are impaired at GAL1 in a pob3 mutant (15, 40); therefore, FACT is a good candidate to mediate chromatin disassembly at GAL1. In addition, the X-ray crystal structure of the middle domain of Spt16 (Spt16M) from C. thermophilum with Xenopus H2A-H2B revealed that Spt16M makes contact with residues I39 and Y42 of H2B in the dimer (16). While the corresponding tyrosine at residue 42 is conserved between Xenopus H2B (xH2B) and yH2B, the isoleucine at position 39 of xH2B is a serine in yH2B (S42). However, another study that used different biochemical methods suggests that yeast Spt16M binds H2A-H2B poorly and that H3-H4 is the main interaction site of Spt16M in the nucleosome (41). It is not clear how well conserved the interface between Spt16 and H2B is among different species, but the

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proximity of I39 to the HBR domain makes it possible that the HBR domain is important for FACT activity. FACT is required to reestablish nucleosome positioning in the wake of RNAPII transcription. Inactivation of FACT impairs the fidelity of transcription initiation, leading to the production of cryptic transcripts from the coding region of certain genes, such as FLO8 (42). To provide evidence that FACT activity is impaired in ⌬HBR mutants, the lengths of transcripts produced from FLO8 were examined. Shifting of the temperature-sensitive spt16-197 mutant to 37°C resulted in the appearance of two cryptic transcripts from FLO8, especially the shorter transcript, as described by others (42) (Fig. 4A). Significantly, the same phenotype was also observed in ⌬HBR mutants, albeit to a lesser extent. As controls, we examined the production of cryptic transcripts from FLO8 in two other histone tail mutant strains, H2B ⌬1-32 and H2A ⌬4-20. Even though biochemical studies indicate that the N-terminal tails of histones H2A and H2B are required for optimal FACT binding to recombinant dimers (18), we did not detect an increase in cryptic transcripts in these mutants. The failure to detect cryptic transcripts in the

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FIG 2 The HBR promotes nucleosome disassembly during transcriptional activation. Nucleosome densities under repressed and activated conditions are shown. Cross-linking of H2A (A) and H3 (B) was measured at the GAL1 promoter in cells grown in medium containing 2% dextrose (Dex) or 2% galactose (Gal). (C) Cross-linking of H3 within the ORF of GAL1 in cells grown in medium containing 2% dextrose or 2% galactose. (D) Nucleosome density at the promoters of PMA1 and PYK1 was measured by ChIP with antibodies to H3. The time course of H2A (E) and H3 (F) eviction at the GAL1 promoter during activation is shown. The experiment was performed as described in the legend to Fig. 1B. IP, immunoprecipitation.

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promoter in cells grown in medium containing dextrose (Dex) or galactose (Gal) was analyzed by ChIP. A polyclonal antibody raised against Swi2 was used (66, 67). IP, immunoprecipitation. (B) Nucleosome reconstitution. Chromatin was assembled onto a DNA fragment consisting of the 601 nucleosome-positioning sequence flanked by 24- and 69-bp linker DNA sequences. The resolution of free DNA (left), wild-type nucleosomes (Nuc, middle), and ⌬HBR mutant nucleosomes (⌬30-37, right) on native gels is shown. DNA was visualized by ethidium bromide staining. (C) SWI/SNF remodeling of wild-type or ⌬HBR mutant yeast nucleosomes (nuc). Fifty femtomoles of nucleosomes was incubated with 50 or 150 fmol of SWI/SNF, and the remodeled species were resolved on native gels. The migration of nucleosomes positioned in the middle or at the ends of the DNA fragment is indicated on the left. The arrow marks the location of the fully slid nucleosome. (D) Quantification of the fraction of the total amount of nucleosomes that is remodeled nucleosomes. The signal in the region where the remodeled species migrates (arrow) in each lane of the nucleosome-alone sample (no SWI/SNF) was subtracted from the total. The results represent the means and standard deviations of three independent experiments.

