Histone/Epigenetic Code

Methylation State-Specific Recognition of Lysines on Histone Tails

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 collaborative research program with the C. David Allis laboratory at The Rockefeller University to define the site- and state-specific readout of histone marks by effector modules.

Our laboratory has published the following reviews on epigenetic regulation:

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]

Coupling Histone and DNA Methylation in Gene Silencing

In a collaborative project championed by Quan Zhao in the Stephen Jane laboratory in 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 Gang Wang in the David Allis laboratory, 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]

Lower Lysine Methylation State-Specific Readout by MBT Repeats of L3MBTL1

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.

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]

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]

H3K4me2 Bound to WDR5, a WD40 Protein

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 Alex Ruthenberg during his graduate training in the Gregory L. Verdine laboratory at Harvard 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]

H3K4me3 Bound to PHD Finger of BPTF

We have recently 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 beta-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 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]

More recently, 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.

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]

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 (ã-H2A.X). The goal is to identify and characterize new mechanisms that regulate pathways associated with the DNA damage response.

WSTF Regulates the H2A.X DNA Damage Response via a Novel Tyrosine Kinase Activity

In a collaborative project championed by Andrew Xiao in the David Allis laboratory, 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.

Tas3 C-terminus Alpha Motif Mediates RITS cis-Spreading and Promotes Heterochromatic Gene Silencing

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 at 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]