Plant Homeodomain (PHD) Fingers of CHD4 Are Histone H3-binding Modules with Preference for Unmodified H3K4 and Methylated H3K9

A major challenge in chromatin biology is to understand the mechanisms by which chromatin is remodeled into active or inactive states as required during development and cell differentiation. One complex implicated in these processes is the nucleosome remodeling and histone deacetylase (NuRD) complex, which contains both histone deacetylase and nucleosome remodeling activities and has been implicated in the silencing of subsets of genes involved in various stages of cellular development. Chromodomain-helicase-DNA-binding protein 4 (CHD4) is a core component of the NuRD complex and contains a nucleosome remodeling ATPase domain along with two chromodomains and two plant homeodomain (PHD) fingers. We have previously demonstrated that the second PHD finger of CHD4 binds peptides corresponding to the N terminus of histone H3 methylated at Lys9. Here, we determine the solution structure of PHD2 in complex with H3K9me3, revealing the molecular basis of histone recognition, including a cation-π recognition mechanism for methylated Lys9. Additionally, we demonstrate that the first PHD finger also exhibits binding to the N terminus of H3, and we establish the histone-binding surface of this domain. This is the first instance where histone binding ability has been demonstrated for two separate PHD modules within the one protein. These findings suggest that CHD4 could bind to two H3 N-terminal tails on the same nucleosome or on two separate nucleosomes simultaneously, presenting exciting implications for the mechanism by which CHD4 and the NuRD complex could direct chromatin remodeling. A major challenge in chromatin biology is to understand the mechanisms by which chromatin is remodeled into active or inactive states as required during development and cell differentiation. One complex implicated in these processes is the nucleosome remodeling and histone deacetylase (NuRD) complex, which contains both histone deacetylase and nucleosome remodeling activities and has been implicated in the silencing of subsets of genes involved in various stages of cellular development. Chromodomain-helicase-DNA-binding protein 4 (CHD4) is a core component of the NuRD complex and contains a nucleosome remodeling ATPase domain along with two chromodomains and two plant homeodomain (PHD) fingers. We have previously demonstrated that the second PHD finger of CHD4 binds peptides corresponding to the N terminus of histone H3 methylated at Lys9. Here, we determine the solution structure of PHD2 in complex with H3K9me3, revealing the molecular basis of histone recognition, including a cation-π recognition mechanism for methylated Lys9. Additionally, we demonstrate that the first PHD finger also exhibits binding to the N terminus of H3, and we establish the histone-binding surface of this domain. This is the first instance where histone binding ability has been demonstrated for two separate PHD modules within the one protein. These findings suggest that CHD4 could bind to two H3 N-terminal tails on the same nucleosome or on two separate nucleosomes simultaneously, presenting exciting implications for the mechanism by which CHD4 and the NuRD complex could direct chromatin remodeling. IntroductionThe N-terminal tails of histones are subject to many reversible covalent modifications in vivo, and different modifications have often been associated with either active or repressed chromatin