Publications by authors named "Tomas Stopka"

34 Publications

Aberrantly elevated suprabasin in the bone marrow as a candidate biomarker of advanced disease state in myelodysplastic syndromes.

Mol Oncol 2020 10 11;14(10):2403-2419. Epub 2020 Aug 11.

Department of Genome Integrity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic.

Myelodysplastic syndromes (MDS) are preleukemic disorders characterized by clonal growth of mutant hematopoietic stem and progenitor cells. MDS are associated with proinflammatory signaling, dysregulated immune response, and cell death in the bone marrow (BM). Aging, autoinflammation and autoimmunity are crucial features of disease progression, concordant with promoting growth of malignant clones and accumulation of mutations. Suprabasin (SBSN), a recently proposed proto-oncogene of unknown function, physiologically expressed in stratified epithelia, is associated with poor prognosis of several human malignancies. Here, we showed that SBSN is expressed in the BM by myeloid cell subpopulations, including myeloid-derived suppressor cells, and is secreted into BM plasma and peripheral blood of MDS patients. The highest expression of SBSN was present in a patient group with poor prognosis. SBSN levels in the BM correlated positively with blast percentage and negatively with CCL2 chemokine levels and lymphocyte count. In vitro treatment of leukemic cells with interferon-gamma and demethylating agent 5-azacytidine (5-AC) induced SBSN expression. This indicated that aberrant cytokine levels in the BM and epigenetic landscape modifications in MDS patients may underlie ectopic expression of SBSN. Our findings suggest SBSN as a candidate biomarker of high-risk MDS with a possible role in disease progression and therapy resistance.
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http://dx.doi.org/10.1002/1878-0261.12768DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530796PMC
October 2020

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

The chromatin remodeler Snf2h is essential for oocyte meiotic cell cycle progression.

Genes Dev 2020 02 9;34(3-4):166-178. Epub 2020 Jan 9.

Howard Hughes Medical Institute, Boston Children's Hospital, Boston, Massachusetts 02115, USA.

Oocytes are indispensable for mammalian life. Thus, it is important to understand how mature oocytes are generated. As a critical stage of oocytes development, meiosis has been extensively studied, yet how chromatin remodeling contributes to this process is largely unknown. Here, we demonstrate that the ATP-dependent chromatin remodeling factor Snf2h (also known as Smarca5) plays a critical role in regulating meiotic cell cycle progression. Females with oocyte-specific depletion of are infertile and oocytes lacking fail to undergo meiotic resumption. Mechanistically, depletion of results in dysregulation of meiosis-related genes, which causes failure of maturation-promoting factor (MPF) activation. ATAC-seq analysis in oocytes revealed that Snf2h regulates transcription of key meiotic genes, such as , by increasing its promoter chromatin accessibility. Thus, our studies not only demonstrate the importance of Snf2h in oocyte meiotic resumption, but also reveal the mechanism underlying how a chromatin remodeling factor can regulate oocyte meiosis.
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http://dx.doi.org/10.1101/gad.331157.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7000916PMC
February 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

Paraproteinemic keratopathy associated with monoclonal gammopathy of undetermined significance (MGUS): clinical findings in twelve patients including recurrence after keratoplasty.

Acta Ophthalmol 2019 Nov 2;97(7):e987-e992. Epub 2019 May 2.

Research Unit for Rare Diseases, Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic.

Purpose: To describe the ocular findings of 12 subjects with paraproteinemic keratopathy associated with monoclonal gammopathy of undetermined significance (MGUS).

Methods: Ocular examination included corneal spectral domain optical coherence tomography. In three individuals with an initial diagnosis of a lattice or Thiel-Behnke corneal dystrophy, the TGFBI gene was screened by conventional Sanger sequencing.

