FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2091288903> ?p ?o ?g. }

• W2091288903 endingPage "46" @default.
• W2091288903 startingPage "36" @default.
• W2091288903 abstract "In fission yeast, assembly of centromeric heterochromatin requires the RITS complex, which consists of Ago1, Tas3, Chp1, and siRNAs derived from centromeric repeats. Recruitment of RITS to centromeres has been proposed to depend on siRNA-dependent targeting of Ago1 to centromeric sequences. Previously, we demonstrated that methylated lysine 9 of histone H3 (H3K9me) acts upstream of siRNAs during heterochromatin establishment. Our crystal structure of Chp1's chromodomain in complex with a trimethylated lysine 9 H3 peptide reveals extensive sites of contact that contribute to Chp1's high-affinity binding. We found that this high-affinity binding is critical for the efficient establishment of centromeric heterochromatin, but preassembled heterochromatin can be maintained when Chp1's affinity for H3K9me is greatly reduced. In fission yeast, assembly of centromeric heterochromatin requires the RITS complex, which consists of Ago1, Tas3, Chp1, and siRNAs derived from centromeric repeats. Recruitment of RITS to centromeres has been proposed to depend on siRNA-dependent targeting of Ago1 to centromeric sequences. Previously, we demonstrated that methylated lysine 9 of histone H3 (H3K9me) acts upstream of siRNAs during heterochromatin establishment. Our crystal structure of Chp1's chromodomain in complex with a trimethylated lysine 9 H3 peptide reveals extensive sites of contact that contribute to Chp1's high-affinity binding. We found that this high-affinity binding is critical for the efficient establishment of centromeric heterochromatin, but preassembled heterochromatin can be maintained when Chp1's affinity for H3K9me is greatly reduced. The eukaryotic genome is structurally divided into dense, heterochromatic regions with low transcriptional activity and more permissive, transcriptionally active euchromatic regions. Heterochromatin is critical for genomic integrity and is localized to centromeres and telomeres as well as other repetitive sequences that often contain transposons and other parasitic genomic elements. In the fission yeast S. pombe, assembly of specialized chromatin at the centromere allows faithful segregation of chromosomes. The centromere consists of a central region on which the kinetochore assembles, flanked by pericentromeric regions consisting of repeat sequences that are packaged into heterochromatin (Allshire et al., 1995Allshire R.C. Nimmo E.R. Ekwall K. Javerzat J.P. Cranston G. Mutations derepressing silent centromeric domains in fission yeast disrupt chromosome segregation.Genes Dev. 1995; 9: 218-233Crossref PubMed Scopus (384) Google Scholar, Partridge et al., 2000Partridge J.F. Borgstrom B. Allshire R.C. Distinct protein interaction domains and protein spreading in a complex centromere.Genes Dev. 2000; 14: 783-791PubMed Google Scholar, Takahashi et al., 2000Takahashi K. Chen E.S. Yanagida M. Requirement of Mis6 centromere connector for localizing a CENP-A-like protein in fission yeast.Science. 2000; 288: 2215-2219Crossref PubMed Scopus (316) Google Scholar). Pericentromeric heterochromatin is characterized by high densities of histone H3 lysine 9 (H3K9) methylation and binding of the S. pombe HP1 homolog, Swi6. Its formation relies on an intricate interplay between transcription by RNA polymerase II, the RNAi machinery, and chromatin-modifying enzymes (Buhler and Moazed, 2007Buhler M. Moazed D. Transcription and RNAi in heterochromatic gene silencing.Nat. Struct. Mol. Biol. 2007; 14: 1041-1048Crossref PubMed Scopus (192) Google Scholar). Centromeres are transcribed at low levels in G1/S phase (Chen et al., 2008Chen E.S. Zhang K. Nicolas E. Cam H.P. Zofall M. Grewal S.I.S. Cell cycle control of centromeric repeat transcription and heterochromatin assembly.Nature. 2008; 451: 734-737Crossref PubMed Scopus (285) Google Scholar, Kloc et al., 2008Kloc A. Zaratiegui M. Nora E. Martienssen R. RNA interference guides histone modification during the S phase of chromosomal replication.Curr. Biol. 2008; 18: 490-495Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), and these transcripts provide a source for the production of double-stranded RNAs that are diced to form siRNAs (Volpe et al., 2002Volpe T.A. Kidner C. Hall I.M. Teng G. Grewal S.I.S. Martienssen R.A. Regulation of heterochromatic silencing and histone H3 Lysine-9 methylation by RNAi.Science. 