Nuclear Respiratory Factor 1 Mediates the Transcription Initiation of Insulin-Degrading Enzyme in a TATA Box-Binding Protein-Independent Manner

Lang Zhang,Qingyang Ding,Zhao Wang,Frederick G Hamel

doi:10.1371/journal.pone.0042035

Abstract

CpG island promoters often lack canonical core promoter elements such as the TATA box, and have dispersed transcription initiation sites. Despite the prevalence of CpG islands associated with mammalian genes, the mechanism of transcription initiation from CpG island promoters remains to be clarified. Here we investigate the mechanism of transcription initiation of the CpG island-associated gene, insulin-degrading enzyme (IDE). IDE is ubiquitously expressed, and has dispersed transcription initiation sites. The IDE core promoter locates within a 32-bp region, which contains three CGGCG repeats and a nuclear respiratory factor 1 (NRF-1) binding motif. Sequential mutation analysis indicates that the NRF-1 binding motif is critical for IDE transcription initiation. The NRF-1 binding motif is functional, because NRF-1 binds to this motif in vivo and this motif is required for the regulation of IDE promoter activity by NRF-1. Furthermore, the NRF-1 binding site in the IDE promoter is conserved among different species, and dominant negative NRF-1 represses endogenous IDE expression. Finally, TATA-box binding protein (TBP) is not associated with the IDE promoter, and inactivation of TBP does not abolish IDE transcription, suggesting that TBP is not essential for IDE transcription initiation. Our studies indicate that NRF-1 mediates IDE transcription initiation in a TBP-independent manner, and provide insights into the potential mechanism of transcription initiation for other CpG island-associated genes.

Highlights

DNA methylation, an epigenetic modification that regulates chromatin structure and gene expression [1,2], occurs predominantly at cytosines of CpG dinucleotides in vertebrates [3,4]
Mapping the Core Promoter Region of Mouse insulin-degrading enzyme (IDE) Because the sequence of the mouse IDE promoter was incomplete in the NCBI genomic database (Figure 1A), we cloned and sequenced the unknown region [Genbank: JN038396]
IDE transcription starts with a purine at position +1 at a frequency of 91% and with a pyrimidine-purine dinucleotide at position 21,+1 at a frequency of 67%, which is consistent with previous studies showing that RNA polymerase II-mediated transcription is preferentially initiated at a pyrimidine-purine dinucleotide at position 21,+1 in mammals [16]

Summary

Introduction

DNA methylation, an epigenetic modification that regulates chromatin structure and gene expression [1,2], occurs predominantly at cytosines of CpG dinucleotides in vertebrates [3,4]. The observed rate of CpG sites is approximately one-fifth of the rate expected based on the GC content [6]. This rarity of CpG sites arises from the spontaneous mutation of methylated cytosines to thymidines by deamination, which converts CpG to TpG dinucleotides [7,8]. CpG islands, which have a high GC content and a high observedto-expected CpG ratio relative to the bulk genome, are often associated with mammalian gene promoters, including all housekeeping genes and some tissue-specific genes [10]. It was estimated that approximately 72% of genes are associated with CpG islands [14]

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