The flexible loop L1 of the H3K4 demethylase JARID1B ARID domain has a crucial role in DNA-binding activity.
JARID1B, a member of the JmjC demethylase family, has a crucial role in H3K4me3 demethylation. The ARID domain is a potential DNA-binding domain of JARID1B. Previous studies indicate that a GC-rich DNA motif is the specific target of the ARID domain. However, the details of the interaction between the ARID domain and duplex DNA require further study. Here, we utilized NMR spectroscopy to assign the backbone amino acids and mapped the DNA-binding sites of the human JARID1B ARID domain. Perturbations to (1)H-(15)N correlation spectra revealed that the flexible loop L1 of ARID was the main DNA-binding interface. EMSA and intrinsic fluorescence experiments demonstrated that mutations on loop L1 strongly reduced the DNA-binding activity of JARID1B ARID. Furthermore, transfection of mutant forms resulted in a distinct loss of intrinsic H3K4 demethylase activity, implying that the flexible loop L1 made a major contribution to sustaining the DNA-binding ability of JARID1B ARID domain.
Yao
Wenming
W
Key Laboratory of Optical and Magnetic Resonance Spectroscopy, East China Normal University, Shanghai 200062, China.
Peng
Yu
Y
Lin
Donghai
D
D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004252:DNA Mutational Analysis; D006367:HeLa Cells; D056466:Histone Demethylases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D056484:Jumonji Domain-Containing Histone Demethylases; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D009687:Nuclear Proteins; D012097:Repressor Proteins; D004247:DNA; D056466:Histone Demethylases; D056484:Jumonji Domain-Containing Histone Demethylases; C119611:KDM5B protein, human