states. According to prevailing ideas, the status of the cell is translated to chromatin in the form of specific post-translational modification (PTM) 4The abbreviations used are: PTMpost-translational modificationNuRDnucleosome remodeling and histone deacetylasePHDplant homeodomainPDBProtein Data Bankr.m.s.d.root mean square deviationHSQCheteronuclear single quantum coherence. patterns on histone tails. This tagged chromatin is then recognized by effector proteins and complexes that regulate how the underlying genetic information is used (1Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7538) Google Scholar). The complicated and intertwined processes of tagging the histone tails, recognizing the tags, remodeling chromatin into active (open) or repressed (compacted) states, and removing the tags requires the coordination of multiple protein functions.The nucleosome remodeling and histone deacetylase (NuRD) complex is unique among nucleosome remodeling complexes in that it couples histone deacetylase activity with nucleosome remodeling ATPase activity (although the purpose of this enzymatic combination is currently unclear). The NuRD complex has traditionally been considered a transcriptional corepressor complex, consistent with the repressive function of histone deacetylation (reviewed in Refs. 2Denslow S.A. Wade P.A. Oncogene. 2007; 26: 5433-5438Crossref PubMed Scopus (351) Google Scholar, 3McDonel P. Costello I. Hendrich B. Int. J. Biochem. Cell Biol. 2009; 41: 108-116Crossref PubMed Scopus (101) Google Scholar). Several key NuRD complex components have been shown to play a role in development and cell lineage commitment in multiple contexts. For example, in Caenorhabditis elegans, the CHD4 homologue let-418 is required for the repression of germ line cell markers in differentiated cells (4Unhavaithaya Y. Shin T.H. Miliaras N. Lee J. Oyama T. Mello C.C. Cell. 2002; 111: 991-1002Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Similarly, in Arabidopsis thaliana, mutants of the CHD4 homologue PICKLE develop embryo-like characteristics after germination, indicating a role for CHD4 in the repression of embryonic genes (5Ogas J. Cheng J.C. Sung Z.R. Somerville C. Science. 1997; 277: 91-94Crossref PubMed Scopus (262) Google Scholar, 6Ogas J. Kaufmann S. Henderson J. Somerville C. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 13839-13844Crossref PubMed Scopus (407) Google Scholar). Conditional CHD4 inactivation in the mouse has revealed important functions for this protein in both differentiation and homeostasis of hematopoietic stem cells (7Williams C.J. Naito T. Arco P.G. Seavitt J.R. Cashman S.M. De Souza B. Qi X. Keables P. Von Andrian U.H. Georgopoulos K. Immunity. 2004; 20: 719-733Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 8Yoshida T. Hazan I. Zhang J. Ng S.Y. Naito T. Snippert H.J. Heller E.J. Qi X. Lawton L.N. Williams C.J. Georgopoulos K. Genes Dev. 2008; 22: 1174-1189Crossref PubMed Scopus (146) Google Scholar). Recently, however, a direct role for the NuRD complex in transcriptional activation has been demonstrated, with NuRD shown to be required for FOG-1-dependent activation of hematopoietic genes (9Miccio A. Wang Y. Hong W. Gregory G.D. Wang H. Yu X. Choi J.K. Shelat S. Tong W. Poncz M. Blobel G.A. EMBO J. 2010; 29: 442-456Crossref PubMed Scopus (120) Google Scholar). NuRD has also been identified as a modulator of aging-associated chromatin defects (10Pegoraro G. Kubben N. Wickert U. Göhler H. Hoffmann K. Misteli T. Nat. Cell Biol. 2009; 