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 factor-like domain-containing protein highly expressed in 3T3-L1 preadipocytes, but is undetectable in mature fat cells; this down-regulation is required for adipocyte differentiation. We show here that pref-1 transcription is markedly suppressed during adipose conversion and results in decreased Pref-1 RNA levels. Using 3T3-L1 cells stably transfected with Pref-1 5'-deletion constructs truncated at -6000, -2100, -1300, -692, -300, -235, -193, -183, -170, -93, and -45 base pairs, we determined 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 preadipocytes. The placement of a 40-base pair -193 to -154 pref-1 sequence containing the putative SAD (suppression in adipocyte differentiation) element upstream of the SV40 promoter decreased promoter activity by 85% upon 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. Competition with oligonucleotides harboring base substitution mutations identified a core sequence of -183AAAGA-179 as crucial for DNA-protein complex formation. UV cross-linking predicts that an approximately 63-kDa protein specifically binds the SAD element.
Smas
C M
CM
Department of Nutritional Sciences, University of California, Berkeley, California 94720, USA.
Kachinskas
D
D
Liu
C M
CM
Xie
X
X
Dircks
L K
LK
Sul
H S
HS
D016475:3T3 Cells; D017667:Adipocytes / Q000166:cytology* / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002135:Calcium-Binding Proteins; D002454:Cell Differentiation; D005786:Gene Expression Regulation; D036341:Intercellular Signaling Peptides and Proteins; D008156:Luciferases; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D013489:Suppression, Genetic; D014158:Transcription, Genetic; D014162:Transfection
D002135:Calcium-Binding Proteins; C512925:Dlk1 protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D008156:Luciferases