2002; 297: 1833-1837Crossref PubMed Scopus (1629) Google Scholar, Reinhart and Bartel, 2002Reinhart B.J. Bartel D.P. Small RNAs correspond to centromere heterochromatic Repeats.Science. 2002; 297: 1831Crossref PubMed Scopus (414) Google Scholar, Cam et al., 2005Cam H.P. Sugiyama T. Chen E.S. Chen X. FitzGerald P.C. Grewal S.I.S. Comprehensive analysis of heterochromatin- and RNAi-mediated epigenetic control of the fission yeast genome.Nat. Genet. 2005; 37: 809-819Crossref PubMed Scopus (385) Google Scholar). The siRNAs are loaded into the argonaute protein Ago1, a component of the RNAi effector complex RITS (Verdel et al., 2004Verdel A. Jia S. Gerber S. Sugiyama T. Gygi S. Grewal S.I.S. Moazed D. RNAi-mediated targeting of heterochromatin by the RITS complex.Science. 2004; 303: 672-676Crossref PubMed Scopus (949) Google Scholar). In addition to Ago1, the RITS complex contains Chp1, a chromodomain protein that binds methylated H3K9 (Partridge et al., 2002Partridge J.F. Scott K.S.C. Bannister A.J. Kouzarides T. Allshire R.C. cis-Acting DNA from fission yeast centromeres mediates histone H3 methylation and recruitment of silencing factors and cohesin to an ectopic site.Curr. Biol. 2002; 12: 1652-1660Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) and a GW-repeat protein, Tas3 (Verdel et al., 2004Verdel A. Jia S. Gerber S. Sugiyama T. Gygi S. Grewal S.I.S. Moazed D. RNAi-mediated targeting of heterochromatin by the RITS complex.Science. 2004; 303: 672-676Crossref PubMed Scopus (949) Google Scholar, Partridge et al., 2007Partridge J.F. DeBeauchamp J.L. Kosinski A.M. Ulrich D.L. Hadler M.J. Noffsinger V.J. Functional separation of the requirements for establishment and maintenance of centromeric heterochromatin.Mol. Cell. 2007; 26: 593-602Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, Till et al., 2007Till S. Lejeune E. Thermann R. Bortfeld M. Hothorn M. Enderle D. Heinrich C. Hentze M.W. Ladurner A.G. A conserved motif in Argonaute-interacting proteins mediates functional interactions through the Argonaute PIWI domain.Nat. Struct. Mol. Biol. 2007; 14: 897-903Crossref PubMed Scopus (186) Google Scholar). siRNA-loaded RITS targets and slices nascent transcripts (Irvine et al., 2006Irvine D.V. Zaratiegui M. Tolia N.H. Goto D.B. Chitwood D.H. Vaughn M.W. Joshua-Tor L. Martienssen R.A. Argonaute slicing is required for heterochromatic silencing and spreading.Science. 2006; 313: 1134-1137Crossref PubMed Scopus (158) Google Scholar, Buker et al., 2007Buker S.M. Iida T. Buhler M. Villen J. Gygi S.P. Nakayama J. Moazed D. Two different Argonaute complexes are required for siRNA generation and heterochromatin assembly in fission yeast.Nat. Struct. Mol. Biol. 2007; 14: 200-207Crossref PubMed Scopus (94) Google Scholar), thereby amplifying the siRNA response by providing templates for the RNA-dependent RNA polymerase (Motamedi et al., 2004Motamedi M.R. Verdel A. Colmenares S.U. Gerber S.A. Gygi S.P. Moazed D. Two RNAi complexes, RITS and RDRC, physically interact and localize to noncoding centromeric RNAs.Cell. 2004; 119: 789-802Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar, Sugiyama et al., 2005Sugiyama T. Cam H. Verdel A. Moazed D. Grewal S.I.S. RNA-dependent RNA polymerase is an essential component of a self-enforcing loop coupling heterochromatin assembly to siRNA production.Proc. Natl. Acad. Sci. USA. 2005; 102: 152-157Crossref PubMed Scopus (227) Google Scholar). Recruitment of RITS to centromeres promotes the association of the Clr4-containing complex CLRC to the centromere in a positive feedback loop (Partridge et al., 2002Partridge J.F. Scott K.S.C. Bannister A.J. Kouzarides T. Allshire R.C. cis-Acting DNA from fission yeast centromeres mediates histone H3 methylation and recruitment of silencing factors and cohesin to an ectopic site.Curr. Biol. 2002; 12: 1652-1660Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, Verdel et al., 2004Verdel A. Jia S. Gerber S. Sugiyama T. Gygi S. Grewal S.I.S. Moazed D. RNAi-mediated targeting of heterochromatin by the RITS complex.Science. 