Characterization of Caenorhabditis elegans Nucleosome Assembly

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Characterization of Caenorhabditis elegans Nucleosome Assembly Protein 1 Uncovers the Role of Acidic Tails in Histone Binding Prithwijit Sarkar, Naifu Zhang, Sudipta Bhattacharyya, Karlah Salvador, and Sheena D'Arcy Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01033 • Publication Date (Web): 06 Dec 2018 Downloaded from http://pubs.acs.org on December 8, 2018

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Biochemistry

Characterization of Caenorhabditis elegans Nucleosome Assembly Protein 1 Uncovers the Role of Acidic Tails in Histone Binding Prithwijit Sarkar1*, Naifu Zhang2*, Sudipta Bhattacharyya3, Karlah Salvador2, Sheena D’Arcy2** 1Department

of Biological Sciences, 2Department of Chemistry and Biochemistry, University of Texas at Dallas, USA 75080 3Department of Biochemistry and Molecular Biology, University of Melbourne, AUS 3000 *These authors contributed equally to this work. **[email protected] Keywords: nucleosome assembly proteins, histone chaperones, histones, chromatin, light scattering, crystallography Supporting Information Placeholder ABSTRACT: Nucleosome assembly proteins (Naps) influence chromatin dynamics by directly binding to histones. Here we provide a comprehensive structural and biochemical analysis of a Nap protein from Caenorhabditis elegans (CeNap1). CeNap1 naturally lacks the acidic N-terminal tail and has a short C-terminal tail compared to many other Nap proteins. Comparison with full length and a tail-less constructs of Saccharomyces cerevisiae Nap1 (ScNap1) uncovers the role of these tails in self-association, histone-binding, and competing H2A-H2B from DNA. We find that the presence of tails influences the stoichiometry of H2A-H2Bbinding and is required to compete interactions between H2A-H2B and DNA. The absolute stoichiometry of the Nap protein and H2AH2B complex is 2:1 or 2:2, with only a very small population of higher-order oligomers occurring at 150 mM NaCl. We also show that H3-H4 binds differently than H2A-H2B, and that a (H3-H4)2 tetramer can simultaneously bind two Nap2 protein homodimers.

In the nucleus of a eukaryotic cell, genomic DNA is packaged into nucleosomes to form chromatin. Each nucleosome contains 146 bp of DNA wrapped 1.6 times around a histone octamer. The histone octamer is composed of a (H3-H4)2 tetramer and two H2AH2B dimers1. Histone chaperones are proteins that directly bind to histones and thereby regulate chromatin architectural changes important for DNA replication and repair, as well as transcription2. Annotation of histone chaperone function is typically associated with tight histone-binding as well as the ability to interfere with histone-histone or histone-DNA interactions3,4. While histones chaperones in general are structurally diverse, several contain short stretches of acidic residues that contribute to chaperone function5. These acidic regions may play a role in nucleosome dynamics by mimicking the negatively-charged DNA backbone. In this study, we investigate the role of these acidic regions in the Nap-family of histone chaperones. Nap proteins are a subset of histone chaperones defined by their structural similarity6. Sequence analysis shows high conservation across the eukaryotes, with many species having multiple Nap paralogs7. Crystallography of several Nap proteins has identified common elements including the formation of a homodimer by antiparallel packing of a long dimerization helixe.g.8. The constitutive Nap2 dimer can also self-associate in solution to form Nap4 tetramers and, in some cases, small populations of even larger

