Background/Purpose:

Systemic Sclerosis (SSc) is an etiologically mysterious disease, in which adults acquire an inflammatory prodrome with progressive fibrosis of the skin and viscera. In the absence of a strong genetic signature in twin studies (monozygous=dizygous=4% concordance) or genome-wide association studies (only indicative of inflammatory predisposition), it remains unclear as to how dermal fibroblasts from SSc patients maintain a fibrotic synthetic repertoire (FSR) when cultured ex vivo. We posited epigenetic regulation of gene expression as a mechanism that “locks in” the aberrant performance of SSc fibroblasts.

Methods:

Transcriptomes and epigenomes were profiled by RNA-seq and Assay for Transposase-Accessible Chromatin (ATAC)-seq, respectively, using primary dermal fibroblasts (PDF) established from skin biopsies of healthy controls or SSc patients with diffuse cutaneous involvement. PDFs were further evaluated by targeted epigenomic editing, chromatin immunoprecipitation, protein analysis, and pharmacological strategies. Organ cultures of SSc patient skins were performed to assess therapeutic agents in vivo.

Results:

RNA-seq revealed that SSc PDFs maintain a strong FSR after many passages in culture (e.g. COL1A1, SERPINH1). This correlated with specific upregulation of TGFβ2 (but not β1 or β3) mRNA and protein expression that was prone to further amplification by TGFβ treatment. siRNA knockdown of TGFβ2 silenced the FSR in SSc PDFs. Together, these data suggest an epigenetic mechanism to “lock in” the FSR in SSc, with particular relevance for TGFβ2. ATAC-seq revealed an open chromatin conformation for a sequence-constrained region distal to the TGFB2 gene in SSc PDFs, with direct correlation between accessibility and TGFβ2 mRNA levels. This element showed a signature for activated enhancers: acetylated H3K27 and occupancy by the histone acetyltransferase (HAT) EP300. CRISPR-based targeted histone acetylation to this enhancer induced TGFβ2 expression, and consequentially TGFβ target gene expression (COL1A1, SERPINH1), in control and SSc PDFs, validating functional enhancer status. In keeping with this result, targeted histone methylation to the TGFB2 enhancer normalized TGFβ2 expression and the FSR in SSc PDFs. Treatment of SSc PDFs with a HAT inhibitor (HATi) was sufficient to deactivate TGFB2 enhancer activity, but full epigenetic activation of the enhancer rebounded after drug removal. We posited that this epigenetic memory—analogous to regulation of inflammatory enhancers—might be initiated by inflammatory effectors (e.g. NF-kB) and enforced by BRD4 recruitment. In support of this hypothesis, we found high NF-kB and BRD4 occupancy at the TGFβ2 enhancer in SSc PDFs. Treatment with the NF-kB or BRD4 inhibitor normalized TGFβ2 expression and the FSR, which was now refractory to drug removal. Finally, SSc skin maintained a FSR and dense fibrosis in organ culture. Both parameters were strikingly reversed upon incubation with BRD4 inhibitor for ten days.

Conclusion:

These data suggest an epigenetic mechanism for fibrosis in SSc pathogenesis, identify therapeutic targets and biomarkers for use in clinical trials, and inform a new regulatory mechanism of TGFB2.

« Back to 2018 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/pathogenic-and-therapeutic-modulation-of-activating-epigenetic-memory-at-a-novel-enhancer-for-tgf%ce%b22-in-systemic-sclerosis/