The chromatin accessibility and transcriptomic landscape of the aging mice cochlea and the identification of potential functional super-enhancers in age-related hearing loss

Abstract

Background: Presbycusis, also referred to as age-related hearing loss (ARHL), is a condition that results from the cumulative effects of aging on an individual's auditory capabilities. Given the limited understanding of epigenetic mechanisms in ARHL, our research focuses on alterations in chromatin-accessible regions.

Methods: We employed assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) in conjunction with unique identifier (UID) mRNA-seq between young and aging cochleae, and conducted integrated analysis as well as motif/TF-gene prediction. Additionally, the essential role of super-enhancers (SEs) in the development of ARHL was identified by comparative analysis to previous research. Meanwhile, an ARHL mouse model and an aging mimic hair cell (HC) model were established with a comprehensive identification of senescence phenotypes to access the role of SEs in ARHL progression.

Results: The control cochlear tissue exhibited greater chromatin accessibility than cochlear tissue affected by ARHL. Furthermore, the levels of histone 3 lysine 27 acetylation were significantly depressed in both aging cochlea and aging mimic HEI-OC1 cells, highlighting the essential role of SEs in the development of ARHL. The potential senescence-associated super-enhancers (SASEs) of ARHL were identified, most of which exhibited decreased chromatin accessibility. The majority of genes related to the SASEs showed obvious decreases in mRNA expression level in aging HCs and was noticeably altered following treatment with JQ1 (a commonly used SE inhibitor).

Conclusion: The chromatin accessibility in control cochlear tissue was higher than that in cochlear tissue affected by ARHL. Potential SEs involved in ARHL were identified, which might provide a basis for future therapeutics targeting SASEs related to ARHL.

Keywords: Age-related hearing loss; Chromatin accessibility; Epigenetics; Senescence; Super-enhancer.

Fig. 1

Histological characterization changes in aging cochleae and the identification of an age-related hearing loss mouse model. A ABR thresholds were observed in 6 W (n = 12) and 12 M (n = 17) C57BL/6 J mice at 4, 8, 16, 24, 32 kHz and click. B Young adult and old aged C57BL/6 J cochlear representative cross sections stained with H&E showed atrophy of SV with age in all three turns. Section thickness is 5 μm. C The quantified analysis of SV in three turns, which showed the most SV atrophy appear in basal turns. D, E, F The difference in HCs counts of young adult and old mice at same locations was calculated, and dramatic loss of HCs in cochlear basal turn was observed. (n = 3). G, H Apoptotic loss of SGCs was detected in two groups and higher TUNEL fluorescence (green color) ratio of SGCs area was detected in 12 M mice than in 6 W mice between the same turn. Blue shows DAPI staining of the nucleus. Scale bar = 50 μm. I, J Western blot analyses of laminb1 and H3K4me3 in the cochleae from 6 W (n = 6) and12 M (n = 6) mice. The statistical significance was represented as * p < 0.05; **p < 0.01; ***p < 0.001;****p < 0.0001. Bar graph results are means ± SD from 3 independent experiments

Fig. 2

Genome-wide accessible chromatin profiling of cochleae of ARHL and normal mice. A Enrichment peaks around the transcription start sites (TSSs), and comparison of the average sequencing depth in TSS of two groups. (X: the distance from the site to the TSS, Y: the average sequencing depth of the site). B Genome-wide chromatin accessibility of control and aging cochlea. C Volcano plot of differential analysis to the peaks during the aging process of cochlea, which consist of 34,269 increased and 59,511 decreased peaks. D The distribution plot of the fragments length revealed that the fragments detected in aging cochleae were shorter overall than in control cochleae. E Genomic annotation of identified different ATAC-seq peaks. F The enrichment heatmap of nucleosome binding sites revealed a looser or more relaxed chromatin structure in the control cochleae. G The geneplot of Hmgb1 in different bioreplicated samples (the arrow indicates the transcription start site and direction, the green region represents the exonic region, and the higher the peak, the stronger the accessibility of that region)

