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BAC transgene arrays as a model system for studying large-scale chromatin structure

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Title: BAC transgene arrays as a model system for studying large-scale chromatin structure
Author(s): Bian, Qian
Director of Research: Belmont, Andrew S.
Doctoral Committee Chair(s): Belmont, Andrew S.
Doctoral Committee Member(s): Ha, Taekjip; Selvin, Paul R.; Wang, Fei
Department / Program: School of Molecular & Cell Bio
Discipline: Biophysics & Computnl Biology
Degree Granting Institution: University of Illinois at Urbana-Champaign
Degree: Ph.D.
Genre: Dissertation
Subject(s): chromatin large-scale chromatin structure bacterial artificial chromosome (BAC) transgene expression Dihydrofolate reductase (DHFR) intranuclear positioning nuclear periphery beta-globin recombineering
Abstract: The folding of interphase chromatin into large-scale chromatin structure and its spatial organization within nucleus has been suggested to have important roles in gene regulation. In this study, we created engineered chromatin regions consisting of tandem repeats of BAC transgenes, which contain 150-200 kb of defined genomic regions, and used them as a model system to study the mechanisms and functional significance of large-scale chromatin organization. The BAC transgene arrays recapitulated several important features of endogenous chromatin, including transcription level and intranuclear positioning. Using this system, we showed that tandem arrays of housekeeping gene loci form open large-scale chromatin structure independent of their genomic integration sites, including insertions within centromeric heterochromatin. This BAC-specific large-scale chromatin conformation provided a permissive environment for transcription, as evidenced by the copy-number dependent and position independent expression of embedded reporter mini-genes. This leads to the development of a novel method for reliable transgene expression in mammalian cells, which should prove useful in a number of therapeutic and scientific applications. We also demonstrated that BAC transgene arrays can be employed as an effective system for dissecting sequence determinants for intranuclear positioning of gene loci. We showed that in mouse ES and fibroblast cells a BAC carrying a 200 kb human genomic fragment containing the beta-globin locus autonomously targets to the nuclear periphery. Using BAC recombineering, we dissected this 200kb region and identified two genomic regions sufficient to target the BAC transgenes to nuclear periphery. This study represents a first step towards elucidation of the molecular mechanism for the nuclear peripheral localization of genes in mammalian cells.
Issue Date: 2011-05-25
URI: http://hdl.handle.net/2142/24479
Rights Information: Copyright 2011 Qian Bian
Date Available in IDEALS: 2011-05-25
2013-05-26
Date Deposited: 2011-05
 

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