2004; 303: 672-676Crossref PubMed Scopus (949) Google Scholar, Zhang et al., 2008Zhang K. Mosch K. Fischle W. Grewal S.I.S. Roles of the Clr4 methyltransferase complex in nucleation, spreading and maintenance of heterochromatin.Nat. Struct. Mol. Biol. 2008; 15: 381-388Crossref PubMed Scopus (270) Google Scholar). Methylation of nucleosomes on histone H3K9 by Clr4, the Su(var)3-9 homolog, facilitates the robust assembly of centromeric heterochromatin by promoting recruitment of Swi6 and cohesin for proper centromere function (Bannister et al., 2001Bannister A.J. Zegerman P. Partridge J.F. Miska E.A. Thomas J.O. Allshire R.C. Kouzarides T. Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain.Nature. 2001; 410: 120-124Crossref PubMed Scopus (2176) Google Scholar, Nakayama et al., 2001Nakayama J. Rice J.C. Strahl B.D. Allis C.D. Grewal S.I.S. Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly.Science. 2001; 292: 110-113Crossref PubMed Scopus (1372) Google Scholar, Bernard et al., 2001Bernard P. Maure J. Partridge J.F. Genier S. Javerzat J. Allshire R.C. Requirement of Heterochromatin for Cohesion at Centromeres.Science. 2001; 294: 2539-2542Crossref PubMed Scopus (490) Google Scholar, Nonaka et al., 2002Nonaka N. Kitajima T. Yokobayashi S. Xiao G. Yamamoto M. Grewal S.I.S. Watanabe Y. Recruitment of cohesin to heterochromatic regions by Swi6/HP1 in fission yeast.Nat. Cell Biol. 2002; 4: 89-93Crossref PubMed Scopus (377) Google Scholar). Chromodomains, originally identified in heterochromatin-associated factors (Paro and Hogness, 1991Paro R. Hogness D.S. The Polycomb protein shares a homologous domain with a heterochromatin-associated protein of Drosophila.Proc. Natl. Acad. Sci. USA. 1991; 88: 263-267Crossref PubMed Scopus (469) Google Scholar), are recognized as modules used to target proteins to specific chromosomal loci (Brehm et al., 2004Brehm A. Tufteland K.R. Aasland R. Becker P.B. The many colours of chromodomains.Bioessays. 2004; 26: 133-140Crossref PubMed Scopus (147) Google Scholar). The chromodomain family displays a range of activities, including methyl-lysine recognition (Lachner et al., 2001Lachner M. O'Carroll D. Rea S. Mechtler K. Jenuwein T. Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins.Nature. 2001; 410: 116-120Crossref PubMed Scopus (2173) Google Scholar, Bannister et al., 2001Bannister A.J. Zegerman P. Partridge J.F. Miska E.A. Thomas J.O. Allshire R.C. Kouzarides T. Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain.Nature. 2001; 410: 120-124Crossref PubMed Scopus (2176) Google Scholar, Jacobs and Khorasanizadeh, 2002Jacobs S.A. Khorasanizadeh S. Structure of HP1 chromodomain bound to a lysine 9-methylated histone H3 tail.Science. 2002; 295: 2080-2083Crossref PubMed Scopus (647) Google Scholar) and RNA and DNA binding (Akhtar et al., 2000Akhtar A. Zink D. Becker P.B. Chromodomains are protein-RNA interaction modules.Nature. 2000; 407: 405-409Crossref PubMed Scopus (309) Google Scholar, Bouazoune et al., 2002Bouazoune K. Mitterweger A. Längst G. Imhof A. Akhtar A. Becker P.B. Brehm A. The dMi-2 chromodomains are DNA binding modules important for ATP-dependent nucleosome mobilization.EMBO J. 2002; 21: 2430-2440Crossref PubMed Scopus (123) Google Scholar). As shown for HP1 and Polycomb, chromodomains can discriminate between different methyl marks in almost identical sequence contexts (Fischle et al., 2008Fischle W. Franz H. Jacobs S.A. Allis C.D. Khorasanizadeh S. Specificity of the chromodomain Y chromosome family of chromodomains for lysine-methylated ARK(S/T) motifs.J. Biol. Chem. 2008; 283: 19626-19635Crossref PubMed Scopus (83) Google Scholar, Min et al., 2003Min J. Zhang Y. Xu R. Structural basis for specific binding of Polycomb chromodomain to histone H3 methylated at Lys 27.Genes Dev. 2003; 17: 1823-1828Crossref PubMed Scopus (512) Google Scholar). In S. pombe, many of the key factors for the assembly of centromeric heterochromatin have a chromodomain. The chromodomain of Chp1 is crucial for tethering the RITS complex to centromeric regions (Petrie et al., 2005Petrie V.J. Wuitschick J.D. Givens C.D. Kosinski A.M. Partridge J.F. RNA interference (RNAi)-dependent and RNAi-independent association of the Chp1 chromodomain protein with distinct heterochromatic loci in fission yeast.Mol. Cell. Biol. 2005; 25: 