shRNA screen for novel epigenetic regulators of γ-globin silencing

© 2016 Dr. Betty Rui-Yun Kaoβ-thalassemia is a common blood disorder caused by mutations within the β-globin gene that result in insufficient hemoglobin production, reduced red blood cell half-life and life-threatening anemia. High expression of γ-globin has been shown to ameliorate the symptoms of β-thalassemia. However, high expression of γ-globin only persists during fetal development, and is downregulated postnatally. Reactivation of developmentally silenced γ-globin represents an attractive therapeutic strategy for β-thalassemia. The molecular mechanisms underpinning γ-globin silencing remain unclear, but recent studies have revealed epigenetic processes play an important role. Better understanding of the epigenetic mechanisms that control γ-globin silencing could identify novel targets for therapeutic reactivation of γ-globin. This PhD project aimed to identify novel epigenetic regulators of γ-globin silencing. The GG-MEL cell line was generated as a genomic reporter assay suitable for use in an shRNA screen. This cell line contains the human β-globin locus modified to express GFP under the control of the γ-globin promoter. Induction of γ-globin is detected as increased GFP expression. This reporter cell line was used to screen an shRNA library containing 1027 shRNAs targeting 158 genes encoding epigenetic regulators. Knockdown of the top candidate gene resulted in induction of γ-globin expression in primary culture of erythroid cells derived from murine fetal liver. Investigation of the molecular mechanism by which this gene regulates γ-globin silencing is ongoing. To date, we have shown the candidate gene directly binds the β-globin locus and knockdown results in downregulation of β-globin expression. This suggests that this gene may be an activator of β-globin expression and downregulates γ-globin expression through a competitive model. Further investigation, including knockdown of the candidate gene in primary culture of erythroid cells derived from β-thalassemia patients, may confirm it as a novel target for therapeutic reactivation of γ-globin