Nucleosomal DNA binding drives the recognition of H3K36-methylated nucleosomes by the PSIP1-PWWP domain
© van Nuland et al.; licensee BioMed Central Ltd. 2013
Received: 15 March 2013
Accepted: 16 April 2013
Published: 8 May 2013
Recognition of histone modifications by specialized protein domains is a key step in the regulation of DNA-mediated processes like gene transcription. The structural basis of these interactions is usually studied using histone peptide models, neglecting the nucleosomal context. Here, we provide the structural and thermodynamic basis for the recognition of H3K36-methylated (H3K36me) nucleosomes by the PSIP1-PWWP domain, based on extensive mutational analysis, advanced nuclear magnetic resonance (NMR), and computational approaches.
The PSIP1-PWWP domain binds H3K36me3 peptide and DNA with low affinity, through distinct, adjacent binding surfaces. PWWP binding to H3K36me nucleosomes is enhanced approximately 10,000-fold compared to a methylated peptide. Based on mutational analyses and NMR data, we derive a structure of the complex showing that the PWWP domain is bound to H3K36me nucleosomes through simultaneous interactions with both methylated histone tail and nucleosomal DNA.
Concerted binding to the methylated histone tail and nucleosomal DNA underlies the high- affinity, specific recognition of H3K36me nucleosomes by the PSIP1-PWWP domain. We propose that this bipartite binding mechanism is a distinctive and general property in the recognition of histone modifications close to the nucleosome core.
KeywordsHistone-methylation H3K36me Nucleosome Structure NMR Affinity Specificity PSIP1 PWWP
Chemical modifications of nucleosomes, the complex of DNA and histone proteins that packages the eukaryotic genome, are key in the regulation of transcription, maintenance of genomic integrity, chromosome condensation and segregation . Modifications such as acetylation or methylation of lysine residues of histone proteins can serve to recruit effector proteins to specific genomic sites . Methylation of lysine-36 in histone H3 (H3K36me) is conserved from yeast to human and is predominantly associated with actively transcribed chromatin . H3K36me has been implicated in diverse processes including splicing, DNA repair, repression of cryptic transcription and histone exchange . Recently, PWWP domains have been identified as H3K36me recognition domains, based on histone peptide interaction studies . PWWP domains feature an aromatic cage, as in other royal Tudor family proteins  that bind the methylated lysine side chain via cation-π interactions . Interestingly, interaction studies have shown that PWWP domains bind methylated H3K36 histone tail peptides with very low affinity [8, 9], in stark contrast with the high affinity recognition of tri-methylated lysine-4 of H3 (H3K4me3) by PHD fingers [10, 11].
Here, we address the structural basis for H3K36me recognition by PWWP domains in the nucleosomal context. Unlike other methylated lysines in the unstructured N-terminal tail of histone H3, K36 is located at the point where the H3 tail protrudes from the nucleosome core . Since PWWP domains were previously also implicated in DNA-binding [13, 14], we hypothesized that the close proximity of the nucleosomal DNA may critically contribute to binding affinity and/or specificity of PWWP domains for H3K36me. We concentrate on the PWWP domain containing protein PSIP1, as its association with naked and chromatinized DNA has been the focus of previous studies [15, 16] and it was recently identified as a H3K36me-interacting protein using synthetic peptides . PSIP1 (LEDGF/p75) was first isolated as an transcriptional co-activator  and tethers the HIV integrase to active host chromatin dependent on its PWWP domain [19, 20]. PSIP1 is an essential subunit of the MLL complex in MLL oncogenic transformations via HOX gene regulation , and is implicated in the homologous recombination pathway for DNA repair .
Using various experimental approaches, we show that concerted, low-affinity interactions of the PSIP1-PWWP domain with both nucleosomal DNA and methylated histone tail result in specific and high-affinity binding to H3K36-methylated nucleosomes. During the final stages of our work, a similar conclusion was reached in another study . Our study underscores this notion with a NMR analysis of the PWWP-nucleosome complex, a structural model of the complex based on experimental interaction data and an extensive in vitro and in vivo validation. Finally, based on a comparison with other PWWP domains and H3K36me-binding modules, we propose that the bipartite recognition of methylated histone tail and nucleosomal DNA is a general feature of H3K36me recognition.
