Role chromation remoledačné ATPázy SMARCA5 v krvetvorbě vývoji červených krvinek

Jaderný protein Smarca5 (Snf2h) je jeden z nejvíce konzervovaných chromatin remodelačních faktorů a molekulárních motorů u savců. ATPáza Smarca5 (a ostatní příbuzní zástupci proteinové nadrodiny SWI/SNF2) společně se svými vazebnými partnery vytváří několika podjednotkové remodelační komplexy, jež posouvají, odstraňují nebo přináší histonové oktamery na DNA za účelem regulace trankripce, replikace, oprav poškození DNA a jiných nepostradatelných buněčných funkcí. Ve své disertační práci se zabývám studiem myšího modelu delece genu Smarca5 na počátku definitivní krvetvorby. Jedinci s delecí genu Smarca5 v hematopoetické kmenové buňce (aktivní promotor Vav1) umírají během pozdního vývoje in utero v důsledku závažné anémie. Ve fetálních játrech těchto experimentálních zvířat jsme pozorovali akumulaci krvetvorných kmenových a progenitorových buněk (HSPCs) a inhibici jejich dozrávání do erythroidní a myeloidní buněčné řady. Popsané poruchy vývoje byly doprovázeny dysplastickými změnami na úrovni proerytroblastů a poruchami buněčného cyklu na přechodu z G2 do M fáze u bazofilních erytroblastů. Dále jsme pozorovali, že nedostatečná funkce Smarca5 zvyšuje hladinu tumor supresorového proteinu p53 v erythroidních progenitorech, aktivaci jeho transkripčních cílů a k postranslačním modifikacím, jež jsou...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.BIOCEV, First Faculty of Medicine, Charles UniversityBIOCEV, 1. LF UKFirst Faculty of Medicine1. lékařská fakult

Role of Smarca5 (Snf2h) chromation remodeling ATPase in hematopoitic development and erythropoiesis

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

Differential requirements for Smarca5 expression during hematopoietic stem cell commitment

Abstract The formation of hematopoietic cells relies on the chromatin remodeling activities of ISWI ATPase SMARCA5 (SNF2H) and its complexes. The Smarca5 null and conditional alleles have been used to study its functions in embryonic and organ development in mice. These mouse model phenotypes vary from embryonic lethality of constitutive knockout to less severe phenotypes observed in tissue-specific Smarca5 deletions, e.g., in the hematopoietic system. Here we show that, in a gene dosage-dependent manner, the hypomorphic allele of SMARCA5 (S5 tg ) can rescue not only the developmental arrest in hematopoiesis in the hCD2iCre model but also the lethal phenotypes associated with constitutive Smarca5 deletion or Vav1iCre-driven conditional knockout in hematopoietic progenitor cells. Interestingly, the latter model also provided evidence for the role of SMARCA5 expression level in hematopoietic stem cells, as the Vav1iCre S5 tg animals accumulate stem and progenitor cells. Furthermore, their hematopoietic stem cells exhibited impaired lymphoid lineage entry and differentiation. This observation contrasts with the myeloid lineage which is developing without significant disturbances. Our findings indicate that animals with low expression of SMARCA5 exhibit normal embryonic development with altered lymphoid entry within the hematopoietic stem cell compartment

Analysis of 5-Azacytidine Resistance Models Reveals a Set of Targetable Pathways

The mechanisms by which myelodysplastic syndrome (MDS) cells resist the effects of hypomethylating agents (HMA) are currently the subject of intensive research. A better understanding of mechanisms by which the MDS cell becomes to tolerate HMA and progresses to acute myeloid leukemia (AML) requires the development of new cellular models. From MDS/AML cell lines we developed a model of 5-azacytidine (AZA) resistance whose stability was validated by a transplantation approach into immunocompromised mice. When investigating mRNA expression and DNA variants of the AZA resistant phenotype we observed deregulation of several cancer-related pathways including the phosphatidylinosito-3 kinase signaling. We have further shown that these pathways can be modulated by specific inhibitors that, while blocking the proliferation of AZA resistant cells, are unable to increase their sensitivity to AZA. Our data reveal a set of molecular mechanisms that can be targeted to expand therapeutic options during progression on AZA therapy