The DOT1L protein and gene network in chondrocytes identifies H3K79 histone methylation as a key regulator of WNT and other growth factor cascades

Abstract

Purpose: DOT1L is the only known H3K79 histone methyltransferase and has been linked to epigenetic regulation of the Wingless-like (Wnt) signaling pathway. 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 signaling. 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 link these DOT1L targets and interactors with the molecular characteristics of the articular chondrocyte during the loss of phenotype in a dedifferentiation setup. Finally, we aimed for a translational validation of our data in cartilage samples from OA patients. 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 leukemia cells, using a series of specific 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 control. The differentially expressed genes were explored with the limma package. String-DB and Panther were used for gene network analyses. Based on the microarray analysis, selected genes were confirmed by quantitative PCR. 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 (P0 to P5). We analyzed 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 signaling cascades was analyzed by Western blot. For translational validation we analyzed the differential gene expression profile of damaged as compared to intact cartilage areas within the same joint of patients with hip OA. Results: The presence of different DOT1L elongation complexes was confirmed in hACs. The CoIP experiments revealed that DOT1L interacts directly with ENL, AF10 and active beta-catenin (more strongly when cells were treated with LiCl to stabilize beta-catenin) and with AF4 and AF5 (only with cells were treated with LiCl). The formation of these complexes was disrupted when DOT1L was inhibited. In the microarray, 1937 genes significantly changed (p<0.05) in DOT1L inhibited samples compared to vehicle treated samples (1048 genes were down-regulated, 889 up-regulated). In EPZ5676 treated samples, chondrocyte differentiation-associated genes, such as ACAN, RGS5, GDF10 and LOXL2 were down-regulated; 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, in vitro expanded hACs showed progressive changes in gene expression. 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. We successfully validated these targets of interest in samples from patients with OA condition. 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 signaling and other growth factor cascades in the joint.