A3.9 DOT1L, a H3K79 methyltransferase linked to the WNT signallingsignaling cascade, regulates fibrotic responses in lung fibroblasts

Background and objectives Fibrosis is the formation of excess fibrous connective tissue. It disturbs cell, tissue and organ homeostasis, leading to severe disease manifestations in various disorders. Transcriptional regulation of genes and pathways with pro- or anti-fibrotic effects is therefore a therapeutic target. DOT1-like histone H3 methyltransferase (DOT1L) is the only known H3K79 methyltransferase. It functions as a positive mediator of gene transcription, of DNA repair, as a regulator of the cell cycle, and has been associated with Wnt signalling. Our aim is to study the role of DOT1L in downstream events associated with pro-fibrotic cascades, hypothesising that it may act as an epigenetic masterswitch in the fibrotic process. Materials and methods MRC5 cells (a human fetal lung fibroblast cell line) were cultured for up to 22 days with 0 and 3µM of EPZ04777 (DOT1L inhibitor). During that period, the cells were stimulated at different time-ponts, with 0 to 5 ng/mL TGFβ 1 for 24 h. We performed mRNA and protein expression analysis. Total RNA was extracted from cell lysates and reverse-transcribed, and gene expression levels were quantified with real-time quantitative PCR (qPCR). Expression was normalised to Hprt and subsequently normalised to the vehicle condition using the comparative cycle threshold method (ΔΔCT). Total cell lysates were homogenised in Cell Extraction Buffer and proteins were analysed by immunoblot, including assessment of the H3K79 methylation status. Results Long-term treatment progressively decreases the methylation status of H3K79. Upon stimulation with TGFβ1, MRC5 cells show upregulation of type I Collagen mRNA expression and collagen deposition. Moreover, αSMA mRNA expression is also increased, indicating a fibroblast to myofibroblast differentiation. TGFβ1 stimulation also increased DOT1L expression and activity in the absence of EPZ04777, as shown by qPCR and H3K79 methylation status. Upon treatment with EPZ04777, we also observed an increase in collagen and αSMA expression, suggesting that loss of DOT1L activity results in pro-fibrotic changes in lung fibroblasts. Conclusions DOT1L has homeostatic effects in the healthy lung fibroblast, since its inhibition triggers a pro-fibrotic transcriptional response. Its upregulation by TGFβ1 could represent a regulatory feedback loop that requires further investigation.