Topic Introduction Confirming the Functional Importance of a Protein–DNA Interaction Michael F. Carey, Craig L. Peterson, and Stephen T. Smale Identifying DNA-binding proteins that interact with a control region of interest has become quite straightforward. However, the functional relevance of a given protein–DNA interaction is difficult to establish. The hypothesis that an interaction is relevant can be tested by several different experiments, 12 of which are outlined in this article. It must be remembered that none of these experiments by itself is conclusive. The information gained from each approach is described and explanations are given for why each yields useful but inconclusive results. The approaches vary widely with respect to the amount of effort required and the quality of information obtained. INTRODUCTION AND OVERVIEW In the modern era of molecular biology, identifying DNA-binding proteins that interact with a control region of interest is relatively straightforward, but establishing definitively that a specific transcription factor directly regulates a target gene by binding to a defined control element can be among the most difficult of tasks. There are experimental strategies to identify important DNA sequence elements, as well as proteins that bind those elements. In most instances, a transcription factor will have been implicated as a potential gene regulator by a TRANSFAC or JASPAR database search, by mass spectrometry analysis of proteins that bind the element in vitro, by a genome-wide chromatin im- munoprecipitation (ChIP) analysis of a transcription factor’s binding sites, or perhaps by a yeast one- hybrid screen. The identification of candidate DNA-binding proteins provides a significant advance because it allows one to hypothesize that the protein is responsible for the function of the control element in regulating the nearby gene. By itself, however, identification does not prove biological relevance. For example, detecting a protein–DNA interaction in a nuclear extract reflects many factors: (1) the abundance of the protein in the cells from which the extract was prepared, (2) the efficiency with which the protein was extracted from the cells, (3) the stability of the active protein within the extract, (4) the maintenance of essential posttranslational modifications during extract preparation, (5) the conditions used for the in vitro DNA-binding assay, and (6) the affinity of the protein for the isolated control element (Table 1). The criteria for detecting protein–DNA interactions in vitro are very different from those that determine which protein interacts functionally with the control element in vivo (i.e., which protein regulates the endogenous gene by binding to the control element) (Table 1). These criteria include (1) the abundance and stability of the protein in the cell nucleus, (2) the affinity of the protein for the site, (3) the ability of the protein to perform appropriate interactions with other proteins bound to adjacent Adapted from Transcriptional Regulation in Eukaryotes: Concepts, Strategies, and Techniques, 2nd edition, by Michael F. Carey, Craig L. Peterson, and Stephen T. Smale. CSHL Press, Cold Spring Harbor, NY, USA, 2009. © 2012 Cold Spring Harbor Laboratory Press Cite this article as Cold Spring Harb Protoc; 2012; doi:10.1101/pdb.top070060 733 Cold Spring Harbor Laboratory Press on June 12, 2020 - Published by http://cshprotocols.cshlp.org/ Downloaded from