single mutants may be due to functional redundancy of the H2A and H2B tails. This was observed in a study measuring replication-dependent chromatin assembly, which is thought to require FACT (43). It has been reported that FACT dysfunction triggers the accumulation of free histones within the cell because they are not assembled into chromatin efficiently. This leads to an increase in the association of free histones with other histone chaperones in the soluble fraction. For example, an increase in the association of histone H3-H4 with Asf1 was observed in FACT mutants (44). Since our results thus far suggest that deletion of the HBR domain may impair FACT activity in vivo, we asked if there was also an increase in the association of histones with Asf1. Myc-tagged Asf1 was immunoprecipitated from cell extracts prepared from wildtype and HBR mutant cells, and the levels of H3 were analyzed by Western blotting. Deletion of the HBR domain did not affect the level of Asf1-Myc or H3 within the cells, but substantially more histone H3 was associated with Asf1 in ⌬HBR mutant cells than in wild-type cells (Fig. 4B). The accumulation of nonnucleosomal histones is toxic to cells and leads to DNA replication defects and HU sensitivity (45). We examined the sensitivity of the HBR domain mutants to HU, a drug that blocks DNA replication. Similar to FACT mutants (13), both ⌬HBR mutants were highly sensitive to HU, implying a de-

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fect in the replication stress response (Fig. 4C). In contrast, deletion of amino acids 1 to 32 of H2B did not result in HU sensitivity (46). Collectively, our phenotypic analyses suggests that the HBR domain may be important for FACT activity and that residues located between 32 and 37 in the H2B tail are particularly important for this function. The HBR domain is important for FACT histone chaperone activity. The similar phenotypes of the ⌬HBR and FACT mutants prompted us to determine if the HBR domain is required for FACT activity. FACT is recruited to GAL1 under inducing conditions (47, 48), and the histone eviction defect in the ⌬HBR mutant cells could be caused by reduced recruitment of the chaperone to genes. Mutations in histone H3 adversely affected FACT recruitment, and deletion of the HBR domain could do the same (48, 49). We examined FACT recruitment with the ChIP assay and found that FACT is recruited to GAL1 in the ⌬HBR mutants, as well as in the wild-type strain (Fig. 5A). Since DNA binding proteins and transcription factors can recruit FACT, just getting FACT to promoters cannot ensure that it can recognize or act on ⌬HBR mutant nucleosomes. We turned to more direct assays to measure FACT-nucleosome interactions with purified components. We analyzed the ability of FACT to bind to ⌬HBR mutant nucleosomes with pulldown and gel mobility shift assays. Purified nucleosomes isolated from wild-type or

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FIG 3 Deletion of the HBR domain does not affect SWI/SNF recruitment or ATP-dependent chromatin remodeling. (A) Recruitment of SWI/SNF to the GAL1

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⌬HBR deletion mutant cells were incubated with immobilized FACT, and the amount of nucleosomes in the bound fraction was detected by Western blotting. Immobilized FACT bound equivalent amounts of the wild-type and ⌬HBR mutant nucleosomes (Fig. 5B), suggesting that the HBR domain is dispensable for FACT-nucleosome interactions. The association between FACT and nucleosomes was also measured by an electrophoretic gel mobility shift assay with radiolabeled NCPs. yFACT is composed of

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FIG 4 HBR mutants display FACT mutant phenotypes. (A) Northern blot analysis of FLO8 mRNA. Blots were probed with sequences complementary to the 3= region of FLO8. The full-length (F.L.) and short cryptic transcripts produced from FLO8 are indicated. The scR1 signal was used as a loading control. The histone tail mutant strains (first five lanes) were grown at 30°C. All samples were analyzed on the same gel, but redundant samples were removed from the image by splicing, indicated by the black lines. (B) Analysis of the amount of histone H3 associated with Asf1. Asf1-myc/H3-H4 complexes were immunoprecipitated (IP) with 9E10 antibody, and the amounts of Asf1 and H3 were detected by Western blotting. Two amounts of each sample were loaded, 1⫻ and 2⫻. (C) Spot tests for growth on HU. Serial dilutions of wild-type and mutant strains were spotted onto YPAD medium (YP medium supplemented with adenine sulfate and 2% dextrose) or YPAD medium plus 50 mM HU and incubated at 30°C for 48 h.

Spt16 and Pob3. The Spt16-Pob3 complex does not bind stably to nucleosomes in gel mobility shift assays but needs the addition of Nhp6 to do so (26, 50). Nhp6 provides the DNA-binding activity for the yeast Spt16-Pob3 complex, and when they are combined, they are referred to as SPN (Spt16, Pob3 and Nhp6). First, we confirmed the known NCP-binding activities of FACT, Nhp6, and SPN. Incubation of FACT alone with NCPs failed to produce a FACT-NCP complex (Fig. 5C, lane 2), but Nhp6 alone supershifted the nucleosome (Fig. 5C, lane 3). Addition of FACT and Nhp6 formed two NCP-containing complexes, Nhp6-NCP and NCP-SPN (Fig. 5C, lane 4). The NCP complexes formed with these combinations of proteins are the same as those reported by others (26). Having validated our system, we then analyzed the binding of FACT to wild-type and ⌬HBR mutant NCPs. FACT and Nhp6 were mixed in a 1:1 ratio and titrated into a binding assay with wild-type and mutant nucleosomes. The FACT-Nhp6 complex (SPN) bound wild-type and mutant nucleosomes equally well (Fig. 5D). The cause of the progressively retarded mobility of the complexes with the addition of more FACT-Nhp6 is unclear, but this effect has been observed by others (26). Nonetheless, the results suggest that the HBR domain has no detectable effect on the ability of SPN (FACT plus Nhp6) to recognize the nucleosome. Although we found that FACT bound ⌬HBR mutant, as well as wild-type, NCPs (Fig. 5B and D), it is well documented that FACT recognizes multiple features of the nucleosome, including the Nterminal tails of histones H3 and H4 and a stirrup-like “docking domain” located in the C-terminal domain of histone H2A (12, 13, 51). These interactions could compensate for reduced interaction between FACT and HBR domainless H2B in biochemical assays, so we next examined the ability of FACT to bind recombinant H2A-H2B dimers. TAP-tagged, purified FACT was incubated with recombinant wild-type and ⌬HBR mutant dimers in solution, and then the complexes were captured on calmodulinSepharose beads. The beads were washed, and the amount of dimer retained was analyzed by SDS-PAGE. Strikingly, the results show that FACT interacted poorly with the ⌬HBR mutant dimers (Fig. 6A). The small amount of mutant dimer in the bound fraction was equivalent to background binding (last two lanes). Pulldown assays are a tried-and-true method for analyzing the binding of histone chaperones to nucleosomal components. However, they are not very effective at measuring affinity constants and the obligatory washing step takes the binding components out of equilibrium conditions. Therefore, we also measured the affinity of FACT for H2A-H2B dimers with a recently developed fluorescence-based dequenching assay that has been used to measure histone-chaperone affinity constants more accurately (30). yFACT was titrated into a binding assay with recombinant dimers containing Alexa 488-conjugated histone H2A. yFACT bound wild-type yeast H2A-H2B with a Kd of 21.4 ⫾ 2.1 nM (Fig. 6B), a value close to that reported by others (41). The affinity of FACT for ⌬HBR mutant dimers was only slightly lower; the Kd was 25.4 ⫾ 5.9 nM. The difference between the equilibrium constants of wild-type and mutant dimers was smaller than expected on the basis of the pulldown assay results but not unprecedented. The equilibrium dissociation constants of Spt16M and H2A-H2B in the presence and absence of the H2B tail (residues 1 to 23) are similar, but removal of the tail disrupted the association between the dimer and Spt16M during size exclusion chromatography and greatly reduced the stability of the Spt16M-dimer interaction

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or galactose (Gal). IP, immunoprecipitation. (B) FACT binding to nucleosomes. Spt16-TAP bound to IgG or IgG resin only (control) was incubated with wild-type or ⌬HBR mutant nucleosomes prepared from cells. The bound proteins were analyzed by SDS-PAGE. The amount of Spt16/Pob3 in the upper panel was visualized by silver staining; the lower panel is a Western blot of the same samples probed with anti-H3 antibody. The band marked by an asterisk in the upper panel is the eluted IgG heavy chain. (C). Histones were reconstituted into NCPs onto a radiolabeled 147-bp 601 sequence. FACT, Nhp6, and SPN (FACT and Nhp6) were added, and the complexes were resolved on native gels. The weak-intensity retarded species observed with FACT alone (lane 2) most likely resulted from FACT binding to the naked DNA remaining in the nucleosome reconstitution. (D) Wild-type and ⌬HBR mutant nucleosomes were incubated with increasing amounts of FACT and Nhp6 at a 1:1 ratio.