Results: We confirmed a diagnosis of MGUS by systemic examination and serum protein electrophoresis in 12 individuals (9 males and 3 females), with a mean age at presentation of 52.2 years (range 24-63 years) and mean follow-up 6.4 years (range 0-17 years). The best-corrected visual acuity (BCVA) at presentation ranged from 1.25 to 0.32. In all individuals, the corneal opacities were bilateral. The appearances were diverse and included superficial reticular opacities and nummular lesions, diffuse posterior stromal opacity, stromal lattice lines, superficial and stromal crystalline deposits, superficial haze and a superficial ring of hypertrophic tissue. In one individual, with opacities first recorded at 24 years of age, we documented the progression of corneal disease over the subsequent 17 years. In another individual, despite systemic treatment for MGUS, recurrence of deposits was noted following bilateral penetrating keratoplasties. The three individuals initially diagnosed with inherited corneal dystrophy were negative for TGFBI mutations by direct sequencing.

Conclusion: A diagnosis of MGUS should be considered in patients with bilateral corneal opacities. The appearance can mimic corneal dystrophies or cystinosis. In our experience, systemic treatment of MGUS did not prevent recurrence of paraproteinemic keratopathy following keratoplasty.
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http://dx.doi.org/10.1111/aos.14123DOI Listing
November 2019

BCR-ABL1 mediated miR-150 downregulation through MYC contributed to myeloid differentiation block and drug resistance in chronic myeloid leukemia.

Haematologica 2018 12 26;103(12):2016-2025. Epub 2018 Jul 26.

Institute of Hematology and Blood Transfusion, Prague, Czech Republic

The fusion oncoprotein BCR-ABL1 exhibits aberrant tyrosine kinase activity and it has been proposed that it deregulates signaling networks involving both transcription factors and non-coding microRNAs that result in chronic myeloid leukemia (CML). Previously, microRNA expression profiling showed deregulated expression of miR-150 and miR-155 in CML. In this study, we placed these findings into the broader context of the MYC/miR-150/MYB/miR-155/PU.1 oncogenic network. We propose that up-regulated MYC and miR-155 in CD34 leukemic stem and progenitor cells, in concert with BCR-ABL1, impair the molecular mechanisms of myeloid differentiation associated with low miR-150 and PU.1 levels. We revealed that MYC directly occupied the -11.7 kb and -0.35 kb regulatory regions in the gene. MYC occupancy was markedly increased through BCR-ABL1 activity, causing inhibition of gene expression in CML CD34 and CD34 cells. Furthermore, we found an association between reduced miR-150 levels in CML blast cells and their resistance to tyrosine kinase inhibitors (TKIs). Although TKIs successfully disrupted BCR-ABL1 kinase activity in proliferating CML cells, this treatment did not efficiently target quiescent leukemic stem cells. The study presents new evidence regarding the MYC/miR-150/MYB/miR-155/PU.1 leukemic network established by aberrant BCR-ABL1 activity. The key connecting nodes of this network may serve as potential druggable targets to overcome resistance of CML stem and progenitor cells.
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http://dx.doi.org/10.3324/haematol.2018.193086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6269310PMC
December 2018

Lenalidomide treatment in lower risk myelodysplastic syndromes-The experience of a Czech hematology center. (Positive effect of erythropoietin ± prednisone addition to lenalidomide in refractory or relapsed patients).

Leuk Res 2018 06 27;69:12-17. Epub 2018 Mar 27.

Center of Oncocytogenetic, Institute of Medical Biochemistry and Laboratory Diagnostics, First Faculty of Medicine, Charles University and General University Hospital, U Nemocnice 2, 128 00 Prague, Czech Republic.

Lenalidomide therapy represents meaningful progress in the treatment of anemic patients with myelodysplastic syndromes with del(5q). We present our initial lenalidomide experience and the positive effect of combining erythropoietin and steroids with lenalidomide in refractory and relapsed patients. We treated by lenalidomide 55 (42 female; 13 male; median age 69) chronically transfused lower risk MDS patients with del(5q) (45) and non-del(5q) (10). Response, meaning transfusion independence (TI) lasting ≥ eight weeks, was achieved in 38 (90%) of analyzed patients with del(5q), of whom three achieved TI only by adding erythropoietin ± prednisone. Another five patients responded well to this combination when their anemia relapsed later during the treatment. In the non-del(5q) group only one patient with RARS-T reached TI. Cytogenetic response was reached in 64% (32% complete, 32% partial response). The TP53 mutation was detected in 7 (18%) patients; four patients progressed to higher grade MDS or acute myeloid leukemia (AML). All seven RAEB-1 patients cleared bone marrow blasts during lenalidomide treatment and reached complete remission (CR); however, three later progressed to higher grade MDS or AML. Lenalidomide represents effective treatment for del(5q) group and combination with prednisone and erythropoietin may be used for non-responders or therapy failures.
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http://dx.doi.org/10.1016/j.leukres.2018.03.015DOI Listing
June 2018