Results and discussion
H3K36 methylation-dependent nucleosome binding by PSIP1-PWWP
To determine the contribution of residues neighboring H3K36, mono-nucleosomes carrying point mutations for residues 31 to 39 in histone H3 were used. Of these only V35A, K36A and K36R affect PWWP-binding (Figure 1b), suggesting limited involvement of the H3 amino acid sequence around the K36 methylation site in determining binding specificity. The specific interaction of the PWWP domain with mono-, di-, and tri-methylated H3K36 was confirmed using biotinylated H3 tail peptides (Figure 1c) and mutation of W21 in the hydrophobic pocket of the domain completely abolished binding even in context of full-length PSIP1 (Additional file 1: Figure S1).
Next, immobilized GST-PWWP was used in binding to mono-nucleosomes prepared from mammalian cells. The bound fractions were enriched for H3K36me3 and H3K36me2 modifications, whereas they showed little enrichment for H3K79me3 and H3K4me3. Comparable results were obtained using an extended fragment of the PWWP domain including the flanking AT-hook region (Figure 1d) or full-length PSIP1 protein (Additional file 1: Figure S1b).
Adjacent PWWP surfaces bind weakly to H3K36me3 peptides and DNA
Addition of a H3K36me3 peptide (residues 28 to 41) resulted in clear chemical shift changes for the backbone amide resonances of residues around the aromatic cage and strand β4 of the PWWP domain (Figure 2c,d left panels). The affinity for the H3K36me3 peptide is very low with a KD of 17 mM (Figure 2f left panel). In part, this may be due to the relatively closed conformation of the binding pocket when compared to crystal structures of related PWWP domains bound to methylated peptides (Additional file 1: Figure S2c). Strikingly, this very low-affinity interaction is completely dependent on the methylation of H3K36, as no changes in chemical shift upon addition of non-methylated H3K36 peptide were observed, even at 11 mM of peptide (Additional file 1: Figure S3).
Addition of a dsDNA fragment that was previously suggested as a substrate for PSIP1-PWWP  resulted in chemical shift changes for a distinct set residues localizing to a single basic patch on the PWWP surface (Figure 2c and d, right panel). A KD of 150 ± 50 μM was determined, indicating a low binding affinity for DNA (Figure 2f, right panel). Imino-proton resonances of DNA base pairs did not change in chemical shift or relative intensity during the titration, suggesting that the interaction lacks sequence-specificity (Additional file 1: Figure S4).
The DNA and histone interaction surfaces of the PWWP domain are adjacent and overlap with areas of positive or negative potential, respectively (Figure 2e). This arrangement provides an excellent electrostatic match to the negative phosphate backbone of the DNA and positive histone tail in the context of the nucleosome.
Aromatic cage and basic surface determine binding specificity and affinity
Mutations in the putative nucleosomal DNA-binding surface show a markedly different result. The K70A mutant binds with a lower affinity to nucleosomes, but retains preference for the modified nucleosomes. Strikingly, alanine substitutions of the solvent exposed R74 abolished the interaction with nucleosomes (Figure 4a). In contrast, the K34A mutant showed a comparable binding pattern to wild type PWWP (Figure 4a). Nearly all charge mutants showed severely reduced binding, but retained specificity for H3KC36me3 nucleosomes (Additional file 1: Figure S5a). Of these, K39 (shown in Figure 4a) and K56 were initially not found to interact with a DNA fragment in the NMR titration experiment, suggesting that binding to nucleosomal DNA involves a larger interaction surface.
