Effects of the CTCF transcriptional factor in the erythroid cell differentiation and regulation of erythroid genes

Abstract

CCCTC-binding factor (CTCF) is a highly conserved zinc finger protein, which was first identified as a transcription factor regulating the c-MYC gene. Further studies revealed several new functions of CTCF including regulation of transcription, epigenetics as well as genome architecture. Due to its multivalent functions, it was reported by our group that CTCF also plays a pivotal role during differentiation of human myeloid leukemia cells. In this study, the effects of CTCF on human erythroid differentiation were investigated and CTCF target genes involved in erythroid differentiation were identified and analyzed. As a model system, K562, a pluripotent human leukemia cell line, was utilized. These cells can be induced chemically to differentiation by cytosine arabinoside (Ara-C) and Imatinib. Proliferation, differentiation and apoptosis analysis were performed upon induction. In order to investigate the role of CTCF in differentiation, CTCF was silenced using shRNA. K562 cells were infected with lentiviral particles containing shCTCF and the effect on erythroid differentiation was evaluated. Expression of erythroid markers was analyzed by western blot and RT-qPCR. Furthermore, the binding of CTCF to the regulatory regions of its putative target genes and changes upon differentiation were studied using ChIP (Chromatin Immunoprecipitation). It could be shown that CTCF knock-down inhibited erythroid differentiation of K562 cells. We aimed to extend these studies to primary hematopoietic precursors CD34+ cells purified from cord blood. Erythroid differentiation was induced with erythropoietin. Our data indicate a role of CTCF in the erythroid differentiation of hematopoietic cells.