PRDM proteins have emerged as important regulators of disease and developmental processes. To gain insight into the mechanistic actions of the PRDM family, we have performed comprehensive characterization of a prototype member protein, the histone methyltransferase PRDM9, using biochemical, biophysical and chemical biology techniques. In the present paper we report the first known molecular characterization of a PRDM9-methylated recombinant histone octamer and the identification of new histone substrates for the enzyme. A single C321P mutant of the PR/SET domain was demonstrated to significantly weaken PRDM9 activity. Additionally, we have optimized a robust biochemical assay amenable to high-throughput screening to facilitate the generation of small-molecule chemical probes for this protein family. The present study has provided valuable insight into the enzymology of an intrinsically active PRDM protein.
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July 2014
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Research Article|
June 26 2014
Characterization of the histone methyltransferase PRDM9 using biochemical, biophysical and chemical biology techniques
Xiaoying Koh-Stenta
;
Xiaoying Koh-Stenta
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore
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Joma Joy
;
Joma Joy
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore
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Anders Poulsen
;
Anders Poulsen
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore
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Rong Li
;
Rong Li
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore
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Yvonne Tan
;
Yvonne Tan
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore
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Yoonjung Shim
;
Yoonjung Shim
†Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, U.S.A.
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Jung-Hyun Min
;
Jung-Hyun Min
†Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, U.S.A.
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Liling Wu
;
Liling Wu
‡Institute for Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore
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Anna Ngo
;
Anna Ngo
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore
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Jianhe Peng
;
Jianhe Peng
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore
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Wei Guang Seetoh
;
Wei Guang Seetoh
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore
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Jing Cao
;
Jing Cao
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore
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John Liang Kuan Wee
;
John Liang Kuan Wee
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore
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Perlyn Zekui Kwek
;
Perlyn Zekui Kwek
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore
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Alvin Hung
;
Alvin Hung
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore
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Umayal Lakshmanan
;
Umayal Lakshmanan
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore
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Horst Flotow
;
Horst Flotow
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore
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Ernesto Guccione
;
Ernesto Guccione
‡Institute for Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore
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Jeffrey Hill
Jeffrey Hill
1
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore
1To whom correspondence should be addressed (email jhill@etc.a-star.edu.sg).
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Biochem J (2014) 461 (2): 323–334.
Article history
Received:
March 24 2014
Revision Received:
April 24 2014
Accepted:
May 02 2014
Accepted Manuscript online:
May 02 2014
Citation
Xiaoying Koh-Stenta, Joma Joy, Anders Poulsen, Rong Li, Yvonne Tan, Yoonjung Shim, Jung-Hyun Min, Liling Wu, Anna Ngo, Jianhe Peng, Wei Guang Seetoh, Jing Cao, John Liang Kuan Wee, Perlyn Zekui Kwek, Alvin Hung, Umayal Lakshmanan, Horst Flotow, Ernesto Guccione, Jeffrey Hill; Characterization of the histone methyltransferase PRDM9 using biochemical, biophysical and chemical biology techniques. Biochem J 15 July 2014; 461 (2): 323–334. doi: https://doi.org/10.1042/BJ20140374
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