11: 1261-1267Crossref PubMed Scopus (212) Google Scholar) and implicated in DNA damage signaling and repair (11Larsen D.H. Poinsignon C. Gudjonsson T. Dinant C. Payne M.R. Hari F.J. Danielsen J.M. Menard P. Sand J.C. Stucki M. Lukas C. Bartek J. Andersen J.S. Lukas J. J. Cell Biol. 2010; 190: 731-740Crossref PubMed Scopus (175) Google Scholar, 12Polo S.E. Kaidi A. Baskcomb L. Galanty Y. Jackson S.P. EMBO J. 2010; 29: 3130-3139Crossref PubMed Scopus (251) Google Scholar, 13Smeenk G. Wiegant W.W. Vrolijk H. Solari A.P. Pastink A. van Attikum H. J. Cell Biol. 2010; 190: 741-749Crossref PubMed Scopus (177) Google Scholar, 14Chou D.M. Adamson B. Dephoure N.E. Tan X. Nottke A.C. Hurov K.E. Gygi S.P. Colaiacovo M.P. Elledge S.J. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 18475-18480Crossref PubMed Scopus (405) Google Scholar). Thus, the NuRD complex is likely to perform diverse functions centered on maintaining the balance between repression and activation of genes required for proliferation, differentiation, and homeostasis, as well as in DNA damage-response pathways. The NuRD complex is also strongly implicated in cancer, with the expression levels of a number of NuRD subunits, including MTA1/2 (15Zhang Y. Ng H.H. Erdjument-Bromage H. Tempst P. Bird A. Reinberg D. Genes Dev. 1999; 13: 1924-1935Crossref PubMed Scopus (925) Google Scholar) and RbAp48 (16Pacifico F. Paolillo M. Chiappetta G. Crescenzi E. Arena S. Scaloni A. Monaco M. Vascotto C. Tell G. Formisano S. Leonardi A. J. Clin. Endocrinol. Metab. 2007; 92: 1458-1466Crossref PubMed Scopus (27) Google Scholar), elevated in numerous cancer cell lines and cancer tissues.The 218-kDa CHD4 protein contains an ATP-dependent helicase domain and is one of the defining components of the NuRD complex. In addition to the ATPase domain, CHD4 contains two plant homeodomains (PHDs) and two chromodomains (Fig. 1A). Chromodomains are emerging predominantly as methyl-lysine-binding domains (17Fischle W. Wang Y. Jacobs S.A. Kim Y. Allis C.D. Khorasanizadeh S. Genes Dev. 2003; 17: 1870-1881Crossref PubMed Scopus (784) Google Scholar, 18Jacobs S.A. Khorasanizadeh S. Science. 2002; 295: 2080-2083Crossref PubMed Scopus (638) Google Scholar, 19Min J. Zhang Y. Xu R.M. Genes Dev. 2003; 17: 1823-1828Crossref PubMed Scopus (505) Google Scholar, 20Flanagan J.F. Mi L.Z. Chruszcz M. Cymborowski M. Clines K.L. Kim Y. Minor W. Rastinejad F. Khorasanizadeh S. Nature. 2005; 438: 1181-1185Crossref PubMed Scopus (417) Google Scholar), although the chromodomains of the Drosophila homologue of CHD4 (Mi-2) (21Bouazoune K. Mitterweger A. Längst G. Imhof A. Akhtar A. Becker P.B. Brehm A. EMBO J. 2002; 21: 2430-2440Crossref PubMed Scopus (121) Google Scholar) and of MSL3 (22Kim D. Blus B.J. Chandra V. Huang P. Rastinejad F. Khorasanizadeh S. Nat. Struct. Mol. Biol. 2010; 17: 1027-1029Crossref PubMed Scopus (80) Google Scholar) have been shown to bind DNA.The PHD is an ∼50-residue module characterized by a conserved Cys4-His-Cys3 motif that coordinates two zinc ions in a “cross-brace” configuration, where each zinc ion is coordinated by alternate pairs of Cys/His ligands. The human genome contains ∼150 PHDs, occurring in a wide variety of mostly nuclear proteins (23Bienz M. Trends Biochem. Sci. 2006; 31: 35-40Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar), and a subset of PHDs have been found to bind N-terminal histone tails, including the PHDs of bromodomain PHD finger transcription factor and ING2 (inhibitor of growth family member 2), which recognize H3K4me3 and thereby facilitate the interaction of bromodomain PHD finger transcription factor, ING2, and their associated corepressor complexes (NURF and Sin3, respectively) with chromatin (24Li H. Ilin S. Wang W. Duncan E.M. Wysocka J. Allis C.D. Patel D.J. Nature. 