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This dissertation, "The Study of the Regulatory Elements of the Human {221}-globin Gene" by Ping-kei, Chan, 陳炳基, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled THE STUDY OF THE REGULATORY ELEMENTS OF THE HUMAN β-GLOBIN GENE Submitted by Chan Ping Kei For the degree of Doctor of Philosophy at The University of Hong Kong in June 2005 The human β-globin locus contains five developmentally regulated β-like globin G A genes arranged in the order of their developmental expression (5' ε-γ-γ-δ-β 3'). All five genes are regulated by an element located far upstream of the locus called the Locus Control Region (LCR). High level of β-globin gene expression is regulated by the LCR which consists of erythroid specific hypersensitive sites HS1 to HS5. Transcriptional regulation of the globin genes occurs through the cooperation of the LCR, the promoters of the globin genes and the interactions of trans-acting proteins. Recent studies show that activation of the β-globin locus involves the spatial formation of a LCR holocomplex that directly interacts with the transcribed genes. This complex is called the Active Chromatin Hub (ACH) (Palstra et al., 2003;Drissen et al., 2004). The objective of this project is to study the functional roles of two regulatory elements, namely, hypersensitive site 5 (HS5) of the LCR and the (AC) (AT) T motif n x y at the promoter of the β-globin gene. We study the enhancer blocking function of HS5 in transgenic mice by analyzing the conformational change in the context of the β-gene locus using the Chromatin Conformation Capture (3C) technique. The results show that HS5 functions as an enhancer blocking element in embryonic blood but not in adult erythroid cells. Interestingly, when HS5 is deleted form the locus, elements located upstream of the LCR are able to interact with the downstream regulatory elements within the β-globin gene locus. These results suggest that HS5 is a developmental stage specific border element. We determine whether CTCF, the only defined insulator protein in mammalian cells, binds to HS5 by using the CTCF-Chromatin immunoprecipitation (ChIP) assay. Our results demonstrate that CTCF binds to human HS5 and may mediate enhancer blocking activity in erythroid cell in vivo. In the second part of the thesis, we have examined the (AC) (AT) T motif n x y residing -530bp 5' upstream of the β-globin gene in Chinese thalassaemic patients. This motif is a putative binding site for a repressor protein, namely beta protein 1 (BP1) (Berg et al., 1989). It has been shown that variations in the (AC) (AT) T n x y repeats affect the binding affinity of BP1 thereby altering the expression of the β-globin gene. Eight different configurations of this repeat motif are identified in our population of Chinese β-thalassaemia patients in Hong Kong. A novel (AC) (AT) T 3 7 5 motif is identified among the thalassaemia patients and its influence in β-globin gene expression is studied using stable transfection assay in murine erythroleukemia (MEL) cells. Our results demonstrate that this motif has moderately strong repressor effect on the expression of the cis-linked β-globin gene. This may be due to the higher affinity of BP1 for the motif resulting in the suppression of the transcription of the β-globin gene. We conclude that the proper developmental expression pattern of the β-like globin gene cluster is absolutely dependent on the presence of both the LCR and the proximal cis-regulatory elements of the globin gene. DOI: 10.5353/th_b