Cooperative Repression of γ-Globin by LYAR and NONO

Developmental silencing of γ-globin is a central event in hemoglobin switching, and reactivation of γ-globin expression effectively alleviates the severity of β-thalassemia and sickle cell disease (SCD). However, the precise regulatory mechanisms governing γ-globin repression remain incompletely defined. Here, we report that LYAR (Ly-1 antibody reactive clone, human homologue) and NONO (non-POU domain-containing octamer-binding protein) physically interact and co-occupy the γ-globin promoter to jointly repress its transcription. Knockdown of LYAR significantly reduced the recruitment of NONO to the γ-globin promoter. Notably, co-depletion of LYAR and NONO in K562 and HUDEP-2 cells resulted in additive induction of γ-globin expression that exceeded the effects of individual knockdowns and was comparable to BCL11A depletion, the established master regulator of HbF silencing. To validate these findings in vivo, we generated a conditional double knockout mouse model using IFN-inducible Mx1-Cre transgenic mice. Induced hematopoietic deletion of both LYAR and NONO in adult β-YAC transgenic mice led to significant reactivation of both murine embryonic globin genes and human γ-globin expression, indicating evolutionary conservation of this regulatory mechanism across mammals. Collectively, our findings identify LYAR and NONO as a functionally cooperative repressor complex in γ-globin silencing and establish co-targeting of these factors as a novel, and potent therapeutic strategy for elevating HbF levels in hemoglobinopathies.