investigator s abstract) The overall goal of the research is to study the mechanisms which control globin gene switching. The main tool consists of hybrids produced by fusing human fetal erythroid cells with mouse erythroleukemia cells (HFE x MEL hybrids). These hybrids initially express predominantly or exclusively human fetal globin (""""""""gamma hybrids"""""""") and 20 to 50 weeks later they switch to exclusive human adult globin formation (""""""""beta hybrids"""""""") providing a model system for the analysis of the control of switching.
The specific aims are: 1) To use the HFE x MEL hybrids, to investigate mechanisms of human globin gene switching. a) Investigate the relationship between globin chromatin acetylation and globin gene switching; expand the findings that the gamma gene expression is associated with a specific pattern of histone acetylation; refine the histone acetylation mapping in the regions of DNAse 1 hypersensitive sites of the LCR and the gamma and beta promoters; identify sequences which harbor nucleosomes with hyperacetylated histones; test whether inhibition or overexpression of histone deacetylase in hybrids or in transgenic mice modulates the rate of gamma to beta switch. b) Using transfers of beta locus YACs with marked beta and gamma genes into HFE x MEL hybrids, test the hypothesis that the rate of gamma to beta gene switch is controlled by a trans acting developmental clock type of mechanism; identify, by transferring into HFE x MEL cell hybrids beta- YACs with truncated beta-loci, the sequences of the beta locus which respond to the postulated developmental clock of switching. 2) Investigate a transcriptional factor preliminarily called FGIF (for fetal globin increasing factor) which increases gamma gene expression in transfected HFE x MEL hybrids. a) Test whether this factor acts by inhibiting gamma gene silencing or by activating gamma gene expression. b) Identify the sequences of the beta locus on which FGIF acts. c) Investigate the effects of FGIF on gamma gene expression in transgenic mice carrying human beta globin genes. d) Investigate gamma gene expression in BFUe colonies transduced with FGIF retroviral vectors. e) Clone the human and mouse FGIF genes. f) Produce erythroid lineage- specific FGIF knockout mice and examine the effects of FGIF deficiency on gamma gene expression. 3) Using transfers of beta locus YACs into non-erythroid and erythroid cells, test the hypothesis, based on preliminary data using somatic cell hybrids, that the beta locus control region is activated stepwise and that normal LCR function requires pre- activation of the LCR in a non-erythroid environment.
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