measured by pulldown assays (16). An explanation for our results is that the HBR domain is required for the kinetic stability of the H2B-FACT interaction. Decreased stability may negatively impact the ability of FACT to compete with DNA within the nucleosome (see Discussion). To test for defects in FACT chaperone activity, we established an in vitro histone chaperone/eviction assay with recombinant yeast nucleosomes immobilized on magnetic beads and purified FACT. FACT was incubated with immobilized nucleosomes, and the amounts of H2B and H3 released were monitored by Western blotting (Fig. 6C). Consistent with previous observations suggesting that FACT is an H2A-H2B chaperone during transcription (12–15, 52), a concentration-dependent release of H2B from wildtype nucleosomes was observed (Fig. 6C and D). In contrast, no release of H3 was detected (Fig. 6C). Even though FACT binds free H3-H4 (12, 13), the combined effects of tighter tetramer-DNA interactions and the lower affinity of FACT for the tetramer may prevent FACT from removing tetramers from DNA (18). Strikingly, while some mutant H2A-H2B dimer was released from nucleosomes in the presence of FACT, the amount was 3- to 5-fold less than what was released from wild-type nucleosomes (Fig. 6C

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and D). These results suggest that the HBR domain is required for the histone chaperone activity of FACT, and deleting it impairs nucleosome eviction from promoters and the restoration of chromatin during transcription. DISCUSSION

The N-terminal tails of histones H2A and H2B show considerable sequence divergence among species (5, 6). The HBR is the only conserved region in the N terminus of H2B, suggesting that it has structural and functional importance. Why this region was so well conserved throughout evolution is not known. The HBR has been implicated in both repression and activation. Even here we report that deletion of the HBR domain causes both derepression of GAL1 under noninducing conditions and defective activation and nucleosome disassembly. These opposite effects can be explained by the difference between the effects of the mutation on positioning and occupancy. Deletion of the HBR domain and alteration of contacts between the dimer and DNA could weaken the positioning of nucleosomes over the promoters, causing a shift in a promoter nucleosome, exposing it to the general transcription machinery in a fraction of the cells in the population. The increase in

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FIG 5 FACT binds ⌬HBR mutant nucleosomes. (A) FACT (Spt16-myc) recruitment to the GAL1 promoter in cells grown in medium containing dextrose (Dex)

HBR Mediates FACT Activity

mRNA would arise from this small population. However, in this scenario, nucleosome occupancy is not strongly affected in most cells. Full activation of a gene requires extensive disassembly of nucleosomes over the promoter, which would require FACT. Weakened dimer-DNA interactions cannot explain the impaired disassembly of nucleosomes at promoters or the reduced removal of dimers from DNA by FACT in vitro. One would expect that a weakening dimer-DNA interaction would make it easier for FACT to remove dimers. This is not the case. We propose that the HBR domain is important for the histone chaperone FACT to remove and replace H2A-H2B dimers from nucleosomes at promoters and ORFs, respectively. Our study has uncovered a previously unappreciated role for the HBR domain in regulating chromatin structure, which involves, at least in part, reducing the ability of FACT to disrupt nucleosomes. While deletion of the HBR domain could affect other chromatin remodeling complexes not tested here, we make a case for its role in the regulation of FACT activity below. The HBR is important for FACT activity. FACT binds to multiple surfaces on the nucleosome, including the flexible tails and

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histone surfaces buried under DNA (12, 13). There are multiple models proposed for how FACT functions; most require contact between FACT and histones, especially the H2A-H2B dimer. Genetic studies have identified mutations in H3 or H4 that implicate FACT in disruption of the nucleosome structure, including some that alter the binding of FACT to genes (49, 53). Histone mutations weakening the association of the dimer with the nucleosome suppress FACT defects, highlighting the importance of FACT’s action on the dimer specifically (54). Removal of the N-terminal tails of Xenopus H2A (residues 1 to 14) and H2B (residues 1 to 23) reduced the binding affinity of human FACT and the dimer approximately 7-fold, suggesting that the tails are important for FACT function (18). However, it still not clear if the flexible regions of the tail are important for FACT function in vivo. Deletion of most of the tail (residues 1 to 32) causes no known phenotypes, let alone FACT phenotypes (this study; 6, 46). Likewise, FACT was able to stimulate RNAPII transcription through a nucleosome lacking the N-terminal tail of H2B (residues 1 to 23), suggesting that it acts on surfaces buried under the DNA (55). Together, this suggests that a region(s) other

