Background and objectives DOT1L is the only known H3K79 histone methyltransferase. Genome-wide association and functional studies identified the DOT1L gene to be associated with cartilage thickness and hip osteoarthritis (OA) and showed an interaction of DOT1L with canonical Wnt signalling. Here, we further investigated the biology of DOT1L in cartilage health and disease. Specifically, our objective was to define the transcriptional complexes and transcriptome associated with DOT1L in articular cartilage, and to investigate the role of DOT1L during the loss of phenotype of the articular chondrocyte.
Methods Human articular chondrocytes (hACs) stimulated or not with LiCl were used to carefully map the presence or absence of DOT1L protein complexes suggested earlier in leukaemia cells, using immunoprecipitation experiments. To elucidate the transcriptional network of DOT1L, we performed a microarray of hACs from 5 non-OA fracture patients treated with a specific DOT1L inhibitor (EPZ5676) or vehicle. To establish the role of DOT1L in the maintenance of hACs phenotype, hACs were cultured in monolayer in the presence or absence of EPZ5676, and RNA and protein were isolated at serial passages. We analysed by quantitative PCR the gene expression of known genes important for chondrocyte biology and/or genes that appeared in the microarray of DOT1L inhibition. Activation of the Wnt canonical and non-canonical signalling cascades was analysed by Western blot.
Results The presence of different DOT1L elongation complexes was confirmed in hACs. In the microarray, chondrocyte differentiation-associated genes, such as ACAN, RGS5, GDF10 and LOXL2 were down-regulated in EPZ5676 treated samples; OA-related genes, such as MMP1, CCL7, CCL8 and GPNMB were up-regulated; and WNT target genes, ligands and antagonists such as LEF1, WNT5A and DKK1 were significantly up-regulated. In the de-differentiation experiment, genes involved in relevant pathways for cartilage biology such as the WNT, NOTCH and BMP pathways exhibited significant changes that were highly accentuated by DOT1L inhibition.
Conclusions Our transcriptomic, protein and gene interaction approach provides novel insights into the DOT1L molecular network and its putative role in osteoarthritis and cartilage. These data further support an important role for DOT1L in joint homeostasis as a key regulator of WNT signalling and other growth factor cascades in the joint.