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

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.

Karin and Greten. Nature Immun. 2005

NFkappaB-Signaling

NFkappaB Dimer

Experimental identification of NFkB binding sites in MAD3 promoter (Le Bail, 1993).

Therefore we decided to use 500bp of upstream sequence

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

MAD3NFKB1C-RelRelBNFKB2RelA

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]

TRES Transcription Regulatory Element Search

a tool for Comparative Promoter Analysis -Uses TRANSFAC database, which contains 3980 consensus 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/

TRES output

MAD3 related sequences control sequences

http://www.cbrc.jp/research/db/TFSEARCH.html

Maximum length: 9,999bp analysis

NFkappa-B family promoters

Control sequences

Boxes that exist in all sequences

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:

oPOSSUM web tool

oPOSSUM output

SummaryWe 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)

Program Advantage Disadvantage binding-site database used

TFSearch 10 kbp of sequence possible for analysis just one sequence to analyse TRANSFACPROMO multiple sequence analysis possible; fancy output;

many information. Filipe`s favouritethe output is a bit "unuebersichtlich"

TRANSFAC

TRES multiple sequence analysis possible; short clear output

not much additional information

TRANSFAC/ ooTFD/PLACE

oPOSSUM multiple sequence analysis possible; easy input (gene Ids), analysis of orthologous sequences

One cannot change sequence input

JASPAR

Toucan easy visualisation of multiple sequences promoter prediction is not working

TRANSFAC/ JASPAR