Background/Purpose: Fibrosis is a key feature of systemic sclerosis, a devastating disease that is not well understood. The role of epigenetic factors in the pathophysiology is increasingly explored. Histone modifications such as histone methylation are dynamic epigenetic events that regulate signaling pathways and cellular processes. DOT1L is the unique H3K79-methyltransferase and methylates histone 3 at the Lysine residue at position 79. Inhibition of DOT1L in cartilage and bone has cell-type specific effects on Wnt-signaling, a pathway suggested to play an important role in fibrosis. We aim to study the role of DOT1L in fibrosis using different in vitro and in vivo models.

Methods: Primary cell cultures of healthy human dermal fibroblasts (breast and abdomen) were treated with DOT1L-inhibitor EPZ-5676 or vehicle for 14 days and stimulated with TGF-β after starvation. Smooth muscle alpha 2 actin (ACTA2) gene expression was measured by RT-qPCR. Western Blot was performed for dimethylated H3K79. Picrosirius Red staining measured collagen deposition. Proliferation was analyzed with 5-Bromo-2’-deoxy-uridine (BrdU) labeling. Flow cytometry was done using Propidium Iodide to analyze the cell cycle phases. Col1a2;Cre-ERT²;DOT1l^fl/flmice (tamoxifen-inducible fibroblast specific DOT1L knockout mice) were injected subcutaneously with bleomycin (0.1mg) or vehicle (5 days/week, 4 weeks). Injected skin was analyzed by OH-proline assay for collagen content, and by histology for dermal thickness.

Results: The DOT1L-inhibitor EPZ-5676 reduced H3K79 dimethylation in all samples. DOT1L inhibition had a differential effect on ACTA2 expression: in breast dermal fibroblasts the increase of ACTA2 after TGF-β stimulation was reduced, while in abdominal dermal fibroblasts the increase of ACTA2 was more pronounced. After 48 hours of TGF-β, collagen deposition was higher in DOT1L-inhibitor exposed fibroblasts. After 72 hours of TGF-β stimulation however, collagen deposition was comparable between control and DOT1L inhibition. BrdU labeling assay showed more fibroblast proliferation with inhibition of DOT1L. Analysis of the cell cycle using flow cytometry revealed that DOT1L inhibition increased the proportion of cells in the G1/G0 phase, with fewer cells in S and M/G2 phase. In vivo, subcutaneous bleomycin increased dermal thickness and skin collagen content in mice. No difference was observed between mice with a conditional fibroblast-specific deletion of DOT1L or wild type mice.

Conclusion: In an in vitro model of fibrosis, the induction of ACTA2 with TGF-β in DOT1L-inhibited primary human dermal fibroblasts was dependent on the site of origin of the fibroblasts. DOT1L inhibition resulted in a more rapid increase in collagen deposition, but this did not result in detectable differences in collagen deposition at the end point of the experiments. DOT1L inhibition increased fibroblast proliferation but also led to a higher proportion of cells in the G0/G1 phase. In an in vivo murine model of skin fibrosis, no difference in bleomycin-induced skin thickness and collagen content was found when the DOT1L gene was deleted in fibroblasts.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/epigenetic-changes-in-dermal-fibroblasts-by-inhibition-of-dot1l-affect-cell-proliferation-and-cell-cycle-but-have-no-direct-effects-on-collagen-deposition-in-in-vitro-and-in-vivo-models-of-fibrosis/