The overall aim of this Phase I research proposal is to develop a new method for the purification and characterization of nucleoprotein complexes that form active regulatory units in vivo. The method will enable recovery of proteins associated with specific cis-regulatory loci in chromatin, and the subsequent analysis of these proteins using mass spectrometry. To assess the feasibility of this approach, we develop and test a methodology based on the purification of the nucleoprotein complex present in a well-characterized cis- regulatory element, hypersensitive site 2 (HS2) of the human beta-globin 5'-locus control region (5'-LCR). This site has been selected because it will allow a direct correlation between our experimental data and known identities of specific transcription factors bound to HS2 in vivo. In the Preliminary Data (Section C) we present our progress in developing a novel in vivo chromatin-capture method to purify specific nucleoprotein complexes from mammalian cell lines.
The Specific Aims of this project address the optimization of this protocol to provide an optimal substrate for mass spectrometric analysis. If optimization of the purification system is successful, the system will be developed into a generalized platform. In Phase II studies, mass spectrometric analyses will be performed on recovered protein substrates. Analyses will be performed on additional cis-regulatory systems, including those known to be involved in specific diseases.