Somatic mutation dynamics in MDS patients treated with azacitidine indicate clonal selection in patients-responders.

Oncotarget 2017 Dec 6;8(67):111966-111978. Epub 2017 Dec 6.

Department Biocev, First Faculty of Medicine, Charles University, Vestec, Czech Republic.

Azacitidine (AZA) for higher risk MDS patients is a standard therapy with limited durability. To monitor mutation dynamics during AZA therapy we utilized massive parallel sequencing of 54 genes previously associated with MDS/AML pathogenesis. Serial sampling before and during AZA therapy of 38 patients (reaching median overall survival 24 months (Mo) with 60% clinical responses) identified 116 somatic pathogenic variants with allele frequency (VAF) exceeding 5%. High accuracy of data was achieved via duplicate libraries from myeloid cells and T-cell controls. We observed that nearly half of the variants were stable while other variants were highly dynamic. Patients with marked decrease of allelic burden upon AZA therapy achieved clinical responses. In contrast, early-progressing patients on AZA displayed minimal changes of the mutation pattern. We modeled the VAF dynamics on AZA and utilized a joint model for the overall survival and response duration. While the presence of certain variants associated with clinical outcomes, such as the mutations of were adverse predictors while mutations yield lower risk of dying, the data also indicate that allelic burden volatility represents additional important prognostic variable. In addition, preceding 5q- syndrome represents strong positive predictor of longer overall survival and response duration in high risk MDS patients treated with AZA. In conclusion, variants dynamics detected via serial sampling represents another parameter to consider when evaluating AZA efficacy and predicting outcome.
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http://dx.doi.org/10.18632/oncotarget.22957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762372PMC
December 2017

Plasma miR-155, miR-203, and miR-205 are Biomarkers for Monitoring of Primary Cutaneous T-Cell Lymphomas.

Int J Mol Sci 2017 Oct 15;18(10). Epub 2017 Oct 15.

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

Primary cutaneous T-cell lymphomas (CTCL) affect the skin and tend to transform and spread. CTCL involves primarily the Mycosis fungoides (MF) and more aggressive Sezary syndrome (SS). Oncogenic microRNAs (miRs) are stable epigenetic inhibitors often deregulated in the tumour and detectable as biomarkers in non-cellular fractions of peripheral blood. The tumour-specific expression of miR-155, miR-203, and miR-205 was shown to correctly diagnose CTCL. We herein asked whether these microRNAs can be used as plasma biomarkers for clinical CTCL monitoring. Patients with CTCL ( = 10) and controls with non-malignant conditions ( = 11) repeatedly donated plasma samples every ca. five months. MicroRNAs were detected in the plasma samples by specifically-primed RT-PCR followed by multivariate analyses of the miR expression dynamics. We herein established the plasma miR-classifier for detecting CTCL based on the miR-155 upregulation and miR-203/miR-205 downregulation with 100% specificity and 94% sensitivity. The 3-miR-score in the consecutive samples coincided with the clinical outcome of MF and SS patients such as the therapy response or changes in the clinical stage or tumor size. Quantitation of the selected microRNAs in plasma is a specific and straightforward approach for evaluating CTCL outcome representing, thus, a valuable tool for CTCL diagnostics and therapy response monitoring.
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http://dx.doi.org/10.3390/ijms18102136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666818PMC
October 2017

Prediction Potential of Serum miR-155 and miR-24 for Relapsing Early Breast Cancer.