To investigate the in vivo relevance of our findings, we examined the contribution of the PWWP domain to the mobility and distribution of PSIP1 in cells. To this end wt and mutant PWWPs were introduced in the context of full-length PSIP1 fused to GFP and expressed in HeLa cells (Additional file 1: Figure S6). The nuclear mobility of the GFP-PSIP1 proteins was measured by fluorescence recovery after photobleaching (FRAP). HeLa cells expressing NLS-GFP or histone H2B-GFP were included as highly mobile and immobile controls, respectively (Figure 4b left panel). FRAP curves for GFP-PSIP1 are indicative of transient chromatin-binding (Figure 4b green curve), consistent with previously published results . Disruption of domain integrity (W21A), as well as mutations in the aromatic cage (M15A) or the DNA interaction surface (K70A, R74A), resulted in faster recovery of fluorescence after bleaching (Figure 4b center and right panels), demonstrating the requirement of both DNA and histone tail interaction surfaces for stable association with chromatin.
To examine the effect of PWWP mutations on the genomic distribution of PSIP1, the GFP-PSIP1 cell lines were used for chromatin immunoprecipitation followed by high-throughput sequencing (ChIPseq). To correlate binding of the PSIP1 proteins to H3K36 methylation, genes were divided into two groups: high H3K36me3- (red) or low H3K36me3- (blue) containing genes (Additional file 1: Figure S7). Wild type PSIP1 protein was selectively enriched on high H3K36me3 genes (Figure 4c and Additional file 1: Figure S7b), while aromatic cage mutant M15A showed no enrichment, in accordance with our in vitro data. Mutation of K70 did not significantly affect the genomic distribution of PSIP1 in correspondence with the mild effect on the affinity for nucleosomes of this mutant in EMSA.
Concerted binding of methylated histone tail and nucleosomal DNA
The PWWP-H3KC36me3 nucleosome interaction was further analyzed using state-of-the-art solution NMR tailored for large supramolecular complexes. Recently, it was shown that a comprehensive characterization of protein-nucleosome interactions can be obtained  using methyl-group based NMR (methyl-TROSY) , in which only the histone methyl groups of isoleucine, leucine and valine (ILV) residues are observed.
Based on fitting the experimental line shapes of the H3V35a methyl group to a 1:2 (nucleosome:PWWP)-binding model, we find that the dissociation constant of PSIP1-PWWP-binding to the H3KC36me3 side chain within the nucleosome is 1.5 μM (Figure 5b, 5c and Additional file 1: Figure S9). The interaction is highly dynamic: the dissociation rate (koff) is ca. 500 s-1, corresponding to a lifetime of the complex (1/koff) of approximately 2 ms. The affinity found here at physiological ionic strength is comparable to the KD value estimated from the gel-shift essay (ca. 0.5 μM), recorded at lower ionic strength and temperature (Figure 4d). Strikingly, the affinity of PSIP1-PWWP for methylated nucleosomes is four orders of magnitude higher than for a methylated peptide (KD 17 mM) and two orders higher than for isolated DNA (KD 150 μM). The enhanced affinity is due to simultaneous binding of both methylated histone tail (see chemical shift perturbations of H3V35, Figure 5b) and nucleosomal DNA (see the binding defects of DNA interaction surface mutants, Figure 4a). The magnitude of such enhancement in binding affinity upon linking of two binding sites cannot simply be predicted from the affinities for the isolated binding sites, as it depends crucially on the relative orientation of the linked sites and the length and flexibility of the linker [30, 31]. Following the framework of Zhou , the enhancement may be expressed in the form of an effective concentration given by (KD,tail × KD,DNA)/KD,nucleosome, which in our case evaluates to 1.5 × 10− 4 × 1.7 × 10− 2/1.5 × 10− 6 = 1.7 M. This enhancement value is significantly higher than typical values in the mM range as found for linked DNA-binding domains or bivalent pharmaceuticals [31, 32]. This suggests that there are limited entropic losses and structural rearrangements upon binding. Thus, our data indicate that both the DNA and histone interaction surfaces of PSIP1-PWWP domain combine in a concerted manner to result in high-affinity binding to H3K36me3 nucleosomes.