species9–11. The affinity of Nap2 self-association can be modulated by salt concentration and varies between homologs. Self-association of the Nap2 dimer has made it difficult to determine the absolute stoichiometry of histone-binding. Some studies report 2:1 or 2:2 (Nap:histone), while others suggest higherorder oligomerization via either Nap and/or histone contacts4,12–15. A crystal structure of Saccharomyces cerevisiae Nap1 (ScNap1) identifies a conserved α-β domain as the primary histone-binding site12. A secondary histone-binding site may also be present in the N- and/or C-terminal tails that are either disordered or not included in the Nap protein structures15. These tails vary in length and are enriched in acidic amino acids6. We set out to determine the role of these acidic tails in Nap2 self-association and the absolute stoichiometry of histone-binding. To identify the role of the Nap tails we use two strategies. The first strategy compares full-length ScNap1 (ScNap1FL; residues 1417) and a tail-less construct (ScNap1CORE; residues 74-365). ScNap1 is a well-characterized Nap protein involved in nucleocytoplasmic shuttling of histones and genome-wide nucleosome assembly16. It binds both H2A-H2B and H3-H4 with nanomolar affinity and previous work has shown that ScNap1CORE bind H2A-H2B approximately 7.5-fold weaker than ScNap1FL17. The second strategy compares ScNap1FL and a Nap ortholog from Caenorhabditis elegans (CeNap1). We identified CeNap1 in an organism-specific blastp18 search using ScNap1FL as the query protein. The CeNap1 sequence (D2096.8) has been noted in two genetic studies and the CeNap1 knockout suggests a role in transcriptional regulation19,20. A ClustalΩ alignment21 reveals 24% identify and 17% similarity to ScNap1FL (Fig. 1A). Comparison is pertinent to this study as CeNap1 lacks an N-terminal tail and has a short C-terminal acidic region like ScNap1CORE. CeNap1 is thus a naturally-occurring Nap protein with no N-terminal tail and a short C-terminal tail. The high sequence conservation between CeNap1 and ScNap1CORE is reflected in their structural homology (Fig. 1A-C). Recombinant CeNap1 purifies using standard chromatography and can form 3.0 Å diffracting crystals (Fig. S1A). We phased these crystals using molecular replacement with a hybrid model containing the dimerization helix from Plasmodium falciparum Nap1 and the α-β domain from ScNap16,8. Crystallographic statistics are reported in Table S1. The resulting asymmetric unit contains four almost-identical protomers (RMSD < 0.4 Å) arranged as the characteristic Nap homodimer and two monomers (Fig. S1B).

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Figure 1. CeNap1 is structurally similar to ScNap1CORE. (A) Sequence alignment and secondary structure of CeNap1 (rainbow) and ScNap1 (grey). (B) Crystal structure of the CeNap1 homodimer with one chain colored rainbow and the other white. (C) Superposition of CeNap1 (green) and ScNap1CORE (wheat) (RMSD 2.2 Å). Second chain of each homodimer is shown in white. (D) Surface charge distribution of CeNap1 (top) and ScNap1CORE (bottom) homodimers (APBS scale -10 to +10). CeNap1 is PDB 6N2G. ScNap1CORE is PDB 2Z2R. CeNap1 purification, asymmetric unit, crystal packing, and electron density are shown in Fig. S1. CeNap1 crystallographic statistics are in Table S1.


