Cheol Won Park

Cell specific expression of the insulin gene is achieved through transcriptional mechanisms operating on multiple DNA sequence elements located in the 5' flanking region of the gene. Of particular importance in the rat insulin I gene are two closely similar 9 bp sequences (IEB1 and IEB2): mutation of either of these leads to 5-10 fold reduction in transcriptional activity. We have screened an expression cDNA library derived from mouse pancreatic endocrine beta cells with a radioactive DNA probe containing multiple copies of the IEB1 sequence. A cDNA clone (A1) isolated by this procedure encodes a protein which shows efficient binding to the IEB1 probe, but much weaker binding to either an unrelated DNA probe or to a probe bearing a single base pair insertion within the recognition sequence. DNA sequence analysis indicates a protein belonging to the helix-loop-helix family of DNA-binding proteins. The ability of the protein encoded by clone A1 to recognize a number of wild type and mutant DNA sequences correlates closely with the ability of each sequence element to support transcription in vivo in the context of the insulin 5' flanking DNA. We conclude that the isolated cDNA may encode a transcription factor that participates in control of insulin gene expression.

Transcription of a number of mammalian genes is controlled in part by closely-related DNA elements sharing a CAxxTG consensus sequence (E boxes). In this report, we survey cell extracts from a variety of mammalian cell lineages for ability to bind to the E box denoted IEB1/kappa E1, which plays an important role in expression of both insulin and immunoglobulin kappa genes. Insulin enhancer factor 1 (IEF1), a binding activity previously identified in beta cells, was also present in pituitary endocrine cells but absent in 7 other mammalian cell lines tested. A distinct binding activity, lymphoid enhancer factor 1 (LEF1), was observed in several lymphoid cell lines, but was absent from all nonlymphoid cells tested. IEF1 and LEF1 were distinct according to electrophoretic mobility, and DNA binding specificity. As previously reported, both beta cell and lymphoid cell factors are recognized by antibodies to helix-loop-helix (HLH) proteins, indicating that they may contain functional helix-loop-helix dimerization domains. To directly demonstrate this, we showed that the binding factors are able to interact in vitro with the HLH domain of a characterized HLH protein. These results support the notion that HLH proteins play a key role in cell-specific transcriptional regulation in cells from endocrine and lymphocyte lineages.

Expression of insulin and immunoglobulin genes is dependent on the presence of E boxes (consensus sequence CAXXTG) within the enhancer regions. These sequences are recognized by cell-specific nuclear factors IEF1 (insulin enhancer factor 1) and LEF1 (lymphoid enhancer factor 1). Although IEF1 and LEF1 are distinct by several parameters, they are both recognized by antisera to the mouse helix-loop-helix (HLH) protein A1 (a homolog of the human protein E47, product of the E2A gene). This suggests that A1/E47 or a close relative is a component of both complexes. In order to further characterize the complexes, we have used in vitro translated DNA-binding proteins of known size to verify that electrophoretic mobility shift analysis can be used to estimate the molecular weight of DNA-binding proteins from both the HLH family and the leucine zipper family. Under the conditions used, migration is relatively insensitive to changes in protein charge. This analysis, in combination with mixing experiments between nuclear extracts and in vitro translated HLH proteins, indicates that IEF1 and LEF1 are dimeric complexes. IEF1 behaves as a complex of two proteins, one of which is 67 kDa and is recognized by antibodies to A1, and the second of which is 25 kDa. LEF1 on the other hand, appears to be a complex of two proteins of 67 kDa. The size of the 67-kDa subunits is consistent with that reported for the full-length E2A gene products. The 25-kDa subunit of IEF1 forms DNA-binding heterodimers with A1 but not MyoD and is present in a limited range of cell types, features characteristic of class B HLH proteins such as MyoD and achaete-scute. Taken together, the data support the idea that the E2A gene products are involved directly in regulation of insulin and immunoglobulin gene expression; regulation of the insulin gene apparently requires, in addition, the 25-kDa HLH protein (designated IESF1 for insulin enhancer-specific factor 1).