Int J Mol Sci 2017 Oct 10;18(10). Epub 2017 Oct 10.

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

Oncogenic microRNAs (oncomiRs) accumulate in serum due to their increased stability and thus serve as biomarkers in breast cancer (BC) pathogenesis. Four oncogenic microRNAs (miR-155, miR-19a, miR-181b, and miR-24) and one tumor suppressor microRNA (let-7a) were shown to differentiate between high- and low-risk early breast cancer (EBC) and reflect the surgical tumor removal and adjuvant therapy. Here we applied the longitudinal multivariate data analyses to stochastically model the serum levels of each of the oncomiRs using the RT-PCR measurements in the EBC patients ( = 133) that were followed up 4 years after diagnosis. This study identifies that two of the studied oncomiRs, miR-155 and miR-24, are highly predictive of EBC relapse. Furthermore, combining the oncomiR level with Ki-67 expression further specifies the relapse probability. Our data move further the notion that oncomiRs in serum enable not only monitoring of EBC but also are a very useful tool for predicting relapse independently of any other currently analyzed characteristics in EBC patients. Our approach can be translated into medical practice to estimate individual relapse risk of EBC patients.
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http://dx.doi.org/10.3390/ijms18102116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666798PMC
October 2017

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

Distinct and overlapping DNMT1 interactions with multiple transcription factors in erythroid cells: Evidence for co-repressor functions.

Biochim Biophys Acta 2016 12 28;1859(12):1515-1526. Epub 2016 Sep 28.

Division of Molecular Oncology, Biomedical Sciences Research Center "Alexander Fleming", Vari, Greece. Electronic address:

DNMT1 is the maintenance DNA methyltransferase shown to be essential for embryonic development and cellular growth and differentiation in many somatic tissues in mammals. Increasing evidence has also suggested a role for DNMT1 in repressing gene expression through interactions with specific transcription factors. Previously, we identified DNMT1 as an interacting partner of the TR2/TR4 nuclear receptor heterodimer in erythroid cells, implicated in the developmental silencing of fetal β-type globin genes in the adult stage of human erythropoiesis. Here, we extended this work by using a biotinylation tagging approach to characterize DNMT1 protein complexes in mouse erythroleukemic cells. We identified novel DNMT1 interactions with several hematopoietic transcription factors with essential roles in erythroid differentiation, including GATA1, GFI-1b and FOG-1. We provide evidence for DNMT1 forming distinct protein subcomplexes with specific transcription factors and propose the existence of a "core" DNMT1 complex with the transcription factors ZBP-89 and ZNF143, which is also present in non-hematopoietic cells. Furthermore, we identified the short (17a.a.) PCNA Binding Domain (PBD) located near the N-terminus of DNMT1 as being necessary for mediating interactions with the transcription factors described herein. Lastly, we provide evidence for DNMT1 serving as a co-repressor of ZBP-89 and GATA1 acting through upstream regulatory elements of the PU.1 and GATA1 gene loci.
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http://dx.doi.org/10.1016/j.bbagrm.2016.09.007DOI Listing
December 2016

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

GATA-1 Inhibits PU.1 Gene via DNA and Histone H3K9 Methylation of Its Distal Enhancer in Erythroleukemia.

PLoS One 2016 24;11(3):e0152234. Epub 2016 Mar 24.

Biocev and Pathological Physiology, 1st Faculty of Medicine, Charles University in Prague, Czech Republic.

GATA-1 and PU.1 are two important hematopoietic transcription factors that mutually inhibit each other in progenitor cells to guide entrance into the erythroid or myeloid lineage, respectively. PU.1 controls its own expression during myelopoiesis by binding to the distal URE enhancer, whose deletion leads to acute myeloid leukemia (AML). We herein present evidence that GATA-1 binds to the PU.1 gene and inhibits its expression in human AML-erythroleukemias (EL). Furthermore, GATA-1 together with DNA methyl Transferase I (DNMT1) mediate repression of the PU.1 gene through the URE. Repression of the PU.1 gene involves both DNA methylation at the URE and its histone H3 lysine-K9 methylation and deacetylation as well as the H3K27 methylation at additional DNA elements and the promoter. The GATA-1-mediated inhibition of PU.1 gene transcription in human AML-EL mediated through the URE represents important mechanism that contributes to PU.1 downregulation and leukemogenesis that is sensitive to DNA demethylation therapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0152234PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4807078PMC
August 2016

Oncogenic microRNA-155 and its target PU.1: an integrative gene expression study in six of the most prevalent lymphomas.

