A pancreatic islet cell-specific enhancer-like element in the glucagon gene contains two domains binding distinct cellular proteins.
Interactions of nuclear proteins with cis-control elements are involved in the programmed developmental expression of the islet polypeptide hormone genes. Three transcriptional control elements within 300 base pairs of the 5'-flanking region of the rat glucagon gene interact with regulatory cellular proteins and direct transcription only in glucagon-producing islet cells. Two islet cell-specific enhancer-like elements (G2, G3) act together with the glucagon promoter (including the G1 element), which confers A cell specificity of glucagon gene expression. In the present study, the G3 element was analyzed in detail by protein binding and in vivo and in vitro transcription assays. Mutational analyses showed that the sequence of the G3 element comprises two distinct protein-binding domains: a more upstream domain A (5'-CGCCTGA-3'), and a more downstream domain B (5'GATTGAAGGGTGTA-3'). Binding of proteins to these two domains is mutually exclusive. Domain A, but not domain B, is responsible for both functional protein binding and the enhancement of transcription from the glucagon or thymidine kinase gene promoter chloramphenicol acetyltransferase reporter gene transfected in vivo into glucagon-producing islet cells (InR1-G9) and transcribed in vitro in a HeLa cell-free transcription system. In islet cell extracts, the Southwestern blot technique labeled a protein of 45 kDa binding to domain A within G3. We conclude that although the G3 sequence contains two protein-binding motifs, the organization of the G3 enhancer-like element is not bipartite. The islet cell specificity of the G3 element is conferred by a tissue-specific transcription factor or protein complex interacting with domain A of G3. This protein or protein complex recognizes different DNA sequences and provides promoter as well as enhancer activity because it binds also to the apparently unrelated sequence of the G1 promoter element.
Knepel
W
W
Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Boston.
Jepeal
L
L
Habener
J F
JF
D007516:Adenoma, Islet Cell / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003851:Deoxyribonucleases; D004742:Enhancer Elements, Genetic; D005934:Glucagon / Q000235:genetics*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007340:Insulinoma / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009710:Nucleotide Mapping; D015345:Oligonucleotide Probes; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D051381:Rats; D014158:Transcription, Genetic
D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D005934:Glucagon; D003851:Deoxyribonucleases