In Vivo Mapping of FACTâHistone Interactions ... - ACS Publications

Article

In vivo mapping of FACT-histone interactions identifies a role of Pob3 C-terminus in H2A-H2B binding ACS Chemical Biology is published by the Chemical Society. 1155 Subscriber access provided by American UNIV MASSACHUSETTS BOSTON Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Christian Hoffmann, and Heinz Neumann ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b00493 • Publication Date (Web): 28 Sep 2015 Downloaded from http://pubs.acs.org on September 30, 2015 ACS Chemical Biology is published by the Chemical Society. 1155 Subscriber access provided by American UNIV MASSACHUSETTS BOSTON Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical ACS Chemical Biology is published by the Chemical Society. 1155 Subscriber access provided by American UNIV MASSACHUSETTS BOSTON Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all ACS Chemical is published American Chemical Society. 1155 readers and citable by the Digital Object Identifier (DOI®). “JustBiology Accepted” is by anthe optional service offered Subscriber access provided by UNIV MASSACHUSETTS BOSTON Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers ACS Chemical Biology is published by the Chemical Society. 1155 Subscriber access provided by American UNIV MASSACHUSETTS BOSTON Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Biology is published by the Chemical Society. 1155 Subscriber access provided by American UNIV MASSACHUSETTS BOSTON Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Pob3-CTD Page 1 of 35

- disordered -

Pob3

Spt16 1 FACT histone chaperone 2 3 4 Chromatin rearrangments 5 6 7 8 9Replication Repair Transcription

Pob3ACS Chemical Biology

UV crosslinking in yeast

Impact of Pob3-CTD on FACT function

+ hν (~ 360nm)

H2

Pob3CTD

Importin-α

A-

O NH

HN

HO

R

O H N

H2

B

dim

er

Po CT b3 D

Pob3 Spt16

Pob3-CTD - structured -

ACS Paragon Plus Environment Yeast

ACS Chemical Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

In vivo mapping of FACT-histone interactions identifies a role of Pob3 C-terminus in H2A-H2B binding

Christian Hoffmann and Heinz Neumann1,*

1

Free Floater (Junior) Research Group “Applied Synthetic Biology”

Georg-August University Göttingen Institute for Microbiology and Genetics Justus-von-Liebig Weg 11 37077 Göttingen, Germany

*Contact: H. Neumann, [email protected], Tel.: +49-551-39-14088

Keywords: FACT complex, genetic code expansion, crosslinking, nuclear transport, replication

1 ACS Paragon Plus Environment

Page 2 of 35

Page 3 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ABSTRACT Histone chaperones assist nucleosomal rearrangements to facilitate the passage of DNA and RNA polymerases through chromatin. The FACT (facilitates chromatin transcription) complex is a conserved histone chaperone involved in transcription, replication and repair. The complex consists of two major subunits, Spt16 and SSRP1/Pob3 in mammals and yeast, which engage histones and DNA by multiple contacts. However, the precise mechanism of FACT function is largely unclear. Here, we used the genetically installed UV-activatable crosslinker amino acid pBPA (pbenzoylphenylalanine) to map the interaction network of FACT in living yeast. Unexpectedly, we found the acidic C-terminus of Pob3 forming crosslinks to histone H2A and H2B most efficiently. This observation was independent of the performed crosslinking chemistry since similar histone-crosslinks were obtained using pazidophenylalanine (pAzF). Further analyses identified a C-terminal nuclear localization sequence in Pob3. Its interaction with Importin-α interfered with H2A-H2B binding, which suggests a possible regulatory role in FACT recruitment to chromatin. Deletion of acidic residues from the Pob3 C-terminus creates a hydroxyurea sensitive phenotype in budding yeast, suggesting a potential role for this domain in DNA replication.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

INTRODUCTION Eukaryotes package their genomes into chromatin using the four core histones H2A, H2B, H3 and H4 as building blocks. Two copies of each core histone complex 147 base pairs and a variable amount of linker DNA to form the nucleosome, the basic repeating unit of chromatin. Open chromatin fibers are able to fold into higher order structures, thereby condensing the genome into the tiny dimensions of the nucleus. Simultaneously, chromatin must facilitate access to the information stored in the genome by enabling polymerases to transcribe, replicate or repair DNA. Nucleosomes present a natural barrier to these processes, and sophisticated molecular machineries exist to open and reorganize chromatin on demand. These include ATP-dependent chromatin remodeling factors that reposition nucleosomes along the DNA (1), enzymes that modify histones posttranslationally (2) and ATPindependent histone chaperones (3). Histone chaperones catalyze the removal and (re)assembly of nucleosomes during the passage of polymerases. This structurally diverse class of proteins shares the ability to bind histones usually as dimers of H2A-H2B or H3-H4 and shields them from unspecific electrostatic interactions. The "facilitates chromatin transcription" (FACT) complex promotes RNA polymerase II (Pol II) dependent transcription in vitro by altering the accessibility of nucleosomal DNA (4-5). FACT is involved in all nuclear processes that require the passage of polymerases through chromatinized DNA such as replication, transcription and repair (6). Its role in transcription elongation is especially well documented by the sensitivity of yeast SPT16 mutants towards elongation inhibitors (7) and by the physical and genetic interactions of FACT with various components of the transcriptional machinery (8-12). Mechanistically, FACT is proposed to associate with nucleosomes to destabilize the interaction of histones with DNA, thereby facilitating the passage of RNA Pol II (6, 13). Eventually, FACT is


Page 4 of 35

Page 5 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


required to restore an intact chromatin landscape and thus, to prevent spontaneous initiation of transcription from normally inactive, cryptic promoters (14-16). Early experiments demonstrated that FACT facilitates in vitro transcription by RNA Pol II by removing H2A-H2B dimers from nucleosomes immediately downstream of the transcription start site (4-5, 10). This led to the hypothesis that FACT acts generally by H2A-H2B dimer displacement (dimer eviction model). An alternative model postulates that FACT tethers the octameric core during polymerase passage, thereby preserving the modification state of the nucleosome (17) (Figure 1A). The requirement of FACT for nucleosome survival is supported by the observation that FACT stabilizes the proximal or distal H2A-H2B dimer in an alternating fashion while RNA Pol II progresses around the nucleosome within a DNA-loop (18). However, the precise mechanism of FACT action is still largely unclear.

The human FACT complex is composed of two subunits, Spt16 (suppressor of Ty 16) and SSRP1 (structure-specific recognition protein 1) (Figure 1B). In yeast, the SSRP1 subunit is genetically split into Pob3 and the HMG-1 domain protein Nhp6. Spt16 is a multi-domain protein, comprising an N-terminal domain (NTD, aa 1–447),

a dimerization domain (DD, aa 527–630) and a middle domain (MD, aa 630–959).

