Nucleic acid binding of multi-zinc finger transcription factors

Abstract

A method of identifying transcription factors comprising providing cells with a nucleic acid sequence at least comprising a sequence CACCT (SEQ ID NO: ______ ) 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: ______ ) sequence, more particularly the bait comprises one of the sequences CACCT-N-CACCT(SEQ ID NO: ______ ), CACCT-N-AGGTG(SEQ ID NO: ______ ), AGGTG-N-CACCT(SEQ ID NO: ______ ), or AGGTG-N-AGGTG (SEQ ID NO: ______ ) 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.

Description

[0001] This application is a continuation of International Appln. PCT / EP00 / 05582 (International Publ. No. WO 01 / 00864, published Jan. 4, 2001, the contents of the entirety of which is incorporated by this reference, filed on Jun. 9, 2000, designating the United States of America.[0002] The invention relates to biotechnology generally, and more specifically to a method of identifying transcription factors.[0003] Zinc fingers are among the most common DNA binding motifs found in eukaryotes. It is estimated that there are 500 zinc finger proteins encoded by the yeast genome and that perhaps 1% of all mammalian genes encode zinc finger containing proteins. These proteins are classified according to the number and position of the cysteine and histidine residues available for zinc coordination.[0004] The CCHH class, typified by the Xenopus transcription factor IIIA (19), is the largest. These proteins contain two or more fingers in tandem repeats. In contrast, the steroid receptors contai...

AI Technical Summary

Benefits of technology

[0009] The invention relates to a method of identifying transcription factors involving providing cells with a nucleic acid sequence including a sequence CACCT (the first 5 nucleotides of SEQ ID NO: 1) as bait for the screening of a library encoding potential transcription factors and performing a specificity test to isolate the factors. Transcription factors identified using the method include separated clusters of zinc fingers such as, for example, a two-handed zinc finger transcription factor. At least one such zinc finger transcription factor, denominated "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.

[0011] This binding may be generalized to other transcription factors that contain separated clusters of zinc fingers and may be applied to other Smad-binding proteins. Moreover, the Smad-interacting protein SIP1 shows high expression in E-cadherin-negative human carcinoma cell lines, resulting in down regulation of E-cadherin transcription. Conditional expression of SIP1 in E-cadherin-positive MDCK cells also abrogates E-cadherin-mediated intercellular adhesion and simultaneously induced invasion. Hence, SIP1 can considered as a potent invasion promoter molecule and compounds, such as anti-SIP1 antibodies, small molecules specifically binding to SIP, anti-sense nucleic acids and ribozymes, which interfere with SIP1 production or activity can prevent tumor invasion and metastasis.

[0022] In all current schemes, the cDNA-encoded proteins are expressed with an activation domain at the amino terminus. The activation domains used include the strong activation domain from Gal4, the very strong activation domain from the Herpes simplex virus protein VP16, or a weaker activation domain derived from bacteria, called B42. The activation-tagged cDNA-encoded proteins are expressed either from a constitutive promoter, or from a conditional promoter such as that of the GAL1 gene. Use of a conditional promoter makes it possible to quickly demonstrate that activation of the reporter gene is dependent on expression of the activation-tagged cDNA proteins.

[0045] With regard to the above-mentioned small molecules, the term `medicament` relates to a composition comprising small molecules as described above and a pharmaceutically acceptable carrier or excipient (both terms can be used interchangeably) to treat diseases as indicated above. Suitable carriers or excipients known to the skilled man are saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers and preservatives. Other suitable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids and amino acid copolymers.

Problems solved by technology

However, the mechanism underlying the cooperative binding of NZF-3 to the bipartite element is currently unknown.

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