Protein-protein and DNA-protein interactions involved in regulation of eukaryotic gene expression

The basic-helix-loop-helix (bHLH) proteins constitute a rapidly growing family of transcription factors; the HLH motif mediates protein-protein interactions, while an adjacent basic region binds DNA sequences (CANNTG) known as E box motifs. It has been shown that bHLH proteins of class B require a class A bHLH partner, in order to exert their regulatory effects on tissue-specific gene expression. It has been proposed that TAL1, which is expressed in a restricted set of cell types during hematopoietic differentiation, represents a bHLH protein of class B. To test this hypothesis, experiments were performed to examine the interactions of selected E box motifs with complexes containing the DNA binding domains of TAL1 and representative class A proteins (HTF4a and E47). It was found that, unlike other class B proteins, TAL1 attenuated the binding of HTF4a and E47 to DNA. This analysis was extended by functional assays, which showed that TAL1 could inhibit the activation effects produced by HTF4a and E2-5, another bHLH protein of class A. The results of additional experiments, using nuclear extracts from lymphoid cell lines, indicated that in some cases the requirement for class B proteins could be obviated by heterodimeric complexes of class A bHLH proteins. Computer analysis revealed several E box motifs in the long terminal repeat (LTR) of human immunodeficiency virus type 1. Band-shift assays showed that representative class A proteins could bind an E box motif located downstream of the TATA element. Site-directed mutagenesis and transient transfection assays revealed that HTF4a could repress transcription from the LTR, suggesting that bHLH proteins binding to this E box could interfere with efficient assembly of the general transcriptional machinery. In contrast, TAL1 activated transcription from the LTR. Since transcription factors play an important role in regulating gene expression, a strategy was designed for isolating control regions in the human genome. A preliminary analysis used human genomic DNA clones selected by their ability to bind nuclear proteins. The method of analysis provides a convenient strategy for detecting eukaryotic regulatory DNA sequences and identifying their associated transcription factors.