Our laboratory has published the following reviews on epigenetic regulation mediated by histone marks:
Patel, D. J. (2015). A structural perspective on readout of epigenetic histone and DNA methylation marks. Epigenetics 2nd Edition. Allis, C. D., Jenuwein, T., and Reinberg, D., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Long Island, NY. in press.
Du, J. and Patel, D. J. (2014). Structural biology-based insights into combinatorial readout and crosstalk amongst epigenetics marks. Biochem. Biophys. Acta. 1839, 719-727.
Wang, Z. and Patel, D. J. (2013). Small molecule epigenetic inhibitors targeted to histone lysine methyltransferases and demethylases. Quart. Rev. Biophys. 46(4), 349-73.
Patel, D. J. and Wang, Z. (2013). A structural perspective of readout of epigenetic posttranslational modifications. Ann. Rev. Biochem. 82, 81-118.
Wang, Z. & Patel, D. J. (2011). Combinatorial readout of dual histone modifications by paired chromatin-associated modules. J. Biol. Chem. 286, 8363-18368.
Ruthenburg, A. J., Li, H., Patel, D. J. & Allis, C. D. (2007). Multivalent engagement of chromatin modifications by linked binding modules. Nat. Rev. Mol. Cell Biol. 8, 983-994. [PubMed Abstract]
Taverna, S. D., Li, H., Ruthenburg, A. J., Allis, C. D. & Patel, D. J. (2007). How chromatin-binding modules interpret histone modifications: Lessons from professional pocket pickers. Nat. Struct. Mol. Biol. 14, 1025-1040. [PubMed Abstract]
Erasure of Methylated-Lysine Marks
It has long been thought that histone lysine methylation marks are stable and irreversible. The discovery of histone lysine demethylases (KDMs) that belong either to the amine oxidase or the Jumonji C-terminal domain (JmjC) families have now established lysine methylation as a dynamic mark that can be written, read and erased. The various KDMs exhibit exquisite specificity for lysine demthylation of a particular histone, lysine and methylation state, the molecular basis of which is incompletely understood.
A molecular threading mechanism underlies jumonji lysine demethylase KDM2A regulation of methylated H3K36
The dynamic reversible methylation of lysine residues on histone proteins is central to chromatin biology. Key components are demethylase enzymes, which remove methyl moieties from lysine residues. KDM2A, a member of the jumonji C domain-containing histone lysine demethylase family, specifically targets lower methylation states of H3K36. Here, as part of a collaborative effort with the Or Gozani laboratory (Stanford University, CA), structural studies reveal that H3K36-specificity for KDM2A is mediated by the U-shaped threading of the H3K36 peptide through a catalytic groove within KDM2A. The side chain of methylated K36 inserts into the catalytic pocket occupied by Ni2+ and cofactor, where it is positioned and oriented for demethylation. Key residues contributing to K36me specificity on histone H3 are G33 and G34 (positioned within a narrow channel), P38 (a turn residue) and Y41 (inserts into its own pocket). Given that KDM2A was found structurally to also bind H3K36me3 peptide, we postulate that steric constraints could prevent a-ketogluterate from undergoing an ‘off-line’ to ‘in-line’ transition necessary for the demethylation reaction. Further, structure-guided substitutions of residues in the KDM2A catalytic pocket abrogate KDM2A-mediated functions important for suppression of cancer cell phenotypes. Together, our results deduce insights into the molecular basis underlying KDM2A regulation of the biologically important methylated H3K36 mark.
Cheng, Z., Cheung, P., Kuo, A. J., Yuki, E. T., Wilmot, C. M., Gozani, O. and Patel, D. J. (2014). A molecular threading mechanism underlies jumonji lysine demethylase KDM2A regulation of methylated H3K36. Genes Dev. 28, 1758-1771.
