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

Disruption of the C/EBPα-miR-182 balance impairs granulocytic differentiation

Transcription factor C/EBPα is a master regulator of myelopoiesis and its inactivation is associated with acute myeloid leukemia. Deregulation of C/EBPα by microRNAs during granulopoiesis or acute myeloid leukemia development has not been studied. Here we show that oncogenic miR-182 is a strong regulator of C/EBPα. Moreover, we identify a regulatory loop between C/EBPα and miR-182. While C/EBPα blocks miR-182 expression by direct promoter binding during myeloid differentiation, enforced expression of miR-182 reduces C/EBPα protein level and impairs granulopoiesis in vitro and in vivo. In addition, miR-182 expression is highly elevated particularly in acute myeloid leukemia patients with C-terminal CEBPA mutations, thereby depicting a mechanism by which C/EBPα blocks miR-182 expression. Furthermore, we present miR-182 expression as a prognostic marker in cytogenetically high-risk acute myeloid leukemia patients. Our data demonstrate the importance of a controlled balance between C/EBPα and miR-182 for the maintenance of healthy granulopoiesis

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