Promoter-specific transcriptional interference and c-myc gene silencing by siRNAs in human cells

Abstract

Small interfering RNAs (siRNAs) directed to gene promoters can silence genes at the transcriptional level. siRNA-directed transcriptional silencing (RdTS) was first described in plants and yeasts and more recently in mammalian cells. RdTS has been associated with the induction of epigenetic changes and the formation of complexes containing RNA interference and chromatin-remodelling factors. Here, we show that a promoter-targeted siRNA inhibits transcription of the c-myc gene. Transcriptional silencing of c-myc did not involve changes of known epigenetic marks. Instead, the c-myc promoter-targeted siRNA interfered with transcription initiation blocking the assembly of the pre-initiation complex. Transcriptional interference depended on Argonaute 2 and a noncoding promoter-associated RNA initiated upstream and overlapping the transcription start site. Silencing of c-myc led to growth arrest, reduced clonogenic potential and senescence of c-myc over-expressing prostate cancer cells with minimal effect on normal cells. RNA-directed transcriptional interference may be a natural mechanism of transcriptional control and siRNAs targeting noncoding RNAs participating in this regulatory pathway could be valuable tools to control expression of deregulated genes in human diseases.

The molecular dynamics of long noncoding RNA control of transcription in PTEN and its pseudogene

“…1C), but had little to no effect on downstream PTEN mRNA expression (Fig. 1D), suggesting there may be unique 5′ UTRassociated transcripts overlapping the PTEN promoter, similar to previous observations with small RNA-targeted transcriptional regulatory mechanisms (11,12).…”

“…By using RNase A and RNase H treatment, we determined that ODN2 binds a PTEN 5′ UTR-associated transcript, which interestingly extends the full length of the PTEN mRNA (Fig. 1E), similar to previous observations involved in the mechanism of small noncoding RNA transcriptional gene silencing (11,12). Notably, although ODN2 also appeared to bind to the PTENpg1 transcript (Fig.…”

“…Collectively, these observations, along with others (10), suggest PTEN promoter-associated 5′ UTR-containing transcripts are required for PTENpg1 asRNA α-and DNMT3a-based epigenetic regulation of PTEN. Previous studies with small antisense RNAs targeted to gene promoters have demonstrated that the small antisense RNAs interact directly with a transcript at the promoter (11,12). The observations presented here (Fig.…”

“…Previous studies with small noncoding RNAs (ncRNAs) demonstrated that an expressed low-copy transcript spanning the 5′ UTR of protein coding genes, designated a promoter-associated RNA (paRNA), was required for small ncRNA-directed epigenetic regulation in human cells (11)(12)(13). Transcriptomic data suggest there are several expressed sequence tags (ESTs) spanning the PTEN promoter [including BG772190 (Fig.…”

Deregulation of ETS1 and FLI1 contributes to the pathogenesis of diffuse large B-cell lymphoma

“…27 After sonication, chromatin was immunoprecipitated with antibody for ETS1 (C-20 X, Santa Cruz Biotechnology). DNA-protein cross-links were reversed and DNA purified from total cell lysates (input) and immunoprecipitated fractions.…”