A Selective H3K27-Specific Jmj Family Demethylase Inhibitor Modulates the Proinflammatory Macrophage Response
The Jumonji (Jmj) family of histone demethylases are Fe2+ and α-ketoglutarate dependent multi-domain oxygenases that constitute essential components of regulatory transcriptional chromatin complexes. These enzymes demethylate methylated-lysine residues found in histones, in a methylation-state and sequence-specific context. Here in a project championed by the GlaxoSmithKline group, we present a structure-guided small molecule and chemoproteomics approach to the H3K27me3-specific demethylase KDM6 subfamily (members JmjD3 and UTX). The liganded structures of human and murine JmjD3 provide novel insights into the specificity determinants for cofactor, substrate and inhibitor recognition of the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small molecule catalytic site inhibitor that is selective for the H3K27me3 Jmj subfamily. We demonstrate that this inhibitor binds in a novel manner, and reduces lipopolysaccharide-induced proinflammatory cytokine production in human primary macrophages, a process that depends on both JmjD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific Jmj’s in regulating disease relevant inflammatory responses, and provide encouragement for designing small molecule inhibitors to enable selective pharmacological intervention across the Jmj family.
Kruidenier, L., Chung, C., Cheng, Z., Liddle, J., Bantscheff, M., Bountra, C., Bridges, A., Che, K., Diallo, H., Eberhard, D., Hutchinson, S., Joberty, G., Jones, E., Katso, R., Leveridge, M., Mosley, J., Rowland, P., Ramirez-Molina, C., Schofield, C. J., Sheppard, R., Smith, J. E., Swales, C., Tanner, R., Thomas, P., Tumber, A., Drewes, G., Oppermann, U., Patel, D. J., Lee, K., & Wilson, W. (2012). A selective H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. Nature 488, 404-408.
Readout of Methylated-Lysine Marks
Mono-, di- and tri-methylated states of histone lysine residues are selectively found in different regions of chromatin, thereby implying specialized biological functions for these marks ranging from heterochromatin formation to X-chromosome inactivation and transcriptional regulation. A major challenge in chromatin biology has centered on efforts to define the connection between specific methylation states and distinct biological readouts impacting function. For instance, histone H3 trimethylated at lysine 4 is associated with transcription start sites at active genes. We have initiated a research program to define the site- and state-specific readout of histone marks by effector modules.
In a collaborative project championed by the Gang Wang laboratory (University of North Carolina, Chapel Hill), it has been shown that PRC2-associated polycomb-like recognition of H3K36me3 promotes intrusion of PRC2 complexes into active chromatin regions to promote gene silencing and modulate the chromatin landscape during development.
Cai, L., Rothbart, S. B., Lu, R., Xu, B., Tripathy, A., Chen, W-Y., Zheng, D., Patel, D. J., Allis, C. D., Strahl, B. D., Song, J. & Wang, G. (2013). An H3K36me3-containing Tudor motif of polycomb-like proteins mediates PRC2 complex targeting. Mol. Cell 49, 571-582.
Recognition of distinctly modified histones by specialized ‘effector’ proteins constitutes a key mechanism for transducing molecular events at chromatin to biological outcomes. Effector proteins influence DNA-templated processes, including transcription, DNA recombination, and DNA repair; however, no effector functions have yet been identified within the mammalian machinery that regulates DNA replication. Here in collaboration with the Or Gozani laboratory (Stanford University), we show that ORC1 – a component of ORC (origin of replication complex), which mediates pre-DNA replication licensing – contains a BAH (bromo adjacent homology) domain that specifically recognizes histone H4 dimethylated at lysine 20 (H4K20me2). Recognition of H4K20me2 is a property common to BAH domains present within diverse metazoan ORC1 proteins. Structural studies reveal that the specificity of the BAH domain for H4K20me2 is mediated by a dynamic aromatic dimethyllysine-binding cage and multiple intermolecular contacts involving the bound peptide. H4K20me2 is enriched at replication origins and abrogating ORC1 recognition of H4K20me2 in cells impairs ORC1 occupancy at origins, ORC chromatin loading, and cell-cycle progression. Mutation of the ORC1 BAH domain has been implicated in the etiology of Meier-Gorlin syndrome (MGS), a form of primordial dwarfism, and ORC1 depletion in zebrafish results in an MGS-like phenotype. We find that wild-type human ORC1, but not ORC1 H4K20me2-binding mutants, rescues the growth retardation of orc1 morphants. Moreover, zebrafish depleted of H4K20me2 have diminished body size, mirroring the phenotype of orc1 morphants. Together, our results identify the BAH domain as a novel methyllysine-binding module, thereby establishing the first direct link between histone methylation and the metazoan DNA replication machinery, and defining a pivotal etiologic role for the canonical H4K20me2 mark, via ORC1, in primordial dwarfism.
