FIRE3 in the promoter of the rat fatty acid synthase (FAS) gene binds the ubiquitous transcription factors CBF and USP but does not mediate an insulin response in a rat hepatoma cell line

Several putative insulin-responsive elements (IRE) in the fatty acid syntha se (FAS) promoter have been identified and shown to be functional in adipoc ytes and hepatocytes. Here we report on the insulin-responsiveness in the r at hepatoma cell line H4IIE of four cis-elements in the FAS promoter: the F AS insulin-responsive elements, FIRE2 and FIRE3; the inverted CCAAT element , ICE; and the insulin/glucose-binding element, designated hepatic FIRE ele ment, hFIRE, originally identified in rat hepatocytes. Using electrophoreti c mobility shift assay (EMSA) competition experiments together with supersh ifts and in vitro transcription/translation we show that FIRE3 (-68/- 58) b inds not only the upstream stimulatory factors USF-1/USF-2 but also the CCA AT-binding factor CBF, also known as the nuclear factor Y, NF-Y. The putati ve IRE FIRE2, which shows sequence similarity to FIRE3, is located between -267 and -249. Gel retardation experiments indicate that USF-1 and USF-2 al so bind to this element, which contains an imperfect E-box motif. Using the same approach we have shown that hFIRE binds the stimulatory proteins Sp1 and Sp3 in addition to CBE Transient transfection experiments using FAS pro moter constructs deleted for FIRE2 and FIRE3 demonstrate that neither of th ese elements mediates the insulin response of the FAS promoter in the rat h epatoma cell line H4IIE, however, ICE at -103/-87 is responsible for mediat ing the effect of the insulin antagonist cAMP. The hFIRE element located at -57/-34, in spite of its role in the glucose/insulin response in primary r at hepatocytes, is apparently not involved in the insulin regulation of the rat FAS promoter in H4IIE cells. The fact that the topology of the promote rs of the FAS genes in rat, human, goose and chicken is conserved regarding CBF-binding sites and USF-binding sites implies an important role for thes e ubiquitously expressed transcription factors in the regulation of the FAS promoter.