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Biochemistry

Figure 2. Nap2 homodimers exhibit salt-dependent self-association. SEC (left y-axis) and MWs (right y-axis) of CeNap1(A), ScNap1FL (B), and ScNap1CORE (C) at 300 mM NaCl (black) or 150 mM NaCl (grey). Dashed lines show the theoretical MW of a Nap2 homodimer and a Nap4 homotetramer. Measured MWs are listed in Table S2. Crystal packing of the two antiparallel monomers from neighboring asymmetric units constitute the well-preserved Nap2 homodimeric structure (Fig. S1C). Density is not observed for the first nine residues, an eight-residue loop (residues 251-258), nor the short C-terminal acidic tail. The overall structural homology of CeNap1 and ScNap1CORE is evidenced by their superposition (RMSD < 2.2 Å for the dimer) and similar surface distribution of charge (Fig. 1C-D). Both proteins contain an acidic cavity and basic β-hairpin protrusions. The β-hairpins are also involved in CeNap1 crystal contacts and adopt a different orientation in CeNap1 than in ScNap1CORE. Another subtle difference is in the accessory domain that drapes over the dimerization helix (Fig. 1C). This domain is four residues shorter and adopts a more folded conformation in CeNap1 than in ScNap1CORE. The overall similarity to ScNap1CORE makes CeNap1 a valuable tool for uncovering the function of Nap protein tails. To compare dimer self-association and histone-binding of CeNap1, ScNap1FL, and ScNap1CORE, we use size exclusion chromatography coupled to multi-angle light scattering (SECMALS). SEC will fractionate mixtures based on size and shape, while MALS will measure the weight-averaged molecular mass (MW) of specified fractions. Since Nap2 self-association is saltdependent, we perform SEC-MALS at 300 mM and 150 mM NaCl. For CeNap1 at 300 mM NaCl the observed MW is 80.3 kDa indicative of a stable dimeric species (theoretical dimer MW is 79.2 kDa) (Fig. 2A, black), while at 150 mM NaCl the MW is 99.0 kDa suggestive of a dimer-tetramer equilibrium (theoretical tetramer MW is 158.4 kDa) (Fig. 2A, grey). For ScNap1FL at 300 mM NaCl the observed MW is 104.8 kDa indicative of a stable dimeric species (theoretical dimer MW is 103.6 kDa) (Fig. 2B, black), while at 150 mM NaCl the MW is 129.2 kDa suggestive of a dimertetramer equilibrium (theoretical tetramer MW is 207.1 kDa) (Fig. 2B, grey). These results for ScNap1FL are similar to that reported previously11. Unlike ScNap1FL, ScNap1CORE displays self-association at both 300mM and 150 mM NaCl. At 300 mM NaCl the observed MW is 140.2 kDa, which approaches that of a tetramer (theoretical tetramer MW is 151.5 kDa) (Fig. 2C, black). At 150 mM NaCl the MW trace is not flat indicating the presence of a heterogenous population of multimers larger than a tetramer (Fig. 2C, grey). This is consistent with prior analytical ultracentrifugation data9, but inconsistent with native mass spectrometry analysis12. It suggests that the tails limit ScNap1 self-association. This is not a general feature of Nap tails, however, as CeNap1 behaves like ScNap1FL and not the ScNap1CORE. The Nap tails thus do not play an obvious role in Nap2 self-association.

Since we are the first to biochemically characterize CeNap1, we next test histone binding in a gel-shift assay. We assay binding to Xenopus laevis H2A-H2B, H3-H4, or a mutant of H3-H4 (DM-H3H4) that is exclusively dimeric due to three point mutations in H3 (L126A, I130A, C110E)22. CeNap1 has retarded mobility in the presence of each histone type, indicating binding like that known for ScNap1FL and ScNap1CORE (Fig. S2A-C). To determine the absolute stoichiometry of these complexes, we perform a comprehensive SEC-MALS analysis where histones are titrated against a constant amount of Nap protein (Fig. 3). Molar ratios are calculated using a Nap monomer and histone dimer, and stoichiometries are reported as Nap:histone. Experiments with H2A-H2B uncover a correlation between stoichiometry and Nap tails. For CeNap1, addition of 0.5 to 2.0 molar equivalents of H2A-H2B results only in a 2:1 complex with a MW of 107.5 kDa (theoretical 2:1 MW is 107.0 kDa) (Fig. 3A, left). Free H2A-H2B is also detected at 1.5 and 2.0 molar equivalents. The presence of excess H2A-H2B thus cannot force a second copy of H2A-H2B to bind the CeNap12 homodimer. This contrasts ScNap1FL, where addition of 1.0 to 2.0 molar equivalents of H2A-H2B results in an equilibrium between 2:1 and 2:2 complexes (Fig. 3A, middle). The observed MWs (139.0 kDa, 150.0 kDa, and 153.4 kDa) approach that of a 2:2 complex (theoretical 2:2 MW is 159.2 kDa) and almost no free H2A-H2B is detected. The ScNap1FL-2 homodimer must contain two H2A-H2B binding sites with the second site more transiently populated than the first. Our result that the complex between ScNap1FL and 1 or 2 copies of H2A-H2B does not oligomerize is apparently contradictory to published mass spectrometry data12. It is feasible, however, that interactions observed in the mass spectrometer are not maintained in SEC-MALS at 300 mM NaCl. Similar SECMALS experiments at 150 mM NaCl in fact give MWs slightly above a 2:1 complex for CeNap1 and a 2:2 complex for ScNap1FL (Fig. S3A). This may indicate a small population of oligomeric complex, as well as stabilization of the second H2A-H2B-binding site in ScNap1FL. Analysis of ScNap1CORE at 300 mM NaCl further suggests that the second H2A-H2B-binding site in ScNap1FL is in the tails. Addition of H2A-H2B to ScNap1CORE results in a mix of oligomers larger than a 2:2 complex, indicative of an equilibrium between many self-associated states (Fig. 3A, right). Since this is not observed for ScNap1FL with H2A-H2B under similar conditions (Fig. 3A, middle), the tails may be interfering with oligomeric interactions. This is not a conserved role for the tails as H2A-H2Bbound CeNap1 also does not self-associate at 300 mM NaCl (Fig. 3B, left). Although the heterogeneity of ScNap1CORE with H2AH2B precludes absolute stoichiometric determination, the molar


