Use of Saccharomyces cerevisiae in the identification of novel transcription factor DNA binding specificities

Members of the steroid/hormone nuclear receptor superfamily regulate target gene transcription via recognition and association with specific cis-actin g sequences of DNA, called hormone response elements (HREs), The identifica tion of novel HREs is signalling pathways, A number of these receptors are transcriptionally active, or can be induced to an active state, when expres sed in the yeast strain Saccharomyces cerevisiae. This aspect of nuclear re ceptor activity was used to screen random rat genomic DNA fragments for the ir ability to function as a HRE for the farnesoid X-activated receptor (FXR ), An isolated genomic fragment mediated FXR transcriptional activation wit hout the co-expression of the retinoid-X receptor (RXR), a receptor previou sly thought to be an obligate heterodimer partner for FXR function. This ge nomic sequence of DNA contained a pair of highly conserved HRE half-sites a rranged in an everted orientation and separated by 3 bp (ER3), Furthermore, it was located 240 bp from a highly conserved TATA box motif, A minimal ER 3 sequence of DNA was further demonstrated to function as a FXR HRE and was bound in vitro by FXR-expressing yeast extracts, Using RT-PCR, an expresse d mRNA fragment was identified within an 8 lib region don:a stream of the p utative TATA box motif, This sequence of DNA was observed to bear homology to a cDNA found in mouse blastocyst, These findings define a novel FXR DNA binding specificity but, more importantly, these data suggest that this str ategy might be universally applied to any transcription system that can be reconstituted in 3 east, Copyright (C) 2001 John Wiley & Sons, Ltd.