The overall aim of this Phase I research proposal is to develop a novel cell-based system to enable efficient recovery and quantitative analysis of the regulatory proteins complexed at a specific, well-characterized human cis-regulatory site. The proposal comprises 3 Specific Aims: Specialized vectors will be synthesized (Specific Aim 1). Each vector comprises 3 main components: (i) a well-characterized cis-regulatory element (human beta-globin 5'HS2); (ii) a quantitative selectable marker (secreted alkaline phosphatase); and (iii) paired chromatin insulator elements (either human HS5 or chicken HS4). A qualitative selectable marker (neo) is also included. Two vectors will be produced that are identical save for the choice of insulator sequence. These vectors will be stably transfected into human erythroleukemia cells (K562), a well established system, and high copy cell lines will be selected (Specific Aim 2). The high-copy cell lines will be further examined to confirm functional activity of the HS2 element through quantitative assessment of DNaseI hypersensitivity (McArthur et al. 2001). Preliminary studies will be undertaken in a selected multi-copy line to determine the optimal conditions for excision of the intact HS2 nucleoprotein complex from crosslinked chromatin (Specific Aim 3). The vectors are specifically designed to take advantage of the fact that DNA sequences immediately flanking the protein-binding core regions of DNaseI hypersensitive sites are accessible to the action of sequence-specific restriction endonucleases in the context of nuclear chromatin. In the vector, the HS2 element is flanked by restriction sites for rare cutters that will excise a specific approximate 600bp fragment. Purification of this fragment and its associated proteins may be thus effected solely on the basis of size and solubility fractionation. Fractionation over sucrose gradients has the added advantage of providing a clean reagent for downstream mass spectrometric studies. Such studies are envisioned to form the basis of a follow-on Phase II proposal.
