The Chromatin Accessibility Landscape of Nonalcoholic Fatty Liver Disease Progression

Abstract

The advent of the assay for transposase-accessible chromatin using sequencing (ATAC-seq) has shown great potential as a leading method for analyzing the genome-wide profiling of chromatin accessibility. A comprehensive reference to the ATAC-seq dataset for disease progression is important for understanding the regulatory specificity caused by genetic or epigenetic changes. In this study, we present a genome-wide chromatin accessibility profile of 44 liver samples spanning the full histological spectrum of nonalcoholic fatty liver disease (NAFLD). We analyzed the ATAC-seq signal enrichment, fragment size distribution, and correlation coefficients according to the histological severity of NAFLD (healthy control vs steatosis vs fibrotic nonalcoholic steatohepatitis), demonstrating the high quality of the dataset. Consequently, 112,303 merged regions (genomic regions containing one or multiple overlapping peak regions) were identified. Additionally, we found differentially accessible regions (DARs) and performed transcription factor binding motif enrichment analysis and de novo motif analysis to determine new biomarker candidates. These data revealed the generegulatory interactions and noncoding factors that can affect NAFLD progression. In summary, our study provides a valuable resource for the human epigenome by applying an advanced approach to facilitate diagnosis and treatment by understanding the non-coding genome of NAFLD.

Keywords: ATAC-sequencing; biomarker; chromatin accessi­bility; epigenome analysis; nonalcoholic fatty liver disease.

Fig. 1. Chromatin accessibility landscape of NAFLD progression.

(A) The workflow overview of the experiment. Forty-four liver samples obtained from the NAFLD study cohort were collected for ATAC-seq profiling. (B) The ATAC-seq signal enrichment around the transcription start sites (promoters), gene bodies and merged peak regions for 3 representative samples (N4, S5, F17). Tag distributions across target regions such as promoters (transcription start sites), gene bodies, or merged peak regions were determined and presented as heatmaps (values in z-axis/color, regions in y-axis). The data were clustered (5 clusters; indicated by C1-C5) and sorted for the heatmaps. (C) The average plot of ATAC-seq profiles for the 14 representative samples (N4, S3, S5, S9, F8, F12-14, F16-21). Tag distributions across target regions such as promoters, gene bodies, or merged peak regions were determined and presented as average plots (average of values for all target regions). (D) The insert size distribution of ATAC-seq profiles for the same samples shown in Fig. 1B.

Fig. 2. Evaluation of the ATAC-seq data sets.

(A) Heatmap clustering of correlation coefficients across 14 ATAC-seq profiles. This heatmap shows the Pearson Correlation coefficients of all pairwise comparisons. (B) Principal component analysis (PCA) results of 14 samples (>20 FRiP). Each point represents an ATAC-seq sample. Samples with similar chromatin accessibility profiles are clustered together. (C) Peak correlation scatterplot. For each pairwise comparison, scatter plots were generated plotting the tag numbers of sample S9 against S3, F19 against F20 for each merged region. In addition, the slope is a measure for the average ratio in tag numbers between the two samples. (D) Genome browser views of ATAC-seq signal for the indicated fibrosis signature genes (Col1a1, Acta2).

Fig. 3. Identification of DARs associated with NAFLD progression.

(A) Number of DARs (up-/down-regulated regions) when comparing disease stages one another. Threshold: FDR < 0.05; FDR: false discovery rate (adjusted P value, Benjamini-Hochberg procedure). (B) Heatmap of ATAC-seq signal around DARs when comparing fibrotic NASH to steatosis. (C) Average profile of ATAC-seq signal around DARs when comparing fibrotic NASH to steatosis. (D) Functional enrichment analysis. Fourteen regions (1.2%) are not associated with any genes. GO Biological Process (down-regulated in fNASH: 1,137 regions) was shown.

Fig. 4. Transcription factor binding motif analysis and de novo motif analysis.

(A) Enrichment analysis of JASPAR transcription factor (TF) binding site. The top 10 TF were indicated. (B) Top 1 motif/4 motifs from de novo motif analysis at the 272/1,137 differentially accessible chromatin regions which are more open/close in fibrotic NASH when compared to steatosis.