1.14.11.69 D191A site-directed mutagenesis, the mutant shows about 95%reduced activity with H3K9me3 compared to wild-type, and no activity with H3K36me3 754871 1.14.11.69 G133A site-directed mutagenesis, the mutation in the catalytic domain abrogates enzyme activity 753371 1.14.11.69 G138A site-directed mutagenesis, the mutation in the catalytic domain abrogates enzyme activity 753371 1.14.11.69 G165A site-directed mutagenesis, the mutation in the catalytic domain abrogates enzyme activity 753371 1.14.11.69 G170A site-directed mutagenesis, the mutation in the catalytic domain abrogates enzyme activity 753371 1.14.11.69 H195A construction of a mutant dKDM4A in which a conserved amino acid in the iron-binding site is mutated to alanine, the mutant dKDM4A has no demethylation activity on histones H3K36me3 and H3K36me2 700209 1.14.11.69 H195A site-directed mutagenesis 724813 1.14.11.69 H195A site-directed mutagenesis, mutation of an Fe2+ binding residue, abolishes demethylase activity of dJMJD2(1)/CG15835 754921 1.14.11.69 H235A catalytically deficient mutant -, 726071, 754922 1.14.11.69 additional information construction of a P-element insertion mutant of dKDMA4, the mutant is homozygous viable, the P element insertion elevates the bulk level of histone H3K36me3 in mutant embryos. Overexpression of dKDM4A seems to only lead to demethylation of histone H3K36, since the level of histone H3K9me3 and H3K4me2 remains unchanged 700209 1.14.11.69 additional information construction of transgenic lines carrying a UASGAL4-CG15835-Flag construct, where expression of dJMJD2(1)/CG15835 is under the control of the yeast activator GAL4, allowing its overexpression upon crossing with lines expressing GAL4. Overexpression of CG15835 results in spreading of HP1 into euchromatin and a strong decrease on the levels of H3K9me3 and H3K36me3 754921 1.14.11.69 additional information enzyme engineering and swapping of the C-terminus region containing the distal Tudor domain between isozymes KDM4C and KDM4A, construction of diverse chimeric enzyme mutants, overview. Chimera5, which encodes the first 934 amino acids of KDM4C fused with the last 129 amino acid containing the distal Tudor domain of KDM4A, is excluded from mitotic chromatin. On the other hand, chimera6 that encodes the first 954 amino acids of KDM4A fused to 101 amino acids of KDM4C, which includes its distal Tudor domain, remains excluded from chromatin. The C-terminus of KDM4C containing the distal Tudor domain is essential but not sufficient for its mitotic chromatin localization 745919 1.14.11.69 additional information enzyme engineering and swapping of the C-terminus region containing the distal Tudor domain between isozymes KDM4C and KDM4A, construction of diverse chimeric enzyme mutants, overview. Chimera5, which encodes the first 934 amino acids of KDM4C fused with the last 129 amino acid containing the distal Tudor domain of KDM4A, is excluded from mitotic chromatin. On the other hand, chimera6 that encodes the first 954 amino acids of KDM4A fused to 101 amino acids of KDM4C, which includes its distal Tudor domain, remains excluded from chromatin. The C-terminus of KDM4C containing the distal Tudor domain is essential but not sufficient for its mitotic chromatin localization. EGFP-KDM4CRDTF/DNLY mutant is excluded from mitotic chromatin. For isozyme knockout, U2OS cells are transfected with KDM4B-C siRNA sequences 745919 1.14.11.69 additional information generation of a rph1DELTA deletion mutant, phenotype overview 744589 1.14.11.69 additional information generation of conditional Jmjd2a/Kdm4a, Jmjd2b/Kdm4b and Jmjd2c/Kdm4c/Gasc1 single, double and triple knockout mouse embryonic stem cells (ESCs). While individual Jmjd2 family members are dispensable for ESC maintenance and embryogenesis, combined deficiency for specifically Jmjd2a and Jmjd2c leads to early embryonic lethality and impaired ESC self-renewal, with spontaneous differentiation towards primitive endoderm under permissive culture conditions. Increased H3K9me3 levels in knockout ESCs compromise the expression of several Jmjd2a/c targets, including genes that are important for ESC self-renewal. Thus, continual removal of H3K9 promoter methylation by Jmjd2 demethylases represents a novel mechanism