Pob3 consists of an N-terminal domain (NTD, aa 1–220) involved in dimerization with

Spt16 and a middle domain (MD, aa 237–447). Both subunits possess an intrinsically disordered acidic C-terminal domain (CTD) (6, 13, 19). Structures of individual domains were solved by X-ray crystallography. The Spt16NTD displays structural homology to bacterial aminopeptidases but lacks peptidase activity (19-20). Both MDs contain a tandem pleckstrin homology domain (21-23). According to biochemical and structural analyses, FACT interacts with histones via several distinct contact sites. The Spt16-MD of Chaetomium thermophilum (ctSpt16) 4 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

was co-crystallized as a chimeric fusion of ctSpt16 connected to Xenopus laevis (xl) H2B by a serine-glycine linker in the presence of xlH2A from and shown to bind H2AH2B dimers with nanomolar affinity (21). The same domain also binds H3-H4 tetramers with low micromolar affinity but using a different interface (21-22). H3-H4 tetramers, but not H2A-H2B dimers, are also bound by the Spt16-NTD (20). Binding of human Spt16 to H2A-H2B dimers is enhanced by its acidic CTD (24), generating at least three sites of contact to the nucleosome by this FACT subunit alone. Pob3/SSRP1 also contributes to the binding of histones, although the roles of individual domains have not been mapped so far (24-25). Together with the HMG-1 domain of SSRP1 or the Nhp6 subunit in yeast, this creates a complex that binds nucleosomes with high affinity and avidity in a multi-dentate manner. Both subunits of the FACT complex possess an unstructured C-terminal acidic tail region. Such negatively charged regions, which occur in many histone chaperones, are often involved in binding and shielding of the positively charged histones against unspecific electrostatic interactions. The histone chaperone Asf1 binds histones H3H4 using its folded core (26), an interaction that is enhanced by its acidic C-terminal tail (27). Similar acidic CTDs occur in other histone chaperones such as Rtt106 or Nap1 (13). Deletions of the Spt16-CTD or Pob3-CTD are lethal in yeast (28-29). How these tails interact with histones, and whether this is a defined or rather an unspecific interaction, is presently little understood.

Besides histones, a plethora of further proteins were identified to interact with the FACT complex. Pob3 was initially identified through its interaction with DNA polymerase α (30) and interacts with replication factor RPA via its MD (23). Human FACT is targeted to origins of replication by its interaction with the MCM2-7 helicase (31), an association that is enhanced by Rtt101-dependent ubiquitylation of FACT in S. cerevisiae (32). Spt16 associates also with components of the transcriptional


Page 6 of 35

Page 7 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


machinery, e.g. the Chd1 chromatin remodeler and the PAF1-complex (11, 33), and co-purifies with the 19S particle of the proteasome (34).

Here, we address the relevance of these interactions in vivo by performing a crosslinking scan of the FACT complex using site-specific genetic installation of pbenzoylphenylalanine (pBPA) in Saccharomyces cerevisiae. The UV-activatable crosslinker

amino

acid

pBPA

is

encoded

by

an

evolved

orthogonal

tRNACUA/aminoacyl-tRNA synthetase pair (35). This facilitates the suppression of amber stop codons, resulting in the formation of a full-length protein containing the unnatural amino acid at the encoded site (36). We have performed a crosslinking survey encompassing more than 200 amber mutants, covering the entire length of the Spt16 and Pob3 subunits of the FACT complex. We identified several sites in the acidic C-terminus of Pob3 that crosslink to H2A or H2B. The distinct interaction pattern assigns a putative, important role in histone binding to the structurally disordered Pob3-CTD in vivo. In addition, a deletion of amino acids S491–E543 from Pob3 creates a hydroxyurea sensitive phenotype, suggesting a potential role in DNA replication. We further show that the binding of H2A-H2B by the Pob3 tail is negatively coupled to binding of Importin-α to a nuclear localization sequence (NLS) present in the C-terminus of Pob3.

RESULTS AND DISCUSSION pBPA-crosslinking scan of the FACT complex. To map the interaction network of the FACT complex in living yeast, we created a library of amber mutants in SPT16 and POB3. Therefore, the genes were cloned as fusion constructs to a C-terminal 9x-myc tag in yeast 2µ shuttle vectors (SPT16 under the control of its endogenous promoter and POB3 expression driven by the inducible GAL1 promoter). Using 6 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

sequence alignments and X-ray structural data as a guide, we introduced individual amber codons preferentially at surface-exposed, non-conserved sites of the FACT complex, creating a library of 214 amber mutants (128 in SPT16; 86 in POB3). This library can be used to map the interactome of FACT to different domains of the complex with single amino acid residue resolution. Yeast cells expressing additional plasmid borne SPT16:9myc or POB3:9myc with pBPA genetically encoded at different positions were exposed to UV-light and total protein extracts analyzed by Western blot against the myc epitope (Figure 2). We observed reproducible banding patterns of crosslink products (migrating at higher molecular weight than the free protein) for most sites of pBPA incorporation in both proteins, Spt16 and Pob3. Fulllength protein levels depended on pBPA addition to the medium and were comparable for most of the sites. The formation of the crosslink products was explicitly UV-dependent (Supplementary Figure 1). Crosslinks at adjacent sites frequently showed similar banding patterns, indicating putative interaction surfaces to other proteins (e.g. Spt16: R875–D908; S975–S1011). In some cases, distinct crosslinks formed only from individual sites of the protein, e.g. Spt16-F250, F596, E976 (Fig 1A) and Pob3-I180, R254, K271 (Figure 2B). We compared the abundance of crosslink product formation at specific sites with available structural data. As expected, positions buried in the inner core of the structure (e.g. Spt16-NTD: F117, F143, F432) were less likely to form crosslink products than surface-exposed residues (e.g. K108, K229, F250 and E426), indicating that the crosslinks are formed by folded proteins. Sites, which were recently shown to form a structurally important hydrophobic motif for histone binding (Spt16 U-turn, aa 927–956) (21), showed reduced Spt16:9myc protein levels (presumably due to interference of pBPA residues with protein folding and stability at these sites). Similar effects could be observed for a segment from Y810 to D850.


Page 8 of 35

Page 9 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Phenotyping of FACT-pBPA mutants. We tested the activity of the Spt16- and Pob3-pBPA proteins in pBPA-dependent rescue experiments of yeast strains with temperature-sensitive SPT16/POB3 alleles. Therefore, we plated serial dilutions of each amber mutant strain on media with or without pBPA and incubated them at restrictive or permissive temperatures. Plasmid borne SPT16:9myc was able to rescue growth at restrictive temperature (37°C), while the SPT16 amber mutants displayed different levels of pBPA-dependent growth (Figure 3A and Supplementary Figure 2A, Supplementary Table 1). This demonstrates that the majority of the pBPAcontaining Spt16 proteins can rescue the temperature-sensitive phenotype and are therefore functional. The variability in the level of rescue may reflect different efficiencies of pBPA incorporation at these sites. Some mutants rescued the temperature-sensitive growth phenotype in the absence of pBPA. This may be explained either by a positive effect of the truncated protein resulting from termination at the amber codon or by incorporation of natural amino acids, such as tryptophan, by EcBPA-tRNA synthetase (37), resulting in low levels of full-length protein even in the absence of the unnatural amino acid (Figure 5B and Supplementary Figure 1C). Incorporation of pBPA in many positions of Spt16-MD did not rescue the temperature-sensitive phenotype, in agreement with the reduced protein levels observed by Western blot for these sites (e.g. Y810–D850 and T925–

Y972) (Figure 2A). Amber mutants of the Spt16-CTD rescued spt16-ts strains very efficiently even in the absence of pBPA, indicating that truncated Spt16 can complement the defects of this allele. Also, plasmid borne POB3:9myc was able to rescue a strain harboring a pob3-L78R temperature-sensitive mutation, which causes loss of Spt16-Pob3 heterodimers due to Pob3 protein instability (Figure 3B) (29). However, growth of these cells expressing wild-type Pob3:9myc was impaired at permissive temperature (25°C), 8 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

indicating that overexpression of wild-type POB3 by the strong inducible galactose promoter affects cellular growth, as previously described (38). This effect was also observed for pBPA incorporation in parts of Pob3-MD (D240–Q278) and Pob3-CTD

(L470–S491). However, the efficiency of pBPA incorporation lowers the amount of full-length pBPA-containing Pob3:9myc protein to approximately 10% compared to plasmid-borne wild-type Pob3:9myc (Supplementary Figure 1C). This estimation is supported by a crosslinking study expressing pBPA containing histones in yeast (39). Hence, most of the mutants showed normal growth at permissive temperature and additionally rescued the pob3-L78R mutation at restrictive temperature. The rescue by approximately half of the mutants depended on pBPA addition to the medium (Supplementary Figure 2B, Supplementary Table 2). POB3-CTD amber mutants displayed a similar pBPA-independent rescue as SPT16-CTD mutants. From these experiments we conclude that most of the FACT-pBPA mutants can rescue a temperature-sensitive phenotype and, hence, are functional in vivo.

