The transcription factor C/EBPα is essential for myeloid differentiation and is frequently dysregulated in acute myeloid leukemia. Although studied extensively, the precise regulation of its gene by upstream factors has remained largely elusive. Here, we investigated its transcriptional activation during myeloid differentiation. We identified an evolutionarily conserved octameric sequence, CCCAGCAG, ∼100 bases upstream of the CEBPA transcription start site, and demonstrated through mutational analysis that this sequence is crucial for C/EBPα expression. This sequence is present in the genes encoding C/EBPα in humans, rodents, chickens, and frogs and is also present in the promoters of other C/EBP family members. We identified that ZNF143, the human homolog of the Xenopus transcriptional activator STAF, specifically binds to this 8-bp sequence to activate C/EBPα expression in myeloid cells through a mechanism that is distinct from that observed in liver cells and adipocytes. Altogether, our data suggest that ZNF143 plays an important role in the expression of C/EBPα in myeloid cells.

• MeSH
• aktivace transkripce * MeSH
• buněčné linie MeSH
• hematopoéza MeSH
• konzervovaná sekvence MeSH
• lidé MeSH
• myeloidní buňky cytologie   metabolismus   MeSH
• promotorové oblasti (genetika) * MeSH
• protein alfa vázající zesilovač transkripce CCAAT genetika   MeSH
• sekvence nukleotidů MeSH
• trans-aktivátory metabolismus   MeSH
• vazba proteinů MeSH
• vývojová regulace genové exprese MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Development of hematopoietic populations through the process of differentiation is critical for proper hematopoiesis. The transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) is a master regulator of myeloid differentiation, and the identification of C/EBPα target genes is key to understand this process. Here we identified the Ecotropic Viral Integration Site 2B (EVI2B) gene as a direct target of C/EBPα. We showed that the product of the gene, the transmembrane glycoprotein EVI2B (CD361), is abundantly expressed on the surface of primary hematopoietic cells, the highest levels of expression being reached in mature granulocytes. Using shRNA-mediated downregulation of EVI2B in human and murine cell lines and in primary hematopoietic stem and progenitor cells, we demonstrated impaired myeloid lineage development and altered progenitor functions in EVI2B-silenced cells. We showed that the compromised progenitor functionality in Evi2b-depleted cells can be in part explained by deregulation of cell proliferation and apoptosis. In addition, we generated an Evi2b knockout murine model and demonstrated altered properties of hematopoietic progenitors, as well as impaired G-CSF dependent myeloid colony formation in the knockout cells. Remarkably, we found that EVI2B is significantly downregulated in human acute myeloid leukemia samples characterized by defects in CEBPA. Altogether, our data demonstrate that EVI2B is a downstream target of C/EBPα, which regulates myeloid differentiation and functionality of hematopoietic progenitors.

• MeSH
• akutní myeloidní leukemie metabolismus   patologie   MeSH
• apoptóza MeSH
• buněčná diferenciace účinky léků   MeSH
• buňky kostní dřeně cytologie   MeSH
• down regulace účinky léků   MeSH
• estradiol farmakologie   MeSH
• faktor stimulující kolonie granulocytů farmakologie   MeSH
• granulocyty cytologie   metabolismus   MeSH
• hematopoetické kmenové buňky cytologie   metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• membránové glykoproteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• membránové proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• proliferace buněk účinky léků   MeSH
• promotorové oblasti (genetika) MeSH
• protein alfa vázající zesilovač transkripce CCAAT genetika   metabolismus   MeSH
• RNA interference MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Lung cancer is the most common cause of cancer deaths. The expression of the transcription factor C/EBPα (CCAAT/enhancer binding protein α) is frequently lost in non-small cell lung cancer, but the mechanisms by which C/EBPα suppresses tumor formation are not fully understood. In addition, no pharmacological therapy is available to specifically target C/EBPα expression. We discovered a subset of pulmonary adenocarcinoma patients in whom negative/low C/EBPα expression and positive expression of the oncogenic protein BMI1 (B lymphoma Mo-MLV insertion region 1 homolog) have prognostic value. We also generated a lung-specific mouse model of C/EBPα deletion that develops lung adenocarcinomas, which are prevented by Bmi1 haploinsufficiency. BMI1 activity is required for both tumor initiation and maintenance in the C/EBPα-null background, and pharmacological inhibition of BMI1 exhibits antitumor effects in both murine and human adenocarcinoma lines. Overall, we show that C/EBPα is a tumor suppressor in lung cancer and that BMI1 is required for the oncogenic process downstream of C/EBPα loss. Therefore, anti-BMI1 pharmacological inhibition may offer a therapeutic benefit for lung cancer patients with low expression of C/EBPα and high BMI1.

