Inhibition of G9a methyltransferase stimulates fetal hemoglobin production by facilitating LCR/g-globin looping

In humans, the b-globin cluster contains fetal gand g-globin and adult dandb-globin genes. Around the timeof birth, fetal hemoglobin (HbF) is almost completely replaced by adult hemoglobin (HbA) containing 2 b-globin chains. Based upon this developmental transition in hemoglobin production, mutations in the b-globin gene locus can cause a variety of hemoglobinopathies including sickle cell disease and b-thalassemia. One longstanding goal for developing treatments for these b-hemoglobinopathies is the reactivation and increased expression of HbF in adult erythroid cells. Therefore, considerable research effort has been focused upon understanding the mechanisms that underlie g-globin gene repression during the developmental switch between HbF and HbA that could suggest new therapeutic approaches for these diseases. Expression of b-globin genes is regulated by physical interactions betweengenepromoters and the locus control region (LCR)enhancer. Experiments using RNA interference have shown that this interaction is facilitated by the LDB1/LMO2/GATA-1/TAL1 erythroid-specific protein complex (LDB1 complex). The LDB1 complex occupies the LCR and the b-globin gene promoter and provides chromatin loop formation between them through interaction between LDB1 homodimerization domains. Mouseb-globingenes are also regulatedby theG9a/EHMT2H3K9 histone methyltransferase. G9a contains a SET domain responsible for histone H3K9 monoand di-methylation associated with repression of gene expression. Interestingly, recent observations support the view that G9a can play a role in activation of gene expression independently from its repressive methyltransferase activity. In human cells, G9a functions as a stable heteromeric complex with a related protein, GLP (EHMT1). UNC0638 specifically inhibits methyltransferase activity of G9a and GLP, causing a strong decrease in bulk H3K9me2 and reactivation of G9a-silenced genes in mouse embryonic stem cells. UNC0638 treatment of CD34 hematopoietic progenitor cells delayed adoption of differentiated phenotypes, suggesting an important role for G9a in lineage specification. Moreover, brief treatment of these cells with UNC0638 activated fetal g-globin genes in parallel with repression of adult dand b-globin genes, reversing the normal sequence of events that occurs late in erythroid differentiation. The mechanistic role of G9a in epigenetic regulation of the b-globin locus remains unclear. Here, we investigated the role of G9a in silencing fetal g-globin genes and activation of adult dand b-globin genes during ex vivo differentiation of CD34 adult hematopoietic progenitor cells.We found thatUNC0638 treatment acts primarily upon erythroblasts as they acquire a glycophorin A positive (GPA) phenotype in response to erythropoietin, and we show that G9a is directly involved in epigenetic repression of the human g-globin genes.