Characterization of the histone methyltransferase PRDM9 using biochemical, biophysical and chemical biology techniques.
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.
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore.
Xiaoying
X
Koh-Stenta
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore.
Joma
J
Joy
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore.
Anders
A
Poulsen
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore.
Rong
R
Li
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore.
Yvonne
Y
Tan
†Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, U.S.A.
Yoonjung
Y
Shim
†Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, U.S.A.
Jung-Hyun
JH
Min
‡Institute for Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore.
Liling
L
Wu
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore.
Anna
A
Ngo
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore.
Jianhe
J
Peng
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore.
Wei Guang
WG
Seetoh
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore.
Jing
J
Cao
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore.
John Liang Kuan
JL
Wee
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore.
Perlyn Zekui
PZ
Kwek
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore.
Alvin
A
Hung
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore.
Umayal
U
Lakshmanan
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore.
Horst
H
Flotow
‡Institute for Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore.
Ernesto
E
Guccione
*Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore.
Jeffrey
J
Hill
D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D057166:High-Throughput Screening Assays; D011495:Histone-Lysine N-Methyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006657:Histones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008163:Luminescent Measurements; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011392:Proline / Q000737:chemistry / Q000235:genetics; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014982:Xenopus laevis
D006657:Histones; D011994:Recombinant Proteins; D011392:Proline; D011495:Histone-Lysine N-Methyltransferase; C534829:prdm9 protein, mouse; D003545:Cysteine