• MeSH
• adenokarcinom genetika   metabolismus   patologie   terapie   MeSH
• lidé MeSH
• mutace genetika   MeSH
• myši knockoutované MeSH
• myši MeSH
• nádory plic genetika   metabolismus   patologie   terapie   MeSH
• PRC1 genetika   metabolismus   MeSH
• protein alfa vázající zesilovač transkripce CCAAT genetika   metabolismus   MeSH
• protoonkogenní proteiny genetika   metabolismus   MeSH
• regulace genové exprese u nádorů genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

The CCAAT/enhancer-binding protein alpha, encoded by the intronless CEBPA gene, is a transcription factor that induces expression of genes involved in differentiation of granulocytes, monocytes, adipocytes and hepatocytes. Both mono- and bi-allelic CEBPA mutations were detected in acute myeloid leukaemia and myelodysplastic syndrome. In this study we also identified CEBPA mutations in healthy individuals and in patients with peripheral artery disease, ischaemic heart disease and hyperlipidaemia. We found 16 various deletions with the presence of two direct repeats in CEBPA by analysis of 431 individuals. Three most frequent repeats included in these deletions in CEBPA gene are CGCGAG (493- 498_865-870), GG (486-487_885-886), and GCCAAGCAGC (508-517_907-916), all according to GenBank Accession No. NM_004364.2. In one case we identified that a father with ischaemic heart disease and his healthy son had two identical deletions (493_864del and 508_906del, both according to GenBank Accession No. NM_004364.2) in CEBPA. The occurrence of deletions between two repetitive sequences may be caused by recombination events in the repair process. A double-stranded cut in DNA may initiate these recombination events in adjacent DNA sequences. Four types of polymorphisms in the CEBPA gene were also detected in the screened individuals. Polymorphism in CEBPA gene 690 G>T according to GenBank Accession No. NM_004364.2 is the most frequent type in our analysis. Statistical analysis did not find significant differences in the frequency of polymorphisms in CEBPA in patients and in healthy individuals with the exception of P4 polymorphism (580_585dup according to GenBank Accesion No. NM_004364.2). P4 polymorphism was significantly increased in ischaemic heart disease patients.

• MeSH
• financování organizované MeSH
• hyperlipidemie genetika   MeSH
• ischemická choroba srdeční genetika   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• mutační analýza DNA MeSH
• onemocnění periferních cév genetika   MeSH
• polymorfismus genetický MeSH
• protein alfa vázající zesilovač transkripce CCAAT genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• Check Tag
• lidé MeSH

In recent years, several independent prognostic factors in cytogenetically normal acute myeloid leukemia (CN-AML) have been reported. Mutations or the expression levels of certain genes have been often used as molecular markers for prediction of a patient's outcome or for evaluation of treatment outcome. One of them, the gene encoding CCAAT/enhanced binding protein alpha (CEBPA), plays an important role in myeloid differentiation and, when mutated, confers a favorable prognosis for patients with CN-AML. Complete mutation screening of the CEBPA gene is therefore beneficial and requires fast, precise, and sensitive diagnostic tools. Thus, for routine diagnostics, we developed a screening method using high-resolution melt curve analysis prior to direct sequencing, where only positive samples (according to reference) are further sequenced. With this approach, all positive and negative patients were successfully distinguished, and the results obtained were in absolute concordance with the direct sequence analysis.

• MeSH
• denaturace nukleových kyselin MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutace genetika   MeSH
• mutační analýza DNA metody   MeSH
• protein alfa vázající zesilovač transkripce CCAAT genetika   MeSH
• sekvence nukleotidů MeSH
• tranzitní teplota MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

• MeSH
• finanční podpora výzkumu jako téma MeSH
• financování organizované MeSH
• hematopoéza MeSH
• inhibitory růstu genetika   chemie   metabolismus   MeSH
• leukemie metabolismus   patologie   MeSH
• lidé MeSH
• nádorové supresorové proteiny metabolismus   MeSH
• onkogenní proteiny genetika   metabolismus   MeSH
• protein alfa vázající zesilovač transkripce CCAAT genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH