Identification of an essential cis-acting element (TR2-PACE) in the 5′ promoter of human TR2 orphan receptor gene

The human TR2 orphan receptor (TR2) is a member of the steroid/thyroid hormone receptor superfamily, It has been shown to be expressed in a wide variety of tissues during development. Using deletion mutation analyses and transient transfection CAT assays, we demonstrated here that a DNA fragment of 103 bp, with a sequence from +65 to -38, containing an initiator is capable of serving as a core promoter to initiate basal level transcription; further extending of this core promoter sequence up to -441 maximizes the reporter gene expression. Within this positive regulatory region (-441/+65), we were able to narrow the regulation-responsible sequence down to a small 64-bp (-263/-201) DNA fragment named the TR2 promoter activating cis-element (TR2-PACE). Further deletion mutagenesis and shifting of the insert position followed by reporter assays demonstrated that this TR2-PACE is essential for high-level induction of a heterologous core promoter's activity in a position-dependent nature. In addition, orientation tests indicated that the sense, but not antisense orientation increased the TR2 core promoter activity. Moreover, electrophoresis mobility shift assays and Southwestern analyses suggested that TR2-PACE may interact with un known specific nuclear proteins for its enhancer activity. Together, our data suggest that TR2-PACE is a position-dependent and, in the case of TR2 core promoter (TATA-less), an orientation-dependent cis-activating element required for maximal expression of the TR2 gene.