The jumonji (JMJ) family of histone demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes1–4. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context5. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance2,4,6, as well as in development, physiology and disease3,7. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX)8. The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.

Objective. TSG-6 (the product of tumor necrosis factor [TNF]-stimulated gene 6) has a potent inhibitory effect on RANKL-mediated bone erosion. The aim of this study was to compare the activity of TSG-6 with that of osteoprotegerin (OPG) and to investigate its role as an autocrine modulator of cytokine-mediated osteoclast formation/activation. We also determined TSG-6 expression in inflammatory joint disease.Methods. The effects of TSG-6, OPG, and the inflammation mediators TNF␣, interleukin-1 (IL-1), and IL-6 on the formation of osteoclasts from peripheral blood mononuclear cells and synovial fluid (SF) macrophages were determined by tartrateresistant acid phosphatase staining. Lacunar resorption and filamentous actin ring formation were measured as indicators of osteoclast activity. The amount of TSG-6 in culture media or SF was quantified by enzyme-linked immunosorbent assay, and expression of TSG-6 in synovial tissue was assessed by immunohistochemistry.Results. TSG-6 acted in synergy with OPG to inhibit RANKL-mediated bone resorption and was produced by osteoclast precursors and mature osteoclasts in response to TNF␣, IL-1, and IL-6. Expression of TSG-6 correlated with inhibition of lacunar resorption; this effect was ameliorated by an anti-TSG-6 antibody. The level of TSG-6 protein was determined in SF from patients with various arthritides; it was highest in patients with inflammatory conditions such as rheumatoid arthritis, in which it correlated with the amount of TSG-6 immunostaining in the synovium. TSG-6 inhibited the activation but not the formation of osteoclasts from SF macrophages.Conclusion. In the presence of inflammatory cytokines, osteoclasts produced TSG-6 at concentrations that are sufficient to inhibit lacunar resorption. This may represent an autocrine mechanism to limit the degree of bone erosion during joint inflammation.

Two distinct areas of cerebellar cortex, vermal lobule VII and the dorsal paraflocculus (DPFl) receive visual input. To help understand the visuomotor functions of these two regions, we compared their afferent and efferent connections using the tracers wheatgerm agglutinin horseradish peroxidase (WGA-HRP) and biotinilated dextran amine (BDA). The sources of both mossy fibre and climbing fibre input to the two areas are different. The main mossy fibre input to lobule VII is from the nucleus reticularis tegmenti pontis (NRTP), which relays visual information from the superior colliculus, while the main mossy fibre input to the DPFl is from the pontine nuclei, relaying information from cortical visual areas. The DPFl and lobule VII both also receive mossy fibre input from several common brainstem regions, but from different subsets of cells. These include visual input from the dorsolateral pons, and vestibular-oculomotor input from the medial vestibular nucleus (MVe) and the nucleus prepositus hypoglossi (Nph). The climbing fibre input to the two cerebellar regions is from different subdivisions of the inferior olivary nuclei. Climbing fibres from the caudal medial accessory olive (cMAO) project to lobule VII, while the rostral MAO (rMAO) and the principal olive (PO) project to the DPFl. The efferent projections from lobule VII and the DPF1 are to all of the recognised oculomotor and visual areas within the deep cerebellar nuclei, but to separate territories. Both regions play a role in eye movement control. The DPFl may also have a role in visually guided reaching.

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