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 (S5tg) 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 S5tg 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.

• MeSH
• adenosintrifosfatasy metabolismus   MeSH
• buněčná diferenciace genetika   MeSH
• hematopoetické kmenové buňky * metabolismus   MeSH
• hematopoéza * genetika   MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Chromatin remodeling complexes are required for many distinct nuclear processes such as transcription, DNA replication, and DNA repair. However, the contribution of these complexes to the development of complex tissues within an organism is poorly characterized. Imitation switch (ISWI) proteins are among the most evolutionarily conserved ATP-dependent chromatin remodeling factors and are represented by yeast Isw1/Isw2, and their vertebrate counterparts Snf2h (Smarca5) and Snf2l (Smarca1). In this study, we focused on the role of the Snf2h gene during the development of the mammalian retina. We show that Snf2h is expressed in both retinal progenitors and post-mitotic retinal cells. Using Snf2h conditional knockout mice (Snf2h cKO), we found that when Snf2h is deleted, the laminar structure of the adult retina is not retained, the overall thickness of the retina is significantly reduced compared with controls, and the outer nuclear layer (ONL) is completely missing. The depletion of Snf2h did not influence the ability of retinal progenitors to generate all the differentiated retinal cell types. Instead, the Snf2h function is critical for the proliferation of retinal progenitor cells. Cells lacking Snf2h have a defective S-phase, leading to the entire cell division process impairments. Although all retinal cell types appear to be specified in the absence of the Snf2h function, cell-cycle defects and concomitantly increased apoptosis in Snf2h cKO result in abnormal retina lamination, complete destruction of the photoreceptor layer, and consequently, a physiologically non-functional retina.

• MeSH
• adenosintrifosfatasy * metabolismus   MeSH
• buněčné jádro metabolismus   MeSH
• chromatin * metabolismus   MeSH
• chromozomální proteiny, nehistonové * metabolismus   MeSH
• myši knockoutované MeSH
• myši MeSH
• proliferace buněk MeSH
• restrukturace chromatinu * MeSH
• retina MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

ISWI chromatin remodeling ATPase SMARCA5 (SNF2H) is a well-known factor for its role in regulation of DNA access via nucleosome sliding and assembly. SMARCA5 transcriptionally inhibits the myeloid master regulator PU.1. Upregulation of SMARCA5 was previously observed in CD34+ hematopoietic progenitors of acute myeloid leukemia (AML) patients. Since high levels of SMARCA5 are necessary for intensive cell proliferation and cell cycle progression of developing hematopoietic stem and progenitor cells in mice, we reasoned that removal of SMARCA5 enzymatic activity could affect the cycling or undifferentiated state of leukemic progenitor-like clones. Indeed, we observed that CRISPR/cas9-mediated SMARCA5 knockout in AML cell lines (S5KO) inhibited the cell cycle progression. We also observed that the SMARCA5 deletion induced karyorrhexis and nuclear budding as well as increased the ploidy, indicating its role in mitotic division of AML cells. The cytogenetic analysis of S5KO cells revealed the premature chromatid separation. We conclude that deleting SMARCA5 in AML blocks leukemic proliferation and chromatid cohesion.

• MeSH
• adenosintrifosfatasy nedostatek   metabolismus   MeSH
• akutní myeloidní leukemie * enzymologie   genetika   patologie   MeSH
• buňky K562 MeSH
• chromatidy * genetika   metabolismus   MeSH
• chromozomální proteiny, nehistonové nedostatek   metabolismus   MeSH
• genový knockout * MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádorové proteiny * nedostatek   metabolismus   MeSH
• proliferace buněk * MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

The imitation switch 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 accumulated but their maturation toward 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 (S15Ph°s) 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-hydroxytamoxifen-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. Stem Cells 2017;35:1614-1623.

• MeSH
• adenosintrifosfatasy nedostatek   metabolismus   MeSH
• anemie patologie   MeSH
• buněčná diferenciace * MeSH
• buněčný cyklus MeSH
• chromozomální proteiny, nehistonové nedostatek   metabolismus   MeSH
• delece genu MeSH
• erytroidní buňky cytologie   MeSH
• erytropoéza MeSH
• genotyp MeSH
• hematopoetické kmenové buňky cytologie   metabolismus   MeSH
• hematopoéza MeSH
• messenger RNA genetika   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• poškození DNA genetika   MeSH
• proliferace buněk MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Ocular lens morphogenesis is a model for investigating mechanisms of cellular differentiation, spatial and temporal gene expression control, and chromatin regulation. Brg1 (Smarca4) and Snf2h (Smarca5) are catalytic subunits of distinct ATP-dependent chromatin remodeling complexes implicated in transcriptional regulation. Previous studies have shown that Brg1 regulates both lens fiber cell differentiation and organized degradation of their nuclei (denucleation). Here, we employed a conditional Snf2h(flox) mouse model to probe the cellular and molecular mechanisms of lens formation. Depletion of Snf2h induces premature and expanded differentiation of lens precursor cells forming the lens vesicle, implicating Snf2h as a key regulator of lens vesicle polarity through spatial control of Prox1, Jag1, p27(Kip1) (Cdkn1b) and p57(Kip2) (Cdkn1c) gene expression. The abnormal Snf2h(-/-) fiber cells also retain their nuclei. RNA profiling of Snf2h(-/) (-) and Brg1(-/-) eyes revealed differences in multiple transcripts, including prominent downregulation of those encoding Hsf4 and DNase IIβ, which are implicated in the denucleation process. In summary, our data suggest that Snf2h is essential for the establishment of lens vesicle polarity, partitioning of prospective lens epithelial and fiber cell compartments, lens fiber cell differentiation, and lens fiber cell nuclear degradation.

