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US 2009/0269751 A1Published application
Dot1 histone methyltransferases as a target for identifying therapeutic agents for leukemia
AssigneeThe University of North Carolina at Chapel Hill
InventorsYI ZHANG, QIN FENG, YUKI OKADA, GUOLIANG XU
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Abstract
The present invention provides polypeptides with histone H3 lysine 79 methyltransferase activity as well as nucleic acids encoding the same. Also provided are methods of using the polypeptides and nucleic acids of the invention in screening assays to identify compounds of interest. Further provided are diagnostic methods for leukemia and prognostic methods to predict the course of the disease in a subject.
Claims20
An isolated nucleic acid comprising a nucleotide sequence encoding a DOT1L polypeptide, said nucleotide sequence selected from the group consisting of:
- (a) a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:1;
- (b) a nucleotide sequence that hybridizes to the complete complement of a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:1 under stringent conditions, wherein said nucleotide sequence encodes a polypeptide having histone H3 lysine 79 (H3-K79) methyltransferase activity;
- (c) a nucleotide sequence having at least 95% nucleotide sequence similarity with the nucleotide sequence of SEQ ID NO:1, wherein said nucleotide sequence encodes a polypeptide having H3-K79 methyltransferase activity;
- (d) a nucleotide sequence that differs from the nucleotide sequence of (a) above due to the degeneracy of the genetic code;
- (f) a nucleotide sequence comprising a functional fragment of SEQ ID NO:1, wherein said functional fragment comprises at least the coding region of the histone methyltransferase catalytic domain of SEQ ID NO:1 or a nucleotide sequence that hybridizes to the complete complement of the coding region of the histone methyltransferase catalytic domain of SEQ ID NO:1 under stringent conditions, and wherein said functional fragment encodes a polypeptide having H3-K79 methyltransferase activity;
- (g) a nucleotide sequence comprising a functional fragment of SEQ ID NO:1, wherein said functional fragment comprises at least the coding region of the histone methyltransferase catalytic domain of SEQ ID NO:1 or a nucleotide sequence having at least 95% nucleotide sequence similarity thereto, and wherein said functional fragment encodes a polypeptide having H3-K79 methyltransferase activity;
- and
- (h) a nucleotide sequence comprising a functional fragment of SEQ ID NO:1, wherein said functional fragment comprises at least the coding region of the histone methyltransferase catalytic domain of SEQ ID NO:1 or a nucleotide sequence that differs therefrom due to the degeneracy of the genetic code, and wherein said functional fragment encodes a polypeptide having H3-K79 methyltransferase activity.
5 more elements
A method of identifying a compound that modulates DOT1L histone H3 lysine 79 (H3-K79) methyltransferase activity, comprising:
- contacting a DOT1L polypeptide with a nucleosome substrate comprising H3 in the presence of a test compound;
- wherein the DOT1L polypeptide comprises an amino acid sequence selected from the group consisting of:
- (a) the amino acid sequence of SEQ ID NO:2;
- (b) an amino acid sequence having at least 95% amino acid sequence similarity with the amino acid sequence of SEQ ID NO:2, wherein said DOT1L polypeptide has H3-K79 methyltransferase activity;
- and
- (c) a functional fragment of at least 400 amino acids of the amino acid sequence of (a) or (b) above, wherein said functional fragment comprises a DOT1L histone methyltransferase catalytic domain and has H3-K79 methyltransferase activity;
- detecting the level of H3-K79 methylation of the nucleosome substrate under conditions sufficient to provide H3-K79 methylation,
- wherein an elevation or reduction in H3-K79 methylation in the presence of the test compound as compared with the level of histone H3-K79 methylation in the absence of the test compound indicates that the test compound modulates DOT1L H3-K79 methyltransferase activity.
5 more elements
A method of identifying a compound that inhibits binding of DOT1L polypeptide to a second polypeptide, comprising:
- contacting a DOT1L polypeptide with a second polypeptide in the presence of a test compound;
- wherein the DOT1I polypeptide comprises an amino acid sequence selected from the group consisting of:
- (a) the amino acid sequence of SEQ ID NO:2;
- (b) an amino acid sequence having at least 95% amino acid sequence similarity with the amino acid sequence of SEQ ID NO:2, wherein said DOT1L polypeptide has H3-K79 methyltransferase activity;
- and
- (c) a functional fragment of at least 400 amino acids of the amino acid sequence of (a) or (b) above, wherein said functional fragment comprises a DOT1L histone methyltransferase catalytic domain and has H3-K79 methyltransferase activity;
- detecting the level of binding between DOT1L polypeptide and the second polypeptide under conditions sufficient for binding of DOT1L polypeptide to the second polypeptide,
- wherein a reduction in binding between DOT1L polypeptide and the second polypeptide in the presence of the test compound as compared with the level of binding in the absence of the test compound indicates that the test compound inhibits binding of DOT1L polypeptide to the second polypeptide.
5 more elements
Dependencies are inferred from the claim text.