2331-2346Crossref PubMed Scopus (67) Google Scholar). The chromodomain of Clr4 links the deposition of methyl marks to their readout and thereby provides a feed-forward mechanism for amplification and spreading of the initial signal (Zhang et al., 2008Zhang K. Mosch K. Fischle W. Grewal S.I.S. Roles of the Clr4 methyltransferase complex in nucleation, spreading and maintenance of heterochromatin.Nat. Struct. Mol. Biol. 2008; 15: 381-388Crossref PubMed Scopus (270) Google Scholar). Swi6 uses its chromodomain to bind to methylated H3K9, and since it is dimeric, it could tether adjacent nucleosomes, thereby inducing a specialized chromatin structure (Cowieson et al., 2000Cowieson N.P. Partridge J.F. Allshire R.C. McLaughlin P.J. Dimerisation of a chromo shadow domain and distinctions from the chromodomain as revealed by structural analysis.Curr. Biol. 2000; 10: 517-525Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, Bannister et al., 2001Bannister A.J. Zegerman P. Partridge J.F. Miska E.A. Thomas J.O. Allshire R.C. Kouzarides T. Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain.Nature. 2001; 410: 120-124Crossref PubMed Scopus (2176) Google Scholar, Nakayama et al., 2001Nakayama J. Rice J.C. Strahl B.D. Allis C.D. Grewal S.I.S. Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly.Science. 2001; 292: 110-113Crossref PubMed Scopus (1372) Google Scholar). The second HP1 homolog in S. pombe, Chp2, is similarly localized to regions of heterochromatin and has recently been found to be part of the SHREC2 complex (Sadaie et al., 2008Sadaie M. Kawaguchi R. Ohtani Y. Arisaka F. Tanaka K. Shirahige K. Nakayama J. Balance between distinct HP1 family proteins controls heterochromatin assembly in fission yeast.Mol. Cell. Biol. 2008; 28: 6973-6988Crossref PubMed Scopus (82) Google Scholar, Motamedi et al., 2008Motamedi M.R. Hong E.E. Li X. Gerber S. Denison C. Gygi S. Moazed D. HP1 proteins form distinct complexes and mediate heterochromatic gene silencing by nonoverlapping mechanisms.Mol. Cell. 2008; 32: 778-790Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). The RITS complex is critical for the assembly of heterochromatin; thus, determination of the mechanism by which RITS is recruited to centromeres is of great interest (Verdel et al., 2004Verdel A. Jia S. Gerber S. Sugiyama T. Gygi S. Grewal S.I.S. Moazed D. RNAi-mediated targeting of heterochromatin by the RITS complex.Science. 2004; 303: 672-676Crossref PubMed Scopus (949) Google Scholar, Partridge et al., 2007Partridge J.F. DeBeauchamp J.L. Kosinski A.M. Ulrich D.L. Hadler M.J. Noffsinger V.J. Functional separation of the requirements for establishment and maintenance of centromeric heterochromatin.Mol. Cell. 2007; 26: 593-602Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Earlier models proposed that siRNAs bound by Ago1 in the RITS complex targeted RITS to either nascent transcripts or centromeric DNA and that once RITS facilitated recruitment of Clr4 activity to centromeres, di- or trimethylated H3K9 (H3K9me) stabilized association of RITS by providing a binding site for the chromodomain of Chp1 (Verdel et al., 2004Verdel A. Jia S. Gerber S. Sugiyama T. Gygi S. Grewal S.I.S. Moazed D. RNAi-mediated targeting of heterochromatin by the RITS complex.Science. 2004; 303: 672-676Crossref PubMed Scopus (949) Google Scholar). However, strains lacking components of the RNAi pathway maintain low levels of H3K9 methylation (Volpe et al., 2002Volpe T.A. Kidner C. Hall I.M. Teng G. Grewal S.I.S. Martienssen R.A. Regulation of heterochromatic silencing and histone H3 Lysine-9 methylation by RNAi.Science. 2002; 297: 1833-1837Crossref PubMed Scopus (1629) Google Scholar, Sadaie et al., 2004Sadaie M. Iida T. Urano T. Nakayama J. A chromodomain protein, Chp1, is required for the establishment of heterochromatin in fission yeast.EMBO J. 2004; 23: 3825-3835Crossref PubMed Scopus (170) Google Scholar, Partridge et al., 2007Partridge J.F. DeBeauchamp J.L. Kosinski A.M. Ulrich D.L. Hadler M.J. Noffsinger V.J. Functional separation of the requirements for establishment and maintenance of centromeric heterochromatin.Mol. Cell. 2007; 26: 593-602Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) that might initially target Chp1 to the centromere. Indeed, we previously demonstrated that