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Direct Visualization of Transcription Factor Binding to Regulatory Elements in Living Cells.

Authors

Hager GL.

Source

Mapping Protein/DNA Interactions by Cross-Linking [Internet]. Paris: Institut national de la santé et de la recherche médicale; 2001.

Excerpt

Transcription factors bind to regulatory elements in eukaryotic chromosomes and modify the rate of promoter utilization for target genes. These regulatory processes are among the most important, and most intensively studied, mechanisms in modern biology. Methodology to characterize gene regulatory events has largely fallen into two general categories. One avenue is to introduce genetically modified copies of a given promoter into cells and ascertain information regarding the function of a given factor binding site through the functional consequences of mutations introduced in that site. A second general approach is to isolate the factors and the template and attempt to reconstruct regulatory events with purified components in cell-free systems. Each of these approaches is susceptible to unique shortcomings. In vivo transfection methods provide information from the actual transcriptional environment of the living cell, but serious problems are encountered in controlling the dose level and structure of introduced templates. A large component of regulation involves modification of the chromatin template, and transiently introduced templates are not appropriately organized at the nucleoprotein level (Smith and Hager 1997). Furthermore, it is difficult to reduce and control the complexity of the transcriptional apparatus in this approach, although minimal efforts can be made in this direction (gene knockouts, antisense nucleic acids, antibody microinjection, etc.) Cell-free systems offer the considerable advantage of reduced complexity and control over constituents, but these systems are notoriously susceptible to artifact. It is often difficult to discern in the in vitro approach whether the result of an experimental manipulation arises from the identification of a mechanism that functions in the living cell or simply an "effect" in the cell-free environment. We have developed an alternate methodology that permits the direct analysis of factor binding to regulatory elements in living cells in real time. This approach derives, first, from the ability to tag protein factors with a fluorescent label, express the tagged protein at physiological levels in target cells, and observe direct localization of the protein. The tags are provided by the rapidly growing family of fluorescent proteins isolated from naturally luminescent organisms, including the green fluorescent protein (GFP) family from the jellyfish, Aequorea victoria (Cubitt et al. 1995), and more recent isolates from other organisms, such as the red protein (DsRed). Secondly, we have devised an amplified gene target system that permits real-time localization of the fluorescent receptors on actual regulatory sites in living cells.

Copyright © 2001, Institut national de la santé et de la recherche médicale (INSERM)

PMID:
21413372
[PubMed]
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