1GBF - German Research Center for Biotechnology / Epigenetic Regulation,
D-38124 Braunschweig
2University Davis, California
3Genomatix Software GmbH,
Karlstr.55, D-80333 München
4Wayne State University School of Medicine,
Detroit, MI 42801, USA
S/MARs are structural DNA elements which associate to protein components of the nuclear matrix whereby they effect an important level of chromatin organization. This association effects a specific cell nuclear architecture which enables the differential gene expression during development and in the mature cell. A classical approach for the definition of S/MARs involves the preparation of nuclear scaffolds by LIS-mediated extraction of histones and soluble protein components. Years of analyses of a number of S/MAR-elements of different origin has shown that binding and the associated biological activities are the result of specific structural features rather than of sequence characteristics in the narrow sense. DNA-strand separation but also secondary structures forming under superhelical tension are important contributants to the underlying mechanisms.
A prominent biomathematical prediction scheme is based on the calculation of the potential for DNA strand separation (SIDD-profile) or on the frequency of certain sequence motifs related to special DNA structures (MAR-Finder). A third approach uses sequence composition analyses and comparision to known elements (S/MARt-Test).
We developed a number of experimental methods to verify the potential of biomathematical S/MAR-predictions with the final goal of improving the computational programs. Based on classical reassociation experiments with LIS-extracted scaffolds we started a screening of sequences in order to precisely quantify S/MAR-activities.
In a more refined experimental set-up we established a method for the analysis of endogenously bound S/MARs using quantitative real-time PCR techniques. This approach allows the estimation of cell specific S/MAR-binding strength combined with the advantage of low amounts of starting material.
A relatively new approach deals with the analysis of endogenous S/MAR-binding. This concept includes FISH-analysis of DNA-domains formed by virtue of S/MARs and will be extended by crosslinking studies which are suited to unambiguously demonstrate S/MAR occupancy in vitro (cis platinum crosslinking).
In conclusion we have developed, and discuss, a series of complementing experimental approaches to verify the accuracy and potential of biomathematical profiling.