Int J Hematol 2015 Oct 11;102(4):441-50. Epub 2015 Aug 11.

Institute of Pathological Physiology, 1st Medical Faculty, Charles University in Prague, U Nemocnice 5, 128 53, Prague, Czech Republic.

The transcription factor PU.1 and its inhibitory microRNA-155 (miR-155) are important regulators of B-cell differentiation. PU.1 downregulation coupled with oncogenic miR-155 upregulation has been reported in lymphoid malignancies; however, these data have not been studied across different subtypes in relation to clinical outcomes. We studied expression of miR-155 and PU.1 in the six most prevalent human B-cell lymphomas (n = 131) including aggressive (DLBCL, HL, MCL) and indolent (B-CLL/SLL, MZL, FL) types. Levels of miR-155 and PU.1 inversely correlated in DLBCL, B-CLL/SLL, and FL tumor tissues. In HL tissues, an exceptionally high level of miR-155 was found in patients with unfavorable responses to first-line therapy and those who had shorter survival times. PU.1 downregulation was noted in B-CLL/SLL samples positive for the adverse prognostic markers CD38 and ZAP-70. Upregulation of miR-155 and downregulation of PU.1 expression are integral aspects of lymphoma biology that could mark aggressive behavior of some, but not all, lymphoma types.
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http://dx.doi.org/10.1007/s12185-015-1847-4DOI Listing
October 2015

Next-generation deep sequencing improves detection of BCR-ABL1 kinase domain mutations emerging under tyrosine kinase inhibitor treatment of chronic myeloid leukemia patients in chronic phase.

J Cancer Res Clin Oncol 2015 May 4;141(5):887-99. Epub 2014 Nov 4.

Institute of Hematology and Blood Transfusion, Prague, Czech Republic,

Purpose: Here, we studied whether amplicon next-generation deep sequencing (NGS) could improve the detection of emerging BCR-ABL1 kinase domain mutations in chronic phase chronic myeloid leukemia (CML) patients under tyrosine kinase inhibitor (TKI) treatment and discussed the clinical relevance of such sensitive mutational detection.

Methods: For NGS data evaluation including extraction of biologically relevant low-level variants from background error noise, we established and applied a robust and versatile bioinformatics approach.

Results: Results from a retrospective longitudinal analysis of 135 samples of 15 CML patients showed that NGS could have revealed emerging resistant mutants 2-11 months earlier than conventional sequencing. Interestingly, in cases who later failed first-line imatinib treatment, NGS revealed that TKI-resistant mutations were already detectable at the time of major or deeper molecular response. Identification of emerging mutations by NGS was mirrored by BCR-ABL1 transcript level expressed either fluctuations around 0.1 %(IS) or by slight transcript level increase. NGS also allowed tracing mutations that emerged during second-line TKI therapy back to the time of switchover. Compound mutants could be detected in three cases, but were not found to outcompete single mutants.

Conclusions: This work points out, that next-generation deep sequencing, coupled with a robust bioinformatics approach for mutation calling, may be just in place to ensure reliable detection of emerging BCR-ABL1 mutations, allowing early therapy switch and selection of the most appropriate therapy. Further, prospective assessment of how to best integrate NGS in the molecular monitoring and clinical decision algorithms is warranted.
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http://dx.doi.org/10.1007/s00432-014-1845-6DOI Listing
May 2015

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

Oncogenic microRNAs: miR-155, miR-19a, miR-181b, and miR-24 enable monitoring of early breast cancer in serum.

BMC Cancer 2014 Jun 18;14:448. Epub 2014 Jun 18.

