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Title: Comparative DNA Sequence Analysis for the Detection of Regulatory Elements
P45
Gausmann, Ulrike (1); Jahn, Niels (1); Platzer, Cornelia (2); Szafranski, Karol (1); Platzer, Matthias (1)

ugau@imb-jena.de, cplatzer@mti-n.uni-jena.de, mplatzer@imb-jena.de
(1) Department of Genome Analysis, Institute of Molecular Biotechnology, Postfach 100 813, 07708 Jena, Germany; (2) Institute of Anatomy II, Medical School, Friedrich Schiller University Jena, Teichgraben 7, 07740 Jena, Germany

The most common method for the prediction of putative regulatory regions in promoter sequences is based on the comparison of the nucleotide sequence with known binding matrices for transcription factor binding sites (MatInspector). However, these predictions still appear to be insufficient in sensitivity and specificity. Several improvements are on the way namely data basis expansion for matrix computation (TransFac), building of higher order binding site models (e.g. TransCompel) and the consideration of comparative approaches (e.g. rVISTA).
Within the newly founded Sonderforschungsbereich "Multifunctional Signaling Proteins" (http://www.imb-jena.de/SFB604/) we deal with groups of proteins which are physically interacting and may be potentially co-regulated. Comparative analysis of the 5' upstream gene regions should reveal regulatory elements common for all members of one group. However, the interactions are not exactly known for all of these groups and posttranscriptional mechanisms may mask expression effects. To enhance the data basis for comparative analysis we include paralogous and orthologous gene sequences of interest.
The in silico analysis requires test- and training sets and the automated data transfer from data storage to data analysis programs. Hence, we started to build a relational database which will also provide the experimental researchers within the SFB with data they need for their experiments, i.e. sequences with corresponding annotations derived from public references and from own analyses.
Comparative analysis of non-coding regions is based on the fact that functional elements are more strictly conserved and can be identified by alignment procedures. Sensitive pairwise alignment algorithms are the first steps but will not give clear results in many cases. Therefore, methods based on multiple comparisons will enhance the significance of the identified regions. For some purposes specialized programs can be used, e.g. 'DNA block aligner' (DBA) for the detection of conserved blocks in human and rodent orthologous non-coding sequences (1).
As an example for the applicability of this approach we present the analysis of the Interleukin-10 (IL-10) promoter. IL-10 is a cytokine with immunosuppressive and anti-inflammatory properties and its synthesis can be induced or enhanced by various stimuli, particularly via a cAMP-dependent signaling pathway. Two functionally active CRE motifs, CRE1 and CRE4, were identified in the promoter region (2). It was suspected that other, non-CRE sites play also a role in this pathway. Comparative analysis of three orthologous promoter sequences (H. sapiens, M. musculus, M. monax) revealed that two binding sites for the CCAAT enhancer binding protein, C/EBP3 and 5, and the previously identified CRE4 site, are located in conserved regions (DBA). Using EMSA and reporter gene assays the prominent functional role of these sites was experimentally confirmed. In contrast, the weakly conserved sites CRE1 and C/EBP1 were shown to be less important.
[1] Jareborg N., E. Birney, R. Durbin (1999) Comparative analysis of noncoding regions of 77 orthologous mouse and human gene pairs. Genome Res 9:815-824.
[2] Platzer C., E. Fritsch, T. Elsner, M.H. Lehmann, H.D. Volk, S. Prosch (1999) Cyclic adenosine monophosphate-responsive elements are involved in the transcriptional activation of the human IL-10 gene in monocytic cells. Eur J Immunol 29:3098-3104.