Haemoglobin is a tetramer consisting of two α-globin and β-globin like chains. During development, the globin genes are sequentially expressed, giving rise to embryonic, foetal and finally adult haemoglobin. This process of globin switching is of much interest, as research has shown that beneficial mutations causing the persistent activation of foetal haemoglobin are able to ameliorate the symptoms inherited blood disorders like β-thalassemia and sickle cell disease, which are caused by defective adult globin genes.

KLF3, KLF8, CtBP1, CtBP2 and SOX6 are transcriptional repressors and co-repressors that play roles in red blood cell development. Kruppel-like Factor 3 (KLF3) is a transcription factor that is abundantly expressed in erythroid cells and operates in a feedback mechanism with a related protein, KLF1, that is essential for erythropoiesis. Klf3 knockout mice exhibit enlarged spleens, abnormal erythrocytes in peripheral blood and impaired erythroid maturation in the liver. When both KLF3 and another related protein KLF8 are knocked out, mice shows de-repression of embryonic globin genes.

C-terminal Binding Protein (CtBP) plays an important role in many developmental and biological pathways. CtBP2 is a corpressor that is recruited by both KLF3 and KLF8 through binding to a PIDLS motif. SOX6 also contains a PIDLS motif that allows it to recruit CtBP2 to repress gene transcription. SOX6 knockout mice show persistent expression of embryonic globins and knock downs in human erythroblasts causes an up-regulation of foetal globin.

To further investigate the role of these transcription factors in globin gene expression, we used CRISPR-Cas9 technology to generate knockout cell lines, Klf3-/-, Klf8-/-, CtBP2-/- and Sox6-/-. We also examined the importance of the PIDLS motif in globin gene regulation by mutating the PIDLS motif to a PIASS motif (∆DL) in Klf3 and Sox6. We have used microarrays to examine how loss of these repressors and co-repressors affects gene expression, with a particular focus on genes in the globin locus. We are now following up our findings from mouse erythroid cells lines in human erythroid cells.