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Abstract: Upregulation of the non-clustered homeobox transcription factor HLX (H2.0-like Homeobox) is frequently observed in patients with acute myeloid leukemia. Since developmental pathways are often reactivated in cancer, we asked whether hlx1 plays a role during haematopoietic development and blood cell differentiation in zebrafish. During the transient wave of haematopoiesis, hlx1 knock-down leads to diminished numbers of erythrocytes and increased numbers of myeloid cells. From RNA-seq analysis of sorted control or hlx1 morphant endothelial cells we could see that many erythroid genes were downregulated upon hlx1 knock-down. Indeed, ChIP-seq analysis of human blood cell lines confirmed that HLX binds to the regulatory region of one of the main regulators of erythropoiesis, namely SCL, suggesting that Hlx1 genetically participates in maintaining a balance between erythroid and myeloid cells during the transient wave of haematopoiesis in zebrafish.〈br〉As for the definitive wave of haematopoiesis, tissue specific overexpression of human HLX leads to aberrant production of hematopoietic stem and progenitor cells (HSPCs) and increased numbers of immature myeloid cells at 48 hours post fertilization. On the other hand, hlx1 morphants have diminished numbers of definitive HSPCs, which can be rescued by means if overexpression of the human ortholog. Edu-labelling of endothelial cells, the precursors of HSPCs, from overexpressing or morphant fish showed hyper- or hypo-proliferation of these cells, respectively. To delve into the molecular mechanism of HLX, we used to the expression analysis of sorted endothelial cells from zebrafish that overexpress HLX in endothelial cells and hlx1 morphants. We found that overexpression of HLX leads to mitochondrial dysfunction and downregulation of oxidative phosphorylation. On the other hand, down-regulation of hlx1 nicely corroborated the overexpression data showing the opposite results. ATAC-seq performed on hlx1 morphant endothelial cells confirmed chromatin accessibility changes of mitochondrial electron transport chain genes and ppard genes, which are important for lipid metabolism and fatty acid oxidation.〈br〉Firstly, we verified our zebrafish HSPC phenotype in human cells, by performing a Colony Forming Unit assays (CFU) in primary CD34+ progenitor hematopoietic cells. Indeed, overexpression of HLX results in an increased number of colonies, whereas down regulation greatly compromises the ability of CD34+ cells to form colonies. To further verify our results, we assesed metabolism both in zebrafish and in CD34+ cells. Seahorse metabolic assays revealed that overexpression of HLX reduces the oxidative phosphorylation capacity in both systems. Moreover, HLX overexpression leads to decreased mitochondrial potential, as detected by TMRM staining, which suggests that HLX can regulate the HSPC metabolic state. From ChIP-seq in HLX overexpressing blood cell lines, we figured that HLX directly regulates metabolic genes by binding to regulatory regions of mitochondrial genes (e.g., NDUFS4, OPA1, and SDHC), as well as PPARδ.〈br〉Finally, experiments showed that zebrafish hlx1 morphant treatment with a PPARδ agonist can rescue the HSPC phenotype, while treatment with an antagonist can rescue the immature myeloid phenotype in HLX overexpressing fish. These results were recapitulated in CD34+ cells. In summary, our findings establish HLX as a novel regulator of metabolism in haematopoietic stem cells
Note:
Dissertation Universität Freiburg 2018
Language:
English
Keywords:
Hochschulschrift
URN:
urn:nbn:de:bsz:25-freidok-160166
URL:
https://nbn-resolving.org/urn:nbn:de:bsz:25-freidok-160166
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