526 A potent PBD-heterocyclic polyamide conjugate targeting an ICB2 transcription factor binding site

Abstract

The rat tyrosine aminotransferase gene (TAT) is a glucocorticoid-inducible gene, specifically expressed in liver. Using gel retardation assays, we have shown that its promoter (nt + 1 to −350; TAT35) binds a combination of both ubiquitous and liver-specific trans-acting factors. Cis-acting sequences spanning: (i) nt −65 to −85 bound NF-Y, an ubiquitous “AACCAAT” box binding factor; (ii) nt −157 to −171 bound a liver-enriched member of the NF1 gene family [NF1Liver(NF1L hereafter)]; (iii) nt −266 to −281 bound the liver specific factor HNF1; and (iv) nt −283 to −288 bound ubiquitous “CCAAT” box binding factor(s). Moreover, the TAT gene promoter was able to drive liver-specific basal transcription, even in an in vitro assay using TAT-expressing (liver) vs non-expressing (spleen) crude nuclear extracts (NEs). Competition studies in transcription with both unmutated and mutated ds-oligonucleotides (ds-oligos) demonstrated that NF1L and HNF1 supported approx. 60 and 25% of the basal transcriptional activity sustained by TAT·35 in the liver, respectively. Neither of these oligos affected the very low level of transcription sustained by spleen NEs. This suggests a minor role for HNF1 in liver-specific basal TAT gene expression, consistent with previous observations with dedifferentiated C2 hepatoma cells (which does not express HNF1) [Deschatrette and Weiss. Biochemie56 (1974) 1603–1611 and Cereghini et al. EMBO Jl9 (1990) 2257–2263]. Competition studies in liver-specific in vitro transcription with ds-oligo −265/−290 yielded a 90% inhibition, suggesting either that sequences spanning nt −283 to −288 sequester “CCAAT-box” binding factor(s) that may be relevant elsewhere for TAT promoter function (e.g. NF-Y which interacts with nt −65 to −85), or that such a factor interacts functionally with HNF1.