H3K9 methylation by Clr4 acts upstream of siRNA production by Dcr1 during the establishment of heterochromatin, suggesting that Chp1's binding to H3K9me might play a crucial role in the initial targeting of RITS to centromeric sequences (Partridge et al., 2007Partridge J.F. DeBeauchamp J.L. Kosinski A.M. Ulrich D.L. Hadler M.J. Noffsinger V.J. Functional separation of the requirements for establishment and maintenance of centromeric heterochromatin.Mol. Cell. 2007; 26: 593-602Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Here, we demonstrate that heterochromatin establishment depends on Chp1's high affinity for H3K9me. Our crystal structure of the Chp1 chromodomain bound to an H3K9me3 peptide highlights residues that may contribute to this high affinity interaction. Mutation of these sites reduced Chp1's binding affinity 2- to 500-fold, and in vivo studies demonstrate that a 5-fold reduction in Chp1's affinity for H3K9me is sufficient to abolish the establishment of centromeric heterochromatin. Interestingly, these mutants express robust levels of centromeric siRNAs, which cannot promote heterochromatin establishment when Chp1's H3K9me binding affinity is reduced. Together, these results reveal that Chp1's high-affinity binding to H3K9me is critical for the establishment of heterochromatin. To investigate the role of Chp1's chromodomain in targeting RITS to centromeres, we determined the binding affinities of Chp1, Swi6, and Clr4 for H3K9-methylated peptides by fluorescence polarization (Figures 1A and 1B). All proteins bound specifically to the di- or trimethylated peptides, but not to unmethylated peptide. Interestingly, the Chp1 chromodomain bound both H3K9me2 and H3K9me3 peptides with significantly higher affinity than either Clr4 or Swi6 (Table 1), and all proteins bound H3K9me3 more tightly than H3K9me2.Table 1Binding Constants, Kd, to Di- and Trimethylated H3 Peptides in μM for the Constructs Used in this StudyConstructKd for H3K9me2 (μM)Kd for H3K9me3 (μM)Chp1 CD (15–76)0.55 ± 0.090.19 ± 0.02Chp1 F61A1.02 ± 0.280.54 ± 0.20Chp1 V21A3.38 ± 0.601.46 ± 0.40Chp1 E23V4.10 ± 0.373.16 ± 0.32Chp1 N59A6.22 ± 1.125.95 ± 1.83Chp1 V24M9.58 ± 0.555.00 ± 0.45Chp1 E23V V24M22.13 ± 1.7014.67 ± NAChp1 V24R>500>500Swi610.28 ± 1.693.34 ± 0.55Swi6 V82E2.19 ± 0.131.04 ± 0.10Swi6 E80V V82E1.07 ± 0.030.61 ± 0.12Clr4 CD (1-70)2.86 ± 0.270.60 ± 0.08Kd values represent mean of three or more experiments ± SD. Open table in a new tab Kd values represent mean of three or more experiments ± SD. We further characterized the binding of Chp1 and Swi6 to H3K9me peptides by isothermal titration calorimetry (ITC) experiments (Figure S1; Table S1 available online). This analysis showed Chp1 binding H3K9me3 with at least 5-fold higher affinity than Swi6 and confirmed the unusually tight association of Chp1 CD to H3K9me. To identify the source of Chp1's increased affinity for H3K9me, we solved the crystal structure of the Chp1 chromodomain (CD) in complex with an H3K9me3 peptide (Figure 1C; Table 2). The Chp1 CD structure shows the typical chromodomain architecture with a three-stranded β sheet supporting the peptide-binding groove and a C-terminal α-helix packing against the β sheet. Forming a β sheet with the N-terminus of the chromodomain, the H3 peptide sits across the chromodomain with the methylated lysine bound by the “aromatic cage” residues. When compared with the cocrystal structure of the Swi6 homolog HP1 (Jacobs and Khorasanizadeh, 2002Jacobs S.A. Khorasanizadeh S. Structure of HP1 chromodomain bound to a lysine 9-methylated histone H3 tail.Science. 2002; 295: 2080-2083Crossref PubMed Scopus (647) Google Scholar) (Figure 1D), it is evident that Chp1 interacts with the H3 peptide more tightly than HP1. This is reflected in the larger interface between the chromodomain and the peptide for the Chp1 complex (625 Å2 versus 521 Å2 for HP1). Most of the difference between the two complexes can be attributed to a better ordered N terminus of the H3 peptide when bound to Chp1. We clearly observe K4 of H3 engaging in a salt bridge and van der Waals contacts with E23 of Chp1, whereas K4 is disordered when bound to HP1. E23 is not conserved in the chromodomains of the HP1 class in which the corresponding residue, valine or alanine, cannot form such a salt