CtBP represses adult β-like globin expression in haematopoiesis

Abstract

Haemoglobin consists of two α- and two β-like globin chains, responsible for oxygen transport through the body. During development, different haemoglobin types are produced, giving rise to first embryonic haemoglobin followed by foetal and finally adult haemoglobin. Understanding this switch in expression and the mechanisms regulating β-like globin genes is of interest, not only as a model system for understanding gene regulation, but also since research has shown that beneficial mutations causing the persistent activation of foetal haemoglobin are able to ameliorate symptoms of defective adult globin genes. Transcription factors Kruppel-like Factor 3 (KLF3), Kruppel-like Factor 8 (KLF8), and Sry-related HMG box 6 (SOX6) are expressed in erythroid cells. In Klf3 and Klf8 double knockout mice, moderate de-repression of embryonic β-like globin genes is seen. Sox6 null mice show persistent expression of embryonic β-like globin. KLF3, KLF8, and SOX6 all contain a PIDLS motif which enables binding of the transcriptional co-repressor C-terminal Binding Protein (CtBP). CtBP has an important role in many transcriptional pathways. To investigate the role of KLF3, KLF8, SOX6, and CtBP in globin gene expression, we used CRISPR/Cas9 technology to generate, Klf3-/-, Klf8-/-, CtBP1-/-, CtBP2-/-, CtBP1CtBP2-/- and Sox6-/- erythroid cell lines. We also examined the importance of the PIDLS motif in β-like globin gene regulation by mutating the PIDLS motif to a PIASS motif in Klf3 and Sox6. We found that β-like globin expression was deregulated by the ablation of KLF3, KLF8 and SOX6 with good correlation to previous studies. Our major novel finding was of the importance of CtBP1 and CtBP2 in regulating β-like globin expression. Undifferentiated CtBP1CtBP2-/- MEL exhibited de-repression of the adult β-like globin genes. It is possible that the deregulation of β-like globin gene expression in the CtBP1CtBP2-/- MEL was due in part to the concurrent de-repression of Klf8 and Sox6 and their role in silencing embryonic β-like globin genes. Microarray and further analysis of CtBP1CtBP2-/- MEL revealed deregulation of erythropoiesis regulatory gene, Id2. Overall, our data suggests that CtBP plays an interesting role in haematopoiesis; in normal undifferentiated erythroid progenitor cells, CtBP co-represses the expression of key erythroid regulatory transcription factors, and regulates the β-like globin gene locus.