Understanding the mechanisms that regulate the developmental switch from fetal to adult haemoglobin has been fundamental to the emergence of novel therapeutic approaches for haemoglobinopathies that affect adult haemoglobin, such as sickle-cell disease, by reactivating fetal gamma-globin. DNA methylation is an epigenetic mark that has received much attention for its putative roles in gene regulation in recent years, however its role in haemoglobin switching has remained largely unexplored. Recent work has implicated Methyl-Binding Domain protein 2 (MBD2), a part of the Nucleosome Remodelling and Deacetylase complex (NuRD) that reads DNA methylation, as an important component in the repression of the gamma-globin gene.
To study the contribution of DNA methylation in haemoglobin switching, we have applied CRISPR-Cas9 genome editing in the adult-like Human Umbilical cord Derived Erythroid Progenitor 2 (HUDEP-2) cell line to precisely impair MBD2’s DNA methylation reading function. In a complementary approach, we have used dCas9-TET1cd based epigenome editing to investigate the impact of DNA methylation directly at the gamma-globin promoter. Gene expression analysis including RNA-seq and qPCR have allowed us to gain insights into the importance of DNA methylation in haemoglobin switching.
Our work has shown that interfering with DNA methylation both at the gamma-globin promoter, and through manipulating MBD2, results in strong de-repression of the gamma-globin gene, while not significantly altering the expression of other factors involved in haemoglobin switching. This suggests that DNA methylation is directly involved in regulating gamma-globin. Ongoing work aims to further characterise this mechanism by investigating the impact of MBD2’s methylation reading functions on chromatin localisation of the NuRD complex, and how this alters nucleosome positioning at affected sites.
This study has provided direct evidence for the involvement of DNA methylation in haemoglobin switching, with results having therapeutic potential if this mechanism could be further understood and targeted.