Structure of the Catalytic Domain of Human DOT1L, a Non-SET Domain Nucleosomal Histone Methyltransferase

Dot1 is an evolutionarily conserved histone methyltransferase that methylates lysine-79 of histone H3 in the core domain. Unlike other histone methyltransferases, Dot1 does not contain a SET domain, and it specifically methylates nucleosomal histone H3. We have solved a 2.5 A resolution structure of the catalytic domain of human Dot1, hDOT1L, in complex with S-adenosyl-L-methionine (SAM). The structure reveals a unique organization of a mainly alpha-helical N-terminal domain and a central open alpha/beta structure, an active site consisting of a SAM binding pocket, and a potential lysine binding channel. We also show that a flexible, positively charged region at the C terminus of the catalytic domain is critical for nucleosome binding and enzymatic activity. These structural and biochemical analyses, combined with molecular modeling, provide mechanistic insights into the catalytic mechanism and nucleosomal specificity of Dot1 proteins.

[1]  D. Reinberg,et al.  Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails. , 2001, Genes & development.

[2]  Thomas C. Terwilliger,et al.  Automated MAD and MIR structure solution , 1999, Acta crystallographica. Section D, Biological crystallography.

[3]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[4]  Karolin Luger,et al.  Structure of the yeast nucleosome core particle reveals fundamental changes in internucleosome interactions , 2001, The EMBO journal.

[5]  W Furey,et al.  PHASES-95: a program package for processing and analyzing diffraction data from macromolecules. , 1997, Methods in enzymology.

[6]  Xiaodong Cheng,et al.  Crystal structure of the conserved core of protein arginine methyltransferase PRMT3 , 2000, The EMBO journal.

[7]  Youngchang Kim,et al.  The active site of the SET domain is constructed on a knot , 2002, Nature Structural Biology.

[8]  J. Boeke,et al.  A core nucleosome surface crucial for transcriptional silencing , 2002, Nature Genetics.

[9]  B M Turner,et al.  Histone acetylation and an epigenetic code. , 2000, BioEssays : news and reviews in molecular, cellular and developmental biology.

[10]  C. Allis,et al.  Methylation of Histone H4 at Arginine 3 Facilitating Transcriptional Activation by Nuclear Hormone Receptor , 2001, Science.

[11]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[12]  Paul Tempst,et al.  PR-Set7 is a nucleosome-specific methyltransferase that modifies lysine 20 of histone H4 and is associated with silent chromatin. , 2002, Molecular cell.

[13]  Zu-Wen Sun,et al.  Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast , 2002, Nature.

[14]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[15]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[16]  P. Grant,et al.  Set2 Is a Nucleosomal Histone H3-Selective Methyltransferase That Mediates Transcriptional Repression , 2002, Molecular and Cellular Biology.

[17]  T. Richmond,et al.  Crystal structure of the nucleosome core particle at 2.8 Å resolution , 1997, Nature.

[18]  S. Grewal,et al.  Heterochromatin: new possibilities for the inheritance of structure. , 2002, Current opinion in genetics & development.

[19]  James H. Hurley,et al.  Structure and Catalytic Mechanism of a SET Domain Protein Methyltransferase , 2002, Cell.

[20]  R J Roberts,et al.  AdoMet-dependent methylation, DNA methyltransferases and base flipping. , 2001, Nucleic acids research.

[21]  Brian D. Strahl,et al.  Gene silencing: Trans-histone regulatory pathway in chromatin , 2002, Nature.

[22]  T. Yeates,et al.  Crystal structure of a protein repair methyltransferase from Pyrococcus furiosus with its L-isoaspartyl peptide substrate. , 2001, Journal of molecular biology.

[23]  Anders Liljas,et al.  Crystal structure of catechol O-methyltransferase , 1994, Nature.

[24]  K. Sharp,et al.  Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons , 1991, Proteins.

[25]  M. Dlakić,et al.  Chromatin silencing protein and pachytene checkpoint regulator Dot1p has a methyltransferase fold. , 2001, Trends in biochemical sciences.

[26]  E. Selker,et al.  Structure of the Neurospora SET Domain Protein DIM-5, a Histone H3 Lysine Methyltransferase , 2002, Cell.

[27]  Kevin Struhl,et al.  Ubiquitination of Histone H2B by Rad6 Is Required for Efficient Dot1-mediated Methylation of Histone H3 Lysine 79* , 2002, The Journal of Biological Chemistry.

[28]  T. Jenuwein,et al.  The many faces of histone lysine methylation. , 2002, Current opinion in cell biology.

[29]  M. Grunstein,et al.  25 years after the nucleosome model: chromatin modifications. , 2000, Trends in biochemical sciences.

[30]  Steven J. Gamblin,et al.  Crystal Structure and Functional Analysis of the Histone Methyltransferase SET7/9 , 2002, Cell.

[31]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[32]  Stuart L. Schreiber,et al.  Active genes are tri-methylated at K4 of histone H3 , 2002, Nature.

[33]  Kevin Struhl,et al.  Lysine methylation within the globular domain of histone H3 by Dot1 is important for telomeric silencing and Sir protein association. , 2002, Genes & development.

[34]  W A Hendrickson,et al.  Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three‐dimensional structure. , 1990, The EMBO journal.

[35]  Jinrong Min,et al.  Structure of the SET domain histone lysine methyltransferase Clr4 , 2002, Nature Structural Biology.

[36]  Hengbin Wang,et al.  Purification and Functional Characterization of SET8, a Nucleosomal Histone H4-Lysine 20-Specific Methyltransferase , 2002, Current Biology.

[37]  Mark Johnston,et al.  Methylation of Histone H3 by COMPASS Requires Ubiquitination of Histone H2B by Rad6* , 2002, The Journal of Biological Chemistry.

[38]  Philip R. Gafken,et al.  Dot1p Modulates Silencing in Yeast by Methylation of the Nucleosome Core , 2002, Cell.

[39]  G. Roeder,et al.  Role for the silencing protein Dot1 in meiotic checkpoint control. , 2000, Molecular biology of the cell.

[40]  G. Poirier,et al.  Disruptor of Telomeric Silencing-1 Is a Chromatin-specific Histone H3 Methyltransferase* , 2002, The Journal of Biological Chemistry.

[41]  C. Allis,et al.  The language of covalent histone modifications , 2000, Nature.

[42]  D. Gottschling,et al.  Identification of high-copy disruptors of telomeric silencing in Saccharomyces cerevisiae. , 1998, Genetics.

[43]  E A Merritt,et al.  Raster3D: photorealistic molecular graphics. , 1997, Methods in enzymology.

[44]  Kevin Struhl,et al.  Methylation of H3-Lysine 79 Is Mediated by a New Family of HMTases without a SET Domain , 2002, Current Biology.