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Figure 3. Stoichiometry of complexes between Nap protein and H2A-H2B, H3-H4, or DM-H3-H4 at 300 mM NaCl. SEC (left y-axis) and MWs (right y-axis) of CeNap1 (left), ScNap1FL (middle) and ScNap1CORE (right) with H2A-H2B (A), H3-H4 (B), or DM-H3-H4 (C) at 300 mM NaCl. Nap protein (black) is held constant and histones are titrated at 0.5 (blue), 1.0 (green), 1.5 (orange), and 2.0 (grey) molar equivalents. Dashed lines show theoretical MWs of indicated complexes. Native-PAGE of the complexes are shown in Fig. S2. Similar experiments with H2A-H2B at 150 mM NaCl are shown in Fig. S3A. SEC profiles with a larger volume range are shown in Fig. S3B. Measured MWs are listed in Table S3. ratio of the complex is indicated by the free H2A-H2B profile. This profile matches CeNap1 rather than ScNap1FL, suggesting a molar ratio of 2:1. This is consistent with previous data using ScNap1CORE12. Hence, CeNap1 and ScNap1COREbind H2A-H2B at 2:1, while the tail-containing ScNap1FL exhibits an equilibrium between 2:1 and 2:2 complexes. The 2:1 molar ratio of the complex between CeNap1 and H2AH2B is surprisingly not conserved for H3-H4. The addition of 0.5 molar equivalents of H3-H4 to CeNap1 produces a MW of 199.8 kDa, which is slightly below a 4:2 complex (theoretical 4:2 MW is 211.9 kDa) (Fig. 3B, left). The (H3-H4)2 tetramer is bridging two CeNap12 homodimers. A second copy of (H3-H4)2 can bind to each CeNap12 homodimer, as the MW increases with further H3-H4 addition. The MWs measured using DM-H3-H4 also support two H3-H4-binding sites in the CeNap12 homodimer, as addition of 1.0 molar equivalent results in a 2:2 complex with a MW of 133.0 kDa

(theoretical 2:2 MW is 132.6 kDa) (Fig. 3C, left). The fact that a homogeneous 2:4 complex is not observed with 2.0 molar equivalents of wild-type H3-H4 suggests that H3-H4 tetramer formation hinders population of the second site. The ability to force binding of two copies of wild-type H3-H4 or DM-H3-H4 is shared with ScNap1FL, although the dynamics of complex formation are variable (Fig. 3B-C, middle). Artificial oligomerization and the inability to recover free H3-H4 preclude comparison with ScNap1CORE (Fig. 3B-C, right). Regardless, the data show that the CeNap12 homodimer can bind two copies of (H3-H4)2 tetramer or H3-H4 dimer, but only one copy of H2A-H2B. This is surprising given histone structural homology and suggests that Nap proteins have multiple modes of histone interaction. These modes likely involve histone side-chains that differ between H2A-H2B and H3H4.