In vivo analysis of FACT-histone interactions. Biochemical and structural investigations of the FACT complex suggest that it binds nucleosomes in a multidentate fashion (24). Therefore, we analyzed the crosslink maps for bands of approximate mass of Spt16- or Pob3-histone crosslink products. Several sites of the Spt16-NTD, MD and CTD as well as Pob3-MD and CTD gave rise to crosslink products of 10–20 kDa higher molecular weight than the full length protein, matching the additional mass of a histone (Figure 2). To test whether these crosslinks are indeed directed to histones, we carried out band shift assays using yeast strains harboring genomically tagged histones. The additional mass of the tag would result in a supershift of the crosslink to a higher molecular weight. This approach identified strong interactions of the Pob3-CTD with histones H2A and H2B (Figure 4). Twelve 9 ACS Paragon Plus Environment

Page 10 of 35

Page 11 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of the sites tested produced crosslinks to histone H2A (e.g. D490, S500, F527), histone H2B (e.g. E510, S516) or to both histones (e.g. S507, A520). This phenomenon is not tag dependent, as similar results were obtained using Flag or GFP tags (Supplementary Figure 3). A shift experiment in a H3:TAP strain showed no supershift indicating that the Pob3-CTD is specific for H2A-H2B dimer interactions. This site- and histone-specific crosslinking behavior suggests that the intrinsically disordered acidic CTD is engaged in a structurally defined interaction with the H2A-H2B dimer in vivo. This finding was independent of the crosslinking chemistry, as we observed the same Pob3 S500pBPA-histone crosslink using p-azidophenylalanine (pAzF) (35), which is less bulky and hydrophobic than pBPA and reacts by a different mechanism (Figure 5A). The production of full-length Pob3:9myc protein was specific for each evolved tRNA/tRNA-synthetase pair (pAzF/pBPA) and its cognate unnatural amino acid (Figure 5B). The Pob3-H2A crosslink yield was substantially lower using pAzF compared to pBPA at Serine-500, while levels of full-length protein produced were similar. This might be due to the reversible excitability of pBPA in comparison to pAzF, which decomposes upon irradiation and is additionally prone to the reduction of the azide functionality to a primary amine.

The Spt16-CTD also produced crosslink products, which would match the mass of a crosslink to a histone, although with significantly lower efficiency. Surprisingly, none of these crosslinks displayed a reduced migration speed in strain backgrounds with tagged versions of core histones (Supplementary Figure 4). This is in stark contrast with published literature, which suggests that the Spt16-CTD is important for H2AH2B dimer binding. Several of the sites in the Spt16-MD that produced stable protein upon pBPA incorporation also gave rise to crosslinks whose size would match histone interactions (e.g. F878, K881, D908). However, none of these sites reacted



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

to histone tagging. Many of the sites within the U-turn motif, which binds H2Bα1 in the crystal structure, unfortunately did not produce full-length Spt16. We specifically probed Q938, W950 and Y972, but neither of these sites displayed a gel shift indicating the presence of H2A or H2B crosslinks. To determine the subcellular localization of the Pob3-H2A-H2B interaction, we fractionated yeast cells after crosslinking by density gradient centrifugation to separate cytosolic and nuclear proteins (Figure 6A). The Pob3 S500pBPA-H2A crosslink was predominantly found in the nuclear fraction. The H2A-crosslink showed approximately fourfold enrichment in nuclei with respect to the amount of full-length Pob3 S500pBPA protein present (Figure 6B and Supplementary Figure 5). This suggests that the crosslink traps the FACT complex while engaged with chromatin.

The interaction of Pob3-CTD with H2A-H2B dimers competes with binding of its NLS to importin-α α in vitro. In order to further characterize the interaction of Pob3 with the histone H2A-H2B dimer, we reconstituted crosslink formation with purified proteins expressed in E. coli. We expressed Chaetomium thermophilum Pob3 (ctPob3) with an N-terminal His6-tag harboring genetically encoded pBPA at several positions. Crosslink studies were performed in the presence of a 2.5-fold molar excess of recombinantly expressed and refolded Xenopus laevis histone H2A-H2B dimers (Figure 7A). Distinct UV-dependent crosslink products formed when pBPA replaced S482, S496 and Y520 in ctPob3-CTD (corresponding to residues S491, S500 and F527 in S. cerevisiae, see Supplementary Figure 6A). In comparison, pBPA incorporation at position Y181 (corresponding to Y175 in scPob3) did not result in detectable crosslink formation. Titration of H2A-H2B dimers indicates an affinity of the Pob3-CTD for these histones in the low micromolar range (Figure 7B). Similar crosslinking studies with H3-H4 tetramers failed due to precipitation of the histones. These in vitro crosslinking experiments substantiate our in vivo findings and 11 ACS Paragon Plus Environment

Page 12 of 35

Page 13 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


suggest that the Pob3 C-terminus is functionally important for FACT mediated nucleosome rearrangement. Previous studies have revealed an essential role of the CTDs of the FACT complex (4, 24, 28-29). Schlesinger and Formosa isolated heat-sensitive yeast strains with mutations affecting C-terminal residues of Pob3 (K548Stop or K547M). Using bioinformatics (40), we identified a C-terminal NLS in Pob3 residues 544– –552 (RPSKKPKVE), which scored 9.5 out of 10 in an NLS cut-off score, clearly indicating the presence of an Importin-α dependent monopartite nuclear import sequence. To test this prediction, we created plasmid-borne Pob3-GFP fusion constructs with mutations in the CTD and the putative localization signal (Figure 8A). Fluorescent microscopy of living yeast cells expressing plasmid-borne copies of POB3:GFP showed nuclear localization of the fusion construct (Figure 8B, b). This nuclear localization was lost in mutants lacking the CTD including the NLS (c, Δ458–552) as well as in mutants lacking the putative NLS alone (d, Δ544–552). Indeed, a single point mutation (e, K547M) in the Importin-α NLS consensus-sequence (K-[K/R]-X[K/R]) was sufficient to cause delocalization of Pob3-GFP. As expected, the localization was not affected by deleting the acidic residues of the CTD (f, Δ458– 543), leaving the putative NLS untouched (544–552). Thus, nuclear targeting of the FACT complex in S. cerevisiae is directed by an NLS in the very C-terminus of Pob3. Furthermore, recombinant human GST-Importin-α ΔIBB (without auto-inhibitory Nterminal Importin-β-binding domain) formed a stable complex with His6-ctPob3, as demonstrated by analytical size-exclusion chromatography. In contrast, a mutant (His6-ctPob3ΔD467–G571) lacking the NLS (aa 563–571: RPKKKKKTG) did not coelute with human Importin-α (Figure 8C and Supplementary Figure 6B and C). 12 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Since we found interactions of the acidic Pob3-CTD with histone H2A-H2B dimers and identified an NLS in close proximity, we asked whether these interactions are mutually exclusive and might reveal another degree of regulation of FACT binding to histones. Therefore, we performed in vitro crosslinking assays of ctPob3-S496pBPA with histone H2A-H2B dimers in the presence or absence of GST-Importin-α ∆IBB (Figure 8D and Supplementary Figure 7). Binding of the ctPob3-NLS to Importin-α reduced the ability to crosslink to the H2A-H2B dimer by approximately twofold, indicating a mutually inhibitory binding of H2A-H2B dimers or Importin-α by the Pob3CTD.

