5'-Cytosine-phosphoguanine (CpG) methylation impacts the activity of natural and engineered meganucleases

TY - JOUR

T1 - 5'-Cytosine-phosphoguanine (CpG) methylation impacts the activity of natural and engineered meganucleases

AU - Valton, Julien

AU - Daboussi, Fayza

AU - Leduc, Sophie

AU - Molina, Rafael

AU - Redondo, Pilar

AU - Macmaster, Rachel

AU - Montoya, Guillermo

AU - Duchateau, Philippe

PY - 2012/8/31

Y1 - 2012/8/31

N2 - In this study, we asked whether CpG methylation could influence the DNA binding affinity and activity of meganucleases used for genome engineering applications. A combination of biochemical and structural approaches enabled us to demonstrate that CpG methylation decreases I-CreI DNA binding affinity and inhibits its endonuclease activity in vitro. This inhibition depends on the position of the methylated cytosine within the DNA target and was almost total when it is located inside the central tetrabase. Crystal structures of I-CreI bound to methylated cognate target DNA suggested a molecular basis for such inhibition, although the precise mechanism still has to be specified. Finally, we demonstrated that the efficacy of engineered meganucleases can be diminished by CpG methylation of the targeted endogenous site, and we proposed a rational design of the meganuclease DNA binding domain to alleviate such an effect. We conclude that although activity and sequence specificity of engineered meganucleases are crucial parameters, target DNA epigenetic modifications need to be considered for successful gene editions.

AB - In this study, we asked whether CpG methylation could influence the DNA binding affinity and activity of meganucleases used for genome engineering applications. A combination of biochemical and structural approaches enabled us to demonstrate that CpG methylation decreases I-CreI DNA binding affinity and inhibits its endonuclease activity in vitro. This inhibition depends on the position of the methylated cytosine within the DNA target and was almost total when it is located inside the central tetrabase. Crystal structures of I-CreI bound to methylated cognate target DNA suggested a molecular basis for such inhibition, although the precise mechanism still has to be specified. Finally, we demonstrated that the efficacy of engineered meganucleases can be diminished by CpG methylation of the targeted endogenous site, and we proposed a rational design of the meganuclease DNA binding domain to alleviate such an effect. We conclude that although activity and sequence specificity of engineered meganucleases are crucial parameters, target DNA epigenetic modifications need to be considered for successful gene editions.

KW - CpG Islands

KW - Crystallography, X-Ray

KW - DNA/chemistry

KW - DNA Methylation

KW - DNA Restriction Enzymes/chemistry

KW - Epigenesis, Genetic

KW - HEK293 Cells

KW - Humans

KW - Protein Structure, Tertiary

U2 - 10.1074/jbc.m112.379966

DO - 10.1074/jbc.m112.379966

M3 - Journal article

C2 - 22740697

SN - 0021-9258

VL - 287

SP - 30139

EP - 30150

JO - The Journal of Biological Chemistry

JF - The Journal of Biological Chemistry

IS - 36

ER -