Identification of aging-related cell-type-specific chromosomal accessibility changes
After quality control, a total of 74,102 cells (33,004 YA, 41,098 AA) were used to generate a PBMC chromatin-accessibility map. MEGAs, TCs, NKs, BCs and myeloid cells were identified based on the promoter sum of genes specifically upregulated in each cluster. After separately reclustering each lineage population, we identified 3 distinct subsets in CD4+ TCs, 3 distinct subsets in CD8+ TCs, 3 distinct subsets in NKs, 3 distinct subsets in BCs, 3 distinct subsets in DCs and 2 distinct subsets in MCs according to gene peaks and transcription factor (TF) activity using chromVAR (Satpathy et al., 2019) (Fig. 4A, 4B, and S8A–D, see Table S 3C for the details). Consistent with the scRNA-seq and CyTOF data, we observed a decrease in naive TCs and an increase in MCs in the elderly (Fig. 4C).
Figure 4 Changes in chromosomal accessibility during aging. (A) Heatmaps showing scaled expression of discriminative gene sets for each cell type and cell subset. Color scheme is based on z-score distribution from −1.5 (purple) to 1.5 (yellow). (B) t-SNE projections of PBMCs derived from scATAC-seq data. (C) t-SNE plots segregated by YA and AA groups. (D) UpSet plot showing the integrated comparative analysis of upregulated differentially expressed transcription factors (DETs) in major immune cell populations between YA and AA groups. Upregulated DETs: upregulated in AA, downregulated in YA. The count showing the number of DETs. (E) UpSet plot showing the integrative comparative analysis of downregulated DETs in major immune cell populations between YA and AA groups. Downregulated DETs: upregulated in YA, downregulated in AA. The count showing the number of DETs. (F) Venn diagram showing integrated comparative analysis of upregulated DETs in CD4+ T cells between YA and AA groups. Upregulated DETs: upregulated in AA, downregulated in YA. The count showing the number of DETs. (G) Venn diagram showing integrated comparative analysis of upregulated DETs in CD8+ T cells between YA and AA groups. Upregulated DETs: upregulated in AA, downregulated in YA. The count showing the number of DETs. (H) Venn diagram showing integrated comparative analysis of upregulated DETs in NK cells between YA and AA groups. Upregulated DETs: upregulated in AA, downregulated in YA. The count showing the number of DETs. (I) Venn diagram showing integrated comparative analysis of upregulated DETs in B cells (top) and monocytes (bottom) between YA and AA groups. Upregulated DETs: upregulated in AA, downregulated in YA. The count showing the number of DETs. (J) Mean scATAC-seq coverage at FOSL2 loci in CD8+ T cells. (K) Mean scATAC-seq coverage at NFATC2 loci in CD8+ T cells. (L) Mean scATAC-seq coverage at CDKN2B loci in B cells. (M) Mean scATAC-seq coverage at SIRT7 loci in NK1 cells. (N) Mean scATAC-seq coverage at GLI2 loci in CD4 Naive cells. (O) Mean scATAC-seq coverage at IFNG loci in CD8 Naive cells. (P) Mean scATAC-seq coverage at DUSP5 loci in CD8 Memory cells. (Q) Mean scATAC-seq coverage at PDCD1 loci in NK3 cells
Next, we focused on the differentially expressed transcription factors (DETs) in immune cells in the AA group compared to the YA group. At the TF level, MCs were the most affected by aging based on the numbers of upregulated and downregulated DETs (Fig. 4D and 4E). To identify aging-associated TF events, we performed an integrated comparative analysis of these DETs and found that AP-1 family TFs, including FOSL2 and JUNB, were increased in all immune cells during aging (Fig. 4D and 4E). Upregulation of AP-1 family TFs, including FOS, FOSB, FOSL1, FOSL2, JUN, JUNB, and JUND, was also observed in almost all cell subsets during aging (Fig. 4F and 4I). The AP-1 family regulates a wide range of cellular processes, including cell proliferation, death, survival, and differentiation. The effects of the activated AP-1 TFs, associated with the active inflammatory state, are primarily mediated through combinatorial regulation with the NFAT family, both of which are key regulators of TC activation and are enriched in TCs (Fig. 4D) (Shaulian and Karin, 2002). In addition, we visualized the chromosomal accessibility of FOSL2 loci and NFATC2 loci and found that the chromosomal accessibility of the FOSL2 and NFATC2 gene regions was also increased in aged TCs (Fig. 4J and 4K). CDKN2B, an aging hallmark gene, also showed an increase in accessibility with age (Fig. 4L). In parallel, we found 25 common decreased TFs, including nuclear respiratory factor 1 (NRF1) and ELK4, which are involved in antioxidant stress and negatively regulate cell differentiation and proliferation (Figs. 4E and S8E–I). Consistently, we found that chromatin accessibility also decreased at the loci of SIRT7 (Fig. 4M), which coordinates with NRF1 to maintain cellular energy metabolism and proliferation (Mohrin et al., 2015).
In TCs, a series of subset-specific TF changes were observed, such as GLI2 in naive cells, which has been associated with decreased TC function and impaired immune defenses (Fig. 4F and 4G). Consistently, increased chromatin accessibility was detected in GLI2 loci (Fig. 4N). Analysis of differentially accessible regions (DARs) demonstrated that the IFNG, DUSP5, and GZMB loci were highly accessible, which indicated activated CD8+ TC states (Figs. 4O, 4P and S8J). In our analysis of NK status, we identified the key TF changes in NK subsets during aging (Fig. 4H), and found that the chromatin accessibility of the inhibitory receptor gene increased, while that of the activating receptor decreased. These changes may weaken the ability to clear virus-infected cells. For example, the PDCD1 exhibited higher chromatin accessibility in the gene region of the elderly group, which might be part of the reason why older individuals were prone to infection (Fig. 4Q). In our analysis of BCs, we identified aging-related TF changes, such as TBX21, IRF4, which are consistent with our scRNA-seq results (Fig. 4I). Aging-associated TFs and DARs in MCs demonstrated enrichment in inflammatory-related TFs and gene loci in the AA group, such as NF-κB family (REL, RELA), IL1B, TNF and CXCL8 (Figs. 4I and S8K–M). In summary, aging-related chromosomal accessibility changes are associated with an increase in the inflammatory pathway and an impaired immune response.