The 5′‐untranslated RNA of the human dhfr minor transcript alters transcription pre‐initiation complex assembly at the major (core) promoter

Abstract

The human dhfr minor transcript is distinguished from the predominant dhfr mRNA by an approximately 400 nucleotide extension of the 5'-untranslated region, which corresponds to the major (core) promoter DNA (its template). Based on its unusual sequence composition, we hypothesized that the minor transcript 5'-UTR might be capable of altering transcription pre-initiation complex assembly at the core promoter, through direct interactions of the RNA with specific regulatory polypeptides or the promoter DNA itself. We found that the minor transcript 5'-UTR selectively sequesters transcription factor Sp3, and to a lesser extent Sp1, preventing their binding to the dhfr core promoter. This allows a third putative transcriptional regulatory protein, which is relatively resistant to sequestration by the minor transcript RNA, the opportunity to bind the dhfr core promoter. The selective sequestration of Sp3 > Sp1 by the minor transcript 5'-UTR involves an altered conformation of the RNA, and a structural domain of the protein distinct from that required for binding to DNA. As a consequence, the minor transcript 5'-UTR inhibits transcription from the core promoter in vitro (in trans) in a concentration-dependent manner. These results suggest that the dhfr minor transcript may function in vivo (in cis) to regulate the transcriptional activity of the major (core) promoter.

Genome-wide computational analysis of potential long noncoding RNA mediated DNA:DNA:RNA triplexes in the human genome

“…The derived processed data in form of peaks was exactly overlapped on the PTS sites corresponding to the three types of motifs (R, Y & M). The complete list for possible Hi-C interaction domains with PTS sites is given as Additional files 13,14,15,16,17,18,19,20,21. We next analysed enrichment or depletion of the hi-C interactions sites using statistical test.…”

“…In another example, the region between minor and major transcript initiation sites (~400 bp) of DHFR gene gives rise to a noncoding RNA transcript. This lncRNA transcript has shown to repress the transcription of downstream protein coding gene by forming a purine-purine-pyrimidine triplex motif with the DHFR promoter [17,18].…”

Transcription control by long non-coding RNAs

“…1C). 57 In quiescent cells, the cell cycle-regulated DHFR gene is repressed in a manner that correlates with the expression of an ~400-nt lncRNA, referred to as DHFR lncRNA, from the upstream minor promoter, which shuts off transcription from the major promoter. This lncRNA was found to directly bind TFIIB in vitro and reduce its occupancy on the major promoter in vivo, 58 © 2012 Landes Bioscience.…”

Transcriptional interference: an unexpected layer of complexity in gene regulation

“…Martianov et al propose that stable triplex formation between the sense-strand regulatory RNA and the DHFR gene at the promoter contribute to TI and promoter targeting in vivo (Martianov et al, 2007). However, a previous report suggested that triplex formation is not important for preventing recruitment of transcription factors in vitro at the DHFR locus (Blume et al, 2003). It would be interesting to know whether triplex formation is necessary for TI at the DHFR gene in vivo, since >95% of genes in mice and humans have triplex-forming ability in their promoters or transcribed regions (Wu et al, 2007).…”