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FIG 6 The HBR domain is important for FACT chaperone activity. (A) FACT binding to recombinant H2A-H2B dimers. TAP-purified FACT was incubated with recombinant wild-type or ⌬HBR mutant dimers in solution and then captured on calmodulin-Sepharose beads. The control lanes contained dimer but no FACT. The bound proteins were resolved by SDS-PAGE and visualized by Coomassie blue staining. An unloaded lane was removed, which is indicated by the location of the black line. (B) Binding of FACT and recombinant dimers by the HI-FI assay. FACT was titrated into binding assays containing 1 nM dimer containing Alexa 488-labled H2A. The results are presented as the average and standard deviations from six determinations. (C) FACT evicts H2A-H2B dimers from recombinant yeast nucleosomes. Immobilized wild-type and ⌬HBR mutant nucleosomes were incubated with purified FACT. After 60 min, the immobilized nucleosomes were collected and the supernatant was immunoblotted with antibodies to H2B and H3. The asterisk marks the location of the residual endogenous wild-type H2B that copurified with FACT. (D) Quantification of H2B eviction. The amount of FACT-stimulated removal of H2B is plotted as the means and standard deviations from three different experiments. The amount of endogenous H2B copurifying with FACT (asterisk in panel C) was subtracted from the signals of wild-type H2B.

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(residues 1 to 23) and residues 39 to 42. FACT first binds the H2B tail, and as more dimer surface is exposed, it contacts the HBR domain and then finally the residues identified in the X-ray structure (I39 to Y42), locking it into place. In the case of the HBR domain mutant, FACT may be impaired in fully accessing the entire dimer-DNA interface, preventing the full displacement of the DNA from the dimer. A simple analogy to a set of gears with interlocking cogs can be drawn. In the absence of the middle cog (HBR), the gears slip and spin unproductively. This may explain why FACT can engage the ⌬HBR mutant nucleosome but is unable to remove the dimer in vitro. Likewise, the same is observed in cells, where FACT is recruited to GAL1 but removes ⌬HBR mutant nucleosomes poorly. This suggests that the missing component leading to complete removal of the mutant nucleosome in vivo is the reduced ability of FACT to progress far enough into the nucleosome to compete fully with DNA on the dimer surface. Implications for the collaboration with chromatin remodelers in nucleosome disassembly and assembly in the promoter and ORFs. FACT has been implicated in both the removal of nucleosomes from promoters and the reestablishment of their structure in the wake of elongating RNAPII (12, 13). These two activities seem somewhat at odds with each other. Why does FACT act differently at the promoters, where the result is nucleosome removal, and within ORFs, where they are retained? How FACT acts on nucleosomes and the product it generates may depend on how it is recruited to its site of action or the engine that exposes FACT to the dimer. There are a few known factors implicated in collaborating with FACT to remodel nucleosomes during transcription. At promoters, ATP-dependent chromatin remodelers expose histone surfaces to FACT. The species generated by ATP-dependent remodeling could favor dimer eviction rather than retention. Human FACT and SWI/SNF work together to support transcription in vitro (57), and GAL1, HO, and RNR3 require both FACT and SWI/SNF for nucleosome eviction in cells (15, 58, 59; unpublished data). On the other hand, RNAPII drives the reorganization of the nucleosome during transcription. Recent evidence suggests that FACT aids in the survival of the nucleosome by retaining H2A-H2B behind polymerase under some conditions (17). RNAPII may generate nucleosome-DNA structures that favor FACT binding to DNA, RNAPII, and dimer simultaneously to preserve the dimer close to its site of reassembly. Collaboration with ATP-dependent remodelers could occur in the ORFs of genes, as well to reestablish nucleosome positioning or repair partially formed chromatin after polymerase has passed or during the process of histone exchange. A physical interaction between yFACT and Chd1 has been described, and Chd1 is believed to act on chromatin during elongation (60, 61). The ability of FACT to bind multiple nucleosomal surfaces by using a combination of its subunits and domains could allow flexibility in its mechanism of action. This may explain why there is solid support for different models of FACT function. Regulation of FACT activity through the HBR domain. The HBR domain was defined by mutagenesis studies to contain the amino acid sequence KKRSKARK (7). Whether or not this region plays a broader role in chromatin transactions other than just mediating the action of FACT is not clear and is beyond the scope of our study. The development of more powerful methods to detect histone posttranslational modifications has identified residues within or adjacent to the HBR domain that are modified. Methylation and acetylation of lysine 37 have been detected (62,

