The goals of this research proposal are a) to test the hypothesis that the transcription factor milieu in adult erythrocytes is supportive of full level expression of the human gamma gene, b) to identify and purify the putative gamma gene trans activators and to test their effects on gamma-globin gene expression in the mouse model.
Our specific aims are i) to investigate whether the transcription factor milieu in adult erythrocytes is supportive of full level expression of the human gamma gene. This will be achieved by replacing the minimal promoter of the human beta-globin gene with the counterpart of the gamma-globin gene in the context of the yeast artificial chromosome (YAC) carrying the human beta- globin locus and by testing beta-globin expression driven by the gamma promoter in transgenic mice. The results from these studies will be instrumental for designing a feasible strategy for gamma-globin gene transactivation. ii) to identify and purify a putative trans activator associated with the hereditary persistence of fetal hemoglobin (HPFH) -198 mutation and to test if overexpression of the gene encoding this protein will re-activate the human gamma-globin gene in transgenic mouse model. iii) to identify and purify proteins that are responsible for human gamma-globin gene activation in the adult by using comparative study of the protein binding profiles of the human (activated) and galago(silenced) gamma promoters in the context of the muLCR gamma in transgenic mice. iv) to identify novel TATA box-independent transcription factor(s) that activates gamma gene expression in K562 cells. It is expected that these studies will facilitate the designing of a feasible strategy for human gamma-globin gene transactivation and discover novel gamma- globin gene trans activators. Such a development will have important consequences for the treatment of patients with sickle cell disease or beta thalassemia syndromes.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Special Emphasis Panel (ZHL1-CSR-J (S3))
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Badman, David G
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University of Washington
Internal Medicine/Medicine
Schools of Medicine
United States
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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
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
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
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
Fang, Xiangdong; Sun, Jin; Xiang, Ping et al. (2005) Synergistic and additive properties of the beta-globin locus control region (LCR) revealed by 5'HS3 deletion mutations: implication for LCR chromatin architecture. Mol Cell Biol 25:7033-41
Xiang, Ping; Han, Hemei; Barkess, Grainne et al. (2005) Juxtaposition of the HPFH2 enhancer is not sufficient to reactivate the gamma-globin gene in adult erythropoiesis. Hum Mol Genet 14:3047-56

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