Stimulation of the Mouse rRNA Gene Promoter by a Distal Spacer Promoter

Abstract

We show that the mouse ribosomal DNA (rDNA) spacer promoter acts in vivo to stimulate transcription from a downstream rRNA gene promoter. This augmentation of mammalian RNA polymerase I transcription is observed in transient-transfection experiments with three different rodent cell lines, under noncompetitive as well as competitive transcription conditions, over a wide range of template concentrations, whether or not the enhancer repeats alone stimulate or repress expression from the downstream gene promoter. Stimulation of gene promoter transcription by the spacer promoter requires the rDNA enhancer sequences to be present between the spacer promoter and gene promoter and to be oriented as in native rDNA. Stimulation also requires that the spacer promoter be oriented toward the enhancer and gene promoter. However, stimulation does not correlate with transcription from the spacer promoter because the level of stimulation is not altered by either insertion of a functional mouse RNA polymerase I transcriptional terminator between the spacer promoter and enhancer or replacement with a much more active heterologous polymerase I promoter. Further analysis with a series of mutated spacer promoters indicates that the stimulatory activity does not reside in the major promoter domains but requires the central region of the promoter that has been correlated with enhancer responsiveness in vivo.Of the three classes of eukaryotic RNA polymerase studied to date, only RNA polymerase I is committed to transcribing a single type of DNA: the ribosomal DNA (rDNA) that ultimately gives rise to the mature 18S, 5.8S, and 28S rRNAs of the ribosome. Study of the organization of the tandem rRNA genes and of the RNA polymerase I transcription machinery has led to the identification of cis-acting elements and transacting factors involved in the regulated expression of these genes (40,44,46). The best-studied rDNA elements are those of Xenopus laevis, but the rDNA of other metazoans, including mice, rats, Chinese hamsters, and Drosophila melanogaster, appears quite conserved in organization, although greatly diverged in sequence. Fig. 1A illustrates the similarity in the organizations of the X. laevis and mouse rDNA repeats.The conserved organization of rDNA begins with the promoter, the most important regions of which are an ϳ40-bp core element extending upstream from the initiation site and an upstream element from approximately residue Ϫ140 to approximately residue Ϫ120. Sequences upstream of the core element can be made dispensable in vitro but are critical in vivo. Just upstream of this complete gene promoter is a promoter-proximal transcriptional terminator element that is required for efficient initiation at the gene promoter in vivo, evidently by preventing polymerase read-in (2, 18) but possibly also by providing a position-dependent stimulation (28, 29). Immediately upstream of the promoter and terminator region in most animal and plant species examined are multiple copies of a repetitive sequence. In frogs and mice (Fig. 1A), t...

p53 represses ribosomal gene transcription

“…Expression of p53 was monitored by immunoblotting using an anti-p53 monoclonal antibody (Pab421, Dianova) p53+Pol I transcription A Budde and I Grummt respectively (Figure 2a). In support of earlier studies showing transcriptional activation by the repetitive enhancer elements and upstream spacer sequences (Kuhn et al, 1990;Paalman et al, 1995), the relative level of transcription from pMr170-BH was lower than that of pMr5783-BH and pMr1930-BH (Figure 2c, lanes 1, 3 and 5). Signi®cantly, overexpression of wtp53 reduced transcription of all three reporter plasmids to a similar degree (ca.…”

PHF8 is a histone H3K9me2 demethylase regulating rRNA synthesis

“…Mapping of PHF8 binding throughout the rDNA locus showed that PHF8 bound mainly to the promoter and its upstream region of the rDNA gene, with particular enrichment at 36-42 kilobases (kb) (Figure 2E). Since this region was shown to be important for rRNA transcription [19], these results indicate that PHF8 binds to the regulatory elements of the rDNA gene and may regulate rRNA transcription.…”

Virtually the Entire Xenopus laevis rDNA Multikilobase Intergenic Spacer Serves to Stimulate Polymerase I Transcription

“…Following the X. laevis paradigm, the promoter-proximal repetitive elements of the rDNA IGS in mouse (24,25), Arabidopsis (26), and Acanthamoeba (27), the promoter-proximal spacer promoter repeats in Drosophila (28,29) and mouse (30), and the promoter-proximal spacer promoter and/or repetitive elements in rat (31) have been found to stimulate transcription from the respective cis-located gene promoters, much as in frog. Thus, there are many examples of promoter-proximal rDNA repeats acting to stimulate pol I transcription.…”