title: JHDM3A module as an effector molecule in guide-directed modification of target chromatin.
abstract: With the objective of returning cells to their undifferentiated state through alteration of epigenetic states, small molecules have been used that specifically inhibit proteins involved in sustaining the epigenetic system. However, this chemical-based approach can cause chaotic epigenomic states due to random actions of the inhibitors. We investigated whether JHDM3A/JMJD2A, a trimethylated histone H3-lysine 9 (H3K9me3)-specific demethylase, could function as an effector molecule to selectively demethylate target chromatin, with the aid of a guide protein to serve as a delivery vehicle. JHDM3A, which normally locates in euchromatin, spread out to heterochromatin when it was fused to heterochromatin protein-1α (HP1α) or HP1β; in these cells, demethylation efficiency was also markedly increased. Two truncated modules, JHDM3A(GFP)(406) and JHDM3A(GFP)(701), had contrasting modes and efficiencies of H3K9me3 demethylation; JHDM3A(GFP)(406) showed a very uniform rate (∼80%) of demethylation, whereas JHDM3A(GFP)(701) had a broad methylation range of 4-80%. The methylation values were highly dependent on the presence of the guide proteins OCT4, CTCF, and HP1. Chromatin immunoprecipitation detected reduced H3K9me3 levels at OCT4 regulatory loci in the cells expressing OCT4-tagged JHDM3A(GFP)(701). Derepression of the Sox2 gene was observed in JHDM3A(GFP)(701)OCT4-expressing cells, but not in cells that expressed the JHDM3A(GFP)(701) module alone. JHDM3A(GFP)(701)-assisted OCT4 more efficiently turned on stem cell-related microRNAs than GFP-OCT4 itself. These results suggest that JHDM3A(GFP)(701) is a suitable catalytic module that can be targeted, under the control of a guide protein, to specific loci where the chromatin H3K9me3 status and the milieu of gene expression are to be modified.
authors.affiliation: Development and Differentiation Research Center, KRIBB, 111 Gwahangno, Yuseong-gu, Daejeon 305-806, South Korea.
authors.forename: Young Sun
authors.initials: YS
authors.lastname: Jeong
authors.forename: Jung Sun
authors.initials: JS
authors.lastname: Park
authors.forename: Yong
authors.initials: Y
authors.lastname: Ko
authors.forename: Yong-Kook
authors.initials: YK
authors.lastname: Kang
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D000076246:CCCTC-Binding Factor; D000090266:Chromobox Protein Homolog 5; D022041:Euchromatin / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology*; D056426:Genetic Loci / Q000502:physiology; D057809:HEK293 Cells; D006570:Heterochromatin / Q000235:genetics / Q000378:metabolism*; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D056484:Jumonji Domain-Containing Histone Demethylases / Q000235:genetics / Q000378:metabolism*; D008745:Methylation; D051379:Mice; D041681:NIH 3T3 Cells; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: C000715593:CBX1 protein, human; C000715594:CBX5 protein, human; D000076246:CCCTC-Binding Factor; C000619697:CTCF protein, human; C000619698:Ctcf protein, mouse; D022041:Euchromatin; D006570:Heterochromatin; D006657:Histones; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; C494174:Pou5f1 protein, mouse; D012097:Repressor Proteins; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; D000090266:Chromobox Protein Homolog 5; D056484:Jumonji Domain-Containing Histone Demethylases; C494424:KDM4A protein, human