Age-like Methylation Changes of HSCs in Gadd45b knock-out mice

Age-related functional decline of hematopoietic stem cells (HSC) contributes to the decline of the hematopoietic system. Growth Arrest and DNA Damage-inducible proteins (Gadd45a, Gadd45b, and Gadd45g) are robustly expressed early in HSC activation and are known to be important for DNA damage response. Gadd45b has also been shown to promote removal of 5-methylcytosine at specific loci. Given that both DNA methylation (DNAm) alterations and DNA damage have been associated with HSC aging, we sought to examine the role of Gadd45b in HSCs. To examine if Gadd45b is critical for regulating DNAm in HSCs, we performed whole genome bisulfite sequence and RNA sequencing on wild-type (WT) and Gadd45b knock-out (KO) HSCs isolated from young and old mice. Global DNAm of young KO showed increased DNAm compared to young WT, and the DNAm profiles were more similar to old HSCs, from either WT and KO, than to young WT. We examined the DNAm changes common between the Gadd45b KO (YKOvsYWT) or WT aging (OWTvsYWT). 52.5% of differentially methylated cytosines (DMCs) in YKO were also seen in normal aging, and analysis of genes with promoters near these overlapping DMCs are enriched for pathways related to immune response and development processes. We compared the expression of genes with DMCs in the promoter regions, but DMCs did not strongly correspond with transcriptional alterations. To test if these overlapping DNAm changes in young KO mice primed HSCs towards aging phenotypes, we evaluated the composition of peripheral blood and bone marrow in KO, and performed functional evaluation experiments. We found no significant differences between young KO and WT HSCs suggesting these overlapping DMCs do not contribute to aging phenotypes, reducing the potential DNAm changes contributing to aging HSC phenotypes.