Essential cis-Acting Elements in Rat Uncoupling Protein Gene Are in an Enhancer Containing a Complex Retinoic Acid Response Domain

Transgenic mice were generated with a transgene containing the 211-base pair (bp) enhancer and 0.4 kilobase pairs of 5′-flanking DNA of the uncoupling protein (ucp) gene. Expression of this transgene was restricted to brown adipose tissue and was inducible by cold exposure or treatment of transgenic mice by norepinephrine, retinoic acid (RA), or CL-316,243 β3-adrenoreceptor agonist. A search for retinoic acid response elements in the ucp gene enhancer was undertaken using mutagenesis and transfection of cultured cells with chloramphenicol acetyltransferase constructs. Deletion or mutations of several putative retinoic acid response elements were ineffective. Mutations of a TGAATCA region dramatically decreased the transcriptional activity in the presence of RA. In vitro this region was able to bind a complex containing proteins recognized by antibodies against Jun or Fos. Mutations of an adjacent region related to an inverted repeat of type 2 also markedly decreased RA effect. This region was able to bind in vitro retinoid X receptor α and retinoic acid receptor β. The two regions form an activating region between bp −2421 and −2402 (referred to as the ucp gene-activating region), which has an enhancer activity but cannot confer RA response to a promoter. This response was obtained with a larger DNA fragment (bp −2489 to −2398) constituting a complex RA response domain.