Publications by authors named "Juraj Kokavec"

10 Publications

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Loss of ISWI ATPase SMARCA5 (SNF2H) in Acute Myeloid Leukemia Cells Inhibits Proliferation and Chromatid Cohesion.

Int J Mol Sci 2020 Mar 18;21(6). Epub 2020 Mar 18.

Biocev, 1st Medical Faculty, Charles University, 25250 Vestec, Czech Republic.

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 knockout in AML cell lines (S5KO) inhibited the cell cycle progression. We also observed that the 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.
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http://dx.doi.org/10.3390/ijms21062073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139293PMC
March 2020

ISWI ATPase Smarca5 Regulates Differentiation of Thymocytes Undergoing β-Selection.

J Immunol 2019 06 8;202(12):3434-3446. Epub 2019 May 8.

BIOCEV, First Faculty of Medicine, Charles University, Vestec 25250, Czech Republic;

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. 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 -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 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.
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http://dx.doi.org/10.4049/jimmunol.1801684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548592PMC
June 2019

The ISWI ATPase Smarca5 (Snf2h) Is Required for Proliferation and Differentiation of Hematopoietic Stem and Progenitor Cells.

Stem Cells 2017 06 15;35(6):1614-1623. Epub 2017 Apr 15.

BIOCEV, First Faculty of Medicine, Charles University, Czech Republic.

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 (S15 ° ) second with CBP/p300 (K376 ), 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.
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http://dx.doi.org/10.1002/stem.2604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5927548PMC
June 2017

Chromatin remodeling enzyme Snf2h regulates embryonic lens differentiation and denucleation.

Development 2016 06;143(11):1937-47

Department of Ophthalmology & Visual Sciences and Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA

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.
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http://dx.doi.org/10.1242/dev.135285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4920164PMC
June 2016

Snf2h-mediated chromatin organization and histone H1 dynamics govern cerebellar morphogenesis and neural maturation.

Nat Commun 2014 Jun 20;5:4181. Epub 2014 Jun 20.

1] Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada K1H 8L6 [2] Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5 [3] Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5.

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.
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http://dx.doi.org/10.1038/ncomms5181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083431PMC
June 2014

Epigenetic silencing of the oncogenic miR-17-92 cluster during PU.1-directed macrophage differentiation.

EMBO J 2011 Sep 6;30(21):4450-64. Epub 2011 Sep 6.

Institute of Pathological Physiology and Center of Experimental Hematology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.

The oncogenic cluster miR-17-92 encodes seven related microRNAs that regulate cell proliferation, apoptosis and development. Expression of miR-17-92 cluster is decreased upon cell differentiation. Here, we report a novel mechanism of the regulation of miR-17-92 cluster. Using transgenic PU.1(-/-) myeloid progenitors we show that upon macrophage differentiation, the transcription factor PU.1 induces the secondary determinant Egr2 which, in turn, directly represses miR-17-92 expression by recruiting histone demethylase Jarid1b leading to histone H3 lysine K4 demethylation within the CpG island at the miR-17-92 promoter. Conversely, Egr2 itself is targeted by miR-17-92, indicating existence of mutual regulatory relationship between miR-17-92 and Egr2. Furthermore, restoring EGR2 levels in primary acute myeloid leukaemia blasts expressing elevated levels of miR-17-92 and low levels of PU.1 and EGR2 leads to downregulation of miR-17-92 and restored expression of its targets p21CIP1 and BIM. We propose that upon macrophage differentiation PU.1 represses the miR-17-92 cluster promoter by an Egr-2/Jarid1b-mediated H3K4 demethylation mechanism whose deregulation may contribute to leukaemic states.
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http://dx.doi.org/10.1038/emboj.2011.317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3230374PMC
September 2011

MYB transcriptionally regulates the miR-155 host gene in chronic lymphocytic leukemia.

Blood 2011 Apr 4;117(14):3816-25. Epub 2011 Feb 4.

First Faculty of Medicine and Center of Experimental Hematology, Charles University in Prague, U Nemocnice 5, Prague, Czech Republic.

Elevated levels of microRNA miR-155 represent a candidate pathogenic factor in chronic B-lymphocytic leukemia (B-CLL). In this study, we present evidence that MYB (v-myb myeloblastosis viral oncogene homolog) is overexpressed in a subset of B-CLL patients. MYB physically associates with the promoter of miR-155 host gene (MIR155HG, also known as BIC, B-cell integration cluster) and stimulates its transcription. This coincides with the hypermethylated histone H3K4 residue and spread hyperacetylation of H3K9 at MIR155HG promoter. Our data provide evidence of oncogenic activities of MYB in B-CLL that include its stimulatory role in MIR155HG transcription.
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http://dx.doi.org/10.1182/blood-2010-05-285064DOI Listing
April 2011

PU.1 activation relieves GATA-1-mediated repression of Cebpa and Cbfb during leukemia differentiation.

