?-Hemoglobinopathies are among the most common inherited monogenic disorders, yet treatment options remain limited. These disorders are defined by an inability to produce enough functional adult hemoglobin and have a variety of severe health outcomes. One major therapeutic approach which has proven successful is to raise the expression level of fetal hemoglobin, a gene typically repressed over time during the first months after birth. However, treatment methods are severely impeded by a lack of mechanistic knowledge regarding fetal hemoglobin repression and hemoglobin switching. In this work, I propose experiments to investigate the post-transcriptional regulation of adult and fetal ?-type globin transcripts. Post-transcriptional regulation of ?-globin RNA has been observed at multiple levels. First, the biology of the Corfu deletion has shown post-transcriptional silencing of ?-globin transcripts derived from the Corfu allele despite the fact that new transcription of ?-globin is elevated from all Corfu deletion alleles. Additionally, it has been observed that treatment of CD34+ cells with salubrinal enhances the translation of ?-globin without affecting the transcript stability or localization of either HBB or HBG transcripts. Further, studies in reticulocytes which lack nuclei have demonstrated the capacity of butyrate to have similar effects despite its known role as an inhibitor of histone deacetylases. Finally, in a recent CRISPR screen to identify HbF repressors, 20/117 hit genes were associated with RNA binding, processing, or decay. Here, I propose experiments to follow up on DDX6, the RNA-binding protein recently identified in our laboratory as the most potent novel HbF repressor. While DDX6 is central to several distinct stages of RNA processing and decay including its role as a P-body constituent and as part of miRNA-mediated silencing, the role of DDX6 in repression of fetal hemoglobin remains largely unexplored. To identify mechanisms through which DDX6 may influence fetal hemoglobin expression, I will perform DDX6 eCLIP to identify RNA targets of DDX6 which may be relevant to fetal hemoglobin repression. Given the role of DDX6 as a P-body constitutent, I will assess the subcellular localization of DDX6 protein and globin transcripts in adult and fetal erythroid models. Further, I aim to directly test the ability of known DDX6 interacting proteins identified by HbF repressor screen to bind directly to ?-globin transcripts in the presence or absence of DDX6. Finally, I will use anti-sense oligo methods to pull down native hemoglobin transcripts and profile the RNA- binding proteins attached to each transcript. Gaining insight into the post-transcriptional regulation of fetal hemoglobin and to the role of DDX6 in fetal hemoglobin repression has the potential to identify new targets for therapeutic intervention for patients diagnosed with ?-Hemoglobinopathies including ?-Thalassemia and Sickle Cell Disease.
Recent work has identified new factors important for the repression of fetal hemoglobin, though the mechanism of this repression is not yet understood. Notably, long-standing observations from the field suggest post- transcriptional regulation of fetal hemoglobin expression, and several of these recent factors are RNA-binding proteins. This work will focus on unraveling these undescribed mechanisms of fetal hemoglobin repression in the hopes of identifying new druggable targets for ?-thalassemia and sickle cell disease.