Regulation of Fetal Liver Hematopoietic Stem Cell Function By the Dpy30 Subunit of Set1/Mll Complexes

While stem cells undergo phenotypic and functional changes in development, the capacity of self-renewal and differentiation remains the defining property of stem cells throughout life, indicating certain fundamental regulatory mechanisms underlying these cardinal features of stem cells. A profound transition occurs to hematopoietic stem cells (HSCs) from embryonic to adult hematopoiesis, resulting in pronounced distinctions between fetal liver (FL) and adult bone marrow (BM) HSCs in many aspects. While many studies have documented a different dependence of fetal versus adult HSC function on epigenetic modulators including several Polycomb proteins, little is known about if Trithorax proteins play a similar or different role in fetal versus adult HSC function. More specifically, despite being a prominent epigenetic mark associated with gene activation, the role of H3K4 methylation (an activity of many Trithorax proteins) in different stages of HSCs remains unclear.

As the major H3K4 methylases in mammals, the Set1/Mll family complexes play important roles in development and stem cell function, and are extensively associated with diseases including blood cancers. We have previously established a direct role of Dpy30, a core subunit in all Set1/Mll complexes, in facilitating genome-wide H3K4 methylation, and this allows an effective interrogation of the functional role of efficient H3K4 methylation through genetic studies of Dpy30. While dispensable for the self-renewal of embryonic stem cells (ESCs), Dpy30 is crucial for efficient differentiation of ESCs by facilitating the induction of many bivalently marked developmental genes (Jiang et al., Cell, 2011). We have then generated a Dpy30 conditional knockout mouse, and shown that Dpy30 plays a crucial role in the long term maintenance and differentiation of adult BM HSCs, and preferentially controls H3K4 methylation and expression of many hematopoiesis-associated genes in adult BM cells (Yang et al., J Exp Med, accepted). However, the role of Dpy30 and efficient H3K4 methylation in fetal HSCs is still unknown.

To study the role of efficient H3K4 methylation in fetal HSCs, we deleted Dpy30 in fetal hematopoietic cells using VavCre line. VavCre; Dpy30^F/- fetuses are anemic at E14.5 and E15.5, with reduced H3K4 methylation but significantly increased numbers of FL HSCs. However, these FL HSCs were functionally defective in colony formation and blood reconstitution following transplantation. Proliferation of the progenitors, but not HSCs, was significantly (but modestly) reduced. These results suggest a role of Dpy30 in differentiation of HSCs and progenitor proliferation in FL. We also competitively transplanted Mx1Cre; Dpy30^F/- FL and deleted Dpy30 after stable engraftment. Our analysis at an early time point after deletion showed little effect on donor contribution to HSCs, but significant reduction of oligopotent progenitors. Analysis at a later time point after deletion, however, showed marked reduction of all hematopoietic cells including HSCs. These results support a cell-autonomous role of Dpy30 in the differentiation and long term maintenance of FL HSCs.

The phenotypes of FL HSCs are largely similar to those of BM HSCs following Dpy30 loss, suggesting that Dpy30 and certain Dpy30 targets are fundamentally important in regulating HSCs regardless of the developmental stages. To identify these targets, we performed RNA-seq analyses for purified FL HSCs from VavCre; Dpy30^F/- versus VavCre; Dpy30^F/+ littermates. Among hundreds of genes that were significantly changed in FL HSCs, however, only a handful of genes were found to be co-downregulated in both FL and BM HSCs following Dpy30 loss, suggesting that Dpy30 may have different functional targets in different stages of HSCs. To identify Dpy30 targets fundamentally important to HSC regulation, we are now selectively investigating the function of a few common Dpy30 targets in HSCs by colony formation and potentially transplantation assays following their stable knockdown.

The similar requirement of Dpy30 in both fetal and adult HSC differentiation as well as long-term maintenance underscores the fundamental importance of this epigenetic modulator in the central properties of stem cells, and studies of the common Dpy30 targets may identify new regulatory genes controlled by this modulator in fetal and adult HSC function.