Mi2b-mediated silencing of the fetal g-globin gene in adult erythroid cells

Hemoglobinopathies such as sickle cell anemia and b-thalassemia result from among the most common single gene defects worldwide. A promising approach for the treatment of these conditions is through the induction of increased fetal hemoglobin (HbF) expression. Hydroxyurea, which is currently part of the standard treatment of sickle cell anemia, causes increased expression of HbF. However, the level of HbF induced in patients is variable, and hydroxyurea is not effective in the treatment of b-thalassemia. Development of effective and potentially less toxic targeted strategies to induce HbF production will require full understanding of the molecular basis of developmental repression of the fetal g-globin gene. The g-globin gene is located on chromosome 11 within the b-globin gene locus, which consists of a group of 5 b-type globin genes positioned in the order in which they are expressed during development and preceded by a locus control region (59-LCR-e-gg-d-b-39). During the embryonic stage of development, the «-globin gene is expressed in the yolk sac, followed by expression of the g-globin gene in the fetal liver during most of gestation. At birth, g-globin expression declines as the expression of adult b-globin in bone marrow–derived erythroid cells predominates. There are numerous trans-acting factors and associated complexes involved in g-globin gene silencing. These include BCL11A, KLF1/EKLF, MBD2/NuRD, TR2/TR4, and GATA-1/FOG-1/NuRD. Among these, KLF1 (EKLF), a member of the Krüppel family of transcription factors, is critical in the expression of many erythroid-specific genes. KLF1/EKLF binds directly to, and positively regulates, the b-globin gene in adult erythroid cells. It also negatively regulates the g-globin gene indirectly through its role in mediating competition between the gand b-globin promoters for the LCR and by binding to and positively regulating expression of BCL11A, an important g-globin gene silencing factor. Originally identified in a Genome-wide Association Study (GWAS) study, BCL11A is a zinc finger transcription factor that acts as a dominant negative regulator of the embryonic to adult hemoglobin switch during murine development. It binds to the locus control region and to an intergenic region located between the g-globin and d-globin genes. Knockout of BCL11A in a humanized sickle cell transgenic mouse model greatly ameliorates the sickle cell disease phenotype. Epigenetic mechanisms, including DNA methylation and histone modifications, also play an important role in developmental globin gene silencing, and inhibitors of DNA methylation induce increased HbF levels in baboons and in humans. The MBD2/ NuRD complex, which selectively binds to methylated CpG-rich DNA, has been shown to play an important role in the silencing of the human embryonic «and fetal g-globin genes. NuRD corepressor complexes include >1 copy of each of the proteins Mi2a and -b, HDAC-1 and -2, MTA-1 and -2, RbAp46/48, and p66a and p66b. MBD2/NuRD does not appear to interact directly with promoters of human b-type globin genes, suggesting that its silencing effects occur through an indirect pathway.