The Chd8 gene encodes a member of chromodomain helicase DNA-binding (CHD) family of SNF2H-like ATP-dependent chromatin remodeler, mutations of which define a subtype of Autism spectrum disorders. Increasing evidence from recent studies indicates that ATP-dependent chromatin-remodeling genes are involved in the control of crucial gene-expression programs in hematopoietic stem/progenitor cell (HSPC) regulation. Here we identify CHD8 as a specific and essential regulator of normal hematopoiesis. Loss of Chd8 leads to severe anemia, pan-cytopenia, bone marrow failure, and engraftment failure due to a drastic depletion of HSPCs. CHD8 forms a complex with ATM and its deficiency increases chromatin accessibility and drives genomic instability in HSPCs causing an activation of ATM kinase that further stabilizes P53 protein by phosphorylation and leads to increased HSPC apoptosis. Deletion of P53 rescues the apoptotic defects of HSPCs and restores overall hematopoiesis in Chd8-/- mice. Our findings demonstrate that chromatin organization by CHD8 is uniquely required for the maintenance of hematopoiesis by integrating the ATM-P53 mediated survival of HSPCs.
The mechanistic target of rapamycin (mTOR) is a central regulator of cell growth and an attractive anticancer target that integrates diverse signals to control cell proliferation. Previous studies using mTOR inhibitors have shown that mTOR targeting suppresses gene expression and cell proliferation. To date, however, mTOR-targeted therapies in cancer have seen limited efficacy, and one key issue is related to the development of evasive resistance. In this manuscript, through the use of a gene targeting mouse model, we have found that inducible deletion ofmTORin hematopoietic stem cells (HSCs) results in a loss of quiescence and increased proliferation. Adaptive to themTORloss,mTOR−/−HSCs increase chromatin accessibility and activate global gene expression, contrary to the effects of short-term inhibition by mTOR inhibitors. Mechanistically, such genomic changes are due to a rewiring and adaptive activation of the ERK/MNK/eIF4E signaling pathway that enhances the protein translation of RNA polymerase II, which in turn leads to increasedc-Mycgene expression, allowing the HSCs to thrive despite the loss of a functional mTOR pathway. This adaptive mechanism can also be utilized by leukemia cells undergoing long-term mTOR inhibitor treatment to confer resistance to mTOR drug targeting. The resistance can be counteracted by MNK, CDK9, or c-Myc inhibition. These results provide insights into the physiological role of mTOR in mammalian stem cell regulation and implicate a mechanism of evasive resistance in the context of mTOR targeting.
Generation of bone marrow (BM) from embryonic stem cells (ESCs) promises to accelerate the development of future cell therapies for life-threatening disorders. However, such approach is limited by technical challenges to produce a mixture of functional BM progenitor cells able to replace all hematopoietic cell lineages. Herein, we used blastocyst complementation to simultaneously produce BM cell lineages from mouse ESCs in a rat. Based on FACS analysis and single-cell RNA sequencing, mouse ESCs differentiated into multiple hematopoietic and stromal cell types that were indistinguishable from normal mouse BM cells based on gene expression signatures and cell surface markers. Receptor-ligand interactions identified Cxcl12-Cxcr4, Lama2-Itga6, App-Itga6, Comp-Cd47, Col1a1-Cd44 and App-Il18rap as major signaling pathways between hematopoietic progenitors and stromal cells. Multiple hematopoietic progenitors, including hematopoietic stem cells (HSCs) in mouse-rat chimeras derived more efficiently from mouse ESCs, whereas chondrocytes predominantly derived from rat cells. In the dorsal aorta and fetal liver of mouse-rat chimeras, mouse HSCs emerged and expanded faster compared to endogenous rat cells. Sequential BM transplantation of ESC-derived cells from mouse-rat chimeras rescued lethally-irradiated syngeneic mice and demonstrated long-term reconstitution potential of donor HSCs. Altogether, a fully functional bone marrow was generated from mouse ESCs using rat embryos as 'bioreactors'.
As a member of the RUNT domain family core-binding transcription factors, RUNX1 is crucial for multiple stages of hematopoiesis and its mutation can cause familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML). Previous work has established that RUNX1 is involved in the maturation of megakaryocytes and the production of platelets. Recently studies have shown that there exists a subpopulation of hematopoietic stem cells (HSCs) with relatively high expression of vWF and CD41 at the apex of the HSC hierarchy, termed MK-HSCs, which can give rise to megakaryocytes without going through the traditional differentiation trajectory from HSC via MPP and MEP. Here, by using Runx1F/FMx1-Cre mouse model we discovered that the MK-HSC to megakaryocyte direct differentiation can happen within one cell division and RUNX1 is an important regulator in the process. Runx1 knockout results in a drastic decrease in platelet counts and a severe defect in the differentiation from MK-HSCs to megakaryocytes. Single cell RNA sequencing (RNAseq) analysis shows that MK-HSCs have a distinct gene expression signature compared with non-MK-HSCs, and Runx1 deletion alters the platelet and megakaryocyte related gene expression in MK-HSCs. Further bulk RNAseq and Cut & Run analyses show that RUNX1 binds to multiple essential megakaryocyte/platelet developmental genes such as Spi1, Selp and Itga2b and regulates their expressions in MK-HSCs. Thus, by modulating the expression of megakaryocyte-related genes, RUNX1 governs the direct differentiation of MK-HSCs to megakaryocytes and platelets.
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