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Title:Changes in large-scale chromatin structure and dynamics of BAC transgenes during transcriptional activation
Author(s):Hu, Yan
Director of Research:Belmont, Andrew S.
Doctoral Committee Chair(s):Schuler, Mary A.
Doctoral Committee Member(s):Belmont, Andrew S.; Katzenellenbogen, Benita S.; Jones, Peter L.; Mizzen, Craig A.
Department / Program:Cell & Developmental Biology
Discipline:Cell and Developmental Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):large-scale chromatin structure
Bacterial artificial chromosome (BAC)
SC-35 domains
nuclear speckle
Heat shock protein 70 (Hsp70) promoter
promoter specificity
heat shock
Abstract:Mounting evidence suggests that the eukaryotic cell nucleus is a dynamic and complex structure, and that changes in large-scale chromatin structure play an important role in gene regulation. In this study, we utilize the high specificity and affinity of the lacO-lacI tethering system to visualize changes in large-scale chromatin structure that accompany gene activation in an inducible BAC transgene system. Using light microscopy, and electron microscopy combined with in vivo immunogold labeling, we observe a 1.5-3-fold decondensation of a 1-2 Mbp chromatin domain composed of multi-copy BAC transgenes after transcriptional activation in interphase nuclei. Although the BAC transgene transcription levels are close to the levels of their endogenous counterparts after transcriptional activation, the BAC transgenes are folded within large-scale chromatin fibers with DNA compaction levels well above the extended 30 nm chromatin fibers predicted by previous models of the chromosome structure of actively transcribing genes. Furthermore, heat shock induced Hsp70 BAC transgenes, but not other BAC transgenes, associated with the exterior of nuclear speckles with their transcripts accumulating within the speckle. Live cell imaging revealed three distinct dynamic events for Hsp70 trangene nuclear speckle association. Further molecular dissection demonstrated association of Hsp70 transgenes with nuclear speckles is transcription-dependent, independent of the identity of the transcribed sequences, relatively independent of the amount of transcripts, but dependent on the Hsp70 promoter sequences. Moreover, despite similar levels of transcriptional activation, Hsp70 transgene speckle association is observed after heat shock, but not after cadmium treatment. Collectively, this work demonstrates transcription can occur on a still highly condensed, large-scale chromatin template and Hsp70 promoter-specific association of transgenes with nuclear speckles upon transcriptional activation.
Issue Date:2010-05-14
Rights Information:copyright 2010 Yan Hu
Date Available in IDEALS:2010-05-14
Date Deposited:May 2010

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