Functional characterization of elements in a human U6 small nuclear RNA gene distal control region.

Abstract

The promoters of vertebrate U6 small nuclear RNA genes contain a distal control region whose presence results in at least an eightfold level of transcriptional activation in vivo. Previous transfection experiments have demonstrated that most of the distal control region of a human U6 gene resides in a restriction fragment located from -244 to -149 relative to the transcriptional start site. Three octamer-related motifs that bind recombinant Oct-1 transcription factor in vitro exist in this segment of DNA. However, transfection of human 293 cells with various plasmid templates in which these Oct-i binding sites had been disrupted individually or in combination showed that only the consensus octamer motif located between positions -221 to -214 was functional. Even so, the consensus octamer motif mutant template was expressed at only a moderately reduced level relative to the wild-type promoter. When another octamer-related sequence located nearby, one that did not bind Oct-i in vitro, was disrupted along with the perfect octamer site, expression was reduced fivefold in transfected cells. A factor that binds this functional, nonconsensus octamer site (NONOCT) was detected in crude cellular extracts. However, the NONOCT sequence was not essential for activation, since its disruption caused only a 40% reduction in U6 gene expression, and mutagenesis to convert the NONOCT sequence to a consensus octamer motif restored wild-type expression. Furthermore, in vitro transcription of a human U6 proximal promoter joined to a single copy of the octamer motif was stimulated by the addition of recombinant Oct-i protein.Vertebrate U6 small nuclear RNA (snRNA) genes are prototypes of the subclass of RNA polymerase III (Pol III)-transcribed genes that contain upstream promoters and no intragenic control region (10,20,29). The organization of these promoters is remarkably similar to those of genes transcribed by RNA Pol II. A general division of the vertebrate U6 gene promoter differentiates (i) a proximal region within 70 bp of the transcriptional start site consisting of an snRNA gene proximal sequence element (PSE) and a TATA element from (ii) a distal region greater than 200 bp upstream that usually contains an octamer motif. Similarly, promoters of the vertebrate Pol II snRNA genes (e.g., Ul and U2 genes) contain PSE and octamer motifs in nearly identical locations but lack recognizable TATA boxes (9

Conditional inhibition of cancer cell proliferation by tetracycline-responsive, H1 promoter-driven silencing of PLK1

“…A mutational analysis has demonstrated that both the octamer sequence and the Staf binding site contribute to full promoter activity. In addition, DSE-and PSEbound factors seem to contact each other (Murphy et al, 1989;Danzeiser et al, 1993;Mittal et al, 1996).…”

The Small RNA Gene Activator Protein, SphI Postoctamer Homology-binding Factor/Selenocysteine tRNA Gene Transcription Activating Factor, Stimulates Transcription of the Human Interferon Regulatory Factor-3 Gene

“…DNA-protein complex formation, competition with oligonucleotides and electrophoresis on nondenaturing gels were carried out as described previously (32). Preparation of a HeLa cell S100 extract is described in Ref.…”

“…33, and in vitro transcription/translation to produce recombinant SBF-(76 -626) was carried out as before (27). The same Ϫ206/Ϫ129 IRF-3 probe was used for DNase I footprinting following methods described previously (32), with bacterially expressed SBF-(76 -626) protein (27).…”

“…Electrophoretic Mobility Shift Assay and DNase I Footprinting-The radiolabeled IRF-3 probe used for protein-DNA binding assays was prepared by PCR using a kinased, 32 P-end labeled IRF-3-206 primer (5Ј-GGAACGCTGGGTGCACGC), an unlabeled, complementary strand CIRF-3-129 primer (5Ј-CCTATGCCCTTTTTTGGG), and pCR2/IRF3 plasmid DNA. DNA-protein complex formation, competition with oligonucleotides and electrophoresis on nondenaturing gels were carried out as described previously (32).…”

Non-cell autonomous induction of apoptosis and loss of posterior structures by activation domain-specific interactions of Oct-1 in the Xenopus embryo

“…Oct-1 is known as a widely expressed transcription factor with a role in the regulation of house keeping genes like the histone H2B and snRNA genes (Mattaj et al, 1985;Staudt et al, 1986;Fletcher et al, 1987;LaBella et al, 1988;Sturm et al, 1988;Murphy et al, 1989;Parry et al, 1989;Scho È ler et al, 1989;Heintz, 1991;Perry, 1991, 1992;Danzeiser et al, 1993;Kambe et al, 1993). Previous work has shown that Oct-1 is differentially expressed during early development in a pattern distinct from overall cell density or cell proliferation , prompting us to explore developmental functions of this transcription factor by perturbing its expression.…”