HSC Aging Epigenome: Widespread Alterations in DNA Methylation and Transcription.

Abstract 2329

Age is the most important risk factor for myelodysplastic syndrome (MDS), a premalignant state that transforms into acute myelogenous leukemia in one third of cases. Indeed with normal aging, hematopoietic stem cell (HSC) regenerative potential diminishes and differentiation skews from lymphopoiesis toward myelopoiesis. The expansion in the HSC pool with aging provides sufficient but abnormal blood production, and animals experience a decline in immune function. Previous studies from our lab established that the DNA methyltransferase 3a (Dnmt3a) enables efficient differentiation by critically regulating epigenetic silencing of HSC genes (Challen et al. 2012) Interestingly, Dnmt3a expression is decreased in old HSCs, leading us to hypothesize that epigenetic changes in old HSCs may partially mimic the changes seen in Dnmt3a mutant HSCs. We propose that revealing the genome-wide DNA methylation and transcriptome signatures will lead to a greater understanding of HSC aging and MDS, which is characterized by frequent epigenetic abnormalities.

In this study, we investigated genome-wide DNA methylation and transcripts by whole genome bisulfite sequencing (WGBS) and transcriptome sequencing (mRNA-seq)in young and old HSCs. For WGBS, we generated ∼600M raw reads resulting in ∼ 60 raw Gb of paired-end sequence data and aligned them to either strand of the reference genome (mm9), providing an average 40-fold sequencing depth. Globally, there was a 1.1% difference in the DNA methylation between young and old HSCs. Of these differences, 38% (172,609) of the CpG dinucleotides were hypo-methylated, and 62% (275,557) were hyper-methylated in old HSCs. To understand where the methylation changes predominantly occurred, the genome was subdivided into 77 features. Among these features, SINEs, especially Alu elements, exhibited the highest level of DNA methylation (90.94% in young HSCs, and 91.87% in old HSCs). CpG islands (CGIs) adjacent to the transcription start sites (TSS) exhibited the lowest level of DNA methylation (2.02% in young HSCs, and 2.11% in old HSCs). Interestingly strong hypo-methylation was observed in ribosomal RNA regions (68.04% in young HSCs, 59.04% in old HSCs), and hyper-methylation was observed in LINEL1 repetitive elements (88.62% in young HSCs, 90.12% in old HSCs). Moreover, the examination of differentially methylated promoters identified enrichment of developmentally important transcription factors such as Gata2, Runx1, Gfi1b, Erg, Tal1 Eto2, Cebpa and Pu.1. Additionally, we compare our ∼10,000 differentially methylation regions (DMRs, regions with clustered DNA methylation changes) with a chip-seq data set containing binding of 160 ChIP-seq analyses of hematopoietic transcription factors in different hematopoietic cells. We found significant overlaps between DMRs and transcription factor binding regions. We found DMRs which were hypermethylated showed association with differentiation-promoting Ets factors, in particular Pu.1 from a range of different blood cell types. In contrast, hypomethylated DMRs showed associations with HSC-associated transcription factors such as Scl and Gata2. Further examination of the differentially methylated gene bodies, intragenic and intergenic DMRs identified some previously noted targets for epigenetic silencing or alteration in AML and also novel transcripts including long non-coding RNAs (lincRNA) and upstream regulatory elements (URE). We found significant correlation between RNA-seq expression and DMRs within +1kb upstream of TSS. RNA-sequencing provided complementary and distinct information about HSC aging. We identified differentially expressed genes, novel RNA transcripts, differential promoter, coding sequence, and splice variant usage with age. Gene set enrichment analysis of up- and down- regulated genes, revealed ribosomal protein and RNA metabolism as critical contributors to HSC aging.

In conclusion, our study marks a milestone in the mouse HSC epigenome, reporting the first complete methylome and transcriptome of pure HSC using whole-genome bisulfite sequencing and RNA-seq. These provide novel information about the magnitude and specificity of age-related epigenetic changes in a well-defined HSC population. Understanding the roles of DNA methylation and transcription in normal HSC function will allow for greater therapeutic exploitation of HSCs in the clinic.