The Pob3-CTD is involved in DNA replication. To investigate the functional relevance of the Pob3-CTD–H2A-H2B interaction we created yeast strains with genomic deletions in the CTD (Figure 9). These POB3 mutants retained the NLS but lacked parts of the acidic unstructured C-terminus. Deletion of residues Q458–E543 was lethal, probably because of the removal of functionally important residues adjacent to the Pob3-MD. Strains with smaller deletions (ΔS491–E543 or ΔA501– E543) grew normally in rich medium at all temperatures tested. In contrast to pob3L78R cells, both strains displayed wild-type like sensitivity to DNA damaging agent methyl methanesulfonate (MMS), indicating that the Pob3-CTD is not required for DNA damage repair processes (Supplementary Figure 8). Similar observations were made with the transcription inhibitor 6-azauracil. However, pob3 ΔS491–E543 showed more than tenfold higher sensitivity to hydroxyurea (HU) compared to wildtype and pob3 ΔA501–E543. HU inhibits ribonucleotide reductase, thereby depleting dNTP pools, which leads to stalling of replication forks. This replicative stress is less


Page 14 of 35

Page 15 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


well tolerated by pob3 ΔS491–E543, indicating that the Pob3-CTD may have a specific role in DNA replication that is mechanistically distinct from its function in transcription and DNA repair.

CONCLUSION In summary, we performed a crosslinking scan of the FACT complex in vivo using the genetically encoded UV-activatable crosslinker amino acid pBPA. The candidate approach employed in this study, optimized for medium throughput using 48-well blocks for yeast transformation, growth, crosslinking and extraction of total proteins, facilitates the identification of crosslink partners by gel shifts. To accelerate the identification, we are presently establishing the analysis of pBPA-crosslinks by quantitative mass spectrometry. Previously unknown interactions could then be analyzed in the context of mutant strain backgrounds or with respect to their cell cycle dependence with single amino acid resolution (39). Moreover, the library of amber mutants created here is not restricted in its use for the incorporation of pBPA but can also be employed to encode bioorthogonal chemical handles for labeling with fluorophores, posttranslational modifications or to engineer a photo-activatable FACT complex (36). Analyzing our pBPA crosslinking map for FACT-histone interactions we identified, to our surprise, only a single domain, the Pob3-CTD, that efficiently crosslinked to histones H2A-H2B. This is in stark contrast to previous biochemical work that observed numerous interaction sites of the FACT complex with core histones. For example, the Spt16-NTD interacts with H3-H4 (20). A recent crystal structure of ctSpt16-MD identified a U-turn motif required for H2A-H2B binding together with an affinity for H3-H4 (21). The same domain of scSpt16 was shown to bind H3-H4 (22). Several studies described a critical electrostatic interaction of Spt16-CTD with H2A-



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

H2B (4, 21, 24). Genetic studies suggest that Spt16-NTD and Pob3-MD synergistically interact with the H2A docking domain (19). The absence of further FACT-histone crosslinks in our survey might be explained if the interactions of other FACT domains with histones are only transient or regulated by posttranslational modifications (32, 41). Some interactions might be concealed in asynchronous cell populations if an interaction occurs only in a minor fraction of the whole protein population. Posttranslational modifications, both on FACT and on histones, diversify FACT's activity and direct it to different cellular pools. For example, FACT facilitates the removal of H2B K123ub (H2B K120 in human) and thereby promotes transcription elongation (42-43). H3 K36 trimethylation, deposited by Set2 on active genes, promotes FACT recruitment and thereby facilitates transcription (44). FACT itself is modified by serine and threonine phosphorylation, lysine acetylation and sumoylation (45-50). In budding yeast, ubiquitylation of Spt16 by Rtt101 regulates FACT during replication and enhances the association of FACT with MCM helicase at replication origins (32).

This diversification of the FACT pool might explain why we do not observe several expected FACT-histone interactions. A technical reason for the absence of further FACT-histone crosslinks could also be that the fusion tags at the histones used in the shift experiments interfere with the interactions, although this should have resulted in a reduction of the intensity of the crosslink product in comparison to the untagged strain. Regarding the observed FACT-histone interaction, we hypothesize that the Pob3-CTD constitutively binds to H2A-H2B dimers or nucleosomes, perhaps anchoring the FACT complex on chromatin, while other domains bind transiently, for example, during the passage of a polymerase. The Pob3-CTD binds the H2A-H2B dimer in a defined manner since we were able to identify sites that specifically crosslink to either H2A or H2B. The same interaction pattern was observed employing two independent crosslinking chemistries, ruling out 15 ACS Paragon Plus Environment

Page 16 of 35

Page 17 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


potential artifacts introduced by the use of a particular unnatural amino acid. Similar observations were made by Berg et al. for crosslinks formed by Hsp90 co-chaperone Aha1 in vivo (51). The interaction of Pob3-CTD with H2A-H2B dimers is differentially modulated in the cytosol and nucleus. This is probably mediated by Importin-α, which localizes to the cytosol where it interferes with the formation of a FACT-histone complex. Release of Importin-α from the NLS upon binding to Ran-GTP in the nucleus might trigger the binding of FACT to chromatin.

Our findings provide additional mechanistic insights into the role of the FACT complex in DNA replication, which was initially suggested by its identification as an interaction partner of DNA polymerase α (30, 52) and its association with the MCM27 helicase complex (31-32). Furthermore, temperature-sensitive mutants in POB3 cause hypersensitivity to HU (29), and a pob3 Q308K mutation abrogates its interaction with the DNA replication factor RPA (23). The sensitivity to hydroxyurea (but not to DNA damaging agents or inhibitors of transcription) of strains with a Cterminal Pob3 deletion (∆S491–E543) indicates a particular role of the acidic Pob3CTD in DNA replication. In light of the fact that the Pob3-CTD interaction is constitutive, we speculate that it may act as a H2A-H2B dimer reservoir, which promotes de novo assembly of nucleosomes. This would be consistent with its specific role in DNA replication where new histones are deposited on DNA in contrast to transcription or repair processes.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

METHODS Incorporation of unnatural amino acids and UV-crosslinking. Yeast cells transformed with plasmids encoding evolved E. coli TyrRS synthetase/tRNACUA pairs and amber mutants of POB3 or SPT16 were grown in the presence of the respective UAA, harvested and irradiated with UV-light on ice. Proteins were extracted by boiling the cells in SDS sample buffer and analyzed by SDS-PAGE and Western blot.

In vitro crosslinking of proteins. In vitro crosslinking assays were performed by preparing mixtures of pBPA-containing proteins with H2A-H2B dimers and subjecting them to UV crosslinking. Total protein concentrations were determined by Bradford assay. Samples were transferred to disposable UV-transparent cuvettes and subjected to 365 nm UV irradiation on ice for 30 to 45 minutes (Vilber Lourmat VL208.BL, 365 nm tubes, 2x8W). Samples were mixed with 4x Loading buffer and boiled at 95°C for 5 minutes. Samples were clarified by centrifugation for 2 minutes at 13.000 rpm and subjected to SDS-PAGE and Western blot.