• MeSH
• adenosintrifosfatasy metabolismus   MeSH
• autofagie MeSH
• biologické modely MeSH
• buněčná diferenciace * MeSH
• buněčné jádro metabolismus   MeSH
• buněčný cyklus MeSH
• chromozomální proteiny, nehistonové metabolismus   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• DNA-helikasy metabolismus   MeSH
• embryo savčí metabolismus   MeSH
• epitelové buňky cytologie   metabolismus   MeSH
• jaderné proteiny metabolismus   MeSH
• kompartmentace buňky MeSH
• mitofagie MeSH
• mutace genetika   MeSH
• myši knockoutované MeSH
• oční čočka cytologie   embryologie   MeSH
• restrukturace chromatinu * MeSH
• transkripční faktor PAX6 metabolismus   MeSH
• transkripční faktory metabolismus   MeSH
• transkriptom genetika   MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Chromatin compaction mediates progenitor to post-mitotic cell transitions and modulates gene expression programs, yet the mechanisms are poorly defined. Snf2h and Snf2l are ATP-dependent chromatin remodelling proteins that assemble, reposition and space nucleosomes, and are robustly expressed in the brain. Here we show that mice conditionally inactivated for Snf2h in neural progenitors have reduced levels of histone H1 and H2A variants that compromise chromatin fluidity and transcriptional programs within the developing cerebellum. Disorganized chromatin limits Purkinje and granule neuron progenitor expansion, resulting in abnormal post-natal foliation, while deregulated transcriptional programs contribute to altered neural maturation, motor dysfunction and death. However, mice survive to young adulthood, in part from Snf2l compensation that restores Engrailed-1 expression. Similarly, Purkinje-specific Snf2h ablation affects chromatin ultrastructure and dendritic arborization, but alters cognitive skills rather than motor control. Our studies reveal that Snf2h controls chromatin organization and histone H1 dynamics for the establishment of gene expression programs underlying cerebellar morphogenesis and neural maturation.

• MeSH
• adenosintrifosfatasy metabolismus   MeSH
• analýza rozptylu MeSH
• bromodeoxyuridin MeSH
• chromatinová imunoprecipitace MeSH
• chromozomální proteiny, nehistonové metabolismus   MeSH
• fluorescence MeSH
• galaktosidy MeSH
• histony metabolismus   MeSH
• homeodoménové proteiny metabolismus   MeSH
• hybridizace in situ MeSH
• imunohistochemie MeSH
• indoly MeSH
• koncové značení zlomů DNA in situ MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• metoda rotující tyčky MeSH
• mikročipová analýza MeSH
• morfogeneze genetika   fyziologie   MeSH
• mozeček embryologie   MeSH
• myši transgenní MeSH
• myši MeSH
• nervové kmenové buňky metabolismus   fyziologie   MeSH
• počítačové zpracování obrazu MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• Purkyňovy buňky metabolismus   MeSH
• restrukturace chromatinu fyziologie   MeSH
• toloniumchlorid MeSH
• transmisní elektronová mikroskopie MeSH
• vývojová regulace genové exprese genetika   fyziologie   MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

• Publikační typ
• abstrakt z konference MeSH

V této aplikaci navrhuji experimenty ke studiu role Snf2h v normální a leukemické krvetvorbě v myši pomocí vytvoření myšího kmenu, ve kterém Snf2h bude inaktivovatelný kondicionálně ve specifických buněčných typech. Navrhuji dále experimenty zacílené nastudium role Snf2h v udržování fentypu leukemických buněk manipulací genové exprese Snf2h v leukemických blastech.; In this application I propose experiments to further investigate the role of Snf2h in normal hematopoiesis and in the development of acute leukemia in mice using conditional knockout model. In this application I also propose experiments to study the roleof Snf2h in maintenance of leukemic phenotype in malignant blasts.

• MeSH
• adenosintrifosfatasy MeSH
• buněčná diferenciace MeSH
• chromatin MeSH
• chromozomální proteiny, nehistonové MeSH
• genetická transkripce MeSH
• genetické komponenty MeSH
• leukemie MeSH
• myši transgenní MeSH
• proliferace buněk MeSH
• ribozomální DNA MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• hematologie a transfuzní lékařství
• onkologie
• biologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

Nucleosome movement is, at least in part, facilitated by ISWI ATPase Smarca5 (Snf2h). Smarca5 gene inactivation in mouse demonstrated its requirement at blastocyst stage; however its role at later stages is not completely understood. We herein determined nuclear distribution of Smarca5 and histone marks associated with actively transcribed and repressed chromatin structure in embryonic and adult murine tissues and in tumor cells. Confocal microscopy images demonstrate that Smarca5 is localized mainly in euchromatin and to lesser extent also in heterochromatin and nucleoli. Smarca5 heterozygous mice for a null allele display decreased levels of histone H3 modifications and defects in heterochromatin foci supporting role of Smarca5 as a key regulator of global chromatin structure.

• MeSH
• adenosintrifosfatasy genetika   metabolismus   MeSH
• blastocysta metabolismus   MeSH
• buněčné jadérko metabolismus   MeSH
• chromozomální proteiny, nehistonové genetika   metabolismus   MeSH
• euchromatin metabolismus   MeSH
• heterochromatin metabolismus   MeSH
• konfokální mikroskopie MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• umlčování genů MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

• MeSH
• adenosintrifosfatasy farmakokinetika   MeSH
• chromozomální proteiny, nehistonové genetika   MeSH
• embryonální vývoj genetika   MeSH
• genetická transkripce MeSH
• heterochromatin genetika   patologie   MeSH
• techniky in vitro MeSH