2006; 442: 91-95Crossref PubMed Scopus (181) Google Scholar, 25Peña P.V. Davrazou F. Shi X. Walter K.L. Verkhusha V.V. Gozani O. Zhao R. Kutateladze T.G. Nature. 2006; 442: 100-103Crossref PubMed Scopus (547) Google Scholar, 26Shi X. Hong T. Walter K.L. Ewalt M. Michishita E. Hung T. Carney D. Peña P. Lan F. Kaadige M.R. Lacoste N. Cayrou C. Davrazou F. Saha A. Cairns B.R. Ayer D.E. Kutateladze T.G. Shi Y. Côté J. Chua K.F. Gozani O. Nature. 2006; 442: 96-99Crossref PubMed Scopus (2) Google Scholar, 27Wysocka J. Swigut T. Xiao H. Milne T.A. Kwon S.Y. Landry J. Kauer M. Tackett A.J. Chait B.T. Badenhorst P. Wu C. Allis C.D. Nature. 2006; 442: 86-90Crossref PubMed Scopus (858) Google Scholar). Since the initial discovery of the chromatin binding properties of several PHDs, it is becoming clear that PHDs recognize a range of different PTMs within H3. In a recent analysis of the 18 PHD fingers from Saccharomyces cerevisiae, 8 were found to recognize H3 methylated at Lys4 (H3K4me3), and 2 showed preference for methylation at Lys36 (28Shi X. Kachirskaia I. Walter K.L. Kuo J.H. Lake A. Davrazou F. Chan S.M. Martin D.G. Fingerman I.M. Briggs S.D. Howe L. Utz P.J. Kutateladze T.G. Lugovskoy A.A. Bedford M.T. Gozani O. J. Biol. Chem. 2007; 282: 2450-2455Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Other PHDs from mammalian proteins recognize the methylation state of Lys9, including PHD2 from CHD4 and the PHDs from KDM5C and UHRF1 (29Iwase S. Lan F. Bayliss P. de la Torre-Ubieta L. Huarte M. Qi H.H. Whetstine J.R. Bonni A. Roberts T.M. Shi Y. Cell. 2007; 128: 1077-1088Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar, 30Karagianni P. Amazit L. Qin J. Wong J. Mol. Cell. Biol. 2008; 28: 705-717Crossref PubMed Scopus (190) Google Scholar, 31Musselman C.A. Mansfield R.E. Garske A.L. Davrazou F. Kwan A.H. Oliver S.S. O'Leary H. Denu J.M. Mackay J.P. Kutateladze T.G. Biochem. J. 2009; 423: 179-187Crossref PubMed Scopus (93) Google Scholar). To date, however, the best characterized interactions involve H3K4, with all currently available structures of PHD-histone complexes describing recognition of or methylated Lys4 in a on the surface of the PHD the of a PHD finger from which several histone L. Zhang S. Nature. 2010; PubMed Scopus Google In we have previously shown that PHD2 of CHD4 is to the modification state of both Lys4 and of histone H3 C.A. Mansfield R.E. Garske A.L. Davrazou F. Kwan A.H. Oliver S.S. O'Leary H. Denu J.M. Mackay J.P. Kutateladze T.G. Biochem. J. 2009; 423: 179-187Crossref PubMed Scopus (93) Google Scholar). Here, we have used to determine the molecular basis for the recognition of Lys4 and Lys9, by the solution structure of in complex with a structure a unique in which the the binding recognition of the is likely to be by a cation-π interaction with a surface Additionally, we that of CHD4 also the N-terminal of H3, for H3 and at Lys9, and a preference for Lys4 To this is the first of a recognition in which two domains from a single protein bind to two separate histone tails. These on the function of CHD4 and the NuRD complex and of the of the PHD in role as a of chromatin modification IntroductionThe N-terminal tails of histones are subject to many reversible covalent modifications in