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than the flexible tail of H2B is required for FACT activity. We provide evidence that one additional surface is the HBR domain. While this report was in preparation, another group demonstrated that Spt16M binds peptides from xH2B containing residues 14 to 33, which partially overlaps the HBR domain (16). Furthermore, the accompanying cocrystal structure and biochemical analysis in that work provide solid evidence that the middle domain of Spt16 (Spt16M) from C. thermophilum makes direct contact with I39 and Y42 in xH2B (16). While I39 and Y42 are not within the HBR domain, they are adjacent to it. It is worth noting that that Y42 is conserved in yeast (Y45), I39 is not. Yeast has a serine residue in its place. Thus, there are still some unanswered questions about how well conserved these contacts are. Mutations in residues of Spt16M that weakened dimer binding in vitro could not support viability, suggesting that the contacts observed in the crystal structure are important for function. However, mutations in H2B predicted to disrupt Spt16M-H2B interactions were not examined. Despite the growing biochemical evidence suggesting that residues in H2B are important for the recognition of the dimer by FACT, to our knowledge, no H2B mutants have been characterized that provide the “smoking gun” linking the binding of FACT to H2B and its mechanism of action in cells. We provide multiple lines of corroborating evidence indicating that the HBR domain is important for FACT activity in vivo and in vitro. The HBR mutant shows many FACT mutant phenotypes in vivo, namely, a nucleosome removal defect at promoters, an increase in cryptic transcripts, HU sensitivity and increased association of free histones with histone chaperones. The phenotypic similarities between HBR and FACT mutants, together with the biochemical evidence presented here, make a strong case that the HBR domain is important for FACT to act on chromatin. How the HBR domain aids FACT in disrupting the nucleosome structure. A model gaining favor in the field is one where FACT shields surfaces on the dimer from DNA. FACT competes with nonnucleosomal H2A-H2B for DNA, suggesting that binding of the two is not possible (18). Furthermore, evidence was recently provided that human FACT displaces DNA from H2AH2B dimer to aid RNAPII transcription (17). Therefore, a body of evidence is growing that suggests that breaking of dimer-DNA interactions is contributing to FACT’s abilities to disrupt chromatin and act as a chaperone. The HBR is buried by DNA in the nucleosome, passing through the gyres (56). Naturally, the binding of FACT and that of DNA to the HBR domain are mutually exclusive; thus, DNA would need to be removed from the surface of the dimer before FACT could engage the HBR domain. Such competition could explain some of our biochemical results. Deletion of the HBR domain reduces the stability of the FACT-dimer interaction, allowing FACT to compete poorly with DNA and favoring DNA-H2B interaction. In this scenario, FACT would be less effective at stimulating the removal of mutant dimer in chaperone assays, which was observed in this study (Fig. 6). The cocrystal structure of Spt16M with the dimer has given rise to a model where FACT initially makes contact with H2B through interactions with the exposed flexible tails and progressively enters the nucleosome and displaces DNA from the dimer as the end of the DNA breathes off the nucleosome (16). We propose a modified version that introduces the importance of the HBR domain. The location of the HBR domain places it at the center of the two regions implicated in FACT-dimer interactions, the flexible tails

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16.

ACKNOWLEDGMENTS

18.

We thank John Wyrick (Washington State University) and Fred Winston (Harvard Medical School) for strains and plasmids used in this study. Tim Formosa is acknowledged for the Nhp6 expression plasmid and protocol and the histone H2AQ114C plasmid. The members of the Center for Eukaryotic Gene Regulation are recognized for their comments during the completion of this work. This research was supported by funds from National Institutes of Health (GM58672) to J.C.R.

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A highly conserved region within H2B is important for FACT to act on nucleosomes.

Histone N-terminal tails play crucial roles in chromatin-related processes. The tails of histones H3 and H4 are highly conserved and well characterize...
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