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Biochemistry

Figure 4. Nap proteins require acidic tails to compete interactions between DNA for H2A-H2B. Native-PAGE of CeNap1 (A), ScNap1FL (B), or ScNap1CORE (C) titrated against a complex between 147 bp Widom 601 DNA and H2A-H2B mixed 1:7. DNA was visualized with ethidium bromide. Protein was visualized using coomassie blue as shown in Fig. S4. A characteristic function of H2A-H2B chaperones is the ability to compete interactions between H2A-H2B and DNA3. We monitor this activity for CeNap1, ScNap1FL and ScNap1CORE in a nativePAGE assay using a 7:1 mixture of H2A-H2B and 147 bp Widom 601 DNA. Titration of CeNap1 against this DNA complex results in the release of a very small amount of free DNA (Fig. 4A). Even when CeNap1 stoichiometrically binds H2A-H2B (i.e. 2:1) only a small amount of free DNA is released. This contrasts titration of ScNap1FL which, as observed previously3, almost completely resolves the complexes to release free DNA (Fig. 4B). Free DNA is obvious when ScNap1FL stoichiometrically binds H2A-H2B (i.e. 1:1). The ability to compete H2A-H2B from DNA is lost in ScNap1CORE, which behaves like CeNap1, and releases only a small amount of free DNA (Fig. 4C). The absence of tails thus correlates with an impaired ability to compete H2A-H2B from DNA. Mechanistically, CeNap1 and ScNap1CORE may have reduced binding to H2A-H2B in the presence of DNA or may form a ternary complex containing Nap protein, H2A-H2B and DNA. The former seems likely for CeNap1 as protein-staining of the competition gel shows a large amount of free CeNap1 (Fig. S4). The absence of the N-terminal tail and presence of a short C-terminal tail compromises the ability of the Nap protein to compete interactions between H2A-H2B and DNA. It is possible that the acidic nature of the tails allows them to mimic the negatively-charged DNA backbone. Overall, our structural and biochemical characterization of CeNap1 provides molecular insight into Nap proteins. CeNap1 is a valuable homolog as it naturally lacks the N-terminal tail and has a short C-terminal tail. This divergence is complimented by high structural homology in the intervening core region. While our results caution against extrapolations between Nap protein and histone homologs, we do observe some general trends. Nap protein oligomerization is dominated by a dimer-tetramer equilibrium that is salt- and concentration-dependent. While we do not uncover a conserved role for the tails, it is possible that they subtly tune the strength of self-association. The tails however do define the stoichiometry of H2A-H2B interaction at least at 150 mM and 300 mM NaCl. The core of the Nap2 homodimer contains a single high affinity H2A-H2B-binding site that is likely visualized in the crystal structure of ScNap1CORE with H2A-H2B12. The presence of both Nap tails introduces an equilibrium between 2:1 and 2:2 complexes suggesting the presence of a second, lower-affinity site. It seems unlikely that the 2:2 complex is symmetric as previously assumed4. Notably, we also observe very little oligomerization of the H2A-H2B-bound Nap2 homodimer. Oligomeric interactions observed by others may be weak and not maintained in our stringent experimental conditions12,14. Finally, we show that Nap proteins have distinct modes of interaction between H2A-H2B and

H3-H4, and that a (H3-H4)2 tetramer can bridge two Nap2 homodimers. Crystals structures of Nap proteins with H3-H4 are eagerly anticipated. A deep understanding of histone binding by Nap proteins takes us closer to understanding the molecular determinants of histone chaperone function.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Supporting information (SupportingInformation.pdf)

AUTHOR INFORMATION Corresponding Author Sheena D’Arcy, Ph. D. Email: [email protected], Phone: +1 972-883-2915

Present Addresses Karlah Salvador – True Velocity, 1030 Nicholson Rd, Garland TX 75042.