Kuo, A. J., Song, J., Cheung, P., Ishibe-Murakami, S., Yamazoe, S., Chen, J., Patel, D. J. & Gozani, O. (2012). ORC1 BAH domain links dimethylation of H4K20 to DNA replication licensing and Meier-Gorlin syndrome. Nature 484, 115-119.
ATRX ADD Domain Links an Atypical Histone Methylation Recognition Mechanism to Human Mental-Retardation Syndrome
ATR-X (alpha thalassemia/mental retardation, X-linked) syndrome is a human congenital disorder that causes severe intellectual disabilities. Mutations in the ATRX gene, which encodes an ATP-dependent chromatin-remodeler, are responsible for the syndrome. Approximately 50% of the patient missense mutations are clustered in a cysteine-rich domain termed ADD (ATRX-DNMT3-DNMT3L, ADDATRX), indicating its importance. However, the function of ADDATRX has remained elusive. Here in collaboration with the Yang Shi laboratory (Harvard University Medical School), we identify ADDATRX as a novel histone H3 binding module, whose binding is promoted by lysine 9 trimethylation (H3K9me3) but inhibited by H3K4me3. The co-crystal structures of ADDATRX bound to H3(1–15)K9me3 peptide reveals an atypical composite H3K9me3-binding pocket, which is distinct from the conventional trimethyllysine-binding aromatic cage. Importantly, H3K9me3-pocket mutants and ATR-X syndrome mutants are defective in both H3K9me3 binding and localization at pericentromeric heterochromatin. Thus, we have discovered a unique histone recognition mechanism underlying the ATR-X etiology.
Iwase, S., Xiang, B., Ghosh, S., Ren, T., Lewis, P. W., Cochrane, J. C., Allis, C. D., Picketts, D. J., Patel, D. J., Li, H. & Shi, Y. (2011). ATRX links atypical histone methylation recognition mechanisms to human cognitive function. Nat. Struct. Mol. Biol. 18, 769-776.
In a collaborative project championed by the Stephen Jane laboratory (Melbourne, Australia), it has been shown that PRMT5-mediated methylation of histone H4R3 recruits DNMT3A, thereby coupling histone and DNA methylation in gene silencing.
Zhao, Q., Rank, G., Tan, Y. T., Li, H., Moritz, R. L., Simpson, R. J., Cerruti, L., Curtis, D. J., Patel, D. J., Allis, C. D., Cunningham, J. M. & Jane, S. M. (2009). PRMT5-mediated methylation of histone H4R3 recruits DNMT3A coupling histone and DNA methylation in gene silencing. Nat. Struct. Mol. Biol. 16, 304-311. [PubMed Abstract]
Engaging H3K4 Methylation Marks by Aberrant PHD Fingers Perturbs Cellular Identities and Initiates Tumorigenesis
In a collaborative project championed by the David Allis laboratory (Rockefeller University), it has been shown that incorrect interpretation of the “histone code” can perturb epigenetic dynamics on developmentally critical loci, which catastrophizes cell fate decision-making during development.
Wang, G. G., Song, J., Wang, Z., Dormann, H. L., Casadio, F., Li, H., Luo, J., Patel, D. J. & Allis, C. D. (2009). Haematopoietic malignancies initiated by dysregulation of a chromatin-binding PHD finger. Nature 459, 847-851. [PubMed Abstract]
Human L3MBTL1, which contains three-malignant brain tumor (MBT) repeats, binds mono- and di- but not tri-methylated lysines in several histone sequence contexts. In crystal structures of L3MBTL1 complexes, the monomethyl- and dimethyl-lysines insert into a narrow and deep cavity of aromatic residue-lined pocket 2, while a proline ring inserts into shallower pocket 1. In both the “cavity insertion” (L3MBTL1) and “surface groove” (PHD finger) modes of methyl-lysine recognition, a carboxylate group both hydrogen bonds and ion pairs to the methylammonium proton. Our structural and binding studies of these two modules provide insights into the molecular principles governing the decoding of lysine methylation states, thereby highlighting a methylation state-specific layer of histone mark readout impacting on epigenetic regulation.
