PO-104 Role of the Oncosuppressor CTCF in the Erythroid Cell Differentiation and Regulation of Erythroid Genes

Introduction CTCF (CCCTC-binding factor) is a highly conserved 11-zinc finger protein that was identified as a MYC regulator. CTCF has several functions including transcriptional activation or repression, insulator binding properties and global organisation of chromatin. In addition, CTCF is involved in the epigenetic regulation of many cancer related genes and is considered as an oncosuppressor. Erythropoiesis is the process of red blood cell formation originating from hematopoietic stem cells. It is highly regulated by growth factors, transcription factors and miRNAs. Previous results of our group indicate that overexpression of CTCF induces differentiation towards the erythroid lineage in leukaemia cells, accompanied with the differential expression of erythroid specific genes. In this study, we investigate the role of CTCF during erythroid differentiation and the regulation of erythroid genes by CTCF. Material and methods As experimental models we used K562, a multipotent human leukaemia cell line, and primary CD34+ hematopoietic stem cells purified from cord blood. CTCF was silenced using lentiviral particles containing shRNA specific for CTCF. Erythroid differentiation was induced by cytosine arabinoside (Ara-C) or Imatinib in K562 cells and by erythropoietin (EPO) in CD34+ cells. Erythroid differentiation was analysed by cell proliferation assays, percentage of haemoglobin producing cells (benzidine test), colony forming units and expression of erythroid markers (Western-Blot and RT-qPCR). Chromatin immunoprecipitation (ChIP) experiments were performed to study the binding of CTCF to the regulatory regions of genes involved in the erythroid differentiation. Results and discussions CTCF was silenced in K562 and CD34+ cells and erythroid differentiation was induced by Ara-C, Imatinib or EPO. Downregulation of CTCF in both K562 and CD34+ cells reduced the cell proliferation, the percentage of benzidine positive cells and the expression of erythroid marker genes. Additionally, in CD34+ cells a reduction in the number of erythroid colonies was observed. CTCF occupancy in the regulatory regions of erythroid specific genes was found. Moreover, CTCF binding to these genes changed upon induction of erythroid differentiation. These results are in agreement with our previous findings. Conclusion Our results demonstrate that CTCF downregulation inhibits erythroid differentiation through the regulation of erythroid specific genes. We suggest a pivotal role of CTCF during erythroid differentiation.