Purification and characterization of a novel factor which stimulates rat ribosomal gene transcription in vitro by interacting with enhancer and core promoter elements.

Abstract

Previous studies in our laboratory have characterized a 174-base-pair (bp) enhancer sequence in the rat ribosomal DNA spacer region that exhibits all of the characteristics of a polymerase (Pol) II enhancer. Further studies showed that at least half of the enhancer activity resides in a 37-bp motif (E1) within the 174-bp spacer sequence that is located between positions -2.183 and -2.219 kilobase pairs upstream of the initiation site. To identify the factor(s) that binds specifically to the 37-bp enhancer domain, we fractionated whole-cell extract from rat adenocarcinoma ascites cells by chromatography on a series of columns, including an oligodeoxynucleotide affinity column. The final preparation contained two polypeptides of molecular weights 79,400 and 89,100 and was completely devoid of RNA Pol I activity. Electrophoretic mobility shift analysis showed that the polypeptides in the purified preparation (designated E1BF) interacted with both the enhancer element and the core promoter. To determine whether each polypeptide can separately bind to the core promoter and the enhancer, the individual components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, renatured, and subjected to gel retardation analysis. This experiment demonstrated that both polypeptides interacted with the two cis-acting sequences. The specificity of the binding was demonstrated by competition with unlabeled 37-bp and core promoter fragments and lack of competition with nonspecific DNAs in the mobility shift assay. The 37-bp enhancer as well as the downstream sequence of the core promoter were protected by E1BF in the DNase I footprinting assay. Addition of E1BF to limiting amounts of fraction DE-B, which contains all factors essential for Pol I-directed transcription, resulted in three- to fourfold stimulation of ribosomal DNA transcription. Comparison of molecular weights and footprinting profiles did not reveal any relationship between E1BF and other Pol I trans-acting factors.

The dual role of helix – loop – helix-zipper protein USF in ribosomal RNA gene transcription in vivo

“…Recent study has suggested the potential involvement of other factors in the initiation of rDNA transcription (Joost et al, 1994). Two other factors, E 1 BF/Ku and CPBF (core promoter-binding factor), are involved in the basal or initiation of rDNA transcription (Zhang and Jacob, 1990;Ghosh et al, 1993;Ho and Jacob, 1993;Ho et al, 1994;Liu and Jacob, 1994). Antibodies against the Ku protein can inhibit initiation of rDNA transcription and this inhibition is signi®cantly restored following addition of puri®ed E 1 BF/Ku protein (Ho et al, 1994).…”

E1BF is an essential RNA polymerase I transcription factor with an intrinsic protein kinase activity that can modulate rRNA gene transcription.

“…E1BF was purified from rat mammary adenocarcinoma cell extract as described (5). Purification by the oligonucleotide affinity column chromatography was repeated four times to avoid contamination with other polypeptides.…”

“…Purification by the oligonucleotide affinity column chromatography was repeated four times to avoid contamination with other polypeptides. The final preparation contained two polypeptides of Mr 89,000 and 79,000 (5).…”

“…Purified E1BF was suspended in 20 mM Hepes, pH 7.9/20o (vol/vol) glycerol/ 0.1 mM EDTA/2 mM dithiothreitol/0.5 mM phenylmethanesulfonyl fluoride/5 mM MgCl2 and incubated with 0.5 unit of calf intestinal alkaline phosphatase for 10 min at room temperature before addition of sodium vanadate (0.1 mM) to inhibit the phosphatase activity (17,18). The phosphorylation reaction was performed by incubating the dephosphorylated E1BF with [y-32P]ATP (5 ,uCi, 3000 Ci/mmol; 1 Ci = 37 GBq) at 37°C for 30 min, and the reaction was terminated by the addition of an equal volume of 30%o (wt/vol) trichloroacetic acid for precipitation. The pellet was dissolved in SDS loading buffer and kept in a water bath at 100°C for 3 min.…”

“…Transcription of rRNA genes (rDNA) is directed by RNA polymerase I and other accessory transcription factors. E1BF is a factor that can interact with an upstream enhancer element (El) (3,4) as well as the promoter sequence and can stimulate rat rDNA transcription in vitro (5). Recent studies have demonstrated that it can also activate the human rDNA promoter, but not polymerase II promoters (H.N.…”