vivo, and different modifications have often been associated with either active or repressed chromatin states. According to prevailing ideas, the status of the cell is translated to chromatin in the form of specific post-translational modification (PTM) 4The abbreviations used are: PTMpost-translational modificationNuRDnucleosome remodeling and histone deacetylasePHDplant homeodomainPDBProtein Data Bankr.m.s.d.root mean square deviationHSQCheteronuclear single quantum coherence. patterns on histone tails. This tagged chromatin is then recognized by effector proteins and complexes that regulate how the underlying genetic information is used (1Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7538) Google Scholar). The complicated and intertwined processes of tagging the histone tails, recognizing the tags, remodeling chromatin into active (open) or repressed (compacted) states, and removing the tags requires the coordination of multiple protein functions.The nucleosome remodeling and histone deacetylase (NuRD) complex is unique among nucleosome remodeling complexes in that it couples histone deacetylase activity with nucleosome remodeling ATPase activity (although the purpose of this enzymatic combination is currently unclear). The NuRD complex has traditionally been considered a transcriptional corepressor complex, consistent with the repressive function of histone deacetylation (reviewed in Refs. 2Denslow S.A. Wade P.A. Oncogene. 2007; 26: 5433-5438Crossref PubMed Scopus (351) Google Scholar, 3McDonel P. Costello I. Hendrich B. Int. J. Biochem. Cell Biol. 2009; 41: 108-116Crossref PubMed Scopus (101) Google Scholar). Several key NuRD complex components have been shown to play a role in development and cell lineage commitment in multiple contexts. For example, in Caenorhabditis elegans, the CHD4 homologue let-418 is required for the repression of germ line cell markers in differentiated cells (4Unhavaithaya Y. Shin T.H. Miliaras N. Lee J. Oyama T. Mello C.C. Cell. 2002; 111: 991-1002Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Similarly, in Arabidopsis thaliana, mutants of the CHD4 homologue PICKLE develop embryo-like characteristics after germination, indicating a role for CHD4 in the repression of embryonic genes (5Ogas J. Cheng J.C. Sung Z.R. Somerville C. Science. 1997; 277: 91-94Crossref PubMed Scopus (262) Google Scholar, 6Ogas J. Kaufmann S. Henderson J. Somerville C. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 13839-13844Crossref PubMed Scopus (407) Google Scholar). Conditional CHD4 inactivation in the mouse has revealed important functions for this protein in both differentiation and homeostasis of hematopoietic stem cells (7Williams C.J. Naito T. Arco P.G. Seavitt J.R. Cashman S.M. De Souza B. Qi X. Keables P. Von Andrian U.H. Georgopoulos K. Immunity. 2004; 20: 719-733Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 8Yoshida T. Hazan I. Zhang J. Ng S.Y. Naito T. Snippert H.J. Heller E.J. Qi X. Lawton L.N. Williams C.J. Georgopoulos K. Genes Dev. 2008; 22: 1174-1189Crossref PubMed Scopus (146) Google Scholar). Recently, however, a direct role for the NuRD complex in transcriptional activation has been demonstrated, with NuRD shown to be required for FOG-1-dependent activation of hematopoietic genes (9Miccio A. Wang Y. Hong W. Gregory G.D. Wang H. Yu X. Choi J.K. Shelat S. Tong W. Poncz M. Blobel G.A. EMBO J. 2010; 29: 442-456Crossref PubMed Scopus (120) Google Scholar). NuRD has also been identified as a modulator of aging-associated chromatin