Live cell imaging of yeast. Yeast cells were picked from a freshly grown plate and resuspended in a small amount of H2O (2–5 µL). Cells were spread onto a microscope slide (Superfrost Microscope Slides from Thermo Scientific). The cover glass was sealed with nail polish, and the slides were imaged immediately. A commercially available modified platform from 3i was used for imaging (Intelligent Imaging Innovations). It contains the Zeiss AxioObserver.Z1 Inverted Microscope, the spinning disk confocal unit (CSU-X1) connected to the QuantEM:512SC EMCCD Camera. Solid state diode lasers (LaserStack) were used as light source for GFP excitation. All images were taken with the Zeiss Objective Plan-Apochromat 63x/1.40 using immersion oil and were processed using Fiji and Photoshop.


Page 18 of 35

Page 19 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ASSOCIATED CONTENT Supporting Information Detailed information on cloning, mutagenesis, protein expression and purification, and yeast subcellular fractionation can be found in the Supplementary Information. Primers, yeast strains and antibodies used in this study are listed in Supplementary Tables 3–5. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR CONTRIBUTION C.H. and H.N. designed and performed experiments, analyzed data and wrote the manuscript. The authors declare no conflict of interest.

ACKNOWLEDGEMENTS We thank Jason W. Chin and Achim Dickmanns for plasmids and Blanche Schwappach for yeast strains. Research in the laboratory of H.N. is funded by the German Research Foundation (Emmy-Noether Programme) and the FreefloaterProgramme of the University of Göttingen and the Cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain.

REFERENCES 1.

Clapier, C. R., and Cairns, B. R. (2009) The biology of chromatin remodeling complexes, Annu Rev Biochem 78, 273-304.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2. 3. 4. 5. 6. 7. 8. 9.

10. 11.

12.

13. 14.

15.

16. 17. 18.

19.

Yang, X. J., and Seto, E. (2008) Lysine acetylation: codified crosstalk with other posttranslational modifications, Mol Cell 31, 449-461. Avvakumov, N., Nourani, A., and Cote, J. (2011) Histone chaperones: modulators of chromatin marks, Mol Cell 41, 502-514. Belotserkovskaya, R., Oh, S., Bondarenko, V. A., Orphanides, G., Studitsky, V. M., and Reinberg, D. (2003) FACT facilitates transcription-dependent nucleosome alteration, Science 301, 1090-1093. Orphanides, G., LeRoy, G., Chang, C. H., Luse, D. S., and Reinberg, D. (1998) FACT, a factor that facilitates transcript elongation through nucleosomes, Cell 92, 105-116. Formosa, T. (2012) The role of FACT in making and breaking nucleosomes, Biochim Biophys Acta 1819, 247-255. Formosa, T., Eriksson, P., Wittmeyer, J., Ginn, J., Yu, Y., and Stillman, D. J. (2001) Spt16-Pob3 and the HMG protein Nhp6 combine to form the nucleosome-binding factor SPN, EMBO J 20, 3506-3517. Biswas, D., Dutta-Biswas, R., Mitra, D., Shibata, Y., Strahl, B. D., Formosa, T., and Stillman, D. J. (2006) Opposing roles for Set2 and yFACT in regulating TBP binding at promoters, EMBO J 25, 4479-4489. Kang, S. W., Kuzuhara, T., and Horikoshi, M. (2000) Functional interaction of general transcription initiation factor TFIIE with general chromatin factor SPT16/CDC68, Genes Cells 5, 251-263. Orphanides, G., Wu, W. H., Lane, W. S., Hampsey, M., and Reinberg, D. (1999) The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins, Nature 400, 284-288. Squazzo, S. L., Costa, P. J., Lindstrom, D. L., Kumer, K. E., Simic, R., Jennings, J. L., Link, A. J., Arndt, K. M., and Hartzog, G. A. (2002) The Paf1 complex physically and functionally associates with transcription elongation factors in vivo, EMBO J 21, 1764-1774. Stevens, J. R., O'Donnell, A. F., Perry, T. E., Benjamin, J. J., Barnes, C. A., Johnston, G. C., and Singer, R. A. (2011) FACT, the Bur kinase pathway, and the histone co-repressor HirC have overlapping nucleosome-related roles in yeast transcription elongation, PLoS One 6, e25644. Winkler, D. D., and Luger, K. (2011) The histone chaperone FACT: structural insights and mechanisms for nucleosome reorganization, J Biol Chem 286, 18369-18374. Formosa, T., Ruone, S., Adams, M. D., Olsen, A. E., Eriksson, P., Yu, Y., Rhoades, A. R., Kaufman, P. D., and Stillman, D. J. (2002) Defects in SPT16 or POB3 (yFACT) in Saccharomyces cerevisiae cause dependence on the Hir/Hpc pathway: polymerase passage may degrade chromatin structure, Genetics 162, 1557-1571. Jamai, A., Puglisi, A., and Strubin, M. (2009) Histone chaperone spt16 promotes redeposition of the original h3-h4 histones evicted by elongating RNA polymerase, Mol Cell 35, 377-383. Kaplan, C. D., Laprade, L., and Winston, F. (2003) Transcription elongation factors repress transcription initiation from cryptic sites, Science 301, 10961099. Xin, H., Takahata, S., Blanksma, M., McCullough, L., Stillman, D. J., and Formosa, T. (2009) yFACT induces global accessibility of nucleosomal DNA without H2A-H2B displacement, Mol Cell 35, 365-376. Hsieh, F. K., Kulaeva, O. I., Patel, S. S., Dyer, P. N., Luger, K., Reinberg, D., and Studitsky, V. M. (2013) Histone chaperone FACT action during transcription through chromatin by RNA polymerase II, Proc Natl Acad Sci U S A 110, 7654-7659. VanDemark, A. P., Xin, H., McCullough, L., Rawlins, R., Bentley, S., Heroux, A., Stillman, D. J., Hill, C. P., and Formosa, T. (2008) Structural and 19 ACS Paragon Plus Environment

Page 20 of 35

Page 21 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20. 21.

22. 23.

24. 25. 26. 27.

28.

29.

30. 31. 32. 33.

34. 35.

