Common inherited hemoglobin disorders of the beta-globin gene, sickle cell disease and ?-thalassemia, cause significant morbidity and mortality worldwide. It has long been appreciated that gamma globin, which is expressed during fetal development can ameliorate these diseases when it is expressed postnatally in the erythroid precursors. However, the expression of significant levels of gamma postnatally is uncommon. The goal of this application is to reveal the mechanisms by which the gamma genes are silenced postnatally in order to generate molecular targets for pharmacologic activation of the gamma genes when it would be therapeutically appropriate. This project will investigate the hypothesis that gamma is silenced by modification of chromatin around the gamma genes and will attempt to identify the molecules involved in marking that chromatin for silencing. Chromatin structure will be investigated in gamma expressing erythroid precursors from human fetal liver and gamma nonexpressing erythroid precursors from human bone marrow. The hypothesis is that DNA methylation clearly shows that the gamma genes are marked for silencing and this methylation is preceded by methylation of histone H3 on lysine 9 and the binding of HP1 proteins to this methylated histone.
Specific aim 1 will utilize bisulphite genomic sequencing and methylation sensitive restriction enzyme analysis to map the methylation of CpG dinucleotides in the promoters of all the expressed genes and the region around the gamma genes.
Specific aim 2 will use the chromatin immunoprecipitation assay to examine the areas that are differentially methylated between bone marrow erythroid precursors and fetal liver erythroid precursors for histone H3 lysine 9 methylation and HP1 binding.
Specific aim 3 will utilize the Affymetrix genechip microarray system to identify differentially transcribed genes between bone marrow erythroid precursors and fetal liver erythroid precursors. Differentially transcribed genes that are associated with transcriptional activation, transcriptional silencing, chromatin modifications or signaling from the cell surface to the nucleus will be further evaluated for involvement in gamma silencing. This evaluation will vary depending on the gene, but will include genomic assessment for location in the genome, expression information and homology to other proteins, and experimental assessment using biochemical and genetic approaches.
