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-bas e pair (bp) enhancer and 0.4 kilobase pairs of 5'-flanking DNA of the uncoupling protein (ucp) gene. Expression of this transgene was restri cted to brown adipose tissue and was inducible by cold exposure or tre atment of transgenic mice by norepinephrine, retinoic acid (RA), or CL -316,243 beta 3-adrenoreceptor agonist. A search for retinoic acid res ponse elements in the ucp gene enhancer was undertaken using mutagenes is and transfection of cultured cells with chloramphenicol acetyltrans ferase 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 o f RA, In vitro this region was able to bind a complex containing prote ins recognized by antibodies against Jun or Fos. Mutations of an adjac ent region related to an inverted repeat of type 2 also markedly decre ased RA effect. This region was able to bind in vitro retinoid X recep tor alpha and retinoic acid receptor beta. The two regions form an act ivating 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 DN A fragment (bp -2489 to -2398) constituting a complex RA response doma in.