defects (10Pegoraro G. Kubben N. Wickert U. Göhler H. Hoffmann K. Misteli T. Nat. Cell Biol. 2009; 11: 1261-1267Crossref PubMed Scopus (212) Google Scholar) and implicated in DNA damage signaling and repair (11Larsen D.H. Poinsignon C. Gudjonsson T. Dinant C. Payne M.R. Hari F.J. Danielsen J.M. Menard P. Sand J.C. Stucki M. Lukas C. Bartek J. Andersen J.S. Lukas J. J. Cell Biol. 2010; 190: 731-740Crossref PubMed Scopus (175) Google Scholar, 12Polo S.E. Kaidi A. Baskcomb L. Galanty Y. Jackson S.P. EMBO J. 2010; 29: 3130-3139Crossref PubMed Scopus (251) Google Scholar, 13Smeenk G. Wiegant W.W. Vrolijk H. Solari A.P. Pastink A. van Attikum H. J. Cell Biol. 2010; 190: 741-749Crossref PubMed Scopus (177) Google Scholar, 14Chou D.M. Adamson B. Dephoure N.E. Tan X. Nottke A.C. Hurov K.E. Gygi S.P. Colaiacovo M.P. Elledge S.J. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 18475-18480Crossref PubMed Scopus (405) Google Scholar). Thus, the NuRD complex is likely to perform diverse functions centered on maintaining the balance between repression and activation of genes required for proliferation, differentiation, and homeostasis, as well as in DNA damage-response pathways. The NuRD complex is also strongly implicated in cancer, with the expression levels of a number of NuRD subunits, including MTA1/2 (15Zhang Y. Ng H.H. Erdjument-Bromage H. Tempst P. Bird A. Reinberg D. Genes Dev. 1999; 13: 1924-1935Crossref PubMed Scopus (925) Google Scholar) and RbAp48 (16Pacifico F. Paolillo M. Chiappetta G. Crescenzi E. Arena S. Scaloni A. Monaco M. Vascotto C. Tell G. Formisano S. Leonardi A. J. Clin. Endocrinol. Metab. 2007; 92: 1458-1466Crossref PubMed Scopus (27) Google Scholar), elevated in numerous cancer cell lines and cancer tissues.The 218-kDa CHD4 protein contains an ATP-dependent helicase domain and is one of the defining components of the NuRD complex. In addition to the ATPase domain, CHD4 contains two plant homeodomains (PHDs) and two chromodomains (Fig. 1A). Chromodomains are emerging predominantly as methyl-lysine-binding domains (17Fischle W. Wang Y. Jacobs S.A. Kim Y. Allis C.D. Khorasanizadeh S. Genes Dev. 2003; 17: 1870-1881Crossref PubMed Scopus (784) Google Scholar, 18Jacobs S.A. Khorasanizadeh S. Science. 2002; 295: 2080-2083Crossref PubMed Scopus (638) Google Scholar, 19Min J. Zhang Y. Xu R.M. Genes Dev. 2003; 17: 1823-1828Crossref PubMed Scopus (505) Google Scholar, 20Flanagan J.F. Mi L.Z. Chruszcz M. Cymborowski M. Clines K.L. Kim Y. Minor W. Rastinejad F. Khorasanizadeh S. Nature. 2005; 438: 1181-1185Crossref PubMed Scopus (417) Google Scholar), although the chromodomains of the Drosophila homologue of CHD4 (Mi-2) (21Bouazoune K. Mitterweger A. Längst G. Imhof A. Akhtar A. Becker P.B. Brehm A. EMBO J. 2002; 21: 2430-2440Crossref PubMed Scopus (121) Google Scholar) and of MSL3 (22Kim D. Blus B.J. Chandra V. Huang P. Rastinejad F. Khorasanizadeh S. Nat. Struct. Mol. Biol. 2010; 17: 1027-1029Crossref PubMed Scopus (80) Google Scholar) have been shown to bind DNA.The PHD is an ∼50-residue module characterized by a conserved Cys4-His-Cys3 motif that coordinates two zinc ions in a “cross-brace” configuration, where each zinc ion is coordinated by alternate pairs of Cys/His ligands. The human genome contains ∼150 PHDs, occurring in a wide variety of mostly nuclear proteins (23Bienz M. Trends Biochem. Sci. 2006; 31: 35-40Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar), and a subset of PHDs have been found to bind N-terminal histone tails, including the PHDs of bromodomain PHD finger transcription factor and ING2 (inhibitor of growth family member 2), which recognize H3K4me3 and thereby facilitate the interaction of bromodomain PHD finger transcription factor, ING2, and their associated corepressor complexes (NURF and Sin3, respectively) with chromatin (24Li H. Ilin S. Wang W. Duncan E.M. Wysocka J. Allis C.D. Patel D.J. Nature. 