IN-VITRO INTERACTIONS BETWEEN NUCLEAR PROTEINS AND UNCOUPLING PROTEINGENE PROMOTER REVEAL SEVERAL PUTATIVE TRANSACTIVATING FACTORS INCLUDING ETS1, RETINOID-X RECEPTOR, THYROID-HORMONE RECEPTOR, AND A CACCC BOX-BINDING PROTEIN

Previous studies of rat ucp (uncoupling protein) gene organization car ried out in this laboratory identified regulatory sequences located in the 5'-flanking region. In this work, DNase I footprint analysis of t he enhancer revealed two domains at base pairs (bp) -2444 to -2423 and bp -2352 to -2319. The former domain can bind in vitro, in a cooperat ive manner, factors related to nuclear factor 1 and Ets1; the latter d omain contains a type 3 directly repeated sequence that was shown to b e able to bind the retinoid X and triiodothyronine receptors. Moreover , a positive effect of retinoic acid on ucp mRNA levels in immortalize d brown adipocytes was observed. DNase I footprint analysis identified two hypersensitive regions, A and B, at bp -509 to -472 and bp -403 t o -350, respectively; region A contains a repeated CACCC box, and regi on B can bind protein related to Ets1. The A box differentially binds liver and brown adipose tissue nuclear proteins and could be involved in uncoupling protein induction. Further analysis showed three footpri nted boxes, C-E, at bp -182 to -159, -147 to -120, and -111 to -85, ab le to bind in vitro proteins related to nuclear factor 1, cAMP respons e element-binding protein, and Sp1, respectively.