The goals of this research application are a) to test the hypothesis that the variable human gamma-globin gene silencing in the adult is the consequence of a dynamic equilibrium between euchromatin originating in the LCR and heterochromatin originating in the gamma gene promoter, b) to identify gamma-globin gene specific repressors and corepressors.
Our specific aims are i) To test the hypothesis that the gamma gene silencing in the adult is the consequence of a dynamic balance between euchromatin originating in the LCR and heterochromatin originating in the gamma gene promoter. This will be tested in transgenic mice carrying the human beta-globin locus yeast artificial chromosome (betaYAC) and various mutated betaYACs by examining changes of the histone code specific for heterochromatin and euchromatin. This hypothesis can be validated if changes of the histone code are correlated with the phenotypes induced by the various mutations, ii) To test whether the gammaCACCC box causes heterochromatinization in the gamma gene promoter in the adult. This will be done by relocating the gammaCACCC box in the different locations in the beta-globin locus and examining formation of heterochromatin induced by the gammaCACCC box. iii) To develop an oligonucleotide-mediated chromatin immunoprecipitation approach and using this approach to search for gamma gene specific repressors/corepressors. It is expected that these studies will lead to a unifying model explaining variable silencing of human gamma-globin gene in the adult, and will identify gamma gene specific repressors/corepressors. These studies will facilitate designing of a feasible strategy for human gamma-globin gene reactivation. Such a development will have important consequences for the treatment of patients with sickle cell disease or beta thalassemia syndromes.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL073439-01
Application #
6614074
Study Section
Special Emphasis Panel (ZHL1-CSR-B (F2))
Program Officer
Evans, Gregory
Project Start
2003-04-01
Project End
2007-03-31
Budget Start
2003-04-01
Budget End
2004-03-31
Support Year
1
Fiscal Year
2003
Total Cost
$303,200
Indirect Cost
Name
University of Washington
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Fang, Xiangdong; Yin, Wenxuan; Xiang, Ping et al. (2009) The higher structure of chromatin in the LCR of the beta-globin locus changes during development. J Mol Biol 394:197-208
Fang, Xiangdong; Xiang, Ping; Yin, Wenxuan et al. (2007) Cooperativeness of the higher chromatin structure of the beta-globin locus revealed by the deletion mutations of DNase I hypersensitive site 3 of the LCR. J Mol Biol 365:31-7
Olave, Ivan A; Doneanu, Catalin; Fang, Xiangdong et al. (2007) Purification and identification of proteins that bind to the hereditary persistence of fetal hemoglobin -198 mutation in the gamma-globin gene promoter. J Biol Chem 282:853-62
Yin, Wenxuan; Barkess, Grainne; Fang, Xiangdong et al. (2007) Histone acetylation at the human beta-globin locus changes with developmental age. Blood 110:4101-7
Li, Qiliang (2006) A Melanesian alpha-thalassemia mutation suggests a novel mechanism for regulating gene expression. Genome Biol 7:238
Li, Qiliang; Fang, Xiangdong; Olave, Ivan et al. (2006) Transcriptional potential of the gamma-globin gene is dependent on the CACCC box in a developmental stage-specific manner. Nucleic Acids Res 34:3909-16
Yu, Man; Han, Hemei; Xiang, Ping et al. (2006) Autonomous silencing as well as competition controls gamma-globin gene expression during development. Mol Cell Biol 26:4775-81
Li, Qiliang; Barkess, Grainne; Qian, Hong (2006) Chromatin looping and the probability of transcription. Trends Genet 22:197-202
Xiang, Ping; Fang, Xiangdong; Yin, Wenxuan et al. (2006) Non-coding transcripts far upstream of the epsilon-globin gene are distinctly expressed in human primary tissues and erythroleukemia cell lines. Biochem Biophys Res Commun 344:623-30
Yin, Wenxuan; Xiang, Ping; Li, Qiliang (2005) Investigations of the effect of DNA size in transient transfection assay using dual luciferase system. Anal Biochem 346:289-94

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