Li, H., Wang, W. K., Fischle, W., Duncan, E. M., Liang, L., Allis, C. D. & Patel, D. J. (2007). Structural basis for lower lysine methylation state-specific readout by MBT repeats and an engineered PHD finger module. Mol. Cell 28, 677-691. [PubMed Abstract]
Wang, W. K., Tereshko, V., Boccuni, P., MacGrogan, D., Nimer, S., & Patel, D. J. (2003). Malignant brain tumor repeats: A three-leaved propeller architecture with ligand/peptide-binding pockets. Structure 7, 775-789. [PubMed Abstract]
WDR5 is a core component of SET1-family complexes that achieve transcriptional activation via methylation of histone H3 on Nζ of lysine 4 (H3 K4). The role of WDR5 in the MLL1 complex was recently described to be specific recognition of dimethyl lysine 4 in the context of a histone H3 N-terminus; WDR5 is essential for vertebrate development, HOX gene activation, and global H3 K4 trimethylation. We report the high-resolution x-ray structures of WDR5 in the unliganded form and complexed with unmodified, mono-, di- and trimethylated K4 histone H3 peptides, which together provide the first comprehensive analysis of methylated histone recognition by the ubiquitous WD40 repeat fold. Unexpectedly, the structures reveal that WDR5 does not read out the methylation state of K4 directly, but instead serves to present the K4 side chain for further methylation by SET1-family complexes. This research was championed by the Gregory L. Verdine laboratory (Harvard University) and the David Allis laboratory (Rockefeller University).
Ruthenberg, A. J., Wang, W., Graybosch, D. M., Li, H., Allis, C. D., Patel, D. J. & Verdine, G. (2006). Histone H3 recognition and presentation by the WDR5 module of the MLL1 complex. Nat. Struct. Mol. Biol. 13, 704-712. [PubMed Abstract]
We have solved the crystal and NMR structures of H3(1-15)K4me3 peptide bound to the bromodomain-proximal PHD finger of human BPTF, the largest subunit of the ATP-dependent chromatin remodeling complex, NURF. The H3(1-15)K4me3 peptide interacts through anti-parallel ß-sheet formation on the surface of the PHD finger, with the long side chains of R2 and K4me3 fitting snugly in adjacent pockets, and bracketing an invariant tryptophan. The trimethyl group of K4 is positioned within an aromatic amino-acid-lined hydrophobic cage, and stabilized by van der Walls and cation interactions. The state-specific preference for K4me3 over K4me2 reflects the absence of a nearby acidic residue, whereas the observed stapling role by non-adjacent R2 and K4me3 provides a molecular explanation for the H3K4me3 site-specificity. Our detailed structural analysis of the H3(1-15)K4me3 peptide bound to the PHD finger in collaboration with the David Allis laboratory (Rockefeller University) establishes new insights into state- and site-specific readout of histone lysine methylation states, and calls attention to the PHD finger as a previously unrecognized chromatin-binding module found in a large number of chromatin-associated proteins.
Li, H., Ilin, S., Wang, W. K., Wysocka, J., Allis, C. D. & Patel, D. J. (2006). Molecular basis for site- and state-specific readout of histone H3 lysine 4 trimethylation by NURF BPTF PHD finger. Nature 442, 91-95. [PubMed Abstract]
The research on the BPTF PHD finger has been extended to the Yng1 PHD finger as part of a collaboration with the David Allis laboratory (Rockefeller University).