Structure of PSIP1-PWWP-H3K36me3 nucleosome complex
PWWP-DNA contacts made by the basic residues are mainly to the phosphate or sugar backbone (Figure 6c). The sequence-specific contacts of residue R74 seen in the lowest energy-structure are not conserved in the cluster of solutions. Overall, residues K73, R74 and K75 bind the DNA non-specifically around SHL +6, while K39 and K56 interact with the other DNA gyre at SHL −1. Residue K16 and K14 sit in between the two gyres and can interact with either. Notably, residues K67 and K70 do not mediate intermolecular interactions in many structures of this cluster, which may reflect the relatively minor binding defects of their alanine mutants in EMSA (see also Additional file 1: Table S2).
Here we determined the molecular basis of H3K36me nucleosome recognition by the PSIP1-PWWP domain. We show that the interaction with nucleosomal DNA is responsible for an approximately 10,000-fold enhancement in binding affinity into an in vivo relevant range. A similar conclusion was reached in the work of Eidahl et al. from a comparison of an NMR- based estimated binding affinity for H3K36me3 peptides and a pull-down assay-based measurement of the affinity for H3K36Cme3 nucleosomes . While full length PSIP1 contains additional DNA binding domains [16, 35], disruption of the PWWP basic surface markedly reduces in vivo chromatin binding ability of full-length PSIP1 as shown in this work and previously . Moreover, mutations in the DNA interaction surface were previously shown to result in a dramatically reduced HIV-infectivity in cells , underscoring the functional significance of the bipartite nucleosome-binding for HIV integration and other PSIP1-dependent cellular processes.
In conclusion, we propose that recognition of H3K36-methylated chromatin not only occurs through the methylated lysine side chain and its amino acid sequence context, but also through the nucleosomal DNA. We propose that this mechanism also applies to the recognition of other modifications close to the nucleosome core, such as H4K20me and H3K79me. This mechanism testifies to the fact that recognition of histone modifications relies on the binding to modified histone residues embedded in the chromatin fiber. Just as histones are not merely packaging material in chromatin, the nucleosomal DNA is not inert in the readout of the epigenetic modifications.
Protein expression and purification
The PWWP domain of human PSIP1 (3 to 100) or the PWWP+AT (3 to 207) were expressed as GST-fusions in BL21-DE3 or Rosetta 2 bacterial strains at 37°C in either LB medium or M9 minimal medium with 15NH4Cl and/or 13C-glucose. The protein was purified by binding to a glutathione agarose (GA) column (Sigma-Aldrich, St. Louis, MO) and eluted with 50 mM reduced glutathione (Sigma-Aldrich, St. Louis, MO). After thrombin digestion, PWWP was purified over a Sephadex-75 (HiLoad 16/60, GE Healthcare, Uppsale, Sweden) column in buffer A (50mM Tris-HCl pH 7, 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.5 mM PMSF and protease inhibitors), applied to a MonoS HR5/5 in buffer A and eluted using a linear gradient (0.1 to 1 M KCl).
Drosophila histones were expressed, purified and alkylated as previously described [26, 40]. Histones used for NMR studies were produced in M9 minimal medium containing desired isotopes. Methyl-labeling of Ile-δ1-[13CH3] and Val/Leu-[13CH3,12CD3] (ILV-labeling) followed the procedure of Tugarinov .
Antibodies and plasmids
α-GST (SC), α-PSIP1 (A300-848A, Bethyl, Montgomery, TX), α-H4 (07 to 108, Upstate), α-H3K36me2 (9758, Cellsignaling), α-H3K4me3 (ab8580,), α-H3K79me3 (ab2621), α-H3K36me1 (ab9048), α-H3K36me3 (ab9050; all “ab” antibodies obtained from Abcam, Cambridge, UK) and α-GFP (gift from Geert Kops) were used for ChIP and immunoblotting.
All GST fusions were cloned into pRPN265NB. PSIP1 cDNA was introduced into pEGFP-C using the Gateway system (Invitrogen, Carlsbad, CA). All point mutations were created using site directed mutagenesis (Stratagene, Santa Clara, CA). Stable GFP-tagged PSIP1 HeLa lines were created by cloning PSIP1 into pCDNA.5/FRT/TO (Invitrogen, Carlsbad, CA) and subsequent recombination into HeLa FRT cells carrying the Tet repressor for inducible expression .
