Aging Human Hematopoietic Stem Cells Manifest Massive Epigenetic Reprogramming and Altered Gene Splicing of Key Hematopoietic Gene Sets

Aging leads to impairment of hematopoietic stem cell (HSC) function with decreased self-renewal, imbalanced differentiation potential and an increased risk to develop myeloid malignancies. These malignancies are associated with epigenetic deregulation, which contributes to pathogenesis. Notably, studies in murine models have revealed epigenetic changes in aged HSC. However, it is unknown if this occurs in normal human HSC aging and whether it may contribute to HSC dysfunction. Therefore, we performed comprehensive epigenomic and transcriptional profiling in primary human HSC (Lin-, CD34+, CD38-) isolated from young (18-30 yo), mid (45-55 yo) and old (65-75 yo) healthy donors. Using a micro-ChIP-seq protocol we profiled H3K4me1, H3K4me3, H3K27me3 and H3K27ac in 4-7 donors per age group, as well as genome-wide DNA methylation (5mC), hydroxymethylation (5hmC) and RNA-seq.

Analysis of enhancer-associated marks revealed that with age there is marked reduction in both H3K4me1 and H3K27ac (20,783 and 15,625 peaks lost, respectively; log10likelihood ratio >3). Gene ontology analysis of these lost peaks revealed their association with genes involved in hematopoiesis and, RNA splicing and chromatin organization, respectively (ChIPenrich, FDR<0.05). In addition, regions depleted in H3K4me1 are enriched for PU.1, FLI1, ETS, and CTCF binding sites (Homer, q<0.00001). We next asked if aging results in specific remodeling of poised (H3K4me1>H3K4me3, H3K27ac-) and active (H3K4me1>H3K4me3, H3K27ac+) enhancers. We found age-related loss of H3K4me1 enrichment at 10,696 poised enhancers, which are associated with hematopoiesis and T- and B-cell receptor signaling (FDR<0.05). We also identified 17,242 active enhancers in young HSC, 7,057 of which are depleted in old HSC. This loss of active enhancers targets genes associated with hematopoiesis, immune signaling and myeloid malignancies (FDR<0.05). Next we analyzed the impact of aging on promoter-associated marks, H3K4me3 and H3K27me3. Remarkably, while aging leads to loss of 22,689 H3K4me3 peaks, only 1,339 H3K27me3 peaks are lost. Loss of H3K4me3 targets genes involved in inflammatory response, development and WNT signaling (FDR<0.05). Given this uneven change in H3K4me3/H3K27me3 with aging, we hypothesized this may correlate with changes in bivalently marked promoters, which regulate key developmental genes. Out of 3,947 bivalent promoters in young HSC, 842 are lost in aged HSC. This loss of bivalency affects genes involved in WNT, Cadherin and Hedgehog signaling pathways (FDR<0.05). Next we analyzed changes in cytosine modifications. We observe widespread gain of 5hmC (n=14,554 differentially hydroxymethylated regions [DHMR]; FDR <0.005), with specific enrichment at introns and exons (p<2.2e-16), as well as enrichment for GATA and KLF binding sites (Homer, q<0.00001). These DHMR target genes involved in hematopoiesis, proteins regulated by alternative splicing, and pathways associated with cancer (FDR<0.05). In contrast, much more subtle changes are found in 5mC with HSC aging, with only 529 differentially methylated regions (q-value <0.05, meth.diff >20%). However, these subtle changes also target genes associated with cadherin and WNT signaling. Finally, RNA-seq analysis revealed that this age-associated epigenetic reprogramming is accompanied by an overall trend to gene downregulation. Amongst the genes most affected are the nuclear lamin gene LMNA (mutated in progeria syndrome), splicing factors SRSF7 and U2AF1 and, the transcription factors KLF3/6 and HIF1α (FDR <0.05, fold change >1.5). Notably, changes in expression also include significant differential exon usage, which may be mediated by DHMR at intron-exon boundaries: 575 genes show altered exon usage (FDR <0.05, fold change>1.5) including LMNA and the epigenetic modifiers BRD9, CITED2, KDM6A and SETD6.

In summary, we have completed the first comprehensive epigenomic profiling of aging in human HSC. Our findings show massive epigenetic remodeling in aged HSC, consisting of loss of activating histone marks primarily targeting enhancers and bivalent promoters at genes involved in hematopoiesis and developmental pathways. Cytosine modifications show widespread changes in 5hmC, targeting intron-exon boundaries. Globally, this epigenetic reprogramming results in overall gene downregulation and altered splicing of genes important for HSC regulation.