AB0200 INHIBITION OF DOT1L ACTIVITY IN HUMAN SKIN FIBROBLASTS: A TRANSCRIPTOME ANALYSIS. (June 2019)

• Record Type: Journal Article
• Authors: Berghen, Nathalie; Langhe, Ellen De; Lories, Rik
• Is Part Of: Annals of the rheumatic diseases. Volume 78(2019)Supplement 2
• Page Start: 1557
• Page End: 1557
• Publication Date: 2019-06
• Subjects: Rheumatism -- Periodicals; 616.723005
• DOI: 10.1136/annrheumdis-2019-eular.1023
• Languages: English
• ISSNs: 0003-4967

Abstract

Abstract : Background: The pathophysiology of fibrosis, a hallmark of diseases like Systemic Sclerosis, is still not well understood. The role of epigenetic factors is increasingly explored. DOT1L, the unique H3K79-methyltransferase, methylates histone 3 at the Lysine residue at position 79, thereby regulating gene expression programs. Inhibition of DOT1L in cartilage and bone has cell-type specific effects on Wnt signaling, a pathway suggested to play an important role in fibrotic disease. Objectives: To study the changes in the transcriptome of dermal fibroblasts after DOT1L inhibition. Methods: Primary human dermal fibroblasts isolated from abdominal skin were treated with DOT1L-inhibitor EPZ-5676 or vehicle and stimulated or not with TGF-β or CHIR99021 (activating Wnt signaling). RNAseq was performed using the NextSeq500 as a platform and TruSeq as library prep kit. The preprocessed reads were aligned to the reference genome of Homo-sapiens (Ensembl.GRCh38.88(GRCh38)). Statistical comparative analysis for differential expressed genes (DEG) was done using the edge package in R version 3.20.9. The resulting p-values were corrected for multiple testing with Benjamini-Hochberg to control for false discovery rate (FDR). DEG were selected based on FDR-value less than 0.05 and a fold change of an absolute log2 -ratio larger than 1. Pathway analysis was done using PANTHER14.0. Western Blot for di-methylated H3K79 was performed to confirm successful DOT1L inhibition. Results: The DOT1L-inhibitor EPZ-5676 effectively reduced H3K79 di-methylation in the treated samples. RNAseq analysis revealed 663 differentially expressed genes in dermal fibroblasts exposed to the DOT1L inhibitor, based on group analysis: 77 genes were downregulated and 556 upregulated. There was no significant interaction between inhibition of DOT1L and stimulation of the cells with TGF-β or CHIR99021. Pathway analysis showed involvement of primarily the integrin signaling, the cadherin pathway and Wnt signaling. Conclusion: More than 600 differentially expressed genes were discovered by RNA sequencing in human dermal fibroblasts exposed to DOT1L inhibition (independent of TGF-β or CHIR 99021 stimulation). These gene lists and their networks indicate that DOT1L activity affects integrin signaling, the cadherin and Wnt pathway, suggesting a potential impact on the fibrotic process that is leading to pathology in SSc. Acknowledgement: RNA sequencing and analysis was performed by VIB Nucleomics Core (www.nucleomics.be). Disclosure of Interests: Nathalie Berghen: None declared, Ellen De Langhe: None declared, Rik Lories Consultant for: Abbvie, Celgene, Eli-Lilly, Janssen, Merck, Novartis, Pfizer, UCB