Abstract
This study demonstrates that the transcriptional repressor sequence of the rat vasoactive intestinal peptide receptor (VIPR) gene constitutes a 42-base pair core element that is the binding site for a nuclear protein. We showed that this element was able to confer transcriptional repression to a heterologous promoter and that deletion or point mutations within this element resulted in loss of transcriptional repression. Southwestern blot analysis indicated that the VIPR repressor element interacts specifically with a nuclear protein of about 72 kDa. By screening a rat lung expression library coupled with rapid amplification of cDNA ends polymerase chain reactions, we isolated a cDNA clone (designated as VIPR-RP) that contains an open reading frame of 656 amino acids. VIPR-RP is 78% identical to a previously characterized protein, differentiation-specific element-binding protein, which is a member of a family of proteins including components of the DNA replication factor C complex. However, VIPR-RP cDNA encodes for a much smaller protein than differentiation-specific element-binding protein because of a frameshift. VIPR-RP mRNA is expressed in multiple tissues, including lung, liver, brain, heart, kidney, spleen, and testis. VIPR-RP protein specifically interacts with the VIPR repressor element as demonstrated by gel shift assays. Transfection of VIP-RP expression vector into Cos cells resulted in transcriptional repression of a reporter construct containing multiple copies of the VIPR repressor element. These results indicate that VIPR-RP is a novel transcriptional repressor protein that regulates VIPR expression.
Highlights
Transcriptional repression plays a critical role in regulating gene expression
We have restricted the biological activity of the negatively acting element to a 42-bp DNA sequence located between Ϫ773 and Ϫ815 bp that contributes importantly to transcriptional repression of the VIP1 receptor (VIPR) gene
We showed that deletion of this sequence results in loss of transcriptional repression and that this sequence can repress transcription of a heterologous promoter
Summary
Transcriptional repression plays a critical role in regulating gene expression. Different repressors are known to function in a wide variety of biological settings [1,2,3,4]. Eukaryotic repressors that work by competitive DNA binding have been uncovered in the regulation of genes involved in pattern formation in fly embryos [5]. Examples of quenching include transcriptional repression of mouse proliferin gene, where AP-1 and glucocor-. Transcriptional activation of VIPR gene is regulated by multiple factors, including Sp1 and glucocorticoid receptor [13, 15]. Previous work in this lab demonstrated that a segment of VIPR gene, located between Ϫ859 and Ϫ488 bp, represses VIPR promoter activity in lung cells [13, 15]. Expression of VIPR-RP led to transcriptional repression These results indicate that positive and negative factors interact with VIPR promoter to regulate its expression
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