Poster Presentation 43rd Lorne Genome Conference 2022

Exploring the potential of CGATA motif methylation to regulate GATA1 binding in the β-globin locus (#112)

Sonia Goozee 1 , Henry Bell 1 , Kate Quinlan 1 , Merlin Crossley 1
  1. University of New South Wales (UNSW), Chatswood, NSW, Australia

Strict regulation of gene expression in the β-globin locus is essential to properly allow globin switching, a fascinating phenomenon describing the changing expression of haemoglobin throughout development. The reversal of globin switching has been shown to alleviate symptoms of beta-haemoglobinopathies such as sickle cell disease. The master erythroid transcription factor, GATA1, is vital in globin locus regulation. GATA1 typically binds to the DNA motifs AGATA and TGATA, as well as CGATA; but only when the sequence is unmethylated. It was discovered 40 years ago that demethylation of the γ-globin gene promotor was associated with an increase in the gene’s expression. This relationship was later found to be shared by both ε-globin and β-globin, however the reason for this relationship is still unknown. It is currently unexplored whether GATA1 binding to the β-globin locus could be regulated by methylation of CGATA elements. In our work, data from the UCSC Genome Browser and ENCODE database was used to identify CGATA sites on the human β-globin locus and ascertain their methylation level. ChIP-qPCR was then used to assess GATA1 binding to these sites. To further explore methylation in the β-globin locus, a novel ‘methyl-binding’ GATA1 protein was synthesised. An electrophoretic mobility shift assay was used to confirm the preferential binding of methyl-binding GATA1 to methylated consensus sequences. Finally, a human erythroid cell line which stably expresses an inducible methyl-binding GATA1 was created. In this work, we have identified CGATA elements in the locus and shown a potential correlation between low CpG methylation and wildtype GATA1 binding in human erythroid cells. This suggests that CGATA methylation may be a mechanism utilised in red blood cells to block GATA1 binding and regulation of the β-globin locus, providing a potential explanation for the historical link between DNA methylation and globin gene regulation.