The goals of this research proposal are to investigate the control of fetal hemoglobin in humans and to develop approaches for in vivo induction of fetal hemoglobin in the adult.
Our Specific Aims are: 1) Investigate the interaction of two transcriptional factors, FKLF-1 and 2 with the gamma globin gene promoter in vivo using chromatin immunoprecipitation (CHIP) assays. Specifically, a) Determine the in vivo binding of FKLF-1 and FKLF-2 on sequences of the gamma globin promoter and the LCR. b) Investigate whether the interactions between FKLF-1 and FKLF-2 and the globin gene promoters change during development, c) Determine the non-globin gene targets of the two transcriptional factors. 2) Test whether the gamma globin gene can be transactivated in vivo and ex vivo by FKLF-1 and FKLF-2. Specifically, a) Investigate the correlation between FKLF expression and gamma gene expression in BFUe colonies of individuals with heterocellular elevations of Hb F. b) Test whether forced overexpression of FKLF-1 and FKLF-2 induces Hb F in BFUe colonies, c) Test whether erythroid lineage specific FKLF overexpression will induce gamma gene expression in transgenic mice carrying 7 globin gene constructs or [3 locus YACs. 3) Examine the effects of targeted inactivation of FKLF-1 and FKLF-2 on hematopoiesis and on fetal globin expression, a) Effects on fetal and embryonic globin expression will be assessed on FKLF-/- mice carrying the human beta locus YAC. If FKLF inactivation proves to be embryonic lethal, effects on gamma gene expression will be assessed b) in FKLF -/- ES cells produced from blastocysts of beta locus YAC mice and c) in somatic cell hybrids formed by fusing FKLF deficient MEL cells with human fetal erythroblasts. 4) Continue the search for transcriptional factors interacting with the gamma globin gene CACCC box, using a one hybrid assay we have developed. 5) Investigate mechanisms of gamma gene silencing using beta locus YAC transgenic mice with emphasis on the function and analysis of a silencing element we have previously identified to be located in the upstream 3' globin gene promoter. 6) Investigate the induction of Hb F in the adult by histone deacetylase (HDAC) inhibitors. Specifically, a) Test the in vivo induction of y gene by HDAC inhibitors we have shown to be potent Hb F inducers in screening assays, b) Search for new Hb F inducers among HDAC inhibitors used in cancer clinical trials, c) Continue the search for new gamma gene inducers among HDAC inhibitors.
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