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Epigenetic and genetic changes during mouse hematopoietic stem cell aging [Bisulfite-Seq]

ABSTRACT: To investigate the global DNA methylation changes in mouse hematopoietic stem cell aging, we performed whole-genome bisulfite sequencing (WGBS). We generated 1,494 million (4mo HSCs), and 1,493 million (24mo HSCs) raw reads; about 82.6% and 84.3%, respectively, were successfully aligned to either strand of the reference genome (mm9). Of all the cytosines present in the reference genome sequence, about 93% of Cs and 99% of CGs were covered in both datasets, with an average coverage of 46-fold (4mo) and 50-fold (24mo). In contrast to the age-associated hypomethylation observed in studies of somatic cells, mentioned above, HSCs showed an increase of methylation with age. The average methylation level over all 16 million covered CpGs increased from 83.5% in young (4mo) HSC to 84.6% in old (24mo) HSC. We observed a total of 448,166 differentially methylated CpGs (DMCs), defined as those having a 20% or more difference in methylation ratio, of which 38.5% (172,609) were hypomethylated (hypo-DMCs) and 61.5% (275,557) were hypermethylated (hyper-DMCs) with aging. For different genomic features, a slightly greater DNA methylation ratio increase was observed for the gene body, LINEs and SINEs, while CGIs and promoters showed balanced increases and decreases. Localization analysis of DMCs indicates that DNA encoding for ribosome RNA (rDNA) is primarily a hotspot for hypo-DMCs, while promoters without CpG islands, CpG island shores and LINE repetitive elements exhibit both hypo- and hyper-DMCs.

ORGANISM(S): Mus musculus

PROVIDER: GSE47815 | GEO | 2014/04/30

SECONDARY ACCESSION(S): PRJNA208011

REPOSITORIES: GEO

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Project description:Since DNMT3A is a de novo DNA methyltransferase, transcriptional differences in WT versus Dnmt3a-/- HSCs upon infection suggest a possible role for epigenetic regulation in the HSC transcriptional response to inflammation. Therefore, we wanted to investigate if differences in stress and presence of Dnmt3a affect the methylome overall, and specific IFNg responses genes. 100000 LT-HSCs (LK CD150+ CD48- were sorted into lysis buffer from the pools of naive or 1-month (M. avium) infected WT/ Dnmt3-/-_x0001_ mice (n = 7-8 per group). Around 300 ng of DNA per group was extracted with AllPrep DNA/RNA Mini Kit. NEBNext Ultra II DNA library prep kit was used for library preparation. Bisulfite conversion step was added after ‘‘methylated’’ adaptor ligation and USER excision. Then, methylated adaptor ligated DNA fragment was treated with bisulfite conversion using EZ DNA methylationlightning kit. These modified DNA fragments were then amplified with index primers with Kapa HiFi urasil+ specialized polymerase for bisulfite converted DNA. For WGBS library sequencing, 150-high output kit from illumine was used. Samples (400 million reads/ sample (15-20X coverage)) were run in NovaSeq S1 FC (2, lanes 1,600 Million reads). Global methylation of Dnmt3a-/- cells was more divergent upon infection compared to WT. There were vastly more hyper- and hypomethylated regions in infected versus naive Dnmt3a-/- HSCs compared to WT. Among the altered regions, both WT and Dnmt3a-/- HSCs showed slightly more hypomethylated than hypermethylated regions, although changes in both directions were present. In the WT background, regions of hyper- and hypomethylation were mostly restricted to CpG islands. By contrast, highly significant differences in hypomethylation were found in the Dnmt3a-/- HSCs in CpG islands, shores, enhancers, and promoters DNMT3A is highly active in HSCs during infection and that the loss of DNMT3A function results in significant global changes in methylation, including both hyper- and hypomethylation, in genome regions that likely affect gene transcription. we examined the methylation of Batf2, Jun, and Fos. All 3 of these prodifferentiation genes showed increased methylation in the promoter region in the Dnmt3a-/- background as compared to the WT background. Hypermethylation of these promoter regions is consistent with their transcriptional repression.

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Epigenetic and genetic changes during mouse hematopoietic stem cell aging [Bisulfite-Seq]

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