Tissue-specific regulation of the insulin gene by a novel basic helix-loop-helix transcription factor.
The insulin gene is one of the best paradigms of tissue-specific gene expression. It is developmentally regulated and is expressed exclusively in the pancreatic beta-cell. This restricted expression is directed by a tissue-specific enhancer, within the promoter, which contains an E-box sequence. The insulin E-box binds an islet-specific protein complex, termed 3a1. E-boxes bind proteins belonging to the basic helix-loop-helix (bHLH) family of transcription factors. The bHLH proteins function as potent transcriptional activators of tissue-specific genes by forming heterodimers between ubiquitous and cell-restricted family members. In addition, the cell-restricted bHLH members play an important role in specifying cell fate. To isolate the tissue-specific bHLH factor controlling insulin gene expression and study its role in islet cell differentiation, a modified yeast two-hybrid system was utilized to clone a novel bHLH factor, BETA2 (beta-cell E-box trans-activator 2), from a hamster insulin tumor (HIT) cell cDNA library. Northern analysis demonstrates that high-level expression of the BETA2 gene is restricted to pancreatic alpha- and beta-cell lines. As expected of tissue-specific bHLH members, BETA2 binds to the insulin E-box sequence with high affinity as a heterodimer with the ubiquitous bHLH factor E47. More importantly, antibody supershift experiments clearly show that BETA2 is a component of the native insulin E-box-binding complex. Transient transfection assays demonstrate that the BETA2/E47 heterodimer synergistically interacts with a neighboring beta-cell-specific complex to activate an insulin enhancer. In contrast, other bHLH factors such as MyoD and E47, which can bind to the insulin E-box with high affinity, fail to do so. Thus, a unique, cooperative interaction is the basis by which the insulin E-box enhancer discriminates between various bHLH factors to achieve tissue-specific activation of the insulin gene.
Naya
F J
FJ
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Stellrecht
C M
CM
Tsai
M J
MJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D018257:Helix-Loop-Helix Motifs; D007328:Insulin / Q000096:biosynthesis* / Q000235:genetics; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D051820:TCF Transcription Factors; D015534:Trans-Activators / Q000235:genetics*; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D014158:Transcription, Genetic; D015003:Yeasts / Q000235:genetics
D004268:DNA-Binding Proteins; D007328:Insulin; D012333:RNA, Messenger; D011994:Recombinant Proteins; D051820:TCF Transcription Factors; D015534:Trans-Activators; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors