Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw

Vlaams Interuniversitair Instituut Voor Biotechnologie

Jacques Remacle

Danny Huylebroeck

Kristin Verschueren

A method of identifying transcription factors comprising providing cells with a nucleic acid sequence at least comprising a sequence CACCT (SEQ ID NO:1) as bait for the screening of a library encoding potential transcription factors and performing a specificity test to isolate said factors. Preferably, the bait comprises twice the CACCT (SEQ ID NO:1) sequence, more particularly the bait comprises one of the sequences CACCT-N-CACCT (a first SEQ ID NO:1 and a second SEQ ID NO:1 separated by N), CACCT-N-AGGTG (SEQ ID NO:1 and SEQ ID NO:3 separated by N), AGGTG-N-CACCT (SEQ ID NO:3 and SEQ ID NO:1 separated by N), or AGGTG-N-AGGTG (a first SEQ ID NO:3 and a second SEQ ID NO:3 separated by N), wherein N is a spacer sequence. The transcription factors identified using the methods of the invention include separated clusters of zinc fingers, such as, for example, a two-handed zinc finger transcription factor. Also, at least one such zinc finger transcription factor, denominated as SIP1, induces tumor metastasis by down regulation of the expression of E-cadherin. Compounds interfering with SIP1 activity can thus be used to prevent tumor invasion and metastasis.

Nucleic acid binding of multi-zinc finger transcription factors

The current invention concerns SMAD-interacting protein(s) obtainable by a two-hybrid screening assay whereby SMAD1 C-domain fused to GAL4 DNA-binding domain as 'bait' and a cDNA library from mouse embryo as 'prey' are used. Some characteristics of a specific SMAD-interacting protein (SIP1) of the family of zinc finger/homeodomain proteins including d-crystallin enhancer binding protein and/orzfh-1 include an inability to interact with full-size XSMAD1 in yeast, SIP1binds to E2 box sites, SIP1binds to the Brachyury protein binding site and interferes with Brachyury-mediated transcription activation in cells and also interacts with the C-domain of SMAD 1, 2 and 5. The minimal length of the amino acid sequence necessary for binding with SMAD appears to be a 51 amino acid domain encompassing amino acids 166-216 of SEQ ID NO: 2 having the amino acid sequence as depicted in the one letter code: QHLGVGMEAPLLGFPTMNSNLSEVQKVLQIVDNTVSRQKMDCKTEDISKLK (SEQ ID NO: 21).

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Hannut, Belgium

Jacques Remacle