JHDM3A module as an effector molecule in guide-directed modification of target chromatin

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_GFP⁴⁰⁶ and JHDM3A_GFP⁷⁰¹, had contrasting modes and efficiencies of H3K9me3 demethylation; JHDM3A _GFP⁴⁰⁶ showed a very uniform rate (∼80%) of demethylation, whereas JHDM3A_GFP⁷⁰¹ 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_GFP⁷⁰¹. Derepression of the Sox2 gene was observed in JHDM3A_GFP⁷⁰¹ OCT4-expressing cells, but not in cells that expressed the JHDM3A _GFP⁷⁰¹ module alone. JHDM3A_GFP ⁷⁰¹-assisted OCT4 more efficiently turned on stem cell-related microRNAs than GFP-OCT4 itself. These results suggest that JHDM3A _GFP⁷⁰¹ 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.