2006; 442: 91-95Crossref PubMed Scopus (181) Google Scholar, 25Peña P.V. Davrazou F. Shi X. Walter K.L. Verkhusha V.V. Gozani O. Zhao R. Kutateladze T.G. Nature. 2006; 442: 100-103Crossref PubMed Scopus (547) Google Scholar, 26Shi X. Hong T. Walter K.L. Ewalt M. Michishita E. Hung T. Carney D. Peña P. Lan F. Kaadige M.R. Lacoste N. Cayrou C. Davrazou F. Saha A. Cairns B.R. Ayer D.E. Kutateladze T.G. Shi Y. Côté J. Chua K.F. Gozani O. Nature. 2006; 442: 96-99Crossref PubMed Scopus (2) Google Scholar, 27Wysocka J. Swigut T. Xiao H. Milne T.A. Kwon S.Y. Landry J. Kauer M. Tackett A.J. Chait B.T. Badenhorst P. Wu C. Allis C.D. Nature. 2006; 442: 86-90Crossref PubMed Scopus (858) Google Scholar). Since the initial discovery of the chromatin binding properties of several PHDs, it is becoming clear that PHDs recognize a range of different PTMs within H3. In a recent analysis of the 18 PHD fingers from Saccharomyces cerevisiae, 8 were found to recognize H3 methylated at Lys4 (H3K4me3), and 2 showed preference for methylation at Lys36 (28Shi X. Kachirskaia I. Walter K.L. Kuo J.H. Lake A. Davrazou F. Chan S.M. Martin D.G. Fingerman I.M. Briggs S.D. Howe L. Utz P.J. Kutateladze T.G. Lugovskoy A.A. Bedford M.T. Gozani O. J. Biol. Chem. 2007; 282: 2450-2455Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Other PHDs from mammalian proteins recognize the methylation state of Lys9, including PHD2 from CHD4 and the PHDs from KDM5C and UHRF1 (29Iwase S. Lan F. Bayliss P. de la Torre-Ubieta L. Huarte M. Qi H.H. Whetstine J.R. Bonni A. Roberts T.M. Shi Y. Cell. 2007; 128: 1077-1088Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar, 30Karagianni P. Amazit L. Qin J. Wong J. Mol. Cell. Biol. 2008; 28: 705-717Crossref PubMed Scopus (190) Google Scholar, 31Musselman C.A. Mansfield R.E. Garske A.L. Davrazou F. Kwan A.H. Oliver S.S. O'Leary H. Denu J.M. Mackay J.P. Kutateladze T.G. Biochem. J. 2009; 423: 179-187Crossref PubMed Scopus (93) Google Scholar). To date, however, the best characterized interactions involve H3K4, with all currently available structures of PHD-histone complexes describing recognition of or methylated Lys4 in a on the surface of the PHD the of a PHD finger from which several histone L. Zhang S. Nature. 2010; PubMed Scopus Google In we have previously shown that PHD2 of CHD4 is to the modification state of both Lys4 and of histone H3 C.A. Mansfield R.E. Garske A.L. Davrazou F. Kwan A.H. Oliver S.S. O'Leary H. Denu J.M. Mackay J.P. Kutateladze T.G. Biochem. J. 2009; 423: 179-187Crossref PubMed Scopus (93) Google Scholar). Here, we have used to determine the molecular basis for the recognition of Lys4 and Lys9, by the solution structure of in complex with a structure a unique in which the the binding recognition of the is likely to be by a cation-π interaction with a surface Additionally, we that of CHD4 also the N-terminal of H3, for H3 and at Lys9, and a preference for Lys4 To this is the first of a recognition in which two domains from a single protein bind to two separate histone tails. These on the function of CHD4 and the NuRD complex and of the of the PHD in role as a of chromatin modification