Enhancer Of Split Proteins Research Articles

Hes-1, a Known Transcriptional Repressor, Acts as a Transcriptional Activator for the Human Acid α-Glucosidase Gene in Human Fibroblast Cells

Hes-1, the mammalian homologue 1 of Drosophila hairy and Enhancer of split proteins, belongs to a family of basic helix–loop–helix proteins that are essential to neurogenesis, myogenesis, hematopoiesis, and sex determination. Hes-1 is a transcriptional repressor for a number of known genes including the human acid α-glucosidase (GAA) gene as we have previously shown in Hep G2 cells. The human GAA gene encodes the enzyme for glycogen breakdown in lysosomes, deficiency of which results in Glycogen Storage Disease type II (Pompe syndrome). Using constructs containing the DNA element that demonstrates repressive activity in Hep G2 cells and conditions in which the same transcription factors, Hes-1 and YY1, bind, we have shown that this element functions as an enhancer in human fibroblasts. Site-directed mutagenesis and overexpression of Hes-1 showed that Hes-1 functions as a transcriptional activator. The dual function of Hes-1 we have found is likely to contribute to the subtle tissue-specific control of this housekeeping gene.

The Mammalian Basic Helix Loop Helix Protein HES-1 Binds to and Modulates the Transactivating Function of the Runt-related Factor Cbfa1

Drosophila Runt is the founding member of a family of related transcription factors involved in the regulation of a variety of cell-differentiation events in invertebrates and vertebrates. Runt-related proteins act as both transactivators and transcriptional repressors, suggesting that context-dependent mechanisms modulate their transcriptional properties. The aim of this study was to elucidate the molecular mechanisms that contribute to the regulation of the functions of the mammalian Runt-related protein, Cbfa1. Here we provide the first demonstration that Cbfa1 (as well as the related protein, Cbfa2/AML1) physically interacts with the basic helix loop helix transcription factor, HES-1, a mammalian counterpart of the Drosophila Hairy and Enhancer of split proteins. This interaction is mediated by the carboxyl-terminal domains of Cbfa1 and HES-1, but does not require their respective tetrapeptide motifs, WRPY and WRPW. Our studies also show that HES-1 can antagonize the binding of Cbfa1 to mammalian transcriptional corepressors of the Groucho family. Moreover, HES-1 can potentiate Cbfa1-mediated transactivation in transfected cells. Taken together, these findings implicate HES-1 in the transcriptional functions of Cbfa1 and suggest that the concerted activities of Groucho and HES proteins modulate the functions of mammalian Runt-related proteins.

The WRPW motif of the hairy-related basic helix-loop-helix repressor proteins acts as a 4-amino-acid transcription repression and protein-protein interaction domain.

Hairy-related proteins include the Drosophila Hairy and Enhancer of Split proteins and mammalian Hes proteins. These proteins are basic helix-loop-helix (bHLH) transcriptional repressors that control cell fate decisions such as neurogenesis or myogenesis in both Drosophila melanogaster and mammals. Hairy-related proteins are site-specific DNA-binding proteins defined by the presence of both a repressor-specific bHLH DNA binding domain and a carboxyl-terminal WRPW (Trp-Arg-Pro-Trp) motif. These proteins act as repressors by binding to DNA sites in target gene promoters and not by interfering with activator proteins, indicating that these proteins are active repressors which should therefore have specific repression domains. Here we show the WRPW motif to be a functional transcriptional repression domain sufficient to confer active repression to Hairy-related proteins or a heterologous DNA-binding protein, Ga14. This motif was previously shown to be necessary for interactions with Groucho, a genetically defined corepressor for Drosophila Hairy-related proteins. Here we show that the WRPW motif is sufficient to recruit Groucho or the TLE mammalian homologs to target gene promoters. We also show that Groucho and TLE proteins actively repress transcription when directly bound to a target gene promoter and identify a novel, highly conserved transcriptional repression domain in these proteins. These results directly demonstrate that Groucho family proteins are active transcriptional corepressors for Hairy-related proteins and are recruited by the 4-amino acid protein-protein interaction domain, WRPW.

Structure, chromosomal locus, and promoter analysis of the gene encoding the mouse helix-loop-helix factor HES-1. Negative autoregulation through the multiple N box elements.

HES-1 is a mammalian helix-loop-helix factor structurally related to the Drosophila hairy and Enhancer of split proteins. It binds more preferentially to the N box (CACNAG) than to the E box (CANNTG) and acts as a negative regulator. In this study, we have isolated and characterized the mouse HES-1 gene. This gene consists of four exons, and the positions of introns are well conserved when compared with those of the Drosophila hairy gene, except for the third intron. Southern blot and interspecies backcross analyses suggest that the mouse HES-1 gene is a single-copy gene and is located around position 26 on chromosome 16. The transcription initiation site, determined by the S1 nuclease and primer extension experiments, is located 31 nucleotides downstream of a TATA box. In the 5'-regulatory region, there are four N box sequences, and the DNase I foot-printing and gel mobility shift analyses show that HES-1 binds to these sequences. Transient transfection assays using C3H10T1/2 cells suggest that there are several positive regulatory regions in the HES-1 gene. However, cotransfection of the HES-1 expression vector leads to approximately 40-fold repression in promoter activity. Furthermore, when the N box sequences are disrupted, this negative regulation is severely impaired. These results raise the possibility that HES-1 gene expression may be negatively autoregulated through the N box sequences.