PROJECT SUMMARY/ABSTRACT The precise temporal and spatial transcription of genes is a fundamental component of most developmental processes in multicellular organisms. Despite its importance, critical details such as the identity of the proteins that occupy specific cis-regulatory elements in their native cellular context are often unknown. In addition, there is a need for methods to robustly evaluate the specific sequence requirements within cis-regulatory elements that are required for DNA-protein interactions. Thus, there exist critical barriers to the identification of the protein and DNA components of gene regulatory networks. The long-term goal of this project is to develop a robust, unbiased method for identification of proteins that occupy specific cis-regulatory elements in their native cellular context. The central hypothesis of this proposal is that the electroporation method for DNA introduction will allow for high sensitivity detection of associated transcription factors and also an experimental avenue to evaluate the sequence requirements of cis-regulatory elements. The rationale to undertake this study is that there is a lack of fundamental information with regards to regulated transcription in the context of development and disease. The approach described here will establish a platform that can serve to overcome this critical barrier and can be adapted to diverse paradigms. To accomplish this goal, the following two aims are proposed: 1) Creation of modified cis-regulatory element plasmids with biotin transfer agents covalently bound to DNA and 2) Evaluation of biotin-labeling of proteins with modified cis-regulatory element plasmids. To provide a basis to evaluate this technique, these aims will use a previously established electroporation paradigm in the developing vertebrate retina and a cis-regulatory element with defined binding sites for known transcription factors. The first aim will use standard DNA and protein modification procedures to generate cis- regulatory element reporter plasmids with the ability to biotinylate proteins in close proximity to the cis- regulatory element. The second aim will determine the effectiveness of this approach to label the known transcription factor interactors using techniques to assess directly these specific proteins as well as an unbiased mass spectrometry approach to determine the sensitivity of this unbiased technique to identify these known DNA-protein interactions. This approach is innovative because it will use cis-regulatory element targets with covalently associated biotinylation agents which are likely to increase the sensitivity to detect protein interactors. In addition, in contrast to current technologies, the proposed system will allow for a robust and facile method to examine the requirements of specific DNA sequences in transcription factor recruitment and to design rigorous controls to confirm interactions. At the completion of the proposed project, a novel methodology that can be used to identify DNA-protein interactions in a native cellular context will have been developed and validated. The application of this method is expected to provide insights into critical parameters of cis-regulation with implications for our understanding of developmental mechanisms and disease etiology.

PROJECT NARRATIVE The identification of the protein constituents that occupy specific cis-regulatory elements is critical to the formulation of integrated models of precise, regulated transcription. This proposal will develop a novel, unbiased approach to identify these protein-DNA interactions in their native cellular context, with the functionality to evaluate the effects of mutations in target cis-regulatory elements. This methodology is expected to have a significant long-term impact on our understanding of regulated transcription in the context of both development and disease.