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Repression of the human dihydrofolate reductase gene by a non-coding interfering transcript


Alternative promoters within the same gene are a general phenomenon in gene expression1,2. Mechanisms of their selective regulation vary from one gene to another and are still poorly understood. Here we show that in quiescent cells the mechanism of transcriptional repression of the major promoter of the gene encoding dihydrofolate reductase depends on a non-coding transcript initiated from the upstream minor promoter and involves both the direct interaction of the RNA and promoter-specific interference. The specificity and efficiency of repression is ensured by the formation of a stable complex between non-coding RNA and the major promoter, direct interaction of the non-coding RNA with the general transcription factor IIB and dissociation of the preinitiation complex from the major promoter. By using in vivo and in vitro assays such as inducible and reconstituted transcription, RNA bandshifts, RNA interference, chromatin immunoprecipitation and RNA immunoprecipitation, we show that the regulatory transcript produced from the minor promoter has a critical function in an epigenetic mechanism of promoter-specific transcriptional repression.

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Figure 1: Organization and regulation of the human DHFR promoter.
Figure 2: Transcriptional interference in vitro.
Figure 3: Dissociation of TBP and TFIIB from the major DHFR promoter under induced transcriptional interference.
Figure 4: Interactions with the regulatory transcript in vitro and in vivo.


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We thank members of the laboratory for advice and support, N. Rust for providing support in flow cytometry analysis, S. Roberts and T. Oelgeschlaeger for suggestions, and J. Svejstrup for advice on the RNA immunoprecipitation procedure. This work was supported by grants from the Wellcome Trust and Medical Research Council. I.M. was supported by EMBO long-term fellowship.

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Correspondence to Alexandre Akoulitchev.

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Martianov, I., Ramadass, A., Serra Barros, A. et al. Repression of the human dihydrofolate reductase gene by a non-coding interfering transcript. Nature 445, 666–670 (2007).

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