bridge (Figure 2A). Additionally, the amide oxygen of H3Q5 is pointing into the N-terminal end of Chp1's C-terminal helix, interacting with the positive end of the helix dipole. A smaller contribution that increases Chp1's binding interface compared to HP1 originates from an increased contact interface with H3R8 and a bridge over H3A7 formed by a van der Waals contact between V21 and F61 (Figure 1D).Table 2Data Collection and Refinement StatisticsChp1 CDData CollectionSpace groupP43212Cell dimensions a, b, c (Å)41.56, 41.56, 87.38 α, β, γ (°)90.0, 90.0, 90.0Resolution (Å)19.3-2.2 (2.4-2.2)aData collected on one crystal; numbers in parentheses are for highest resolution shell.Rmerge (%)4.7 (31.4)I/σ(I)14.9 (3.5)Completeness (%)97.9 (99.8)Redundancy3.4 (3.3)RefinementResolution (Å)19.3-2.2No. reflections7255Rwork/Rfree (%)19.58/23.68No. atomsbNonhydrogen atoms. Riding hydrogens were used in refinement and are included in the deposited structure.576 Protein540 Ligand/ion4 Water29<B-factors> (Å2)cOnly nonhydrogen atoms included. Protein46.80 Peptide51.75 Water53.29rmsdsBond lengths (Å)0.008Bond angles (°)0.798a Data collected on one crystal; numbers in parentheses are for highest resolution shell.b Nonhydrogen atoms. Riding hydrogens were used in refinement and are included in the deposited structure.c Only nonhydrogen atoms included. Open table in a new tab To determine which residues of Chp1's CD contribute to its binding affinity, mutations were generated to specifically perturb interactions identified within the cocrystal structure. The binding affinity of these recombinant mutants was assessed by fluorescence polarization binding assays (Figure 2C; Table 1). The observed affinities ranged from close to wild-type to complete loss of specific binding. The F61A mutant showed little reduction in binding affinity compared with wild-type Chp1. A second class of mutants showed a 5- to 17-fold reduction in binding affinity for H3K9me2 compared with the wild-type Chp1 chromodomain (V21A, E23V, N59A, and V24M). A third class showed a more profound reduction in binding affinity: the E23V,V24M mutant reduced binding affinity ∼40 fold, and the V24R mutant abolished the specificity of the chromodomain interaction for K9 methylated peptides (Kd > 500 μM). The observation that disruption of the salt bridge between E23 of Chp1 and K4 of histone H3 (in the E23V mutant) reduced Chp1's binding affinity for H3K9me peptides was intriguing, since the residue at this position is frequently valine in other chromodomain proteins. To directly assess the importance of this salt bridge for high-affinity binding, we replaced the corresponding valine in the Swi6 chromodomain with glutamate (V82E). The Swi6 V82E mutant bound H3K9me2 with 5-fold higher affinity than wild-type Swi6 (Table 1; Figure S2A), indicating that the presence of glutamate at this position is an important determinant of high-affinity H3K9me association. Introduction of an E80V mutation, corresponding to V21 of Chp1, into Swi6V82E further increased Swi6's affinity by 2-fold (Table 1; Figure S2A). Importantly, these Swi6 mutants showed no increased affinity for the unmethylated histone H3 peptide, indicating that the salt bridge between K4 and a correctly positioned glutamate depends on the specific interaction of the positively charged methyl-lysine with the aromatic cage residues. In our crystal structure, the side chain of K4 lies across the aliphatic portion of E23's side chain and displays an average B-factor significantly higher than the average of the peptide (83.7 versus 51.8 Å2), indicating increased flexibility of this residue. Given the importance of H3K4 methylation in regulation of transcription (Martin and Zhang, 2005Martin C. Zhang Y. The diverse functions of histone lysine methylation.Nat. Rev. Mol. Cell Biol. 2005; 6: 838-849Crossref PubMed Scopus (1591) Google Scholar), it is possible that methylation could act as a modulator of Chp1's affinity for the H3 peptide. However, because of the open binding site and flexibility of H3K4, we think it unlikely that methylation of H3K4 would have a strong effect on binding affinity. It is interesting that although valine is widespread among chromodomains at the position corresponding to Chp1 E23, both Chp1 and Clr4 possess a glutamate