The establishment of long-lasting immunity against pathogens is facilitated by the germinal center (GC) reaction, during which B cells increase their antibody affinity and differentiate into antibody-secreting cells (ASC) and memory cells. These events involve modifications in chromatin packaging that orchestrate the profound restructuring of gene expression networks that determine cell fate. While several chromatin remodelers were implicated in lymphocyte functions, less is known about SMARCA5. Here, using ribosomal pull-down for analyzing translated genes in GC B cells, coupled with functional experiments in mice, we identified SMARCA5 as a key chromatin remodeler in B cells. While the naive B cell compartment remained unaffected following conditional depletion of Smarca5, effective proliferation during B cell activation, immunoglobulin class switching, and as a result GC formation and ASC differentiation were impaired. Single-cell multiomic sequencing analyses revealed that SMARCA5 is crucial for facilitating the transcriptional modifications and genomic accessibility of genes that support B cell activation and differentiation. These findings offer novel insights into the functions of SMARCA5, which can be targeted in various human pathologies.

• MeSH
• adenosintrifosfatasy MeSH
• aktivace lymfocytů imunologie   MeSH
• B-lymfocyty * metabolismus   imunologie   MeSH
• buněčná diferenciace * MeSH
• chromozomální proteiny, nehistonové * metabolismus   genetika   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• přesmyk imunoglobulinových tříd genetika   MeSH
• restrukturace chromatinu * MeSH
• zárodečné centrum lymfatické uzliny * imunologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfatasy MeSH
• chromozomální proteiny, nehistonové * MeSH
• Smarca5 protein, mouse MeSH Prohlížeč

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.

• Klíčová slova
• Smarca5, Snf2h, apoptosis, cell cycle, photoreceptors, 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
• Názvy látek
• adenosintrifosfatasy * MeSH
• chromatin * MeSH
• chromozomální proteiny, nehistonové * MeSH
• Smarca5 protein, mouse MeSH Prohlížeč

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.

• Klíčová slova
• AML, CRISPR, SMARCA5, SNF2H, leukemia, therapeutic target,
• 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
• Názvy látek
• adenosintrifosfatasy MeSH
• chromozomální proteiny, nehistonové MeSH
• nádorové proteiny * MeSH
• SMARCA5 protein, human MeSH Prohlížeč

Development of lymphoid progenitors requires a coordinated regulation of gene expression, DNA replication, and gene rearrangement. Chromatin-remodeling activities directed by SWI/SNF2 superfamily complexes play important roles in these processes. In this study, we used a conditional knockout mouse model to investigate the role of Smarca5, a member of the ISWI subfamily of such complexes, in early lymphocyte development. Smarca5 deficiency results in a developmental block at the DN3 stage of αβ thymocytes and pro-B stage of early B cells at which the rearrangement of Ag receptor loci occurs. It also disturbs the development of committed (CD73+) γδ thymocytes. The αβ thymocyte block is accompanied by massive apoptotic depletion of β-selected double-negative DN3 cells and premitotic arrest of CD4/CD8 double-positive cells. Although Smarca5-deficient αβ T cell precursors that survived apoptosis were able to undergo a successful TCRβ rearrangement, they exhibited a highly abnormal mRNA profile, including the persistent expression of CD44 and CD25 markers characteristic of immature cells. We also observed that the p53 pathway became activated in these cells and that a deficiency of p53 partially rescued the defect in thymus cellularity (in contrast to early B cells) of Smarca5-deficient mice. However, the activation of p53 was not primarily responsible for the thymocyte developmental defects observed in the Smarca5 mutants. Our results indicate that Smarca5 plays a key role in the development of thymocytes undergoing β-selection, γδ thymocytes, and also B cell progenitors by regulating the transcription of early differentiation programs.

• MeSH
• adenosintrifosfatasy genetika   metabolismus   MeSH
• B-lymfocyty fyziologie   MeSH
• buněčná diferenciace MeSH
• chromozomální proteiny, nehistonové genetika   metabolismus   MeSH
• genová přestavba MeSH
• klonální selekce zprostředkovaná antigeny MeSH
• kultivované buňky MeSH
• lymfoidní progenitorové buňky fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• receptory antigenů B-buněk genetika   metabolismus   MeSH
• receptory antigenů T-buněk alfa-beta genetika   metabolismus   MeSH
• receptory antigenů T-buněk gama-delta genetika   metabolismus   MeSH
• T-lymfocyty fyziologie   MeSH
• thymocyty fyziologie   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
• Názvy látek
• adenosintrifosfatasy MeSH
• chromozomální proteiny, nehistonové MeSH
• nádorový supresorový protein p53 MeSH
• receptory antigenů B-buněk MeSH
• receptory antigenů T-buněk alfa-beta MeSH
• receptory antigenů T-buněk gama-delta MeSH
• Smarca5 protein, mouse MeSH Prohlížeč
• Trp53 protein, mouse MeSH Prohlížeč

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.

• Klíčová slova
• Cell cycle progression, Erythroid differentiation, Fetal liver erythropoiesis, Hematopoietic stem and progenitor cells, Hypoxia, Imitation switch, Smarca5, p53 pathway,
• 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
• Názvy látek
• adenosintrifosfatasy MeSH
• chromozomální proteiny, nehistonové MeSH
• messenger RNA MeSH
• nádorový supresorový protein p53 MeSH
• Smarca5 protein, mouse MeSH Prohlížeč

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.

• Klíčová slova
• Brg1, Cataract, Denucleation, Lens, Smarca4, Smarca5, Snf2h, Terminal differentiation,
• 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 tepelného šoku 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
• Názvy látek
• adenosintrifosfatasy MeSH
• chromozomální proteiny, nehistonové MeSH
• DNA vazebné proteiny MeSH
• DNA-helikasy MeSH
• Hsf4 protein, mouse MeSH Prohlížeč
• jaderné proteiny MeSH
• Pax6 protein, mouse MeSH Prohlížeč
• Smarca4 protein, mouse MeSH Prohlížeč
• Smarca5 protein, mouse MeSH Prohlížeč
• transkripční faktor PAX6 MeSH
• transkripční faktory tepelného šoku MeSH
• transkripční faktory MeSH