Author Contributions P.S. and N.Z. contributed equally to this work.

Funding Sources This project was funded by start-up fund to S.D. at The University of Texas at Dallas.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We thank Jay Nix at the ALS for data collection, and Karolin Luger for supporting early stages of the project.

ABBREVIATIONS Nap, Nucleosome assembly protein; Ce, Caenorhabditis elegans; Sc, Saccharomyces cerevisiae; FL, Full-length; SEC-MALS, size exclusion chromatography coupled to multi-angle light scattering; MW, molecular weight.


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UniProt Accession IDs: CeNap1 (Q19007); ScNap1FL (P25293, with point mutations at S2T, C200A, C249A, C272A, E384D); ScNap1CORE (P25293, aa. 74-365 with point mutations at S2T, C200A, C249A, C272A); H2A (P06897, with point mutations at G99R, A123S); H2B (Q92130, deletion of the first three amino acids (SDP) and point mutations at A10P, S22T, and G98V); H3 (P84233, with point mutations at G102A); H4 (P62799).

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Figure 1. CeNap1 is structurally similar to ScNap1CORE. (A) Sequence alignment and secondary structure of CeNap1 (rainbow) and ScNap1 (grey). (B) Crystal structure of the CeNap1 homodimer with one chain colored rainbow and the other white. (C) Superposition of CeNap1 (green) and ScNap1CORE (wheat) (RMSD 2.2 Å). Second chain of each homodimer is shown in white. (D) Surface charge distribution of CeNap1 (top) and ScNap1CORE (bottom) homodimers (APBS scale -10 to +10). CeNap1 is PDB 6N2G. ScNap1CORE is PDB 2Z2R. CeNap1 purification, asymmetric unit, crystal packing, and electron density are shown in Fig. S1. CeNap1 crystallographic statistics are in Table S1. 177x221mm (300 x 300 DPI)


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Biochemistry

Figure 2. Nap2 homodimers exhibit salt-dependent self-association. SEC (left y-axis) and MWs (right y-axis) of CeNap1(A), ScNap1FL (B), and ScNap1CORE (C) at 300 mM NaCl (black) or 150 mM NaCl (grey). Dashed lines show the theoretical MW of a Nap2 homodimer and a Nap4 homotetramer. Measured MWs are listed in Table S2.


Biochemistry 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 3. Stoichiometry of complexes between Nap protein and H2A-H2B, H3-H4, or DM-H3-H4 at 300 mM NaCl. SEC (left y-axis) and MWs (right y-axis) of CeNap1 (left), ScNap1FL (middle) and ScNap1CORE (right) with H2A-H2B (A), H3-H4 (B), or DM-H3-H4 (C) at 300 mM NaCl. Nap protein (black) is held constant and histones are titrated at 0.5 (blue), 1.0 (green), 1.5 (orange), and 2.0 (grey) molar equivalents. Dashed lines show theoretical MWs of indicated complexes. Native-PAGE of the complexes are shown in Fig. S2. Similar experiments with H2A-H2B at 150 mM NaCl are shown in Fig. S3A. SEC profiles with a larger volume range are shown in Fig. S3B. Measured MWs are listed in Table S3.


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Biochemistry

Figure 4. Nap proteins require acidic tails to compete interactions between DNA for H2A-H2B. Native-PAGE of CeNap1 (A), ScNap1FL (B), or ScNap1CORE (C) titrated against a complex between 147 bp Widom 601 DNA and H2A-H2B mixed 1:7. DNA was visualized with ethidium bromide. Protein was visualized using coomassie blue as shown in Fig. S4. 177x56mm (300 x 300 DPI)


Biochemistry 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

TOC 89x35mm (300 x 300 DPI)


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Characterization of Caenorhabditis elegans Nucleosome Assembly

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