functional analysis of the Spt16p N-terminal domain reveals overlapping roles of yFACT subunits, J Biol Chem 283, 5058-5068. Stuwe, T., Hothorn, M., Lejeune, E., Rybin, V., Bortfeld, M., Scheffzek, K., and Ladurner, A. G. (2008) The FACT Spt16 "peptidase" domain is a histone H3-H4 binding module, Proc Natl Acad Sci U S A 105, 8884-8889. Hondele, M., Stuwe, T., Hassler, M., Halbach, F., Bowman, A., Zhang, E. T., Nijmeijer, B., Kotthoff, C., Rybin, V., Amlacher, S., Hurt, E., and Ladurner, A. G. (2013) Structural basis of histone H2A-H2B recognition by the essential chaperone FACT, Nature 499, 111-114. Kemble, D. J., Whitby, F. G., Robinson, H., McCullough, L. L., Formosa, T., and Hill, C. P. (2013) Structure of the Spt16 middle domain reveals functional features of the histone chaperone FACT, J Biol Chem 288, 10188-10194. VanDemark, A. P., Blanksma, M., Ferris, E., Heroux, A., Hill, C. P., and Formosa, T. (2006) 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 22, 363-374. Winkler, D. D., Muthurajan, U. M., Hieb, A. R., and Luger, K. (2011) Histone chaperone FACT coordinates nucleosome interaction through multiple synergistic binding events, J Biol Chem 286, 41883-41892. Zunder, R. M., Antczak, A. J., Berger, J. M., and Rine, J. (2012) Two surfaces on the histone chaperone Rtt106 mediate histone binding, replication, and silencing, Proc Natl Acad Sci U S A 109, E144-153. English, C. M., Adkins, M. W., Carson, J. J., Churchill, M. E., and Tyler, J. K. (2006) Structural basis for the histone chaperone activity of Asf1, Cell 127, 495-508. Dennehey, B. K., Noone, S., Liu, W. H., Smith, L., Churchill, M. E., and Tyler, J. K. (2013) The C terminus of the histone chaperone Asf1 cross-links to histone H3 in yeast and promotes interaction with histones H3 and H4, Mol Cell Biol 33, 605-621. Evans, D. R., Brewster, N. K., Xu, Q., Rowley, A., Altheim, B. A., Johnston, G. C., and Singer, R. A. (1998) The yeast protein complex containing cdc68 and pob3 mediates core-promoter repression through the cdc68 N-terminal domain, Genetics 150, 1393-1405. Schlesinger, M. B., and Formosa, T. (2000) POB3 is required for both transcription and replication in the yeast Saccharomyces cerevisiae, Genetics 155, 1593-1606. Wittmeyer, J., and Formosa, T. (1997) The Saccharomyces cerevisiae DNA polymerase alpha catalytic subunit interacts with Cdc68/Spt16 and with Pob3, a protein similar to an HMG1-like protein, Mol Cell Biol 17, 4178-4190. Tan, B. C., Chien, C. T., Hirose, S., and Lee, S. C. (2006) Functional cooperation between FACT and MCM helicase facilitates initiation of chromatin DNA replication, EMBO J 25, 3975-3985. Han, J., Li, Q., McCullough, L., Kettelkamp, C., Formosa, T., and Zhang, Z. (2010) Ubiquitylation of FACT by the cullin-E3 ligase Rtt101 connects FACT to DNA replication, Genes Dev 24, 1485-1490. Krogan, N. J., Kim, M., Ahn, S. H., Zhong, G., Kobor, M. S., Cagney, G., Emili, A., Shilatifard, A., Buratowski, S., and Greenblatt, J. F. (2002) RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach, Mol Cell Biol 22, 6979-6992. Ferdous, A., Gonzalez, F., Sun, L., Kodadek, T., and Johnston, S. A. (2001) The 19S regulatory particle of the proteasome is required for efficient transcription elongation by RNA polymerase II, Mol Cell 7, 981-991. Chin, J. W., Cropp, T. A., Anderson, J. C., Mukherji, M., Zhang, Z., and Schultz, P. G. (2003) An expanded eukaryotic genetic code, Science 301, 964-967. 20 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

36. 37. 38. 39.

40.

41. 42. 43. 44.

45. 46. 47. 48.

49. 50.

51. 52.

Neumann, H. (2012) Rewiring translation - Genetic code expansion and its applications, FEBS Lett 586, 2057-2064. Chen, S., Schultz, P. G., and Brock, A. (2007) An improved system for the generation and analysis of mutant proteins containing unnatural amino acids in Saccharomyces cerevisiae, J Mol Biol 371, 112-122. Yoshikawa, K., Tanaka, T., Ida, Y., Furusawa, C., Hirasawa, T., and Shimizu, H. (2011) Comprehensive phenotypic analysis of single-gene deletion and overexpression strains of Saccharomyces cerevisiae, Yeast 28, 349-361. Wilkins, B. J., Rall, N. A., Ostwal, Y., Kruitwagen, T., Hiragami-Hamada, K., Winkler, M., Barral, Y., Fischle, W., and Neumann, H. (2014) A cascade of histone modifications induces chromatin condensation in mitosis, Science 343, 77-80. Kosugi, S., Hasebe, M., Tomita, M., and Yanagawa, H. (2009) Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs, Proc Natl Acad Sci U S A 106, 10171-10176. Tessarz, P., Santos-Rosa, H., Robson, S. C., Sylvestersen, K. B., Nelson, C. J., Nielsen, M. L., and Kouzarides, T. (2014) Glutamine methylation in histone H2A is an RNA-polymerase-I-dedicated modification, Nature 505, 564-568. Fleming, A. B., Kao, C. F., Hillyer, C., Pikaart, M., and Osley, M. A. (2008) H2B ubiquitylation plays a role in nucleosome dynamics during transcription elongation, Mol Cell 31, 57-66. Pavri, R., Zhu, B., Li, G., Trojer, P., Mandal, S., Shilatifard, A., and Reinberg, D. (2006) Histone H2B monoubiquitination functions cooperatively with FACT to regulate elongation by RNA polymerase II, Cell 125, 703-717. Carvalho, S., Raposo, A. C., Martins, F. B., Grosso, A. R., Sridhara, S. C., Rino, J., Carmo-Fonseca, M., and de Almeida, S. F. (2013) Histone methyltransferase SETD2 coordinates FACT recruitment with nucleosome dynamics during transcription, Nucleic Acids Res 41, 2881-2893. Albuquerque, C. P., Smolka, M. B., Payne, S. H., Bafna, V., Eng, J., and Zhou, H. (2008) A multidimensional chromatography technology for in-depth phosphoproteome analysis, Mol Cell Proteomics 7, 1389-1396. Choudhary, C., Kumar, C., Gnad, F., Nielsen, M. L., Rehman, M., Walther, T. C., Olsen, J. V., and Mann, M. (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions, Science 325, 834-840. Dephoure, N., Zhou, C., Villen, J., Beausoleil, S. A., Bakalarski, C. E., Elledge, S. J., and Gygi, S. P. (2008) A quantitative atlas of mitotic phosphorylation, Proc Natl Acad Sci U S A 105, 10762-10767. Gocke, C. B., Yu, H., and Kang, J. (2005) Systematic identification and analysis of mammalian small ubiquitin-like modifier substrates, J Biol Chem 280, 5004-5012. Li, Y., Keller, D. M., Scott, J. D., and Lu, H. (2005) CK2 phosphorylates SSRP1 and inhibits its DNA-binding activity, J Biol Chem 280, 11869-11875. Olsen, J. V., Vermeulen, M., Santamaria, A., Kumar, C., Miller, M. L., Jensen, L. J., Gnad, F., Cox, J., Jensen, T. S., Nigg, E. A., Brunak, S., and Mann, M. (2010) Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis, Sci Signal 3, ra3. Berg, M., Michalowski, A., Palzer, S., Rupp, S., and Sohn, K. (2014) An in vivo photo-cross-linking approach reveals a homodimerization domain of Aha1 in S. cerevisiae, PLoS One 9, e89436. Wittmeyer, J., Joss, L., and Formosa, T. (1999) Spt16 and Pob3 of Saccharomyces cerevisiae form an essential, abundant heterodimer that is nuclear, chromatin-associated, and copurifies with DNA polymerase alpha, Biochemistry 38, 8961-8971.


Page 22 of 35

Page 23 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


53.

54.

Masse, J. E., Wong, B., Yen, Y. M., Allain, F. H., Johnson, R. C., and Feigon, J. (2002) The S. cerevisiae architectural HMGB protein NHP6A complexed with DNA: DNA and protein conformational changes upon binding, J Mol Biol 323, 263-284. O'Donnell, A. F., Brewster, N. K., Kurniawan, J., Minard, L. V., Johnston, G. C., and Singer, R. A. (2004) Domain organization of the yeast histone chaperone FACT: the conserved N-terminal domain of FACT subunit Spt16 mediates recovery from replication stress, Nucleic Acids Res 32, 5894-5906.