1st Faculty of Medicine, Institute of Pathological Physiology, Charles University in Prague, Prague, Czech Republic.

Background: MicroRNAs (miRs) represent a distinct class of posttranscriptional modulators of gene expression with remarkable stability in sera. Several miRs are oncogenic (oncomiRs) and are deregulated in the pathogenesis of breast cancer and function to inhibit tumor suppressors. Routine blood monitoring of these circulating tumor-derived products could be of significant benefit to the diagnosis and relapse detection of early-stage breast cancer (EBC) patients.

Methods: Aim of this project was to determine expression of miR-155, miR-19a, miR-181b, miR-24, relative to let-7a in sera of 63 patients with EBC and 21 healthy controls. Longitudinal multivariate data analysis was performed to stochastically model the serum levels of each of the oncomiRs during disease phases: from diagnosis, after surgery, and following chemo/radiotherapy. Moreover, this analysis was utilized to evaluate oncomiR levels in EBC patients subgrouped using current clinical prognostic factors including HER2, Ki-67, and grade III.

Results: EBC patients significantly over-express the oncomiRs at the time of diagnosis. Following surgical resection the serum levels of miR-155, miR-181b, and miR-24 significantly decreased (p = 1.89e-05, 5.41e-06, and 0.00638, respectively) whereas the miR-19a decreased significantly after the therapy (p = 0.00869). Furthermore, in case of high-risk patients serum levels of miR-155, miR-19a, miR-181b, and miR-24 are significantly more abundant in comparison to low-risk group (p = 0.026, 0.02567, 0.0250, and 0.00990) and show a decreasing trend upon therapy.

Conclusions: OncomiRs are significantly more abundant in the sera of EBC patients compared to controls at diagnosis. Differences in oncomiR levels reflecting EBC risk were also observed. Testing the oncomiRs may be useful for diagnostic purpose and possibly also for relapse detection in follow-up studies of EBC.
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http://dx.doi.org/10.1186/1471-2407-14-448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4075993PMC
June 2014

Epigenetic control of SPI1 gene by CTCF and ISWI ATPase SMARCA5.

PLoS One 2014 3;9(2):e87448. Epub 2014 Feb 3.

Department of Pathophysiology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic ; Department of Medicine - Hematology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.

CCCTC-binding factor (CTCF) can both activate as well as inhibit transcription by forming chromatin loops between regulatory regions and promoters. In this regard, Ctcf binding on non-methylated DNA and its interaction with the Cohesin complex results in differential regulation of the H19/Igf2 locus. Similarly, a role for CTCF has been established in normal hematopoietic development; however its involvement in leukemia remains elusive. Here, we show that Ctcf binds to the imprinting control region of H19/Igf2 in AML blasts. We also demonstrate that Smarca5, which also associates with the Cohesin complex, facilitates Ctcf binding to its target sites on DNA. Furthermore, Smarca5 supports Ctcf functionally and is needed for enhancer-blocking effect at ICR. We next asked whether CTCF and SMARCA5 control the expression of key hematopoiesis regulators. In normally differentiating myeloid cells both CTCF and SMARCA5 together with members of the Cohesin complex are recruited to the SPI1 gene, a key hematopoiesis regulator and leukemia suppressor. Due to DNA methylation, CTCF binding to the SPI1 gene is blocked in AML blasts. Upon AZA-mediated DNA demethylation of human AML blasts, CTCF and SMARCA5 are recruited to the -14.4 Enhancer of SPI1 gene and block its expression. Our data provide new insight into complex SPI1 gene regulation now involving additional key epigenetic factors, CTCF and SMARCA5 that control PU.1 expression at the -14.4 Enhancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0087448PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911986PMC
December 2014

Role of epigenetics in chronic myeloid leukemia.

Curr Hematol Malig Rep 2013 Mar;8(1):28-36

Institute of Hematology and Blood Transfusion, U Nemocnice 1, Prague 2, 128 20, Czech Republic.

