Mechanistic and structural studies of KDM-catalysed demethylation of histone 1 isotype 4 at lysine 26.

N-Methylation of lysyl residues is widely observed on histone proteins. Using isolated enzymes, we report mechanistic and structural studies on histone lysine demethylase (KDM)-catalysed demethylation of Nε -methylated lysine 26 on histone 1 isotype 4 (H1.4). The results reveal that methylated H1.4K26 is a substrate for all members of the KDM4 subfamily and that KDM4A-catalysed demethylation of H1.4K26me3 peptide is similarly efficient to that of H3K9me3. Crystallographic studies of an H1.4K26me3:KDM4A complex reveal a conserved binding geometry to that of H3K9me3. In the light of the high activity of the KDM4s on this mark, our results suggest JmjC KDM-catalysed demethylation of H1.4K26 may be as prevalent as demethylation on the H3 tail and warrants further investigation in cells.

KDM3A coordinates actin dynamics with intraflagellar transport to regulate cilia stability.

Cilia assembly and disassembly are coupled to actin dynamics, ensuring a coherent cellular response during environmental change. How these processes are integrated remains undefined. The histone lysine demethylase KDM3A plays important roles in organismal homeostasis. Loss-of-function mouse models of Kdm3a phenocopy features associated with human ciliopathies, whereas human somatic mutations correlate with poor cancer prognosis. We demonstrate that absence of KDM3A facilitates ciliogenesis, but these resulting cilia have an abnormally wide range of axonemal lengths, delaying disassembly and accumulating intraflagellar transport (IFT) proteins. KDM3A plays a dual role by regulating actin gene expression and binding to the actin cytoskeleton, creating a responsive "actin gate" that involves ARP2/3 activity and IFT. Promoting actin filament formation rescues KDM3A mutant ciliary defects. Conversely, the simultaneous depolymerization of actin networks and IFT overexpression mimics the abnormal ciliary traits of KDM3A mutants. KDM3A is thus a negative regulator of ciliogenesis required for the controlled recruitment of IFT proteins into cilia through the modulation of actin dynamics.

Arginine demethylation is catalysed by a subset of JmjC histone lysine demethylases.

While the oxygen-dependent reversal of lysine N(ɛ)-methylation is well established, the existence of bona fide N(ω)-methylarginine demethylases (RDMs) is controversial. Lysine demethylation, as catalysed by two families of lysine demethylases (the flavin-dependent KDM1 enzymes and the 2-oxoglutarate- and oxygen-dependent JmjC KDMs, respectively), proceeds via oxidation of the N-methyl group, resulting in the release of formaldehyde. Here we report detailed biochemical studies clearly demonstrating that, in purified form, a subset of JmjC KDMs can also act as RDMs, both on histone and non-histone fragments, resulting in formaldehyde release. RDM catalysis is studied using peptides of wild-type sequences known to be arginine-methylated and sequences in which the KDM's methylated target lysine is substituted for a methylated arginine. Notably, the preferred sequence requirements for KDM and RDM activity vary even with the same JmjC enzymes. The demonstration of RDM activity by isolated JmjC enzymes will stimulate efforts to detect biologically relevant RDM activity.

A cell-permeable ester derivative of the JmjC histone demethylase inhibitor IOX1.

The 2-oxoglutarate (2OG)-dependent Jumonji C domain (JmjC) family is the largest family of histone lysine demethylases. There is interest in developing small-molecule probes that modulate JmjC activity to investigate their biological roles. 5-Carboxy-8-hydroxyquinoline (IOX1) is the most potent broad-spectrum inhibitor of 2OG oxygenases, including the JmjC demethylases, reported to date; however, it suffers from low cell permeability. Here, we describe structure-activity relationship studies leading to the discovery of an n-octyl ester form of IOX1 with improved cellular potency (EC50 value of 100 to 4 µM). These findings are supported by in vitro inhibition and selectivity studies, docking studies, activity versus toxicity analysis in cell cultures, and intracellular uptake measurements. The n-octyl ester was found to have improved cell permeability; it was found to inhibit some JmjC demethylases in its intact ester form and to be more selective than IOX1. The n-octyl ester of IOX1 should find utility as a starting point for the development of JmjC inhibitors and as a use as a cell-permeable tool compound for studies investigating the roles of 2OG oxygenases in epigenetic regulation.