Nucleosome and peptide pull-downs
Mono-nucleosomes were extracted from HeLa or yeast cells by MNase treatment of lysed cells as previously described . Histone H3 mutants were selected from a mutant library . GA beads (Sigma-Aldrich, St. Louis, MO) were covered with GST-fusion proteins, mixed with mono-nucleosomes and washed. Eluted proteins were analyzed by immunoblotting. HeLa mono-nucleosomes were incubated with premixed GFP-fusion protein and GFP binder beads (ChromoTek, Planegg-Martinsried, Germany) and analyzed in a similar way. Peptide pull-downs were performed as described previously .
Nucleosome reconstitution and EMSA
The 601-DNA ‘Widom’ template was amplified using PCR, purified using anion exchange chromatography and used for reconstitution using salt-gradient deposition. Nucleosomes were incubated with GA purified GST-PWWP protein in 0.2X TBE and analyzed by native 5% 60:1 acryl-bis gel electrophoresis. Either 1.5 or 3 pmol of nucleosome was used in all experiments and up to 3 molar equivalents of protein in 8 μL load volume. All steps were performed at 4°C. Gels were stained with ethidium-bromide and analyzed on a Gel-Doc XR+ system (Bio-Rad, Hercules, CA). If applicable, band densities corresponding to free, singly and doubly bound nucleosomes were quantified using ImageJ software package and subsequently fitted together to a 2:1 binding model using in-house written MatLAB routine (MATLAB version 7.13.0, The MathWorks Inc., Natick, MA).
FRAP studies were performed using a Zeiss 510 META confocal LSM (Zeiss, Oberkocken, Germany) as previously described . GFP protein expression was induced with 0.5 μg/ml doxycycline for five hours.
Chromatin preparation and ChIP were essentially performed as described [43, 44]. Libraries were sequenced on AB/SOLiD 5500XL, producing 48 bp reads. Sequencing reads were mapped with Burrows-Wheeler Aligner (BWA-0.5.8c) (settings: -c -l 25 -k 2 -n 10) . As a gene set, the known protein-coding genes as annotated in Ensembl 67 were used (http://www.ensembl.org). The number of reads mapped to each gene was normalized to the total number of reads mapping inside genes per sample. A separation of H3K36me3 enriched and non-enriched was made based on the density plot of the read density. Genes were filtered to have at least 50 sequencing reads in the GFP tagged PSIP1 ChIP-seq data. All plots were created using the R package (http://www.r-project.org/).
Samples used for assignment and structure calculation contained ca. 1 mM PWWP domain in 90/10% H2O/D2O with 20 mM NaPi buffer at pH 6.2. Interaction studies were done at 0.3 mM PWWP in 20 mM NaPi pH 7.0 with 100 mM NaCl. ILV-labeled H3KC36me3 nucleosome sample contained 116 μM nucleosome in 20 mM NaPi pH 7 with 100 mM NaCl.
Peptides were extensively lyophilized and dissolved in NMR buffer to a stock concentration of 110 mM. Cysteine peptides were alkylated according to the MLA protocol  and purified using a Sephadex G-10 (GE Healthcare, Uppsale, Sweden) column followed by cation exchange chromatography. The purity of the peptide was confirmed by NMR. Annealed DNA oligos (Eurogentec, Liege, Belgium) were lyophilized and dissolved in NMR buffer to a stock concentration of 11.5 mM. Titration of H3KC36me3 nucleosomes was done using a PWWP stock of 1.28 mM.
