Page 65 - Haematologica April 2020

RUNX3 in aging and erythroid-myeloid balance
the signals for Runx3 expression came from multipotent and early committed erythroid progenitors rather than contaminant lymphocytes. Discrimination of HSC versus MPP compartments is not possible by this approach (Online Supplementary Figure S1C).
Because epigenetic changes occur with HSC aging and
participate in regulation of RUNX3,25-27 we investigated the effect of aging on DNA and histone modifications within the murine and human loci. Analysis of comprehensive DNA methylation mapping by whole genome bisulfite sequencing3 (GSE47819) revealed significant increases in P2 promoter methylation in aged murine HSC (Figure 1C
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C
DE
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Figure 1. Hematopoietic stem and progenitor cells (HSPC) and SPC RUNX3 levels decline with aging. (A) Human RUNX3 mRNA levels in Lin–CD34+CD38– bone mar- row (BM) HSPC obtained from healthy young and aged individuals. N=10 per group. (B) Mouse Runx3 mRNA levels in side population, Lin–Sca1+Kit+CD150+ BM HSC obtained from healthy young and aged animals (GSE478193). N=4 per group. (C and D) Tracks for DNA methylation by whole genome bisulfite sequencing (WGBS, red) and RNA-seq read counts (green) within the mouse Runx3 locus in HSC from healthy young and aged animals (GSE478193). The blue box highlights the DNA methylation trough associated with the P2 promoter. (D) Mean methylation density (% of CpG reads with methylation) within trough. N=5-6 per group. (E-G) Histone H3K27 acetylation by chromatin immunoprecipitation (ChIP)-sequencing within the human locus in HSPC from healthy young and aged individuals. Gray shading highlights peaks within the P2 promoter and upstream super-enhancer. (F) H3K27ac score for the P2 promoter peak, and (G) the sum of peak scores across the enhancer. N=4-5 per group. All statistics two-tailed Student t-test, except in F: log likelihood ratio (LLR). Error bars+standard error of mean. FPKM: fragments per kilobase of transcript per million.
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