The Imprinted Dlk1-Gtl2 Locus Epigenetically Regulates Primitive Hematopoietic Stem Cell Mitochondrial Function and Energy Metabolism Via Repression of PI3K/Akt/mTOR Pathway

Balanced regulation is essential for the long-term preservation of stem cells while providing for ongoing tissue maintenance. We and others have previously shown that these dichotomous functions are accomplished through the co-existence of at least two stem cell populations—reserve and primed stem cells. This balance has been shown previously to be regulated by protein-coding genes; however, the potential roles of noncoding RNAs (ncRNAs) and their relationships with protein-coding genes in regulating hematopoietic stem cells (HSCs) remain largely unknown. To systematically identify ncRNAs involved in the murine hematopoiesis, we used RNA sequencing and identified unique, differentially expressed (fingerprint) ncRNAs representing reserve HSCs, primed HSCs, and more active stem/progenitor cells. These were also compared with committed progenitors and all major mature hematopoietic lineages. Intriguingly, all of the fingerprint ncRNAs uniquely expressed in reserve HSCs were derived from the imprinted Dlk1-Gtl2 locus, which spans a 780kb region on the mouse chromosome 12qF1 and is precisely controlled by the Intergenic Germ line-derived Differentially Methylated Region (IG-DMR). The Gtl2 locus contains a large cluster of snoRNAs (23 snoRNAs) and the largest cluster of mammalian miRNAs (57 miRNAs) as part of a single transcript of long length ncRNA downstream of Gtl2. To determine the role of Dlk1-Gtl2 locus in hematopoiesis, we utilized the IG-DMR knockout mouse model and carried out phenotypic and functional assays in E15.0 fetal liver HSCs since the embryos loss of maternal IG-DMR are lethal after E16. We observed that deletion of the maternal IG-DMR (ΔmIG-DMR), but not the paternal one, leads to 2-fold reduction in CD93⁺ fetal liver HSC number and 4-fold decrease of reconstitution ability after tertiary transplantation. Further, we employed RNA-seq using fetal liver HSCs from wt and ΔmIG-DMR and found that several pathways involved in growth control, mitochondrial function and energy metabolism, such as mTOR, PI3K/Akt and Wnt, are significantly enhanced in ΔmIG-DMR HSCs. We also carried out small RNA-seq in both adult HSCs and fetal liver HSCs and identified 13 HSC-specific miRNAs, which are predominantly expressed in reserve HSCs and predicted to target multiple proteins in PI3K/Akt/mTOR pathway. Mechanistically, maternal IG-DMR deletion leads to down-regulation of Gtl2-derived 13 miRNAs and hyperactivation of PI3K/Akt/mTOR pathway, which further enhances mitochondrial activity and biogenesis, increases oxidative phosphorylation (OXPHOS) mediated ATP production and ROS levels, and eventually causes HSC exhaustion. Moreover, either pharmacological inhibition of the mTOR activity by rapamycin or overexpression of Gtl2-derived miRNAs could partially, if not all, rescues the defective HSC phenotype and bioenergetic activities caused by mIG-DMR deletion. Collectively, our work provides a global landscape of murine hematopoietic lncRNAs and demonstrates that Dlk1-Gtl2 locus is critical in maintaining primitive HSCs with a fundamentally epigenetic regulation of mitochondrial function, energy metabolism via repression of PI3K/Akt/mTOR pathway.