Fragile Sites, Bordering Elements and Chromatin Domains: Lessons from the Type 1 Human Interferon Gene Cluster on 9p22

Juergen Bode
Gesellschaft für Biotechnologische Forschung
mbH / EPI
Mascheroder Weg 1
38124 Braunschweig
telephone: +49 531 6181 251
fax: +49 531 6181 262
email: jbo@gbf.de
prestype: Platform
presenter: Juergen Bode

J. Bode 1), A. Baer 1), E. Ernst 1), S. Goetze 1), A. Knopp 1), K. Nehlsen 1), C. Benham2)
1)German Research Center for Biotechnology (GBF)/EPI, D-38124 Braunschweig, jbo@gbf.de;
2)UC Davis,Div. Biol. Saciences, Genome Center benham@blue.ucdavis.edu

S/MARs have been discovered more than a decade ago and have been defined as DNA-elements staying attached to or associating with the nuclear skeleton after the extraction of the histones and soluble factors from eukaryotic nuclei (1). While S/MARs do not conform to any obvious sequence consensus, their recognition is governed by structural features, most significantly a propensity to expose single strands under negative superhelical tension. This property has been used to localize S/MARs in SIDD (stress-induced duplex destabilization) profiles, to explain their illegitimate recombination potential (4) which becomes evident by

• their preference as integration for retroelements
  
• their coincidence with fragile genomic sites.

We are in the process of predicting their participation in transcription and replication processes and to use biomathematics to guide their design for various biological and biotechnological applications.

The first widely accepted activity of S/MARs was the augmentation of transcription initiation rates which is distinct from enhancement (1). Since then a variety of additional functions have been delineated acting on transcriptional competence, and providing enhancer or origin of replication support. This overlap of functions has made difficult the unambiguous demonstration of any of these components. It was originally for this reason that we have refined techniques based on site-specific recombination systems like Flp/FRT (3,7). With these techniques, complete chromatin domains cannot only be decomposed but also be elaborated at a predefined chromosomal locus. Our most advanced system, the recombinase-mediated cassette exchange (Flp-RMCE) permits the mutual exchange of cassettes which are flanked by an FRT-site and an FRT-mutant, resp. (3,7).

So far the stable modification of target cells is mostly achieved by integrating vectors(2). For gene therapy purposes, derivatives of retroviral vectors have found extensive use although their expression may be rapidly silenced. While methylation-dependent silencing can be controlled by the presence of S/MARs(1), major recent efforts have been devoted to the use of S/MAR-ori sequences from the human genome to obtain vectors which replicate autonomously providing a stable and high-level expression. 5,6).

Recent reviews:

1. J. Bode, C. Benham, A. Knopp and C. Mielke (2000). Transcriptional Augmentation: Modulation of Gene Expression by Scaffold/Matrix Attached Regions (S/MAR Elements).Crit. Rev. Eukaryot. Gene Expr. 10, 73-90.
   
2. A. Baer, D. Schübeler and J. Bode (2000). Transcriptional Properties of genomic transgene integration sites marked by electroporation or retroviral infection. Biochemistry 39, 7041-7049.
   
3. J. Bode, T. Schlake, M. Iber, D. Schübeler, J. Seibler, E. Snezhkov and L. Nikolaev (2000). The transgeneticist´s toolbox - Novel methods for the targeted modification of eukaryotic genomes. Biol. Chem. 381, 801-813.
   
4. J. Bode, C. Benham, E. Ernst, A. Knopp, R. Marschalek, R. Strick, and P. Strissel (2000). Fatal connections: When DNA ends meet on the nuclear matrix. J. Cell. Biochem. Suppl. 35, 3-22, http://www3.interscience.wiley.com/cgi-bin/issuetoc?ID=82002725.
   
5. J. Bode, C. P. Fetzer, K. Nehlsen, M. Scinteie, Bok-Hee Hinrich, A.Baiker, C. Piechazcek, C. Benham and H. J. Lipps (2001). The Hitchhiking Principle: Optimizing episomal vectors for the use in gene therapy and biotechnology. Int. J. Gene Ther. Mol. Biol 6, 33-46. www.gtmb.org
   
6. H. J. Lipps and J. Bode (2001). Exploiting chromosomal and viral strategies: The design of safe and efficient non-viral gene transfer systems. Curr. Opin. Mol. Therapeut. 3, 133-141.
   
7. A. Baer and J. Bode (2001). Coping with kinetic and thermodynamic barriers: RMCE, an efficient strategy for the targeted integration of transgenes. Curr. Opin. Biotech. Curr. Opin. Biotech. 12, 473-480.