Taverna, S. D., Illin, S., Rogers, R. S., Tanny, J. C., Lavender, H., Li, H., Baker, L., Boyle, J., Blair, L. P., Chait, B., Patel, D. J., Aitchison, J. D., Tackett, A. J. & Allis, C. D. (2006). Yng1 PHD finger binding to H3 trimethylated at K4 promotes NuA3 HAT activity at K14 of H3 and transcription at a subset of targeted ORFs. Mol. Cell 24, 785-796. [PubMed Abstract]
Combinatorial Readout by PHD-Bromo Cassettes of Methylated-Lysine and Acetylated-Lysine Marks
The PHD-Bromodomain cassette is the most frequently observed dual reader module in eukaryotes. Given that the PHD finger targets methylated-lysine marks and the bromodomain targets acetyl-lysine marks, there is a potential for combinatorial readout and synergistic impact on binding affinities. Structure-function studies have been undertaken on a range of PHD-Bromo cassetes to monitor the potential for multivalent readout at the histone tail and nucleosomal level.
Specific chromatin marks keep master regulators of differentiation silent, yet poised for activation by extracellular signals. Here in a project championed by the Joan Massague laboratory (Memorial Sloan Kettering Cancer Center), we report that nodal TGF-ß signals use the poised histone mark H3K9me3 to trigger differentiation of mammalian embryonic stem cells. Nodal receptors induce the formation of companion Smad4-Smad2/3 and TRIM33-Smad2/3 complexes. The PHD-Bromo cassette of TRIM33 facilitates binding of TRIM33-Smad2/3 to H3K9me3 and H3K18ac on the promoters of mesendoderm regulators Gsc and Mixl1. The crystal structure of this cassette, bound to histone H3 peptides, illustrates that the PHD finger recognizes K9me3, and bromodomain binds an adjacent K18ac. The interaction between TRIM33-Smad2/3 and H3K9me3 displaces the chromatin compacting factor HP1-γ, making nodal response elements accessible to Smad4-Smad2/3 for pol II recruitment. Thus, nodal effectors use the H3K9me3 mark as a platform to switch master regulators of stem cell differentiation from the poised to the active state.
Xi, Q., Wang, Z., Zaromytidou, A., Zhang, X. H., Chow-Tsang, L-F., Liu, J. X., Kim, H., Monova-Todorova, K., Kaartinen, V., Studer, L., Mark, W., Patel, D. J. & Massague, J. (2012). A poised chromatin platform for Smad access to master regulators. Cell 147, 1511-1524.
Little is known how combination of histone marks are interpreted at the level of nucleosomes. The second PHD finger of BPTF is known to specifically recognize histone H3 when methylated on lysine 4 (H3K4me2/3). Here in collaboration with the David Allis laboratory (Rockefeller University), we examine how additional heterotypic modifications influence BPTF binding. Using peptide surrogates, three acetyllysine ligands are identified for a PHD-adjacent bromodomain in BPTF via symmetric screening and biophysical characterization. Although the bromodomain displays limited discrimination among the three possible acetyllysines at the peptide level, marked selectivity is observed for only one of these sites, H4K16ac, in combination with H3K4me3 at the mononucleosomal level. In support, these two histone marks constitute a unique trans-histone modification pattern that unambiguously resides within a single nucleosomal unit in human cells, and this module colocalizes with these marks in the genome. Together, our data call attention to nucleosomal patterning of covalent marks in dictating critical chromosomal associations.
Ruthenburg, A., Li, H., Milne, T., Dou, Y., McGinty, R. K., Yuen, M., Muir, T. W., Patel, D. J. & Allis, C. D. (2011). Recognition of a mononucleosomal histone modification pattern by BPTF via multivalent interactions. Cell 145, 692-706.
The recognition of modified histone species by distinct structural domains within ’reader’ proteins plays a critical role in the regulation of gene expression. Readers that simultaneously recognize histones with multiple marks allow transduction of complex chromatin modification patterns into more specific biological outcomes. Here in collaboration with the Michelle Barton laboratory (M. D. Anderson Cancer Center), we report that the chromatin regulator tripartite motif-containing 24 (TRIM24) functions in humans as a reader of dual histone marks by means of tandem plant homeodomain (PHD) and bromodomain (Bromo) regions. The three-dimensional structure of the PHD-Bromo region of TRIM24 revealed a single functional unit for combinatorial recognition of unmodified H3K4 and acetylated H3K23 within the same histone tail. TRIM24 binds chromatin and oestrogen receptor to activate oestrogen-dependent genes associated with cellular proliferation and tumor development. Aberrant expression of TRIM24 negatively correlates with survival of breast cancer patients. The PHD-Bromo of TRIM24 provides a structural rationale for chromatin activation through a non-canonical histone signature, establishing a new route by which chromatin readers may influence cancer pathogenesis.