Figure Legends Figure 1. Structure and function of FACT. (A) Current working model of FACT. FACT rearranges the nucleosome by stabilizing the histone core and displacing the DNA using its multiple binding sites for histones and DNA. This facilitates the passage of polymerases during transcription, replication and repair. (B) Available information on the structure of individual FACT domains. These domains are assumed to be tethered by flexible linkers to form a complex that binds the nucleosome in a multidentate manner. Scheme represents the domain architecture of the fungal FACT complex (Spt16, Pob3 and Nhp6 (NTD: N-terminal domain in red, DD: dimerization domain in red/violet, MD: middle domain in cyan, CTD: C-terminal domain in yellow, Nhp6A: HMGB (high mobility group box) domain in green)). Domain boundaries are given for the yeast (y) form based upon limited proteolysis and functional studies (19, 21-23, 53-54). Domains are presented as ribbon diagram from several crystal structures. PDB ID in brackets. Organism in bold: S.c. (Saccharomyces cerevisiae), C.t.

(Chaetomium

thermophilum),

X.l.

(Xenopus

laevis).

Numbers

indicate

crystallized residues. PBD 4KHB represents co-crystallization of Spt16-DD with Pob3 NTD/DD. PDB 4KHA represents chimeric Spt16:H2B protein co-crystallized with H2A (U-turn motif for interaction with H2B is indicated).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. In vivo crosslinking survey. FACT subunits Spt16 (A) and Pob3 (B) containing pBPA at indicated positions were expressed in BY4741 cells, crosslinked and proteins analyzed by Western blot against the C-terminal myc-epitope. Positions of full-length protein (FL) and crosslink products (X) are indicated. M: fluorescent protein standard.

Figure 3. Phenotyping of SPT16- and POB3-pBPA mutants. (A) Yeast cells with the indicated temperature-sensitive alleles (spt16-ts or pob3-L78R) were transformed with plasmids to produce pBPA mutants. Tenfold serial dilutions were spotted on SDUra/Leu plates and incubated at the indicated temperatures in the presence or absence of 2 mM pBPA. (B) Scoring table for the plates shown in A. The full data set can be found in the Supplementary Information (Supplementary Figure 2 and Supplementary Tables 1 and 2).

Figure 4. The acidic Pob3-CTD interacts with H2A and H2B in vivo. Three different yeast strains transformed with plasmids to produce the indicated Pob3-pBPA mutants were grown in the presence of pBPA, crosslinked and myc-tagged proteins visualized by Western blot. The primary sequence of Pob3-CTD showing pBPA residues in green and the resulting color-coded interaction profile to histones H2A and H2B is depicted above the Western blot. Full-length Pob3 (FL), Pob3–histone crosslinks (X) and Pob3–histone:3myc crosslinks (X+) are indicated. Black: wild-type (BY4741, Wt), red: BY4741 H2A:3myc, blue: BY4741 H2B:3myc.

Figure 5. The Pob3S500-histone interaction is independent of crosslinking chemistry. BY4741 cells containing tagged alleles of histones as indicated were transformed with plasmids to incorporate tyrosine (YRS), pBPA (pBPA-RS, 1 mM 23 ACS Paragon Plus Environment

Page 24 of 35

Page 25 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


pBPA) or pAzF (pAzF-RS, 2 mM pAzF) at Pob3 S500 (A). Cells were irradiated with UV-light and proteins analyzed by Western blot against the C-terminal myc-epitope. (B) Production of full-length Pob3S500 protein in BY4741 (wild-type) depends on the addition of the cognate UAA. Full-length Pob3 (FL), Pob3–histone crosslinks (X) and Pob3–H2A:3myc crosslinks (X+) are indicated. Black: wild-type (BY4741, Wt), red: BY4741 H2A:3myc, blue: BY4741 H3:TAP.

Figure 6. Interaction of Pob3 with histones H2A-H2B predominately localizes to the nuclear fraction. (A) Yeast cells producing Pob3 S500pBPA were crosslinked and fractionated into cytosol and nuclei. Proteins were visualized by Western blot using the indicated antibodies. Marker proteins (phosphoglycerate kinase (Pgk1) and histone H3) were used to verify the fractionation. WCE: whole cell extract. Truncated forms of Pob3 (T; resulting from termination at the amber codon), full-length Pob3 (FL) and crosslink to H2A (X) are indicated. (B) Quantification of the Pob3-H2A crosslink product in cytosolic and nuclei fractions in A. Three independent experiments were averaged, error bars are standard deviation of the mean (p=2.2x10-9).

Figure 7. Pob3-CTD interacts with H2A-H2B dimers in vitro. (A) Purified recombinant ctPob3 containing pBPA at the indicated positions was incubated with reconstituted H2A-H2B dimers and crosslinked. (B) ctPob3 S496pBPA was crosslinked in the presence of the indicated H2A-H2B concentrations. Proteins were analyzed by SDSPAGE and stained with Coomassie or detected by Western blot against the Nterminal His6-tag on ctPob3. Truncated forms of ctPob3 (T), full-length ctPob3 (FL) and crosslinks to histones (X) are indicated.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 8. Binding of Importin-α to an NLS at the C-terminus of Pob3 is required for nuclear import and interferes with H2A-H2B binding. (A) Schematic representation of plasmid-encoded Pob3-GFP fusion constructs (pMET25 promoter, CEN). (B) Yeast cells (BY4741) transformed with Pob3-GFP variants were visualized by fluorescence microscopy. (C) Analytical gel filtration of ctPob3 in complex with hsImportin-α. Individual proteins or complexes of ctPob3 with or without the C-terminal NLS and hsImportin-α lacking its IBB domain were subjected to analytical Superdex-200 gel filtration. Fractions were analyzed by SDS-PAGE and proteins stained with Coomassie. SDS-PAGE analysis of recombinant protein expression of ctPob3 variants and hsImportin-α including the chromatogram of the analytical S200 gel filtration are shown in Supplementary Figure 6B-C. Imp.: hsImportin-α ∆IBB, ctPob3 FL: ctPob3 FL aa 1–571, ctPob3∆C: ctPob3∆D467–G571. (D) Crosslinking of ctPob3-S496pBPA to histone H2A-H2B dimers is negatively affected by the presence of Importin-α. Three independent experiments were averaged, error bars are standard deviation of the mean (p=0.005). Western blots are shown in Supplementary Figure 7.

Figure 9. Deletion of the acidic Pob3-CTD renders cells hypersensitive to hydroxyurea. Tenfold serial dilutions of yeast strains containing the indicated mutations were incubated on plates containing 130 mM hydroxyurea (HU). Orientation of marker gene downstream of POB3 alleles is indicated (C=collinear, N=non-collinear).


Page 26 of 35

Page 27 of 35 1 A Spt16 2 3 4 5 6 7 8 9 10 Pob3 11 12 13 Destabilization of nucleosomes 14 promotes functions during 15 16 17 Replication Repair Transcription 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


B

5-447 S.c.

Spt16

677-941 S.c.

(3BIQ)

amino-peptidase fold

NTD-1

h431 y447

h606 y630

NTD-2 N

(4IOY)

Double-PH

DD

Spt16:H2B 649-944:1013-1121 C.t. : X.l. + H2A 11-102 X.l. (4KHA)

h933 h1047 y959 y1035

MD

CTD

U-turn motif

C

Pob3

N

C

Pob3 1-185 C.t. + Spt16 527-642 C.t. (4KHB)

y220

NTD/DD

Single-PH 1-111 S.c.

