Epigenetic and transcriptomic profiling of mouse neural stem cells

Neural stem cells (NSCs) in the hippocampus of mammalian brains decline rapidly and persistently with age, eventually leading to impairment of hippocampal memory function in later life. However, the mutual relationship between epigenetic remodeling and transcriptional regulation that initially compromises hippocampal NSC activity during the early stage of chronological aging has not been well elucidated. We here performed single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) for NSCs and newly generated neurons in a chronological manner. Integrated analysis using these datasets revealed that the chromatin profile of hippocampal NSCs and their progeny undergoes continuous alterations from neonatal to mature adult stages, accompanied by considerable changes in transcriptional regulation. We further show that a decrease in the expression of Setd8, encoding the only known enzyme that catalyzes histone H4 monomethylation at lysine 20 (H4K20me1), underlies age-related changes in the characteristics of mouse NSCs in the hippocampus: the downregulation of Setd8 expression elicits age-related changes in gene expression and epigenetic regulation, and impairs NSC activity, leading to functional deficiency in hippocampal memory. Thus, our study identifies a mechanism for an unexpectedly early-onset decline of NSC activity and hippocampal neurogenesis that precedes aging. Overall design: We used 50,000 cultured NSCs to construct a library for each sample. Samples collected from cultured mouse NSCs in Setd8 knock down after selection with puromycin were subjected to RNA-seq, ChIP-seq (H4K20me1) and ATAC-seq. For scRNA-seq and scATAC-seq, the cells were sorted on a FACS Aria II (BD Biosciences), with 7-AAD (559925, BD Pharmingen) added to exclude nonviable cells from the analysis. Cells dissociated from the DG of WT mice were used to draw gates for EGFP+ and EGFP- cell populations. The EGFP+ cells were sorted into N2 medium and centrifuged at 200 x g at 4C for 10 min. The supernatant was carefully removed, leaving 30 uL to resuspend the cell pellet. One microliter of cells was mixed with 10uL Trypan Blue to determine cell viability. For scRNA-seq and scATAC-seq analyses, 700-1200 cells/uL were used.