The efficacy of therapeutic modalities in chronic myeloid leukemia (CML) depends on both genetic and epigenetic mechanisms. This review focuses on epigenetic mechanisms involved in the pathogenesis of CML and in resistance of tumor cells to tyrosine kinase inhibitors leading to the leukemic clone escape and propagation. Regulatory events at the levels of gene regulation by transcription factors and microRNAs are discussed in the context of CML pathogenesis and therapeutic modalities.
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http://dx.doi.org/10.1007/s11899-012-0152-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3578734PMC
March 2013

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

Expression patterns of microRNAs associated with CML phases and their disease related targets.

Mol Cancer 2011 Apr 18;10:41. Epub 2011 Apr 18.

Institute of Hematology and Blood Transfusion, Prague, U Nemocnice 1, 128 20, Czech Republic.

Background: MicroRNAs are important regulators of transcription in hematopoiesis. Their expression deregulations were described in association with pathogenesis of some hematological malignancies. This study provides integrated microRNA expression profiling at different phases of chronic myeloid leukemia (CML) with the aim to identify microRNAs associated with CML pathogenesis. The functions of in silico filtered targets are in this report annotated and discussed in relation to CML pathogenesis.

Results: Using microarrays we identified differential expression profiles of 49 miRNAs in CML patients at diagnosis, in hematological relapse, therapy failure, blast crisis and major molecular response. The expression deregulation of miR-150, miR-20a, miR-17, miR-19a, miR-103, miR-144, miR-155, miR-181a, miR-221 and miR-222 in CML was confirmed by real-time quantitative PCR. In silico analyses identified targeted genes of these miRNAs encoding proteins that are involved in cell cycle and growth regulation as well as several key signaling pathways such as of mitogen activated kinase-like protein (MAPK), epidermal growth factor receptor (EGFR, ERBB), transforming growth factor beta (TGFB1) and tumor protein p53 that are all related to CML. Decreased levels of miR-150 were detected in patients at diagnosis, in blast crisis and 67% of hematological relapses and showed significant negative correlation with miR-150 proved target MYB and with BCR-ABL transcript level.

Conclusions: This study uncovers microRNAs that are potentially involved in CML and the annotated functions of in silico filtered targets of selected miRNAs outline mechanisms whereby microRNAs may be involved in CML pathogenesis.
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http://dx.doi.org/10.1186/1476-4598-10-41DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3102634PMC
April 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

GATA-1 directly regulates p21 gene expression during erythroid differentiation.

Cell Cycle 2010 May 15;9(10):1972-80. Epub 2010 May 15.

1Department of Cell Biology, Montefiore Medical Center, Bronx, NY, USA.

Lineage-determination transcription factors coordinate cell differentiation and proliferation by controlling the synthesis of lineage-specific gene products as well as cell cycle regulators. GATA-1 is a master regulator of erythropoiesis. Its role in regulating erythroid-specific genes has been extensively studied, whereas its role in controlling genes that regulate cell proliferation is less understood. Ectopic expression of GATA-1 in erythroleukemia cells releases the block to their differentiation and leads to terminal cell division. An early event in reprogramming the erythroleukemia cells is induction of the cyclin-dependent kinase inhibitor p21. Remarkably, ectopic expression of p21 also induces the erythroleukemia cells to differentiate. We now report that GATA-1 directly regulates transcription of the p21 gene in both erythroleukemia cells and normal erythroid progenitors. Using reporter, electrophoretic mobility shift, and chromatin immunoprecipitation assays, we show that GATA-1 stimulates p21 gene transcription by binding to consensus binding sites in the upstream region of the p21 gene promoter. This activity is also dependent on a binding site for Sp1/KLF-like factors near the transcription start site. Our findings indicate that p21 is a crucial downstream gene target and effector of GATA-1 during red blood cell terminal differentiation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3019278PMC
http://dx.doi.org/10.4161/cc.9.10.11602DOI Listing
May 2010

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

Nuclear localization of ISWI ATPase Smarca5 (Snf2h) in mouse.

Front Biosci (Elite Ed) 2009 Jun 1;1:553-9. Epub 2009 Jun 1.

