We would like to understand how genes work at a molecular level. Specifically, what is it about the binding of regulatory proteins (and other chemical) that switches certain genes on and others off? It is here that the secret of development lies (i.e., what makes one cell a red-blood cell and another a neuron?). and of the life-cycle of the cell.

The scanning probe microscopes, capable of imaging in fluid at very high resolution, offer the chance of imaging the components of a regulatory complex (DNA and protein) in natural conditions, and possibly even "in action" (that is, it might be possible to make movies of biological processes at the molecular level.

We have developed more sensitive instruments ("A magnetically driven oscillating probe microscope for operation in liquids" W. Han, S.M. Lindsay and T. Jing, Applied Physics Letters 69, 4111-4113 (1996)) capable of gentler imaging at higher resolution and we have used them to study structural transitions in DNA ("Kinked DNA" W. Han, S.M. Lindsay, M. Dlakic and R.E. Harrington, Nature 386, 563-564 (1997).)

We are currently working on two major projects using these instruments:

The first project involves examining kinking and bending in small DNA circles as they bind important proteins. The use of a circle simplifies interpretation of the bending, allowing us to identify "weak spots" along the gene ( see “Conformation and rigidity of DNA microcircles containing waf1 response elements for p53 regulation protein” H. Zhou, Y. Zhang, Z. Ou-Yang, S.M. Lindsay, X.Z. Feng, P. Balaguuurumoorthy and R.E. Harrington, J Mol Biol 306(2), 227-38 (2001)). This work is supported by the NIH and is part of a collaborative program with the Harrington Labs in the Department of Microbiology at Arizona State University.

The second project involves SPM methods to study chromatin remodeling. It is becoming increasingly clear that promoter chromatin structure and the remodeling of that structure in association with gene activation are crucial facets of eukaryotic transcriptional regulation. The recent development of an in vitro MMTV-LTR system (Hager G.L. "Understanding nuclear receptor function: from DNA to chromatin to the interphase nucleus." Prog Nucleic Acid Res Mol Biol 2000;66:279-305) that can reconstitute the correct promoter chromatin structure and the correct remodeling of that structure in vitro presents an unprecedented opportunity to study these important facets of transcriptional regulation. In particular, it will now be possible to study promoter chromatin with and without bound receptor, and thus obtain information on this key first step of promoter recognition. We can then analyze the remodeled chromatin, to characterize the chromatin structure and transcription factor changes that have occured as a result of remodeling.

Because of its scale of imaging, the atomic force microscope (AFM) is well suited for studying the various structural aspects of this process that we want to analyze: the linear organization (nucleosome locations), the higher order structure (conformations of fully hydrated chromatin and transcription factors), and molecular recognition mapping (identifying specific molecules in the spreads based on antibody recognition). These approaches are a blend of established techniques and new AFM techniques that will be developed for this application but will undoubtedly prove useful for other biophysical and biological applications.

This project involves a collaboration with the labs of Dennis Lohr at A.S.U., the labs of Rodney Harrington at A.S.U., and the labs of Gordon Hager at the National Institute of Health. The collaboration also includes biophysicists Hansgeorg Schindler and Peter Hinterdorfer (University of Linz, Austria) pioneers in developing nm-scale molecular recognition techniques that use an antibody attached to an AFM probe.

Specific Aims of this project include:

Develop rapid and reliable antibody-based Moleculer Recognition Mapping.

Characterize and test Molecular Recognition Mapping on known and defined model chromatin.

Carry out Linear Organization, Higher Order Structures and Molecular Recognition Mapping Studies of LTR promoter region chromatin

Study GR binding to this chromatin.

Study chromatin remodeling in relation to Higher Order Structures and Molecular Recognition Mapping.