(3F5R)

ACS Paragon Plus Environment

y477 y552 y21 y89

MD

CTD

Double-PH 237-474 S.c.

(2GCL)

HMG

Nhp6A

HMGB 1-93 S.c.

(1J5N)

Figure 1

Y78 5 E78 8 L79 1 R7 9 9 Y81 0 L82 2 F82 9 R8 4 0 Q8 3 5 F84 4 T84 9 D8 5 0 E86 6 R8 7 5 V87 6 M F87 8 K88 1 V88 8 F89 2 D9 0 8 T92 5 I926 L92 T938 2 I933 S93 6 Y94 2 L94 6 W95 0 L95 3 S96 5 M Y97 2 S97 5 E97 6 S98 0 F98 5 S98 6 D9 9 6 S10 0 Y10 3 06 S10 11 E10 1 D1 0 2 1 W10 6 17 K10 22 A10 2 R1 0 5 2 F10 9 33

K69 2 T71 3 D7 1 8 M L73 6 K74 5 L74 9 K75 2 K75 4 Y76 1 D7 7 0 R7 7 M 6 M Y19 K21 E42 Y45 S78 K80 K82 H8 3 D8 9 K10 8 E11 1 F11 7 S13 8 K14 2 F14 3 M E15 9 V17 8 K21 6 K22 9 L24 1 K24 7 F25 0 D2 5 1 D2 5 4 W25 5 R2 7 0 S27 2 N2 7 7 E31 1 F31 M 9 Y34 8 K36 2 S37 5 D3 8 4 K40 4 E42 6 F43 2 Y43 6 Q4 4 2 Y44 6 F53 2 S54 R552 2 W55 S55 7 9 T56 1 I562 L56 4 R5 6 9 P57 0 V57 1 F57 3 H5 7 4 Y57 8 Y59 1 F59 6 I605 S61 7 T67 7 F68 4

1 2 3 4 5 6 7 kDa 8 225 9 150 10 11 12 13 527 630 Spt16-DD 14 15 16 17 18 19 20 kDa 21 225 22 150 23 24 25 26 27B. Pob3 28 29 1 Pob3-NTD 30 31 32 33 34 kDa 35 36 225 150 37 38 102 39 76 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 M L10 R20 I30 Q40 T50 G60 G80 K90 Q10 0 G11 0 M R12 0 K13 0 E16 0 V17 0 I180 P20 0 D21 0 M22 0 E23 0 D24 0 F25 0 T25 R252 R254 D256 D268 G270 K27 0 T27 1 K272 Q276 R28 8 K29 0 L30 0 E300 3 Q30 M 8 Q31 0 F31 5 F32 K320 2 D32 3 E32 5 L33 0 V36 0 I370 N40 0 K40 L41 7 0 Y41 1 V42 R420 4 T43 0 R43 M 3 D43 V44 6 F45 0 0 L46 L470 0 N47 Q484 D49 0 S50 0 E510 0 A52 0 D53 E53 0 K557 0

A. Spt16 ACS Chemical Biology

1

Spt16-NTD

690 Spt16-MD

220 237 Pob3-MD

Page 28 of 35

447

959

477


527

Spt16-CTD

Pob3-CTD

Spt16-DD 630

X FL

1035

X FL

552

X FL

Figure 2

Page 29 of 35

A

1 2 3 4 Empty 5 Spt16 wt 6 K82TAG 7 D251TAG 8 D718TAG 9 D908TAG 10L928TAG 11 12 13 14 15 16 17 Empty 18 Pob3 wt 19 20 Q40TAG 21 Q308TAG 22 K407TAG 23 S500TAG 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

B


spt16-ts 25°C +pBPA

-pBPA

37°C

+pBPA

Position Empty Wt K82 D251 D718 D908 L928

25°C + pBPA

37°C - pBPA

37°C + pBPA

Position Empty Wt Q40 Q308 K407 S500

25°C + pBPA

33°C - pBPA

33°C + pBPA

++++++ ++++++ ++++++ ++++++ +++++ ++++++ +++++

0 ++++++ ++ ++ 0 0 0

0 ++++++ ++++++ ++++ + ++++ 0

pob3-L78R 25°C +pBPA

-pBPA

33°C

+pBPA


+++++ +++ +++++ +++++ +++++ +++++

0 ++ ++++ 0 0 ++++

0 ++ ++++ +++ +++ ++++

Figure 3


1 2 3 4 5 6 7 8 9 10 11 12 13 Pob3 CTD: Profile: 14 15 Q480 D490 S491 16 kDa 17 18 140 19 115 20 80 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

S500

S507

E510

S516

A520

Page 30 of 35

F527

D530 S534

E537

S546

K550

X+ X FL Wt

H2A:3myc

H2B:3myc


Figure 4

Page 31 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A X-RS: X-AA: UV: strain:

B

A

B

A

B

-

A

+

B

A

B

A

B

A

BAY

+

+

kDa 140 115

X+ X

80

FL

65

B: pBPA-RS

H2A:3myc

wt

B

X-RS: pBPA: pAzF:

Y: YRS

A: pAzF-RS

-

+ -

Y

+

+ +

-

B + -

α-myc

H3:TAP

+

-

A + -

+

kDa

FL

80

α-myc


Figure 5

-

+

-

140

+ 40 kDa

115

X FL

80

α-PGK1

T

65

α-myc

15 kDa

α-H3


histone crosslink / Pob3FL in %

UV: +

kDa

B

cle i

WCE - +

Page 32 of 35

Nu

Cytosol Nuclei UV: - + - +

E

A

WC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Cy tos ol


15 14 12 10 8 6 4 2 0

***

Cytosol Nuclei

Figure 6

7.0

3.5

2.1

0.7

5

c(H2A-H2B) [µM]

0.3

7.0

5

+

3.5

-

2.1

+

0.7

-

BP A

BP A

BP A

+

B 0.3

-

Y5 20

+

S4 96

-

S4 82

A

Y1 81

1 2 3 4 5 6 7 ctPob3: 8 UV: 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


BP A

Page 33 of 35

kDa X FL T

170 130

Coomassie X FL T

Coomassie

α-His

100

X

70

FL

55

T

40

α-His

35


Figure 7


A

B

GFP (a)

Pob3 (b)

Pob3∆458–552 (c)

Pob3∆544–552 (d)

Pob3 K547M (e)

Pob3∆458–543 (f)

C

molecular weight MW in kDa 100

hsImp. (

)

A7 A8 A9 A10 A11 A12 B12 B11 B10 B9

load

70

D

55

1.2

100

ctPob3 FL (

)

Rel. x-linking efficiency

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 35

70 55 100

hsImp ( + ctPob3 FL (

) )

70 55 100

ctPob3∆C (

)

70

) )

0.8 0.6 0.4 0.2 0

+ hsImp-α:

55

hsImp. ( + ctPob3∆C (

1

**

+

100 70 55

Coomassie ACS Paragon Plus Environment

Figure 8

Page 35 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Pob3 genotype


25°C

YPD

32°C

25°C

130 mM HU

32°C

∆A501–E543 (C) ∆A501–E543 (N) ∆S491–E543 (C) Wt control (C) Wt control (N) ∆S491–E543 (N)


Figure 9

In Vivo Mapping of FACTâHistone Interactions ... - ACS Publications

Recommend Documents

In Vivo Mapping of FACTâHistone Interactions ... - ACS Publications