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Identification of new tissue-specific interaction partners of chromatin remodelling ATPase Smarca5
Arishaka, Yuliia ; Kokavec, Juraj (advisor) ; Děd, Lukáš (referee)
The regulation of chromatin structure is fundamental to a wide range of cellular processes, including transcriptional regulation, cell division, differentiation and DNA damage repair, and ATP-dependent chromatin remodeling complexes have been established as essential components of this regulatory network. Smarca5, as an ATPase/Helicase enzyme, has been shown to regulate chromatin structure by interacting with bromodomain and DDT-WHIM domain-containing partners to control the binding of chromatin-associated proteins and transcription factors to their specific DNA target sequences. In this work we identify a previously undescribed protein with a conserved N-terminal bromodomain and ISWI protein binding DDT-WHIM domain through co-immunoprecipitation and mass spectrometry in mammalian cell lines and establish it as a novel interaction partner of chromatin remodeling ATPase Smarca5. Furthermore, we have pinpointed the region required for Smarca5 interaction that corresponds to DDT-WHIM domain. We have furthermore attempted to identify additional interaction partners which may hint on the potential function of this novel chromatin complex and validated its expression in embryonic and postnatal tissues. This discovery represents a unique opportunity for further investigation into its potential function in...
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Hematopoietic stem and progenitor cell defects in transgenic model of Diamond-Blackfan anemia
Holečková, Markéta ; Kokavec, Juraj (advisor) ; Valášek, Leoš (referee)
Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome characterized by deficient development of erythroid progenitors and accompanied by a variable set of developmental defects. About 25 % of patients have mutations of the small ribosomal subunit protein RPS19, and the precise mechanism of single aminoacidic mutations of RPS19 protein in the pathology of Diamond-Blackfan anemia remains largely unknown. To understand the interaction between of genotype and phenotypic variability we have created a mouse model with homozygous mutation in a highly conserved arginine 67 (Rps19R67Δ/R67Δ ). Mouse model with this mutation display many of the same phenotypical trades as patients with DBA. We decided to focus on hematopoiesis and erythropoiesis in this mouse model and tried to characterize those processes. We discovered that Rps19R67Δ/R67Δ mice similarly to DBA patients suffer from anemia and that the erythropoiesis process is disrupted at the stage of proerythroblasts. We also observed changes in hematopoiesis in stages as early as multipotent progenitors. The role of p53 protein as a modifier of DBA phenotype is well known. We created mouse model with p53 depletion to assess the role of p53 protein in relation with mutation in Rps19. Rps19R67Δ/R67Δ Trp53-/- mice show no signs of...
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Role of Smarca5 (Snf2h) chromation remodeling ATPase in hematopoitic development and erythropoiesis
Kokavec, Juraj ; Stopka, Tomáš (advisor) ; Divoký, Vladimír (referee) ; Kořínek, Vladimír (referee)
The Imitation Switch (ISWI) nuclear ATPase Smarca5 (Snf2h) is one of the most conserved chromatin remodeling factors. It exists in a variety of oligosubunit complexes that move DNA with respect to the histone octamer to generate regularly spaced nucleosomal arrays. Smarca5 interacts with different accessory proteins and represents a molecular motor for DNA replication, repair and transcription. We deleted Smarca5 at the onset of definitive hematopoiesis (Vav1-iCre) and observed that animals die during late fetal development due to anemia. Hematopoietic stem and progenitor cells (HSPCs) accumulated but their maturation towards erythroid and myeloid lineages was inhibited. Proerythroblasts were dysplastic while basophilic erythroblasts were blocked in G2/M and depleted. Smarca5 deficiency led to increased p53 levels, its activation at two residues, one associated with DNA damage (S-18) second with CBP/p300 (K376Ac), and finally activation of the p53 targets. We also deleted Smarca5 in committed erythroid cells (Epor-iCre) and observed that animals were anemic postnatally. Furthermore, 4- OHT-mediated deletion of Smarca5 in the ex vivo cultures confirmed its requirement for erythroid cell proliferation. Thus, Smarca5 plays indispensable roles during early hematopoiesis and erythropoiesis.
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The cell cycle and differentiation of haematopoietic stem and progenitor cells.
Páral, Petr ; Šefc, Luděk (advisor) ; Horváthová, Monika (referee) ; Kokavec, Juraj (referee)
Haematopoietic stem and progenitor cells (HSPCs) are crucial for lifelong blood cell production. We analysed the cell cycle and cell production rate in HSPCs in murine haematopoiesis. The labelling of DNA-synthesizing cells by two thymidine analogues, optimized for in-vivo use, enabled the determination of the cell cycle flow rate into the G2-phase, the duration of the S-phase and the average cell cycle time in Sca-1+ and Sca-1- HSPCs. The determination of cells with 2n DNA content and labelled during the preceding S-phase was used to establish the cell flow rates in the G1-phase. Our measurements revealed a significant difference in how Sca-1+ and Sca-1- HSPCs self-renew and differentiate. The division of Sca-1+ progenitors led to the loss of the Sca-1 marker in about half of newly produced cells, corresponding to asymmetric cell division. In contrast both Sca-1- progenitors, arising from mitotic cell division, entered a new round of the cell cycle. This corresponds to symmetric self-renewing cell division. The novel data also enabled us to estimate the cell production rates in the Sca-1+ and in three subtypes of Sca-1- HSPCs. We focused on adult murine erythroid differentiation in the next part of our study. We introduced an original flow cytometry approach for identifying and studying erythroid...
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Role of Smarca5 (Snf2h) chromation remodeling ATPase in hematopoitic development and erythropoiesis
Kokavec, Juraj ; Stopka, Tomáš (advisor) ; Divoký, Vladimír (referee) ; Kořínek, Vladimír (referee)
The Imitation Switch (ISWI) nuclear ATPase Smarca5 (Snf2h) is one of the most conserved chromatin remodeling factors. It exists in a variety of oligosubunit complexes that move DNA with respect to the histone octamer to generate regularly spaced nucleosomal arrays. Smarca5 interacts with different accessory proteins and represents a molecular motor for DNA replication, repair and transcription. We deleted Smarca5 at the onset of definitive hematopoiesis (Vav1-iCre) and observed that animals die during late fetal development due to anemia. Hematopoietic stem and progenitor cells (HSPCs) accumulated but their maturation towards erythroid and myeloid lineages was inhibited. Proerythroblasts were dysplastic while basophilic erythroblasts were blocked in G2/M and depleted. Smarca5 deficiency led to increased p53 levels, its activation at two residues, one associated with DNA damage (S-18) second with CBP/p300 (K376Ac), and finally activation of the p53 targets. We also deleted Smarca5 in committed erythroid cells (Epor-iCre) and observed that animals were anemic postnatally. Furthermore, 4- OHT-mediated deletion of Smarca5 in the ex vivo cultures confirmed its requirement for erythroid cell proliferation. Thus, Smarca5 plays indispensable roles during early hematopoiesis and erythropoiesis.
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