Reactivation of fetal hemoglobin expression: functional analysis of candidate modifiers in vivo.

Conditions affecting the function of beta-globin, known as beta-thalassemias and sickle cell disease, are the most common monogenetic disorders in the human population. It is known that expression of the fetal gamma-globin genes greatly ameliorates the effects of these diseases. Intense research efforts by us and other groups have recently led to the identification of the first transcription factors that normally suppress the human gamma-globin genes when the expression of the fetal gamma-globin genes switches to that of the adult delta- and beta-globin genes. In addition, in a recent shRNA screen we have identified a number of novel potential modifiers of gamma-globin expression. These novel factors not yet been subjected to stringent genetic tests. The aim of this proposal is therefore to functionally characterize these novel factors in vivo, using mice as the model system. We will use compound transgenic mice carrying the complete human beta-globin locus, conditional knockout alleles of potential gamma-globin modifier genes, and a Cre knockin allele in the erythropoietin receptor locus. We will determine the effect of erythroid-specific ablation of the potential gamma-globin modifiers on expression of the globin genes during the transition from fetal to adult erythropoiesis, and in adulthood. In addition, we will assess the properties of the erythroid system using a panel of standardized tests. In the erythropoietic tissues (fetal liver, bone marrow and spleen), we will perform FACS analysis to assess the distribution of erythroid progenitors at various maturation stages. In peripheral blood, we will measure standard hematological parameters and determine the percentage of immature erythrocytes (reticulocytes). Of the mature erythrocytes, we will measure the half life, size distribution, volume, shape, hemoglobin content, osmotic fragility, membrane rigidity and presence of pathological lesions such as nuclear remnants (Howell-Jolly bodies) and membrane blebbing. Molecularly, we will use RNA-seq for transcriptome profiling and ChIP-seq for epigenetic profiling of erythropoietic tissues. Comparative analysis with control fetal (gamma-globin on) and adult (gamma-globin off) samples will allow reconstruction of the pathways regulating globin switching and enable identification of key components serving as targets for gamma-globin reactivation in adults. The long term objective is to develop reagents that interfere with these key components to achieve reactivation of the gamma-globin genes in adult sickle cell disease and beta-thalassemia patients.