Synergistic activation of the insulin gene by a LIM-homeo domain protein and a basic helix-loop-helix protein: building a functional insulin minienhancer complex.
The distal portion of the rat insulin I gene 5'-flanking DNA contains two sequence elements, the Far and FLAT elements, that can function in combination, but not separately, as a beta-cell-specific transcriptional enhancer. We have isolated several cDNAs encoding proteins that bind to the FLAT element. Two of these cDNAs, cdx-3 and lmx-1, represent homeo box containing mRNAs with restricted patterns of expression. The protein encoded by lmx-1 also contains two amino-terminal cysteine/histidine-rich "LIM" domains. Both cdx-3 and lmx-1 can activate transcription of a Far/FLAT-linked gene when expressed in a normally non-insulin-producing fibroblast cell line. Furthermore, in fibroblasts expressing transfected beta-cell lmx-1, the addition of the Far-binding, basic helix-loop-helix protein shPan-1 (the hamster equivalent of human E47) causes a dramatic synergistic activation. ShPan-1 causes no activation in fibroblasts expressing transfected cdx-3 or the related LIM-homeodomain protein isl-1. Deletion of one or both of the LIM domains from the 5' end of the lmx-1 cDNA removes this synergistic interaction with shPan-1 without any loss of basal transcriptional activation. We conclude that beta-cell lmx-1 functions by binding to the FLAT element and interacting through the LIM-containing amino terminus with shPan-1 bound at the Far element. These proteins form the minimal components for a functional minienhancer complex.
Hormone Research Institute, University of California, San Francisco 94143-0534.
M S
MS
German
J
J
Wang
R B
RB
Chadwick
W J
WJ
Rutter
D000595:Amino Acid Sequence; D000682:Amyloid / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D005801:Genes, Homeobox; D018398:Homeodomain Proteins; D007328:Insulin / Q000235:genetics*; D058228:Islet Amyloid Polypeptide; D008647:Mesocricetus; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017386:Sequence Homology, Amino Acid
D000682:Amyloid; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D007328:Insulin; D058228:Islet Amyloid Polypeptide; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D004247:DNA