Chromatin regulators, including both Trithorax-group (trxG) and Polycomb-group (PcG) families, have been reported to maintain hematopoietic stem cells (HSC) self-renewal and differentiation. However, their primary targets in HSC remain to be fully identified. Moreover, those histone modifiers have been recently found to control longevity in C.elegans, which leads us to further investigate their roles in HSC aging. HSCs increase in number, decrease in regeneration capacity, and exhibit a myeloid biased differentiation with age. In this study, we profiled global mRNA (RNA-seq) and chromatin changes (ChIP-seq) in highly purified young (4 month) and old (24 month) murine bone marrow-derived HSCs (SP-KSL-CD150+). One key challenge in this study is that mouse HSCs represent less than 0.01% of all bone marrow cells. Thus, we first developed and optimized a method for successful application of ChIP-seq to a limited number of cells (<20,000 cells). This method allowed us to generate the binding profiles for H3K4me3, H3K27me3 and H3K36me3 in young and old HSCs, differentiated granulocytes (Gr1+) and B (B220+) cells.
In young HSCs, we determined ∼18,000 peaks for the active gene mark H3K4me3. For the repressive mark H3K27me3, we identified ∼6000 peaks across the genome, 2591 of which were present in the promoter region defined as the transcriptional start site (TSS) ± 100 bp. Strikingly, 70.2% (1820 out of 2591) of H3K27me3-enriched genes were also bound by H3K4me3. These so-called “Bivalent genes”, with both H3K4me3 and H3K27me3, are also found in embryonic stem cell (ESC) where they represent master regulators for lineage development. Here we found that these 1820 genes in HSC are prevalent in development, transcriptional regulation and RNA metabolism. They include many lineage-specific transcription factors, such as Cebpa, Ebf-1, Pax5, Gata3, Tbx21, Runx3 and Eomes. During HSC differentiation to granulocytes or B cells, HSC pluripotency regulators acquired the H3K27me3 repressive mark, while lineage regulators lost it in differentiated lineages. In addition, we observed with HSC differentiation alternative promoter usage on many epigenetic modifiers or transcription factors, such as Dnmt3a, Dnmt3b and Kdm6b (Jmjd3) and Runx3. The different isoforms may have different functions in hematopoiesis.
Compared to HSC differentiation, HSC aging showed less extensive chromatin changes. Increased H3K4me3 binding at the TSS was identified for ∼300 genes, including Selp, Nupr1, Sdpr, Plscr2, Slamf1 (CD150) and Mt1. Interestingly, several genes in the Hoxb cluster showed increased H3K4me3 binding and higher expression with age. For H3K27me3, although PcG family member EZh2 expression decreases with HSC aging, we did not detect a global H3K27me3 decrease. On the contrary, H3K27me3 binding increased at ∼500 genes and decreased at ∼100 genes. Among them, the lymphoid transcription factors Pax5 and Ebf1 exhibited increased H3K27me3 binding. The enhanced repression of these lymphoid regulators may explain the myeloid-skewed differentiation that occurs with age. We also observed increased H3K27me3 on several Wnt ligands, including Wnt2a, Wnt8a and Wnt8b. As Wnt signaling is required for active HSC cycling, the increased H3K27me3 binding on these ligands may reinforce HSC quiescence. One well known target of the PcG family in aging is Cdkn2a (p16), which showed progressive loss of H3K27me3 repression with aging in neural stem cell (NSC). In contrast, our results revealed that p16 is repressed by H3K27me3 in both 4M and 24M HSC, and we did not observe its expression increase with aging. However, another cell cycle regulator p21 expression increases with aging, accompanied by a decrease of H3K27me3.
In summary, we mapped chromatin state alterations with HSC differentiation and aging in this study. These findings will advance our fundamental understanding of HSC biology. Furthermore, chromatin regulators, such as Bmi1 and EZh2, have been implicated in leukemia transformation. This study provides a mechanistic link into how deregulation of these factors correlates with cancer progression.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.