Background/Purpose: We previously identified novel non-coding RNAs that were markedly overexpressed in SLE patient monocytes. Among all classes of RNAs, these non-coding RNAs were by far the most highly over-expressed in SLE.  We hypothesized that these novel RNAs represented regulatory RNAs. The purpose of this study was to determine whether the non-coding RNAs mapped to enhancers that regulated gene activity. 

Methods: We used transfection of over-expression vectors and knockdown of non-coding RNAs to test their function. ChIP assays for H3K27ac, as a mark of active enhancers, and H3K4me3, as mark for promoter activity were performed.  qRT-PCR was used to investigate the putative enhancers and to define functionality.

Results: Over-expression of noncoding RNAs in control monocytes could be induced by LPS in a p38-dependent manner.  We examined two clusters of non-coding RNAs in more detail at the IL-1 and the ADAM28 loci.  At both loci, LPS treatment was associated with significant induction of H3K27ac at the site of the non-coding RNA, supporting a role in enhancer activation and defining these specific non-coding RNAs as enhancer RNAs.  Acquisition of H3K27ac at the enhancer site was concordant with induction of the non-coding RNA and preceded changes in H3K4me3 at the promoter.  LPS induced not only the enhancer RNA but also induced expression of the adjacent mRNA.  To investigate the role of the p38 pathway, we examined AP-1, a downstream regulator of p38 activity.  We tested for loading onto the enhancer and promoter regions by ChIP assay for c-jun, one of common group of proteins incorporated into the dimer of AP-1. Both at the IL1 and ADAM28 loci, c-jun was loaded onto the enhancer without increased loading at the promoter at early time points.  Therefore, LPS induced c-jun loading onto the enhancer with concordant changes in enhancer RNA and acquisition of H3K27ac at the enhancer suggesting that this pathway activated the enhancers.  To investigate the function of the enhancer RNAs specifically, we manipulated the amount of expression.  Knockdown of one enhancer RNA within the IL1 cluster, led to diminished levels of multiple enhancer RNAs at that locus and diminished expression of IL1.  Therefore, the enhancer RNA performs a critical function for gene expression. 

Conclusion: These data define a set of disease-specific enhancers that appear to be dysregulated in SLE.  Impressive over-expression of these enhancer RNAs suggests that the regulatory network is highly dysregulated in SLE. Enhancers have not been previously examined in SLE and identification of novel disease-specific enhancers is of critical importance in considering new epigenetically-directed therapeutics. This study identifies a specific pathway that activates these normally latent enhancer RNAs in SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/epigenetic-changes-in-sle-implicate-enhancers-as-a-force-in-pathologic-cell-behavior/