Disruption of the C/EBP? - MiR-182 balance impairs granulocytic differentiation

10.1038/s41467-017-00032-6Nature Communications813

Disturbance of the C/EBP alpha-miR-182 balance impairs granulocytic differentiation and promotes development of acute myeloid leukemia

Introduction: Silencing of major myeloid transcription factor C/EBPa by gene mutation, promoter hypermethylation or posttranslational modifications is well described and occurs in ~50% of acute myeloid leukemia (AML) cases. Deregulation of C/EBPa by microRNAs, a class of small non-coding RNAs, as a substantial event during AML development or during myeloid differentiation has not been studied yet. Methods: We screened for C/EBPa dependent miRNAs in inducible K562-C/EBPa-ER cells using Illumina’s Next Generation Sequencing. For in vitro functional studies including gain-of-function and loss-of-function experiments, we utilized common acute myeloid leukemia cell lines, human hematopoietic stem cells from umbilical cord blood, murine hematopoietic stem cells from mouse bone marrow and primary human cells from patients with acute myeloid leukemia. For in vivo investigations, we manipulated Lin-Sca-1+c-Kit+ (LSK) murine hematopoietic progenitor cells by lentiviral infection and transplanted them into lethally irradiated littermates. The resulting phenotype was analyzed by flow cytometry and morphological staining of blood and bone marrow. Results: We identified oncogenic miR-182 as strong regulator of C/EBPa during myeloid differentiation and in AML. Moreover, we uncovered a novel regulatory loop between C/EBPa and miR-182. While C/EBPa blocks miR-182 expression by direct promoter binding during myeloid differentiation, enforced expression of miR-182 leads to reduced C/EBPa protein levels and impairs granulopoiesis in vitro and in a transplantation based mouse model in vivo. In contrast to this, a knockdown of miR-182 expression enhances C/EBPa protein levels in human AML. Furthermore, we observed highly elevated miR-182 expression levels particularly in AML patients with C-terminal CEBPA mutations and thereby uncovered a mechanism how C/EBPa blocks miR-182 expression. Finally by evaluation of TCGA database, we identified miR-182 expression as a strong adverse prognostic factor in cytogenetically high risk AML patients. Conclusions: Taken together, our data demonstrate the importance of a controlled balance between C/EBPa and miR-182 for the maintenance of healthy granulopoiesis and might uncover a novel mechanism for potential treatment strategies in AML. Disclosure: No conflict of interest disclosed

ZNF143 protein is an important regulator of the myeloid transcription factor C/EBP

10.1074/jbc.M117.811109JOURNAL OF BIOLOGICAL CHEMISTRY2924618924-1893

ZNF143 is an important regulator of the myeloid transcription factor C/EBPα

The transcription factor C/EBP 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, approximate to 100 bases upstream of the CEBPA transcription start site, and demonstrated through mutational analysis that this sequence is crucial for C/EBP expression. This sequence is present in the genes encoding C/EBP 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/EBP 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/EBP in myeloid cells

EVI2B is a C/EBPα target gene required for granulocytic differentiation and functionality of hematopoietic progenitors

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