Tsai, W-W., Wang, Z., Yiu, T. T., Akdemir, K. C., Xia, W., Winter, S., Tsai, C-Y., Shi, X., Schwarzer, D., Plunkett, W., Aronow, B., Gozani, O., Fischle, W., Hung, M. C., Patel, D. J. & Barton, M. C. (2010). TRIM24 links recognition of a non-canonical histone signature to breast cancer. Nature 468, 927-932.
Pro Isomerization in MLL1 PHD3-Bromo Cassette Connects H3K4me Readout to Cyp33 and HDAC-Mediated Repression
The MLL1 gene is a frequent target for recurrent chromosomal translocations, resulting in transformation of hematopoietic precursors into leukemia stem cells. Here in collaboration with the David Allis laboratory (Rockefeller University), we report on structure-function studies that elucidate molecular events in MLL1 binding of histone H3K4me2/3 marks and recruitment of the cyclophilin CyP33. CyP33 contains a PPIase and a RRM domain and regulates MLL1 function through HDAC recruitment. We find that the PPIase domain of CyP33 regulates the conformation of MLL1 through proline isomerization within the PHD3-Bromo linker, thereby disrupting the PHD3-Bromo interface and facilitating binding of the MLL1-PHD3 domain to the CyP33-RRM domain. H3K4me2/3 and CyP33 target different surfaces of MLL1-PHD3 and can bind simultaneously to form a ternary complex. Furthermore, MLL1-CyP33 interaction is required for repression of HOXA9 and HOXC8 genes in vivo. Our results highlight the role of the PHD3-Bromo cassette as a regulatory platform, orchestrating MLL1 binding of H3K4me2/3 marks and cyclophilin-mediated expression through HDAC recruitment.
Wang, Z., Song, J., Milne, T. A., Wang, G. G., Li, H., Allis, C. D. & Patel, D. J. (2010). Pro isomerization in MLL1 PHD3-Bromo cassette connects H3K4me3 readout to CyP33 and HDAC-mediated repression. Cell 141, 1183-1194.
Readout of Unmodified Lysine and Arginine Marks by PHD Fingers
PHD fingers utilize an aromatic cage to recognize methylated-lysines by using cation-π interactions. Recently, PHD fingers have been shown to also target unmodified lysines and unmodified arginines using a hydrogen-bonding interaction network to a combination of basic side chains and backbone carbonyl oxygens.
PHD Finger Recognition of Unmodified Histone H3R2 Links UHRF1 to Regulation of Euchromatic Gene Expression
Histone methylation occurs on both lysine and arginine residues and its dynamic regulation plays a critical role in chromatin biology. Here in collaboration with the Yang Shi laboratory (Harvard Medical School), we identify the UHRF1 PHD finger domain, an important regulator of DNA CpG methylation, as an unanticipated histone H3 unmodified arginine 2 (H3R2)-recognition modality. This conclusion is based on binding studies and co-crystal structures of the PHD finger of UHRF1 bound to histone H3 peptides, where the guanidinium group of unmodified R2 forms an extensive intermolecular hydrogen bond network, with methylation of H3R2, but not H3K4 or H3K9, disrupting complex formation. We have identified direct target genes of UHRF1 from microarray and ChIP studies. Importantly, we show that UHRF1’s ability to repress its direct target gene expression is dependent on the PHD finger of UHRF1 binding to unmodified H3R2, thereby demonstrating the functional importance of this recognition event and supporting the potential for crosstalk between histone arginine methylation and UHRF1 function.
Rajakumara, E., Wang, Z., Ma, H., Hu, L., Chen, H., Lin, Y., Guo R., Wu, F., Li, H., Lan, F., Shi, Y., Xu, Y., Patel, D. J. & Shi, Y. (2011). PHD finger recognition of unmodified histone H3R2 links UHRF1 to regulation of euchromatic gene expression. Mol. Cell 43, 275-284.
Readout of Phosphorylated-Serine/Threonine/Tyrosine Marks
Phosphorylation of serines, threonines and tyrosines represent dynamic marks that are written by kinases and erased by phosphotases. Phosphorylation marks are also read by reader modules that are very specific for both histone and sequence context. Further, phosphorylation marks also play a key role in epigenetic regulation due to cross-talk with adjacent methylated-lysine and acetylated-lysine marks.
Survivin is an inhibitor of apoptosis (IAP) family protein implicated in apoptosis and mitosis. In apoptosis, it has been shown to recognize the Smac/DIABLO protein. It is also a component of the chromosomal passenger complex, a key player during mitosis. Recently, Survivin was identified in vitro and in vivo as the direct binding partner for phosphorylated Thr3 on histone 3 (H3T3ph). Here in collaboration with the Hironori Funabiki laboratory (Rockefeller University), we have undertaken structural and binding studies to investigate the molecular basis underlying recognition of H3T3ph and Smac/DIABLO N-terminal peptides by Survivin. Our crystallographic studies establish recognition of N-terminal Ala in both complexes, and identify intermolecular hydrogen bonding interactions in the Survivin phosphate-binding pocket that contribute to H3T3ph mark recognition. In addition, our calorimetric data establish that Survivin binds tighter to the H3T3ph-containing peptide relative to the N-terminal Smac/DIABLO peptide, and that this preference can be reversed through structure-guided mutations that increase the hydrophobicity of the phosphate-binding pocket.
Du, J., Kelly, A. E., Funabiki, H. & Patel, D. J. (2012). Structural-functional basis for recognition of H3T3ph and Smac/DIABLO N-terminal peptides by human survivin. Structure 20, 185-195.
Histone chaperones represent a structurally and functionally diverse family of histone-binding proteins that prevent promiscuous interactions of histones before their assembly into chromatin. Our understanding of the mechanisms of histone shuttling between different chaperone systems, and histone transfer onto and off DNA, has been hampered due to the availability of only a limited number of histone-chaperone complexes.
DAXX is a metazoan histone chaperone specific to the evolutionary conserved histone variant H3.3. Here in collaboration with the David Allis laboratory (Rockefeller University), we report the crystal structures of the DAXX histone-binding domain with a histone H3.3-H4 dimer, including mutants within DAXX and H3.3, together with in vitro and in vivo functional studies towards elucidation of the principles underlying H3.3 recognition specificity. Occupying 40% of the histone surface-accessible area, DAXX wraps around the H3.3-H4 dimer, with complex formation accompanied by structural transitions in the H3.3-H4 histone fold. DAXX employs an extended α-helical conformation to compete with major inter-histone, DNA and ASF1 interaction sites. Our structural studies identify recognition elements that read out H3.3-specific residues, while functional studies address the contributions of Gly90 in H3.3 and Glu225 in DAXX to chaperone-mediated H3.3 variant recognition specificity.
Elsasser, S. J., Huang, H., Lewis, P. W., Allis, C. D. & Patel, D. J. (2012). DAXX histone chaperone envelops an H3.3/H4 dimer for H3.3-specific recognition. Nature 491, 560-565.
Fusion Proteins in Leukemia
Transcription factors are frequently altered in leukemia through chromosomal translocation, mutation or aberrant expression. Structure-function studies are required to elucidate the interactions between co-regulatory factors that target the oligomerized states of fusion proteins to elucidate their contribution to leukemogenesis and their potential as therapeutic targets.
AML1-ETO, a fusion protein generated by the t(8;21) translocation in acute myeloid leukemia, is a transcription factor implicated in both gene repression and activation. AML1-ETO oligomerization, mediated by the NHR2 domain, is critical for leukemogenesis, making it important to identify co-regulatory factors that ‘read’ the NHR2 oligomerization and contribute to leukemogenesis. Here in a project championed by the Robert Roeder laboratory (Rockefeller University), we show that in human leukemia cells, AML1-ETO resides and functions through a stable AML1-ETO-containing transcription factor complex (AEFTC) that contains several haematopoietic transcription co(factors). These AEFTC components stabilize the complex through multivalent interactions, provide multiple DNA-binding domains for diverse target genes, co-localize genome wide, cooperatively regulate gene expression, and contribute to leukemogenesis. Within the AETFC complex, AML1-ETO oligomerization is required for a specific interaction between the oligomerized NHR2 domain and a novel NHR2-binding (N2B) motif in E proteins. Crystallographic analysis of the NHR2-N2B complex reveals a unique interaction pattern in which the N2B peptide makes direct contact with side chains of two NHR2 domains as a dimer, providing a novel model of how dimeric/oligomeric transcription factors create a new protein-binding interface through dimerization/oligomerization. Intriguingly, disruption of this interaction by point mutations abrogates AML1-ETO-induced haematopoietic stem/progenitor cell self-renewal and leukemogenesis. These results reveal new mechanisms of action of AML1-ETO, and provide a potential therapeutic target in t(8;21)-positive acute myeloid leukemia.
Sun, X-J., Wang, Z., Wang, L., Jiang, Y., Chen, W-Y., Melnick, A., Patel, D. J., Nimer, S. D. & Roeder, R. G. (2013). A stable transcription factor complex nucleated by dimeric AML1-ETO controls leukemogenesis. Nature 500, 93-97.
Mechanisms that Regulate the DNA Damage Response
DNA double-stranded breaks present a serious challenge for eukaryotic cells. The inability to repair breaks leads to genomic instability, carcinogenesis, and cell death. During the double-strand break response, mammalian chromatin undergoes reorganization demarcated by H2A.X Ser 139 phosphorylation (γ-H2A.X). The goal is to identify and characterize new mechanisms that regulate pathways associated with the DNA damage response.
In a collaborative project championed by the David Allis laboratory (Rockefeller University), a new regulatory mechanism has been identified that is mediated by WSTF (Williams-Beuren syndrome transcription factor), a component of the WICH complex (WSTF-ISWI ATP-dependent chromatin-remodeling complex). The results show that WSTF phosphorylates Tyr 142 of H2A.X, and that WSTF activity have an important role in regulating several events that are critical for the DNA damage response.
Xiao, A., Li, H., Shechter, D., Ahn, S. H., Fabrizio, L., Erajument-Bromage, H., Murakami-Ishibe, S., Wang, B., Tempst, P., Hofmann, K., Patel, D. J., Elledge, S. J. & Allis, C. D. (2009). WSTF regulates the DNA damage response of H2A.X via a novel tyrosine kinase activity. Nature 457, 57-62. [PubMed Abstract]
Mechanistic Insights into Gene Silencing along the Chromatin Fiber
Many studies over the past decade have alluded to self-association of silencing proteins as a key event in the spreading of chromatin modifications and gene silencing along the chromatin fiber. Our goal has been to identify a polymeric domain in a silencing complex and demonstrate that this domain is important for spreading of the silencing complex.
RNA interference (RNAi) plays a pivotal role in the formation of heterochromatin at the fission yeast centromeres. The RITS complex, composed of heterochromatic siRNAs, siRNA-binding protein, Ago1, the chromodomain protein, Chp1, and Ago1/Chp1-interacting protein, Tas3, provides a physical tether between the RNAi and heterochromatin assembly pathways. Together with the Danesh Moazed laboratory (Harvard Medical School), we have reported on the structural and functional characterization of a C-terminal Tas3 alpha-helical motif (TAM), which self-associates into a helical polymer and is required for cis-spreading of RITS in centromeric DNA regions. Site-directed mutations of key residues within the hydrophobic monomer-monomer interface disrupt Tas3-TAM polymerization in vitro and result in loss of gene silencing, spread of RITS, and a dramatic reduction in centromeric siRNAs in vivo. These results demonstrate that in addition to the chromodomain of Chp1 and siRNA-loaded Ago1, Tas3 polymerization, mediated via Tas3-TAM, is required for RITS spreading and efficient heterochromatic gene silencing at centromeric repeat regions.
Li, H., Motamedi, M., Wang, Z., Patel, D. J. & Moazed, D. (2009), An alpha motif of Tas3 C-terminus mediated RITS cis-spreading and promotes heterochromatin gene silencing. Mol. Cell 34, 155-167. [PubMed Abstract]