ensuring transcriptional competence and stability of the pluripotent cell identity. Phenotypes, overview -, 744785 1.14.11.69 additional information generation of dkdm4a mutant embryos, the dkdm4a allele contains a P-element inserted within the first exon of the gene and abrogates dKDM4A transcription 726301 1.14.11.69 additional information generation of JmjC protein dKDM4A mutant allele, DELTAdKDM4A, with a deletion spanning from 47 nucleotides downstream of the ATG to the end of the gene by means of imprecise excision of the P-element P{GawB}NP0618 724813 1.14.11.69 additional information introduction of a di-glycine motif at the +3 to +4 positions of the H3K27 sequence, a site which shares sequence homology with the H3K9 sequence, enables JMJD2A to efficiently demethylate H3K27me3, cf. EC 1.14.11.68 752795 1.14.11.69 additional information JMJD2A knockout or overexpression in Hep-3B cells. Construction of JMJD2ADELTA mutant. A 39KD JMJD2A transcript, JMJD2ADELTA, is significantly increased in JMJD2A or miR372 overexpressing Hep3B cell line 745949 1.14.11.69 additional information Jmjd2b knockdown by siRNA, leading to induction of p53 via activation of the DNA damage response pathway. p53 Inhibition significantly restored the clonogenic potential of AGS and HeLa cells treated with JMJD2B siRNA. Increased apoptosis in BGC-823 and HeLa cells but not AGS cells with JMJD2B siRNA knockdown. AGS cells are arrested at the G1 phase, but BGC-823 and HeLa cells are arrested at the S phase 752645 1.14.11.69 additional information overexpression bypasses the requirement for the positive elongation factor gene BUR1 687571 1.14.11.69 additional information overexpression of JmjD2A in fibroblasts specifically depletes H3K9me3 and H3K36me3 753371 1.14.11.69 additional information the kdmA mutant shows a significant increase in H3K36me3 during primary metabolism at the aflR and ipnA locus and some slightly higher levels at the aptA genes, the mutant has reduced levels of sterigmatocystin compared to wild-type, mutant phenotype, overview. Deletion of kdmA in Aspergillus nidulans produces both positive and negative changes in transcriptional readouts and the number of affected genes is different under different conditions. KdmA deletion alters expression pattern of secondary metabolism cluster genes in secondary metabolism phase, analysis of the heat map for mean expression of previously annotated secondary metabolism clusters. Deletion of kdmA causes light lethality and sensitivity to oxidative stress during vegetative growth -, 745803 1.14.11.69 additional information transposition of the Ala-Pro motif of H3K27 to AARK(me3)SPAAT, to mimic the proline position in H3K36, resulted in no detectable activity 754876 1.14.11.69 N290A site-directed mutagenesis, the mutant shows no activity with H3K36me3 and almost no activity with H3K9me3 754871 1.14.11.69 N290D site-directed mutagenesis, the mutant shows about 98%reduced activity with H3K9me3 compared to wild-type, and no activity with H3K36me3 754871 1.14.11.69 R919D site-directed mutagenesis, the mutant is not associated with mitotic chromatin in contrast to the wild-type enzyme 745919 1.14.11.69 S198M site-directed mutagenesis, a KDM4C demethylase dead mutant 745919 1.14.11.69 S288A mutation augments activity, particularly toward H3K9me2 754871 1.14.11.69 S288A mutations of the residues comprising the methylammonium-binding pocket abrogate demethylation by JMJD2A, with the exception of an S288A substitution, which augments activity, particularly toward H3K9me2 754871 1.14.11.69 S288N site-directed mutagenesis, the mutation in the catalytic domain abrogates enzyme activity 753371 1.14.11.69 T289B site-directed mutagenesis, the mutation in the catalytic domain abrogates enzyme activity 753371 1.14.11.69 V423A site-directed mutagenesis, the point mutation at the central valine of PxVxL motif disrupts the interaction between dKDM4A and HP1a in vitro 726301 1.14.11.69 Y175F site-directed mutagenesis, the mutant shows about 90%reduced activity with H3K9me3 compared to wild-type, and no activity with H3K36me3 754871 1.14.11.69 Y177F site-directed mutagenesis, the mutant shows about 90%reduced activity with H3K9me3 compared to wild-type, and no activity with H3K36me3 754871