at this position. We predict that the increased affinity of Clr4 compared with Swi6 for binding H3K9me may be, at least in part, due to glutamates' role in formation of the salt bridge with K4 of the H3K9me peptide. Indeed, a recent report has shown high-affinity binding for the chromodomain Y chromosome (CDY) family of proteins (Fischle et al., 2008Fischle W. Franz H. Jacobs S.A. Allis C.D. Khorasanizadeh S. Specificity of the chromodomain Y chromosome family of chromodomains for lysine-methylated ARK(S/T) motifs.J. Biol. Chem. 2008; 283: 19626-19635Crossref PubMed Scopus (83) Google Scholar), which also have a glutamate in this position. S10 of histone H3 is phosphorylated during mitosis and displaces HP1 proteins (including Swi6) from chromatin (Fischle et al., 2005Fischle W. Tseng B.S. Dormann H.L. Ueberheide B.M. Garcia B.A. Shabanowitz J. Hunt D.F. Funabiki H. Allis C.D. Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation.Nature. 2005; 438: 1116-1122Crossref PubMed Scopus (738) Google Scholar, Hirota et al., 2005Hirota T. Lipp J.J. Toh B. Peters J. Histone H3 serine10 phosphorylation by Aurora B causes HP1 dissociation from heterochromatin.Nature. 2005; 438: 1176-1180Crossref PubMed Scopus (517) Google Scholar, Yamada et al., 2005Yamada T. Fischle W. Sugiyama T. Allis C.D. Grewal S.I. The nucleation and maintenance of heterochromatin by a histone deacetylase in fission yeast.Mol. Cell. 2005; 20: 173-185Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). We investigated whether binding of Swi6 and Chp1 to H3K9me peptides was affected by S10 phosphorylation and found a strong reduction in both Chp1- and Swi6-binding affinity (Table S2; Figure S2B), making it unlikely that Chp1 persists on chromatin during mitosis. To test the physiological consequences of lowering Chp1's binding affinity, mutations were engineered into the chromodomain of the fully functional chp1-6xmyc allele at the normal chromosomal locus (Partridge et al., 2000Partridge J.F. Borgstrom B. Allshire R.C. Distinct protein interaction domains and protein spreading in a complex centromere.Genes Dev. 2000; 14: 783-791PubMed Google Scholar). One set of mutants (V24M, V24R, and N59A) was designed to mimic previously identified HP1 mutants that affect heterochromatic gene silencing in Drosophila by destabilization of the H3-binding interface (Platero et al., 1995Platero J.S. Hartnett T. Eissenberg J.C. Functional analysis of the chromo domain of HP1.EMBO J. 1995; 14: 3977-3986Crossref PubMed Scopus (233) Google Scholar, Jacobs et al., 2001Jacobs S.A. Taverna S.D. Zhang Y. Briggs S.D. Li J. Eissenberg J.C. Allis C. Khorasanizadeh S. Specificity of the HP1 chromo domain for the methylated N-terminus of histone H3.EMBO J. 2001; 20: 5232-5241Crossref PubMed Scopus (330) Google Scholar). We further generated mutants that block the salt bridge between E23 and K4 of the peptide (E23Vchp1), disrupt the V21-F61 “bridge” over the peptide (V21Achp1 and F61Achp1), or block the coordination of zinc by D48 and D51 (D48S; D51Schp1) (see the Supplemental Data). Additionally, a replacement of the entire chromodomain of Chp1 with that of Swi6 (chp1Swi6CD) was included. Western blot analysis with an anti-myc antibody showed that all the mutant proteins were stably expressed in fission yeast (Figure S3). In wild-type cells (chp1+) or cells bearing wild-type chp1-6xmyc, centromeric heterochromatin silences a transgene (cen::ura4+) inserted at the dg region of the outer repeats in centromere 1, allowing growth on media containing FOA, a drug toxic to ura4+-expressing cells (Allshire et al., 1995Allshire R.C. Nimmo E.R. Ekwall K. Javerzat J.P. Cranston G. Mutations derepressing silent centromeric domains in fission yeast disrupt chromosome segregation.Genes Dev. 1995; 9: 218-233Crossref PubMed Scopus (384) Google Scholar, Partridge et" @default.
• W2091288903 created "2016-06-24" @default.
• W2091288903 creator A5020476833 @default.
• W2091288903 creator A5023331856 @default.
• W2091288903 creator A5023541010 @default.
• W2091288903 creator A5049531558 @default.
• W2091288903 creator A5059398577 @default.
• W2091288903 creator A5076746607 @default.
• W2091288903 creator A5086943824 @default.
• W2091288903 date "2009-04-01" @default.
• W2091288903 modified "2023-09-27" @default.
