Ure 1B”, E” and H”), as has previously been shown for a truncated version [22]. Alternatively, we did not observe H3K9 demethylase activity for JMJD1C (Figure 1C”, F” and I”). Next we tested further methylation web-sites, such as H3K4, K27 and K36 marks, also as H4K18 and K20, but once again JMJD1C overexpression did not lead to visible modifications of their methylation levels (Figure S2). To exclude a cell line specific impact, all overexpression analyses had been also performed in the human embryonic kidney cell line HEK293T, exactly where once again KDM3A and KDM3B were enzymatically active although JMJD1C overexpression did not influence H3K9 methylation levels (Figure S3A ). KDM3 subfamily members were additional overexpressed in HeLa, NIH3T3, and TM3 cells, and again, precisely the same final results were obtained (Figure S3 G ). In addition, we extended these observations for the second described splice isoform of JMJD1C which is 219 aa shorter than the initial isoform (Figure 2A construct i; Figure S4 C ). Ultimately, a full-length mouse Jmjd1C construct also failed to minimize H3K9 methylation levels upon overexpression (Figure S4 A ). Taken together, these results show that overexpression of KDM3A and KDM3B strongly decreased international H3K9me1 and ?me2 levels, whilst overexpression of JMD1C/ Jmjd1c did not. JmjC containing proteins function in an iron and a-ketoglutarate dependent mechanism [20]. It has been shown that single amino acid substitutions in the conserved active internet sites are sufficientA Systematic Comparison of KDM3 Subfamily MembersFigure 1. Enzymatic activity of KDM3 subfamily members towards H3K9 methylation. Individual KDM3 subfamily members had been transiently overexpressed in U-2 OS cells.661487-17-8 structure (A-M) DAPI staining indicating cell nuclei. (A’-M’) Cellular expression of Avi-tagged KDM3 subfamily members, as detected by streptavidin-AlexaFluor-488 recognizing the biotinylated Avi-tag.5-(Aminomethyl)picolinic acid site (A”-M”) H3K9me1, -me2 or -me3 groups, respectively, as detected by antibody staining.PMID:24324376 White circles outline the transfected cells within the final panel of each series. Note that cells transfected with KDM3A and KDM3B (A, D, G and B, E, H) abolish H3K9me1 (A” and B”) and -me2 (D” and E”) but not -me3 (G” and H”) staining. Alternatively, JMJD1C transfection (C, F, I) doesn’t lower H3K9me1 (C”), -me2 (F”) or -me3 (I”) levels. The catalytic mutant versions of KDM3A(H1120A) (J, L) and KDM3B(H1560A) (K, M) neither minimize H3Kme1 (J”, K”) nor H3K9me2 (L”, M”) levels. N shows the summary of the enzymatic activity described above. doi:10.1371/journal.pone.0060549.gto completely abrogate enzymatic activity, as shown for instance for KDM7 [23]. To this finish, we mutated one of many histidines involved in iron binding in the active web-site of KDM3A and B (KDM3A(H1120A) and KDM3B(H1560A)) to alanine, each, and tested the activities of these mutants towards H3K9me1/2. As expected, each proteins localized towards the nucleus (Figure 1J’, K’, L’ and M’). Indeed, overexpression of these mutants did not trigger demethylation of H3K9 (Figure 1J”, K”, L” and M”), suggesting that enzymatic activity occurs by the expected co-factor-dependent mechanism. It has previously been suggested that a brief version of mouse Jmjd1c is an active H3K9me1/2 demethylase enzyme [19]. Therefore, we performed a number of experiments to address this discrepancy in comparison to our observations described above. In our experiments, we made use of a full-length JMJD1C expression construct, and we noticed that overexpression of this construct.
