The long term goal of the investigator's laboratory is to define the mechanisms that control gamma to beta globin gene switching during erythroid development. The precise organization of regulatory elements within globin gene clusters may play a crucial role in developmental switching. To better define the cis-acting elements that control developmental switching, the following specific aims are proposed:
Specific Aim 1 : Define cis-acting sequences in artificial chromosomes (PACs) containing the human beta globin gene cluster that contribute to globin gene switching in embryonic stem cells and transgenic mice. The investigators will use a newly described cloning system that allows for the propagation of 80 to 120 kb fragments in plasmid-based P1 vectors carried in E. coli. A series of PAC clones containing the human beta globin gene cluster will be stably transfected into embryonic stem (ES) cells or used to create transgenic mice. Human globin gene switching will be observed in these environments. PAC globin gene clusters will be mutagenized using recA assisted endonuclease cleavage, which will allow them to insert HPFH-associated point mutations into the intact human beta globin cluster, to define their switching behaviors in vitro and in vivo.
Specific Aim 2 : Analyze the role of the murine beta globin locus control region for switching by creating targeted deletions of specific LCR hypersensitive sites in knockout model systems. Standard homologous recombination techniques will be used to create a deletion of murine 5'HS-3 in ES cells. 5'HS-3 +/- ES cells will be used to create mice containing this deletion in the germ line; these mice will be examined during embryonic and adult stages for the abundance of embryonic and adult hemoglobins from the mutated locus. Additional deletions of 5' LCR sites will be created depending on the results with the 5'HS-3 deletion.
Specific Aim 3 : Determine whether gamma globin gene promoter activity is mediated by stochastic or non-stochastic mechanisms. Gamma globin promoter activity will be measured using reporters that can be quantified in individual cells (LacZ or green fluorescent protein). Gamma globin promoter activity will be assessed in transfected K562 cells to determine whether 5'HS-2 increases gamma globin promoter activity, or whether it increases the probability that this promoter will fire at a set level. These reporters will be inserted into wild type or HPFH gamma globin genes in intact PAC/beta globin gene clusters to determine whether gamma globin activity is activated and/or repressed via stochastic mechanisms in vivo.
Specific Aim 4 : Define the role of the cold-shock domain containing proteins dbpB and dbpA for globin gene switching by creating loss-of- function models in mice. They have determined that dbpB and dbpA interact specifically with a secondary structure upstream for the gamma globin genes that is altered by several mutations associated with HPFH. To define the role that these proteins play in globin gene switching, they will determine which members of the dbpB and A gene family are expressed in erythroid precursors. Using this information, they will create null mutations in appropriate dbpA and B family members using homologous recombination in ES cells to asses their role for globin gene switching in ES cells and in knockout mice.
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