PSIP1-PWWP structure determination
NMR experiments for assignment, and structure calculation of the PSIP1-PWWP domain were carried out at 293K on a 600 or 750 MHz Bruker Avance II spectrometer (Bruker Biospin, Rheinstetten, Germany). Processing was done using the NMRPipe package . Spectra were analyzed using Sparky (Goddard and Kneller, UCSF, USA). Backbone assignments were obtained using MARS  based on HNCACB and CBCACONH spectra. Side chain resonances were assigned using CCH-TOCSY, CBHD and NOESY spectra. Overall assignment completeness was 97.1% for all non-labile protons. Backbone dihedral angle restraints were derived using TALOS+ . Distance restraints were derived from 13C- and 15N-edited 3D NOESY spectra (mixing time 120 ms). The NOE cross peaks were assigned and converted into distance restraints using CYANA 3.0 [49, 50]. First, 10 ensembles of 100 structures were calculated by using CYANA using different random number seeds. Out of the 10 resulting distance restraint lists, only the restraints that were reproduced in all cases were retained to produce a final restraint list. This final list was then used to calculate 100 structures in CNS 1.2 , which were subsequently refined in explicit water by using the RECOORD protocol . The final ensemble containing the 20 lowest-energy structures, contained neither distance violations > 0.5 A, nor dihedral angle violation > 5°, and was validated by using the iCing validation suite .
Titration experiments and data analysis
Interaction studies of the PSIP1-PWWP domain were carried out at 293K on a 600 MHz Bruker Avance II spectrometer. Nucleosome spectra were recorded at 308K on a 900 MHz Bruker Avance III spectrometer with a TCI cryo-probe. Titration data were fitted using MatLAB scripts either using the fast-exchange assumption in case of fitting CSP derived binding curves or using explicit evaluation of the exchange matrix, and subsequent calculation of the FID in case of line shape fitting (see supporting materials in Kato et al.  for details).
All molecular graphics were prepared using open-source PyMOL (The PyMOL Molecular Graphics System, Version 1.4, Schrödinger, LLC). Electrostatic surfaces were calculated using the adaptive Poisson-Boltzman solver  and the AMBER force field.
Docking protocol PSIP1-PWWP–nucleosome complex
We used our experimental chemical shift perturbation, transferred-NOESY and mutagenesis data, together with available literature data to create a structural model for the PSIP1-PWWP-nucleosome complex with Haddock version 2.1  and CNS 1.3 [51, 55]. In what follows, we describe the docking procedure.
In short, the docking was divided in two stages: i) docking of the H3 N-terminal tail to the PSIP1-PWWP domain guided by the chemical shift perturbation, transferred-NOESY and mutation data, and using homology-derived interaction restraints from the homologous BRPF1-H3K36me3, HDGF2-H4K20me3/H3K79me3 crystal structures; ii) docking of the PWWP-H3K36me3 complex to the nucleosome, again guided by the chemical shift perturbation and mutation data, together with restraints to enforce the covalent attachment of the H3-tail to the remainder of H3. This approach was based on the flexible multi-domain docking protocol described by Karaca et al. . It allows the efficient docking of the PSIP1-PWWP domain to both to K36me3 side chain in the H3 tail and to the nucleosomal DNA, and at the same time, to sample a large conformational space for the flexible H3 tail. This procedure is described in details in the Supplementary Material.
PDB accession codes
The solution structure of the PSIP1-PWWP domain is accessible from the Protein Data Bank, [PDB: ID 3ZEH]. The structural model of the H3K36me3-nucleosome-PSIP1-PWWP domain complex is deposited under [PDB: ID 3ZH1] and is also available from the author’s website: http://www.nmr.chem.uu.nl/~hugo.
We thank S Taylor, R Roeder, Y Bai and G Kops for reagents; P de Graaf for help with FRAP and the HADDOCK Team (ASJ Melquiond, E Karaca, M van Dijk and Prof. AMJJ Bonvin) for valuable discussions on the docking. This work was supported by the Chemical Sciences division of Dutch Science foundation NWO through a Veni fellowship to HvI (700.59.401), a NWO-Chemical Sciences TOP grant to MT (700.57.302) and by the Netherlands Proteomics Centre. RB was supported by NWO-Chemical Sciences for the 900 MHz NMR and the TCI cryoprobe.
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