OP0184 PROFIBROTIC LNCRNA H19X: UNRAVELLING THE EFFECTS ON CHROMATIN REMODELING IN SYSTEMIC SCLEROSIS FIBROBLASTS
Authors: Elena Pachera, Shervin Assassi, Gloria Salazar, Mojca Frank-Bertoncelj, Jörg Distler, Gabriela Kania, Oliver Distler
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background</h3> Long noncoding RNAs (lncRNAs) are noncoding transcripts involved in the regulation of gene expression in health and disease. LncRNAs function via different mechanisms either acting cis, regulating the expression of nearby genes, or in trans, modulating the expression of distant genes. We identified a novel TGFβ regulated lncRNA, H19X, which was strongly upregulated in a wide variety of fibrotic disorders. Moreover, we were able to demonstrate that H19X is a key driver of myofibroblast formation and extracellular matrix (ECM) overproduction. <h3>Objectives</h3> To define the mode of action of H19X (cis or trans) and to assess H19X function on chromatin rearrangements that may drive myofibroblast differentiation and ECM production. <h3>Methods</h3> The function of H19X was investigated in SSc dermal fibroblasts (n=5) by knocking down H19X with locked nucleic acid oligonucleotides followed by TGF-β stimulation and microarray analysis with Illumina HT-12 arrays. In situ hybridization for H19X was performed using Stellaris FISH probes. Chromatin remodeling was investigated by Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). <h3>Results</h3> As analyzed by microarray, none of the 16 genes belonging to the same genomic region as H19X locus was affected by its downregulation, indicating that in-cis regulation is not a major mechanism of H19X activity. Cellular localization can give a first indication about lncRNA trans function: FISH staining of SSc fibroblasts stimulated with TGF-β revealed tightly localized nuclear foci in TGF-β treated cells. H19X nuclear localization was confirmed by cell fractionation, where H19X expression was peaking in the nucleus of cells after 6h of treatment with TGF-β. We next hypothesized that H19X nuclear expression might induce chromatin rearrangements. We assessed whether chromatin changes caused by H19X knockdown might be correlated with changes in expression profile seen by microarray. Therefore, we analyzed the differences in the chromatin conformation flanking transcription start sites (TSSs) by ATAC-seq. A clear separation between the two experimental groups (scrambled vs H19X knockdown) was observed and 450 differentially accessible regions were identified (p&lt;0.05, Log2FC&gt;0.5). After focusing on genes that displayed significant variations and Log2FC&gt;0.5 for both the array and ATAC-seq analysis, eight candidates genes were found both upregulated by microarray and showed increased accessibility at their TSSs by ATAC-Seq. Similarly, three candidate genes were identified as being downregulated by microarray and showed decreased TSS accessibility by ATAC-Seq. These genes included histones HIST1H2BD and HIST1H1C, suggesting a direct regulation of chromatin organization as a mode of action for H19X. Moreover, the cell cycle progression inhibitors FABP3 and PTTG1 were pointing to a role of H19X in proliferation. In addition, analysis for transcription factor motif enrichment in the differentially accessible peaks of the ATAC-Seq analysis revealed that motifs for TEAD2, RUNX family, and NFKB2 were less accessible, while motifs for JUN::FOS, NF2L2 and EHF were more accessible due to the chromatin changes. These results indicate that H19X could promote or repress the activity of these candidate transcription factors by changing chromatin conformation at the sites of their consensus sequence. <h3>Conclusion</h3> ATAC-Seq in combination with microarray analysis identified candidate mechanism for the TGFβ regulated profibrotic effects of H19 X including direct effects on chromatin organization and on transcription factors associated with fibrotic pathways. <h3>Disclosure of Interests</h3> Elena Pachera: None declared, Shervin Assassi: None declared, Gloria Salazar: None declared, Mojca Frank-Bertoncelj : None declared, Jörg Distler: None declared, Gabriela Kania: None declared, Oliver Distler Grant/research support from: Prof. Distler received research funding from Actelion, Bayer, Boehringer Ingelheim and Mitsubishi Tanabe to investigate potential treatments of scleroderma and its complications, Consultant for: Prof. Distler has/had consultancy relationship within the last 3 years with Actelion, AnaMar, Bayer, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, Italfarmaco, iQvia, Lilly, medac, MedImmune, Mitsubishi Tanabe Pharma, Pharmacyclics, Novartis, Pfizer, Sanofi, Serodapharm and UCB in the area of potential treatments of scleroderma and its complications. In addition, he had/has consultancy relationship within the last 3 years with A. Menarini, Amgen, Abbvie, GSK, Mepha, MSD, Pfizer and UCB in the field of arthritides and related disorders