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

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3556728PMC
http://dx.doi.org/10.2741/e53DOI Listing
June 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

Modifiers of epigenetic reprogramming show paternal effects in the mouse.

Nat Genet 2007 May 22;39(5):614-22. Epub 2007 Apr 22.

Epigenetics Laboratory, Queensland Institute of Medical Research, 300 Herston Road, Herston, Brisbane, Queensland 4006, Australia.

There is increasing evidence that epigenetic information can be inherited across generations in mammals, despite extensive reprogramming both in the gametes and in the early developing embryo. One corollary to this is that disrupting the establishment of epigenetic state in the gametes of a parent, as a result of heterozygosity for mutations in genes involved in reprogramming, could affect the phenotype of offspring that do not inherit the mutant allele. Here we show that such effects do occur following paternal inheritance in the mouse. We detected changes to transcription and chromosome ploidy in adult animals. Paternal effects of this type have not been reported previously in mammals and suggest that the untransmitted genotype of male parents can influence the phenotype of their offspring.
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http://dx.doi.org/10.1038/ng2031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3199608PMC
May 2007

Regulation of alphaA-crystallin via Pax6, c-Maf, CREB and a broad domain of lens-specific chromatin.

EMBO J 2006 May 4;25(10):2107-18. Epub 2006 May 4.

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

Pax6 and c-Maf regulate multiple stages of mammalian lens development. Here, we identified novel distal control regions (DCRs) of the alphaA-crystallin gene, a marker of lens fiber cell differentiation induced by FGF-signaling. DCR1 stimulated reporter gene expression in primary lens explants treated with FGF2 linking FGF-signaling with alphaA-crystallin synthesis. A DCR1/alphaA-crystallin promoter (including DCR2) coupled with EGFP virtually recapitulated the expression pattern of alphaA-crystallin in lens epithelium and fibers. In contrast, the DCR3/alphaA/EGFP reporter was expressed only in 'late' lens fibers. Chromatin immunoprecipitations showed binding of Pax6 to DCR1 and the alphaA-crystallin promoter in lens chromatin and demonstrated that high levels of alphaA-crystallin expression correlate with increased binding of c-Maf and CREB to the promoter and of CREB to DCR3, a broad domain of histone H3K9-hyperacetylation extending from DCR1 to DCR3, and increased abundance of chromatin remodeling enzymes Brg1 and Snf2h at the alphaA-crystallin locus. Our data demonstrate a novel mechanism of Pax6, c-Maf and CREB function, through regulation of chromatin-remodeling enzymes, and suggest a multistage model for the activation of alphaA-crystallin during lens differentiation.
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http://dx.doi.org/10.1038/sj.emboj.7601114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1462985PMC
May 2006

PU.1 inhibits the erythroid program by binding to GATA-1 on DNA and creating a repressive chromatin structure.

EMBO J 2005 Nov 13;24(21):3712-23. Epub 2005 Oct 13.

Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA.

Transcriptional repression mechanisms are important during differentiation of multipotential hematopoietic progenitors, where they are thought to regulate lineage commitment and to extinguish alternative differentiation programs. PU.1 and GATA-1 are two critical hematopoietic transcription factors that physically interact and mutually antagonize each other's transcriptional activity and ability to promote myeloid and erythroid differentiation, respectively. We find that PU.1 inhibits the erythroid program by binding to GATA-1 on its target genes and organizing a complex of proteins that creates a repressive chromatin structure containing lysine-9 methylated H3 histones and heterochromatin protein 1. Although these features are thought to be stable aspects of repressed chromatin, we find that silencing of PU.1 expression leads to removal of the repression complex, loss of the repressive chromatin marks and reactivation of the erythroid program. This process involves incorporation of the replacement histone variant H3.3 into nucleosomes. Repression of one transcription factor bound to DNA by another transcription factor not on the DNA represents a new mechanism for downregulating an alternative gene expression program during lineage commitment of multipotential hematopoietic progenitors.
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http://dx.doi.org/10.1038/sj.emboj.7600834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1276718PMC
November 2005