Background/Purpose: Long noncoding RNAs (lncRNAs) are an emerging class of 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 have identified a novel TGFβ regulated lncRNA, H19X, which was strongly and consistently 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. Here we aimed to define the mode of action of H19X and to assess H19X functions that may drive myofibroblast differentiation and ECM production.

Methods: The function of H19X was investigated in SSc dermal fibroblasts (n=5) by knocking down H19X with locked nucleic acid oligonucleotides (LNA GapmeRs) followed by TGF-β stimulation and microarray analysis with Illumina HT-12 arrays. In situ hybridization for H19X was performed using Stellaris FISH probes. Genomic regions of interaction with H19X were identified by Chromatin Isolation by RNA Purification (ChIRP) using biotinylated probes tiling the transcript, RNA pull-down and sequencing. Chromatin remodeling was investigated by Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). DNA damage-inducible transcript 4-like protein (DDIT4L) function was tested using siRNA in parallel to H19X downregualtion.

Results: As analyzed by microarray, none of the 16 genes belonging to the same genomic region (1 Mb) 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. Moreover, increased numbers of positive nuclei were also recorded after TGF-β stimulation. 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.
 
Next, we identified 58 genomic regions that directly interact with H19X using ChIRP. In order to establish the effect of H19X physical presence at these sites, the expression of the genes with the closest transcription start site was analyzed in the microarray data set. Among these genes, DDIT4L displayed the strongest induction following H19X knockdown. Furthermore, we cloud identify chromatin rearrangements caused by H19X knockdown in close proximity with its interaction site with DDIT4L locus using ATAC-seq. Interestingly, DDIT4L expression was consistently repressed by TGFβ stimulation at mRNA and protein level. Additionally, DDIT4L knockdown was able to restore COL1A1 expression that was reduced by H19X downregulation but only in presence of TGFβ. These data indicate that H19X mediates its effects unabeling DDIT4L transcription which, in turn, is a novel repressor of TGFβ pathway activity.

Conclusion: Our data uncover a novel mechanism for the TGFβ regulated profibrotic effects of H19X including direct effects on chromatin organization associated with fibrotic pathways.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/cracking-a-novel-profibrotic-molecular-mechanism-lncrna-h19x-and-ddit4l-crosstalk/