GADD45β plays an essential role in the G-CSF triggered granulocytic differentiation of human hematopoietic stem cells

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dc.contributor.advisor Skokowa, Julia (Prof. Dr.)
dc.contributor.author Mir, Perihan
dc.date.accessioned 2020-08-07T09:41:02Z
dc.date.available 2020-08-07T09:41:02Z
dc.date.issued 2022-01-01
dc.identifier.uri http://hdl.handle.net/10900/104503
dc.identifier.uri http://nbn-resolving.de/urn:nbn:de:bsz:21-dspace-1045031 de_DE
dc.identifier.uri http://dx.doi.org/10.15496/publikation-45881
dc.description.abstract Dissertation ist gesperrt bis 01. Januar 2022 ! de_DE
dc.description.abstract Congenital neutropenia (CN) is a bone marrow failure syndrome caused by inherited gene mutations in e.g., ELANE or HAX1 leading to markedly low neutrophil numbers in peripheral blood. The mechanism of the “maturation arrest” of myeloid progenitors in CN patients is not fully elucidated. We aimed to shed light on the pathomechanism of this bone marrow failure. Considering the fact that inherited mutations may disturb the fitness of hematopoietic stem and progenitor cells (HSPCs) and may cause deregulated differentiation, we first investigated the CN HSPC composition and stress levels. Indeed, we found that early HSCs showed higher stress levels than later progenitors. Moreover, despite G-CSF treatment, CN HSPCs were committed towards lymphopoiesis, rather than myelopoiesis. We identified the myeloid differentiation primary response (MyD) and stress sensor gene GADD45β that might play a role in the defective stress-induced granulopoiesis: G-CSF induced the expression of GADD45β in healthy individuals, but not in CN patients. We functionally elaborated the role of GADD45β in stress response and granulopoiesis by CRISPR/Cas9-mediated knockout in iPSCs, primary HSPCs, and zebrafish. Since CRISPR/Cas9 gene-editing in iPSCs and HSPCs is challenging, we established a method that allows the fluorescent labeling of CRISPR/Cas9 RNP and thus the enrichment of gene-edited cells. GADD45β knockout cells were significantly more susceptible to UV-mediated DNA damage compared to control cells. Knockout of GADD45β leads to drastic reduction of granulocytes in vitro and in vivo. At the same time, the ectopic expression of GADD45β in CN HSPCs restored granulocytic differentiation. We could show that GADD45β is responsible for the demethylation and thus induction of genes essential for granulocytic differentiation. Among others, GADD45β activates the retinoic acid signaling pathway to induce granulopoiesis. Strikingly, the treatment of CN HSPCs with ATRA could bypass the GADD45β activation and rescued diminished neutrophil differentiation. Taken together, we could show that GADD45β regulates granulopoiesis downstream of G-CSF by modulating retinoic acid signaling. To further elaborate the mechanisms of defective granulopoiesis in CN, we established an iPSC model. Research on rare diseases requires valuable patient samples, which are very restricted in the case of pediatric patients. iPSC models may overcome these limitations. This model allows us to study CN pathogenesis, but also to test novel therapies. Using the iPSC model, we were able to set up a CRISPR/Cas9-based ELANE knockout to restore granulocytic differentiation. This approach could serve as gene therapy for CN patients. en
dc.language.iso en de_DE
dc.publisher Universität Tübingen de_DE
dc.rights ubt-podok de_DE
dc.rights.uri http://tobias-lib.uni-tuebingen.de/doku/lic_mit_pod.php?la=de de_DE
dc.rights.uri http://tobias-lib.uni-tuebingen.de/doku/lic_mit_pod.php?la=en en
dc.subject.classification Granulozytopoese , Neutropenie , Hämatopoese , Differenzierung de_DE
dc.subject.ddc 000 de_DE
dc.subject.other Granulopoiesis en
dc.subject.other Congenital Neutropenia en
dc.subject.other GADD45b en
dc.subject.other Hematopoiesis en
dc.subject.other Differentiation en
dc.title GADD45β plays an essential role in the G-CSF triggered granulocytic differentiation of human hematopoietic stem cells en
dc.type Dissertation de_DE
dcterms.dateAccepted 2020-07-02
utue.publikation.fachbereich Biochemie de_DE
utue.publikation.fakultaet 7 Mathematisch-Naturwissenschaftliche Fakultät de_DE
utue.publikation.noppn yes de_DE

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