Cellular, biochemical and phylogenomic analyses of the mouse Jumonji domain containing 6 protein provide new evidence for functions as a 2-oxoglutarate dependent dioxygenase ; Zelluläre, biochemische und phylogenomische Analysen des murinen Jumonji domain containing 6 Proteins erbringen neue Hinweise für eine Funktion als 2-Oxoglutarat abhängige Dioxygenase

The “jumonji domain containing 6” protein (Jmjd6) is involved in embryogenetic tissue differentiation and cytokine release of activated macrophages. Given the importance of jumonji C (JmjC) domain-containing proteins in oxidative DNA and RNA repair, protein hydroxylation, and histone lysine demethylation, an analysis of the Jmjd6 locus, evolution, and protein function was carried out. Sequences homologous to the mouse Jmjd6 protein were identified in 61 species from all major living phyla. This lead to the identification and characterisation of a bi-directional transcriptional unit comprising Jmjd6 and the neighbouring 1110005A03Rik locus, verification of a novel Jmjd6 exon and two new splice variants in vivo. A calculated and cross-validated structural model of the Jmjd6 protein identified a conserved double stranded ß-helix (DSBH) fold, an 2-His-1-carboxylate facial triad, and a 2-oxoglutarate co-ordination site as structural motifs. Using mAB328, a newly generated monoclonal anti-Jmjd6 antibody, it was shown that endogenous Jmjd6 is a nuclear and nucleolar, cell cycle dependent protein with no co-localisation to heterochromatic DNA. Western blot analyses showed that Jmjd6 forms homo-multimers, that homo-multimerization requires the full-length protein and identified an N-terminal protein multimerization domain. An analysis of H3K4, H3K9, H3K27, H3K36, and H4K20 histone methylation states in wildtype and Jmjd6 -/- cells and overexpression of Jmjd6-reporter constructs excluded a function of Jmjd6 in the demethylation of these residues. Finally, nuclease assays showed a likely association of Jmjd6 to a ribonucleic matrix. In conclusion, additional evidence that Jmjd6 functions most likely as a nonheme-Fe(II)-2-oxoglutarate-dependent dioxygenase and novel insights into the evolution of Jmjd6 and JmjC domain-containing proteins are provided. The results suggest that enzymatic targets of Jmjd6 might be RNA or RNA-associated proteins and not histone lysine residues. ; Das “jumonji domain containing 6”-Protein ...