Abstract
Id family helix-loop-helix (HLH) proteins are involved in the regulation of proliferation and differentiation of several cell types. To identify cis- and trans-acting factors that regulate Id4 gene expression, we have analyzed the promoter regulatory sequences of the human Id4 gene in transient transfections and gel mobility shift assays. We have identified two functional elements, both located downstream from the TATA motif, that control Id4 promoter activity. One element contains a consensus E-box, and we demonstrated that the protein complex binding to the E-box contains the bHLH-zip upstream stimulatory factor (USF) transcription factor. Enforced expression of USF1 leads to E-box-mediated stimulation of promoter activity. The E-box also mediated stimulatory effects of several bHLH transcription factors, and co-expression of Id4 blocked the stimulatory effect mediated by the bHLH factors. A second element is a GA motif, located downstream from the transcriptional start sites, mutation of which resulted in a 20-fold increase in transcriptional activity. Gel-shift analysis and transfections into Drosophila Schneider SL2 cells showed that the repressor element is recognized by both Sp1 and Sp3 factors. These data suggest that Id4 transcription control is highly complex, involving both negative and positive regulatory elements, including a novel inhibitory function exerted by Sp1 and Sp3 transcription factors.
Highlights
The basic-helix-loop-helix1 family of transcription factors has been shown to play a key role in the differentiation processes of a number of cell lineages [1,2,3]
We have demonstrated that the core promoter of the Id4 gene comprises a relatively small region spanning nucleotide residues from Ϫ48 to ϩ32
Apart from the TATAbox, the presence of which is strictly required for promoter activity, we have identified two regions that appear to be important for proper promoter activity, and both cis-acting elements are located downstream from the TATA motif
Summary
The basic-helix-loop-helix (bHLH) family of transcription factors has been shown to play a key role in the differentiation processes of a number of cell lineages [1,2,3] These proteins contain an HLH domain consisting of two amphipathic helixes separated by a loop, which mediates homo- and heterodimerization, plus an adjacent DNA-binding region rich in basic amino acids [4, 5]. Dimerization is essential for binding and transcriptional regulation in vivo, and in general tissue-specific bHLH form heterodimers with a partner from the ubiquitously expressed class A family [13, 14]. These factors form an interacting network that regulate transcription of several genes.
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