TRANSCRIPTIONAL CONTROL OF THE PREF-1 GENE IN 3T3-L1 ADIPOCYTE DIFFERENTIATION - SEQUENCE REQUIREMENT FOR DIFFERENTIATION-DEPENDENT SUPPRESSION

Preadipocyte factor-1 (Pref-1) is a transmembrane epidermal growth fac tor-like domain-containing protein highly expressed in 3T3-L1 preadipo cytes, but is undetectable in mature fat cells; this down-regulation i s required for adipocyte differentiation. We show here that pref-1 tra nscription is markedly suppressed during adipose conversion and result s in decreased Pref-1 RNA levels. Using 3T3-L1 cells stably transfecte d with Pref-1 5'-deletion constructs truncated at -6000, -2100, -1300, -692, -300, -235, -193, -183, -170, -93, and -45 base pairs, we deter mined that the -183 to -170 region is responsible for the suppression of the pref-1 gene during adipogenesis, This is distinct from the -93 to -45 sequence important for pref-1 promoter activity in preadipocyte s, The placement of a 40-base pair -193 to -154 pref-1 sequence contai ning the putative SAD (suppression in adipocyte differentiation) eleme nt upstream of the SV40 promoter decreased promoter activity by 85% up on adipocyte differentiation, compared with 40% observed with the SV40 promoter alone. The SAD element is therefore sufficient for adipocyte differentiation-dependent down-regulation of a heterologous promoter. A DNA-protein complex was observed when the -193 to -174 sequence was used with 3T3-L1 nuclear extracts in gel mobility shift assays. Compe tition with oligonucleotides harboring base substitution mutations ide ntified a core sequence of (-183)AAAGA(-179) as crucial for DNA-protei n complex formation. UV cross-linking predicts that an similar to 63-k Da protein specifically binds the SAD element.