Fig. 3

Motif enrichment analysis and motif-TF-gene prediction associated with ARHL progression. A The top 10 significantly enriched TF binding motifs by decreased peaks from young to aging according to the enrichment p-values. The corresponding binding motif of TFs is shown. B Aggregated footprint plots display the top 10 TFs with reduced footprints, showing an overall footprint for all related transcription factor binding sites for each TF, with individual plots centered on binding motifs. C TF-gene regulatory network involved in ARHL progression extracted from HOMER data based on TOP 10 motifs. D The top 10 enriched GO terms by the genes regulated by predicted TFs indicated that the predicted TF-regulated genes were significantly enriched in different developmental processes

Fig. 4

Genome-wide gene transcriptional difference analysis between the young and aging cochleae and TFs prediction related to ARHL. A Heatmap analysis and hierarchical clustering on transcriptome samples using normalized read counts (TPM values). Each row represents a gene, and the TPM values were Z-scaled by row. The scale bar indicates the Z-scaled TPM values. B The scatter plot of differential expression genes. Gray dots represent genes that are not differentially expressed, blue dots represent genes that are differentially downregulated, and red dots represent genes that are differentially upregulated. C GSEA enrichment analysis of transcriptome with high enrichment scores. D Differential binding TFs prediction using the transcriptomic data. E The footprint of top 5 TFs according to the |logFC| was all significantly decreased from young to aged mice genome-wide

Fig. 5

Integrated analysis of chromatin accessibility and gene expression during ARHL development. A Venn diagram showing the number of genes in different ATAC and mRNA state, providing a more reliable analysis of the regulatory mechanisms underlying age-related changes in gene expression. B GO enrichment analysis of differentially expressed genes with different peaks. C KEGG analysis of differentially expressed genes with different peaks. D Gene element-based correlation analyses between peak intensity and gene expression, suggesting the importance of chromatin accessibility in the promoter and 5’UTR regions for proximal regulation. R is Pearson’s correlation coefficient and P is the significance test for the correlation coefficient. E The number of genes containing different numbers of peaks in the promoter regions in two groups. Most genes in both groups have 1–3 peaks in their promoter regions. F Comparing expression distribution of up or downregulated genes based on whether they contain promoter peaks or not

Fig. 6

Identification of SASEs and prediction of their target genes in mice with ARHL. A, B Western blot analyses of H3K9ac, H3K18ac, and H3K27ac in the cochleae of 6 W and12 M mice. The statistical significance was represented as * p < 0.05; **p < 0.01; ***p < 0.001;****p < 0.0001. Bar graph results are means ± SD from 6 independent experiments. C The distribution of SASEs region peaks around the transcription start sites and average ATAC-seq signal density in the young state and aging state of cochlea surrounding the SASE regions. D Geneplots of identified SASEs. The position of different peaks was labeled with green cubes and peak numbers. E Geneplots of SASE-linked genes. The position of different peaks and gene exons was marked with green cubes. The thick green line represents the location of SASE

Fig. 7

The expression level changes of SASE genes in D-gal-induced aging and JQ-1-treated HEI-OC1 cells. A The CCK8 results showed that the inhibitory rate of HEI-OC1 cells rises as the increased D-gal concentration (n = 6). B The ROS level increased after the HEI-OC1 treated by D-gal (n = 4). C The mRNA level of aging hallmarks in the control and D-gal treated cells (n = 3). D The protein level of p16, p21 increased as the D-gal concentration increased. E The differential expressed genes with different chromatin accessibility state. F The mRNA level changes of the top genes in different four quadrants between normal and mimic-aging cells (n ≥ 4). F The levels of H3K27ac decreased with increasing D-gal concentration. H The mRNA level changes of the SASE-linked genes in D-gal-induced aging cells (n ≥ 3). I The mRNA level changes of the SASE-linked genes after treated by JQ-1(1 μm) (n ≥ 3). J Schematic diagram of possible mechanisms underlying positive role of identified SASEs in ARHL (drawn by Figdraw). During the aging process of the cochlea, the positive regulation of candidate SASE gene expression is inhibited due to decreased chromatin accessibility, and inhibition of SE activity caused by JQ-1 could lead to similar effects. The statistical significance was represented as * p < 0.05; **p < 0.01; ***p < 0.001;****p < 0.0001