1. Finding conserved transcription factor binding sites in promoter sequences NfkappaB motifs in promoters controling human NFkappaB gene family members Markella Katidou Ulf Andersson Orom Carlos Reguenga Filipe Vilas-Boas David Kuttenkeuler

2. The Project The inhibitor of NFkappaB MAD3 in humans has been shown to be regulated by NFkappaB itself. Therefore we asked the question if other NFkappaB family members are regulated in the same manner. Approach: Analysis of promoter sequences of six NFkappaB family members in the human genome using different web-based promoter analysis programs.

3. Karin and Greten. Nature Immun. 2005 NFkappaB-Signaling NFkappaB Dimer

4. Experimental identification of NFkB binding sites in MAD3 promoter (Le Bail, 1993). Therefore we decided to use 500bp of upstream sequence

5. Position of NFkB sites in the MAD3 family upstream regions (500bp) TOUCAN: workbench for regulatory sequence analysis http://homes.esat.kuleuven.be/~saerts/software/toucan.php MAD3 NFKB1 C-Rel RelB NFKB2 RelA

6. Control set of unrelated sequences (500bp) Used our EMBO login numbers to find corresponding NCBI gene IDS: Carlos: adenosine A2b receptor [Homo sapiens] David: alcohol dehydrogenase 1C (class I), gamma polypeptide [Homo sapiens] Ulf: ADAM metallopeptidase domain 10 [Homo sapiens] Filipe: poly (ADP-ribose) polymerase family, member 4 [Homo sapiens] Markella: adipose differentiation-related protein [Homo sapiens]

7. TRES Transcription Regulatory Element Search a tool for Comparative Promoter Analysis -Uses TRANSFAC database, which contains 3980 c onsensus transcription factor binding sites. - multiple promoter sequence analysis with a maximum of 20 sequences at the same time. http://bioportal.bic.nus.edu.sg/tres/

8. TRES output MAD3 related sequences control sequences

9. http://www.cbrc.jp/research/db/TFSEARCH.html Maximum length: 9,999bp analysis

11. NFkappa-B family promoters Control sequences Boxes that exist in all sequences

12. Excluded short boxes with low score consensus seuquence. GCF [T00320] NFI/CTF [T00094] p53 [T00671] Pax-5 [T00070] ENKTF-1 [T00255] IRF-1 [T00423] Sp1 [T00759] NF-kappaB [T00590] c-Jun [T00133] Boxes that exist in NFkappa-B promoters, but not in the control promoters Included: NFkappa-B Pax5 Excluded:

13. oPOSSUM web tool

14. oPOSSUM output

15. Summary We found NFkappaB sites in all human NFkappaB- family gene promoters and not in the control set. Therefore Web tools are usable to find specific sites to a certain extent. A publication search AFTER our analysis revealed this finding is true. (Bren et al. Oncogene 2001)