• W2091288903 title "High-Affinity Binding of Chp1 Chromodomain to K9 Methylated Histone H3 Is Required to Establish Centromeric Heterochromatin" @default.
• W2091288903 cites W1500148822 @default.
• W2091288903 cites W1509466986 @default.
• W2091288903 cites W1603793127 @default.
• W2091288903 cites W160853306 @default.
• W2091288903 cites W1971279688 @default.
• W2091288903 cites W1978197643 @default.
• W2091288903 cites W1985180051 @default.
• W2091288903 cites W1988249075 @default.
• W2091288903 cites W1996567457 @default.
• W2091288903 cites W1997630514 @default.
• W2091288903 cites W1997814909 @default.
• W2091288903 cites W1999284470 @default.
• W2091288903 cites W2004658265 @default.
• W2091288903 cites W2015735754 @default.
• W2091288903 cites W2024295388 @default.
• W2091288903 cites W2026344530 @default.
• W2091288903 cites W2028795836 @default.
• W2091288903 cites W2032187789 @default.
• W2091288903 cites W2037541952 @default.
• W2091288903 cites W2040234712 @default.
• W2091288903 cites W2042812035 @default.
• W2091288903 cites W2046393794 @default.
• W2091288903 cites W2046820586 @default.
• W2091288903 cites W2052770408 @default.
• W2091288903 cites W2053803579 @default.
• W2091288903 cites W2053874183 @default.
• W2091288903 cites W2055223560 @default.
• W2091288903 cites W2056024736 @default.
• W2091288903 cites W2057664120 @default.
• W2091288903 cites W2059999434 @default.
• W2091288903 cites W2064791692 @default.
• W2091288903 cites W2065055008 @default.
• W2091288903 cites W2068986932 @default.
• W2091288903 cites W2070122527 @default.
• W2091288903 cites W208070023 @default.
• W2091288903 cites W2087746700 @default.
• W2091288903 cites W2092592278 @default.
• W2091288903 cites W2102691929 @default.
• W2091288903 cites W2105624980 @default.
• W2091288903 cites W2106639226 @default.
• W2091288903 cites W2107905258 @default.
• W2091288903 cites W2110107454 @default.
• W2091288903 cites W2110399580 @default.
• W2091288903 cites W2117771255 @default.
• W2091288903 cites W2132434654 @default.
• W2091288903 cites W2137824312 @default.
• W2091288903 cites W2140197830 @default.
• W2091288903 cites W2141764062 @default.
• W2091288903 cites W2142414089 @default.
• W2091288903 cites W2146394302 @default.
• W2091288903 cites W2151809557 @default.
• W2091288903 cites W2152092610 @default.
• W2091288903 cites W2152869747 @default.
• W2091288903 cites W2158168507 @default.
• W2091288903 doi "https://doi.org/10.1016/j.molcel.2009.02.024" @default.
• W2091288903 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2705653" @default.
• W2091288903 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19362535" @default.
• W2091288903 hasPublicationYear "2009" @default.
• W2091288903 type Work @default.
• W2091288903 sameAs 2091288903 @default.
• W2091288903 citedByCount "103" @default.
• W2091288903 countsByYear W20912889032012 @default.
• W2091288903 countsByYear W20912889032013 @default.
• W2091288903 countsByYear W20912889032014 @default.
• W2091288903 countsByYear W20912889032015 @default.
• W2091288903 countsByYear W20912889032016 @default.
• W2091288903 countsByYear W20912889032017 @default.
• W2091288903 countsByYear W20912889032018 @default.
• W2091288903 countsByYear W20912889032019 @default.
• W2091288903 countsByYear W20912889032020 @default.
• W2091288903 countsByYear W20912889032021 @default.
• W2091288903 countsByYear W20912889032022 @default.
• W2091288903 countsByYear W20912889032023 @default.
• W2091288903 crossrefType "journal-article" @default.
• W2091288903 hasAuthorship W2091288903A5020476833 @default.
• W2091288903 hasAuthorship W2091288903A5023331856 @default.
• W2091288903 hasAuthorship W2091288903A5023541010 @default.
• W2091288903 hasAuthorship W2091288903A5049531558 @default.
• W2091288903 hasAuthorship W2091288903A5059398577 @default.
• W2091288903 hasAuthorship W2091288903A5076746607 @default.
• W2091288903 hasAuthorship W2091288903A5086943824 @default.
• W2091288903 hasBestOaLocation W20912889031 @default.
• W2091288903 hasConcept C103435993 @default.
• W2091288903 hasConcept C104317684 @default.
• W2091288903 hasConcept C161223559 @default.
• W2091288903 hasConcept C179138559 @default.

• next