Objective 1: Transient inhibition of epigenetics regulators of HSPC self-renewal and differentiation using CRISPRi Several transcription factors involved in the maintenance of HSPC function have been identified and recent studies have suggested involvement of epigenetic mechanisms to orchestrate the activities of these factors to ensure blood homeostasis. DNA methylation of CpG dinucleotides is a key epigenetic modification that influences mammalian gene expression. CpG methylation is catalyzed by a family of DNA methyltransferase (DNMT) enzymes, comprising three members (DNMT1, DNMT3A, and DNMT3B) catalyze DNA methylation of CpG dinucleotides. Conditional ablation of DNMT3a in a mouse model resulted in progressive impairment of HSPC differentiation over serial transplantation, while simultaneously expanding HSPC numbers in the bone marrow. DNMT3a-null HSPCs upregulated HSPC multipotency genes and downregulated differentiation factors, and their progeny exhibited global hypomethylation and incomplete repression of HSC-specific genes. These data established DNMT3a as a critical participant in the epigenetic silencing of HSPC multipotency genes. Ten-Eleven Translocation-2 (TET2) is one of the three proteins of the TET (Ten-Eleven Translocation) family, which are evolutionarily conserved dioxygenases that catalyze the conversion of 5-methyl-cytosine (5-mC) to 5-hydroxymethyl-cytosine (5-hmC), thus resulting in DNA demethylation. TET2 has pleiotropic roles in hematopoiesis, including stem-cell self-renewal, lineage commitment and terminal differentiation of specific lineages. The TET2 gene is highly expressed in HSPCs, and is downregulated with differentiation. Tet2 depletion in primary murine bone marrow cells results in an increase in the percentage of immature c-Kit+, Lin cells and their replating ability, suggesting that Tet2 silencing could impair HSPC differentiation and favor self-renewal. Consistent with these observations, loss-of-function somatic mutations in DNMT3A and TET2 have been shown to confer a proliferative advantage on HSPCs, resulting in age-related clonal hematopoiesis. Permanent inactivation of DNMT3A and TET2 favors HSPC self-renewal but may also predispose to the development of malignancies in cooperation with secondary mutations that drives the phenotype of the disease. Therefore, permanent inactivation of these genes would not be desirable for HSPC expansion. However, we hypothesized that transient inactivation of DNMT3A and TET2 activity might allow HSPC expansion in vitro without increasing susceptibility to malignancies. To investigate this possibility, studies are ongoing to: 1) Optimize position and sequence of gRNA for optimal repression; 2) Determine kinetics and specificity of expression repression; 3) Determine the impact of transient gene expression repression on HSPC function. Objective 2: Potentiation of known and new pathways involved in HSC self-renewal with hypoxia. Ex vivo culture of human hematopoietic stem/progenitor cells (HSPCs) under either low O2 tension or Notch signal activation driven by Delta1 ligand (Delta1) have been independently shown to primarily expand short-term HSPCs. In contrast, we have previously (FY17) demonstrated a significant (5-fold) expansion of long-term repopulating HSPCs upon combination of these two strategies, suggesting synergy between Notch and hypoxia signaling pathways. Understanding the molecular mechanisms underlying this synergy in HSPCs could provide new insights to further enhance expansion by combined activation of these pathways. Given the stoichiometric relationship between the number of Notch receptors (Notch 1-4) and Notch signaling intensity, we assessed whether hypoxia regulated Notch receptor expression. G-CSF mobilized human CD34+ cells from 3 healthy subjects were cultured for 0, 4, 24, or 48hrs in the presence of cytokines (SCF, FLT3L, TPO), under hypoxic (1.5-2.0% O2) or normoxic (21% O2) conditions. Using flow cytometry, Notch 3 expression was not detected in CD34+ cells and no differences in the expression level of any of the other Notch receptor isoforms (Notch 1, 2 and 4) were observed regardless of O2 tension. Another unique aspect of Notch signaling is that the receptor must undergo enzymatic cleavage to initiate signal transduction. These cleavage events culminate in the nuclear translocation of the intracellular domain of notch (ICDN) where it binds to Notch promoter regions, leading to the expression of Notch regulated genes. It has been suggested that a shift in energy metabolism from aerobic to glycolytic may lead to an enhancement of these cleavage events. To directly measure the differences in Notch cleavage events, CD34+ cells from 2 healthy donors were cultured for 24 hours under hypoxic or normoxic conditions, followed by a 1-hour exposure to Delta1. Cells were then collected, processed and stained with antibodies specific to the cleaved ICDN of Notch 1, 2, and 4. Using ImageStream analysis, preliminary data suggest that hypoxia accelerates the rate of Notch 2 receptor intracellular signaling by as much as 2-fold at this early time point. Another potential mechanism by which hypoxia may modulate Notch signaling is via protein-protein interaction of the ICDN and HIF1, a master transcriptional regulator of cellular response to hypoxia. To assess this possible interaction, CD34+ cells were cultured in chamber slides as described above. After exposure to Delta1, cells were fixed, permeabilized, and labeled with antibodies specific for cleaved ICDN and HIF1. Ten cells per condition (i.e. normoxia or hypoxia, +/- Delta1) were imaged with a Zeiss 780 inverted confocal microscope. Using Imaris and Interactive Data Language (IDL) software, all high resolution images were analyzed to determine the subcellular localization of each protein. As expected, exposure to Delta1 increased the levels of cleaved ICDN, and hypoxia increased the levels of HIF1 detected. Interestingly, the combination of hypoxia and Delta1 exposure resulted in a significant increase in the ICDN-HIF1 Pearsons coefficient of colocalization (PCC = 0.51) compared to control groups without Delta1 (PCC normoxia = 0.17, p<0.0001; PCC hypoxia = 0.26, p<0.05). Finally, to assess whether the observed ICDN-HIF1 interaction led to a recruitment of HIF1 to Notch promoter regions and thereby upregulate Notch-driven genes, ChIP-qPCR was performed on cells cultured under the same conditions described above. Samples were fixed, lysed, and immunoprecipitated through an anti-HIF1 column. Selected protein/DNA conjugates were then eluted and qPCR was performed on the captured DNA fragments. In cultures exposed to hypoxia and Delta1, ChiP-qPCR results indicated a significant enhancement of HIF1 /promoter interaction at the Hey-2 gene, a major downstream target of HIF1. No DNA from this region was detected in the normoxia+Delta1 control sample (0.02% of input vs. below the level of background, p<0.05). Taken together, these data suggest that the synergy between hypoxia and Notch in human HSPCs may be driven by an enhancement of Notch signaling events, a protein-protein interaction between ICDN and HIF1, and a recruitment of HIF1 to the promoter region of Hey-2, a gene traditionally thought to be regulated by Notch alone. Experiments are ongoing to further understand these mechanisms and their relationship to HSPC expansion.
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