Developmental regulation of human beta-like globin gene switching is controlled by several parameters, primarily the trans-acting transcriptional milieu and cis-acting DNA elements. These cis motifs include, but are not limited to, individual gene associated sequences involved in activation, silencing and competition for interaction with the locus control region (LCR), gene order, and distance from the LCR. Other potential points of cis-regulation may comprise intergenic sequences such as domain boundaries or barriers and chromatin architecture. The goals of this grant proposal are to delineate the cis-control of human beta-globin gene switching and the structure/function relationships of the beta-globin LCR. The proposed study will utilize transgenic mice produced with beta-globin locus yeast artificial chromosomes (beta-YACs) so that the effect of mutations introduced into cis-linked sequences may be analyzed in the context of the entire locus throughout development.
Specific Aim 1 will examine the structure/function relationship of the LCR and globin gene switching by: a) determining the function of the individual DNAseI-hypersensitive sites (HSs), and b) testing whether formation of an LCR complex is required for LCR function.
Specific Aim 2 will identify Agamma-globin gene proximal sequences responsible for autonomous silencing of gamma-globin gene expression during development.
Specific Aim 3 will employ constructs to: a) determine whether a putative delta-beta boundary element demarcates embryonic and fetal chromatin domains from an adult chromatin domain and b) test the role of this delta-beta boundary element in regulating embryonic, fetal and adult globin gene expression during development.
Specific Aim 4 will investigate the role of the human beta-globin LCR in globin gene switching when the human locus is integrated in place of the murine beta-globin locus by: a) deleting one copy of the endogenous locus to provide a globin locus insertion site, b) testing whether the locus is open or closed in the absence of the beta-LCR after passage through the mouse germ line, and c) testing whether the human beta-globin LCR functions exclusively as an enhancer or has additional roles in the temporal regulation of downstream genes by replacing it with alternate enhancers.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL067336-03
Application #
6638774
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Evans, Gregory
Project Start
2001-06-15
Project End
2005-05-31
Budget Start
2003-06-01
Budget End
2004-05-31
Support Year
3
Fiscal Year
2003
Total Cost
$337,500
Indirect Cost
Name
University of Kansas
Department
Biochemistry
Type
Schools of Medicine
DUNS #
016060860
City
Kansas City
State
KS
Country
United States
Zip Code
66160
Peterson, Kenneth R; Costa, Flávia C; Fedosyuk, Halyna et al. (2014) A cell-based high-throughput screen for novel chemical inducers of fetal hemoglobin for treatment of hemoglobinopathies. PLoS One 9:e107006
Peterson, Kenneth R; Fedosyuk, Halyna; Harju-Baker, Susanna (2012) LCR 5' hypersensitive site specificity for globin gene activation within the active chromatin hub. Nucleic Acids Res 40:11256-69
Costa, Flávia C; Fedosyuk, Halyna; Chazelle, Allen M et al. (2012) Mi2? is required for ?-globin gene silencing: temporal assembly of a GATA-1-FOG-1-Mi2 repressor complex in ?-YAC transgenic mice. PLoS Genet 8:e1003155
Harju-Baker, Susanna; Costa, Flavia C; Fedosyuk, Halyna et al. (2008) Silencing of Agamma-globin gene expression during adult definitive erythropoiesis mediated by GATA-1-FOG-1-Mi2 complex binding at the -566 GATA site. Mol Cell Biol 28:3101-13
Fedosyuk, Halyna; Peterson, Kenneth R (2007) Deletion of the human beta-globin LCR 5'HS4 or 5'HS1 differentially affects beta-like globin gene expression in beta-YAC transgenic mice. Blood Cells Mol Dis 39:44-55
Blau, C Anthony; Peterson, Kenneth R (2006) Establishment of cell lines that exhibit correct ontogenic stage-specific gene expression profiles from tissues of yeast artificial chromosome transgenic mice using chemically induced growth signals. Methods Mol Biol 349:163-73
Harju, Susanna; Navas, Patrick A; Stamatoyannopoulos, George et al. (2005) Genome architecture of the human beta-globin locus affects developmental regulation of gene expression. Mol Cell Biol 25:8765-78
Blau, C Anthony; Barbas 3rd, Carlos F; Bomhoff, Anna L et al. (2005) {gamma}-Globin gene expression in chemical inducer of dimerization (CID)-dependent multipotential cells established from human {beta}-globin locus yeast artificial chromosome ({beta}-YAC) transgenic mice. J Biol Chem 280:36642-7
Harju, Susanna; Fedosyuk, Halyna; Peterson, Kenneth R (2004) Rapid isolation of yeast genomic DNA: Bust n' Grab. BMC Biotechnol 4:8
Peterson, Kenneth R (2003) Transgenic mice carrying yeast artificial chromosomes. Expert Rev Mol Med 5:1-25

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