Mol Cancer Res 2009 Oct 13;7(10):1693-703. Epub 2009 Oct 13.

Institute of Pathological Physiology and Center of Experimental Hematology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.

Hematopoietic transcription factors GATA-1 and PU.1 bind each other on DNA to block transcriptional programs of undesired lineage during hematopoietic commitment. Murine erythroleukemia (MEL) cells that coexpress GATA-1 and PU.1 are blocked at the blast stage but respond to molecular removal (downregulation) of PU.1 or addition (upregulation) of GATA-1 by inducing terminal erythroid differentiation. To test whether GATA-1 blocks PU.1 in MEL cells, we have conditionally activated a transgenic PU.1 protein fused with the estrogen receptor ligand-binding domain (PUER), resulting in activation of a myeloid transcriptional program. Gene expression arrays identified components of the PU.1-dependent transcriptome negatively regulated by GATA-1 in MEL cells, including CCAAT/enhancer binding protein alpha (Cebpa) and core-binding factor, beta subunit (Cbfb), which encode two key hematopoietic transcription factors. Inhibition of GATA-1 by small interfering RNA resulted in derepression of PU.1 target genes. Chromatin immunoprecipitation and reporter assays identified PU.1 motif sequences near Cebpa and Cbfb that are co-occupied by PU.1 and GATA-1 in the leukemic blasts. Significant derepression of Cebpa and Cbfb is achieved in MEL cells by either activation of PU.1 or knockdown of GATA-1. Furthermore, transcriptional regulation of these loci by manipulating the levels of PU.1 and GATA-1 involves quantitative increases in a transcriptionally active chromatin mark: acetylation of histone H3K9. Collectively, we show that either activation of PU.1 or inhibition of GATA-1 efficiently reverses the transcriptional block imposed by GATA-1 and leads to the activation of a myeloid transcriptional program directed by PU.1.
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http://dx.doi.org/10.1158/1541-7786.MCR-09-0031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3193075PMC
October 2009

Chromatin remodeling and SWI/SNF2 factors in human disease.

Front Biosci 2008 May 1;13:6126-34. Epub 2008 May 1.

Pathologic Physiology and Center for Experimental Hematology, Charles University in Prague, First Faculty of Medicine, U nemocnice 5, Prague 12853, Czech Republic.

Chromatin structure and its changes or maintenance throughout developmental checkpoints play indispensable role in organismal homeostasis. Chromatin remodeling factors of the SWI/SNF2 superfamily use ATP hydrolysis to change DNA-protein contacts, and their loss-of-function or inappropriate increase leads to distinct human pathologic states. In this review, we focus on the translational view of human pathologic physiology involving SWI/SNF2 superfamily, combining latest finding from basic and clinical research. We discuss in mechanistic terms the consequences resulting from dose alteration of the SWI/SNF2 superfamily ATPases and emphasize the necessity of future human subject-based studies.
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http://dx.doi.org/10.2741/3142DOI Listing
May 2008

Analysis of the tellurite resistance determinant on the pNT3B derivative of the pTE53 plasmid from uropathogenic Escherichia coli.

Biometals 2006 Oct;19(5):453-60

Department of Molecular Biology, Comenius University, Mlynská dolina B2-137, 842 15, Bratislava, Slovak Republic.

We have found and sequenced a significant part of the previously described tellurite resistance determinant on mini-Mu derivative pPR46, named pNT3B, originally cloned from a large conjugative plasmid pTE53, found in Escherichia coli. This plasmid contains genes essential for tellurite resistance, together with the protective region bearing genes terX, Y, W, and the conserved spacing region bearing several ORFs of unknown function. Computer analysis of obtained sequence revealed a close similarity to the formerly described ter operons found on the Serratia marcescens plasmid R478 and the chromosome of Escherichia coli O157:H7. This finding confirms the presence of a whole region on the large conjugative plasmid that pTE53 originated from a uropathogenic E. coli strain, and suggests its possible role in horizontal gene transfer, resulting in the development of new pathogenic E. coli strains.
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http://dx.doi.org/10.1007/s10534-005-4862-8DOI Listing
October 2006