Background/Purpose: Although the majority of SSc research has focused on differential gene expression, recent studies havedemonstrated that non-coding epigenetic changes in chromatin accessibility are likely play a key role in SSc biology. In this study we developed a system for analyzing differentially accessible regulatory regions in SSc-derived cell lines using Assay for Transposase Accessible Chromatin with sequencing (ATAC-seq) in a novel 3D in vitro skin-like tissue model.

Methods: SSc and healthy control (HC) dermal fibroblast (FB) lines were isolated and expanded from skin biopsies. All patients met 2013 ACR/EULAR criteria for SSc.FBs were seeded in transwell chambers and cultured for 5 weeks to obtain self-assembled stromal (SAS) tissues in a simple 3D tissue model that recapitulates SSc biology. ATAC-seq was performed on both monolayer/2D fibroblast cultures and 3D tissues following tissue dissociation. ATAC-seq libraries were sequenced. Paired end reads were aligned to the hg19 reference genome and 300bp peaks were called in ZINBA. Data underwent quality control analyses and unsupervised hierarchal clustering. Genomic location of peaks was determined using ChIPseeker. The integrative genomics viewer (IGV) was used to analyze the genomic context of specific differentially accessible regions.

Results: ATAC-seq libraries contained a large number of reads (~30 million) and had comparable accessibility profiles to standard data sets (Fig. 1A-D). SSc FBs contained significantly more open chromatin (Fig. 1E) and the percentage of peaks within distal/intergenic regions was significantly higher than in HC FBs (Fig. 1F). In unsupervised hierarchal clustering of the top 50 differential 300bp peaks in both 2D and 3D tissues, SSc and HC tissues clustered by disease state. A differentially accessible region of significant interest was identified in both 2D and 3D individual heatmaps as well as a combined 2D/3D heatmap in which it was the only genomic peak which distinguished between SSc and HC samples (Fig. 1G). This region falls within an intron of a gene on chromosome 8 and contains a putative enhancer predicted to contain a binding site for the transcription factor STAT3.

Conclusion:

We were able to produce high quality data from both 2D and 3D cultures. Preliminary analysis of ATAC-seq data demonstrates that SSc fibroblasts maintain a distinct chromatin accessibility profile as compared to HC fibroblasts, characterized by increased global accessibility. The majority of these differentially accessibility peaks reside in genomic regions commonly associated with regulatory elements. Additionally, we were able to identify a region of significant interest containing a putative enhancer region and predicted binding site for STAT3, a transcription factor known to be dysregulated in SSc.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-of-differential-chromatin-accessibility-using-atac-seq-in-a-novel-3d-tissue-culture-system-of-systemic-sclerosis/