Molecular and CellularBiochemistry 104: 155-162, 1991. © 1991KluwerAcademicPublishers. Printedin the Netherlands.
Multiple functional enhancer motifs of rat ribosomal gene Samson T. Jacob, Ji Zhang, Lalit C, Garg ~ and Carol-Beth Book 2 Department of Pharmacology and Molecular Biology, The Chicago Medical School, 3333 Green Bay Road, North Chicago, IL 60064, USA; 1Present address: National Institute of Immunology, Shahid Jeel-Sing Marg, New Delhi 110067, India; 2Present address: Department of Pharmacology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA
Key words: multiple functional motifs, enhancer, rat ribosomal gene Abstract
Previous studies from this laboratory have characterized a 174 bp enhancer element which is located 2 kb upstream of the initiation site. Half of the enhancer action is controlled by a 37 bp element at the 3' end of the 174 bp region. We now report that a 43 bp adjacent domain which is located upstream of the 37 bp element constitutes an additional motif of the rDNA enhancer. When the plasmid consisting of the 43 bp DNA upstream of the rDNA core promoter was transcribed in a fractionated rat tumor cell extract (fraction DE-B), transcription of rDNA was augmented 4 fold. Electrophoretic mobility shift and DNAase I footprinting analyses showed that the purified 37bp enhancer (E0-binding protein, (E1BF) not only interacted with the enhancer motif E~ but also interacted with the neighbouring 43 bp enhancer domain E 2. The specificity of the binding was demonstrated by competition with unlabeled 37 bp and 43 bp fragment and lack of competition with nonspecific DNAs in the mobility shift assay. These studies have shown that a single pol I transcription factor can bind to multiple enhancer domains with no significant sequence homologies and such multiple interactions may result in maximal transcription of ribosomal gene from the core promoter.
Regulation of eukaryotic gene expression is accomplished by two basic types of components, namely cis-acting elements and trans-acting factors. In addition to the promoter, the enhancer constitutes another major cis-acting element. The enhancer, first identified in Simian virus 40, could stimulate transcription from homologous or heterologous promoters irrespective of their orientation or distance from the initiation site [1, 2]. Several viral and cellular enhancers have since been identified (for reviews, see references 3-6). The mechanism of enhancer action has not been completely elucidated. The enhancers appear to stimulate transcription by forming a tight complex with their putative binding factor(s) that causes stable activa-
tion of the promoter. A similar enhancer element has also been reported for ribosomal genes from Xenopus  and . In X-laevis, enhancer elements share stretches of nucleotides with the promoter , and thus may act as a loading site for the essential transcription factors. However, the rat rDNA enhancer (174bp) characterized earlier in our laboratory  lies far upstream (between positions - 2.357 and - 2.183 kb) of the promoter and does not share any detectable sequence homology with the promoter. The rat rDNA enhancer exhibits all the characteristics of the RNA polymerase II enhancer . Thus, it can function in either orientation or when inserted downstream of the start site or independent of the distance from nucleotide + 1. Further analysis of this 174 bp enhancer region revealed that one element lies within the 37bp
156 from the 3' end ( - 2.219 kb to - 2.183 kb)  and binds to a trans-acting factor E1BF . However, the stimulation of transcription by he 37 bp element was only half of that obtained with the 174bp spacer . This finding suggested that other enhancer motif(s) must be present upstream of the 37 bp region. The present study was undertaken to delineate the additional motifs of the rat rDNA enhancer element.
Materials and methods
Reagents Restriction endonuclease, DNA modification enzymes, and DNA size markers (OX 174 RF DNA digested with Hae III) were purchased from Bethesda Research Laboratories. All enzymes were used as directed by the manufacturers. E. Coli HB101 (rec A) frozen competent cells were also obtained from Bethesda Research Laboratory and were used as a host for subcloning.
Plasmids Plasmids pB7-2.0  and pEH-5.0  were provided by Dr. Lawrence Rothblum (Geisinger Clinic, Sigfried and Janet Weis Center for Research) and Dr. Christina Harrington (Tufts University School of Medicine) respectively. These plasmids were used to construct plasmid pDJ4-3' ASK which contained 174 bp upstream region of rDNA spacer ( - 2 . 3 5 7 k b to - 2 . 1 8 3 k b ) ligated to - 1 6 7 b p to + 2.0 kb region of the rat rDNA . A plasmid containing 43 bp of rDNA spacer (spanning from - 2.262 kb to - 2.220 kb), rDNA core promoter and part of the coding region ( - 167 bp to + 2.0 kb) was constructed. (Figs. 1A, B, C). For this purpose a 43-mer oligonucleotide was synthesized. A 43-met ladder was made by standard ligation method. The ends of multimers were repaired by using DNA polymerase I (Klenow), ligated to BamHI linkers at both ends and cloned into pB72.0 linearized with Barn HI. E Coli HB101 cells were transformed with this DNA and the positive
clones were identified by standard colony lift and hybridization with nick translated 32p-labeled mixture of 43-mer multimers. Restriction digestion of the positive clones with Barn HI showed the size of insert. A clone containing the shortest insert (a 86 bp dimer of 43-mer) was selected in the present study and is designated plasmid pR43X2 (Fig. 1C). In vitro transcription assay In vitro transcription was carried out with plasmids linearized with Xho I, which should yield 635 nucleotides-long transcripts following a run-off transcription assay . The transcription reaction was carried out for 30 minutes at 30° C and the reaction products were analyzed as described . The amount of the transcript was determined by densitometric analysis of the autoradiograms .
Purification of E1BF E1BF (El binding factor which interacts with the enhancer motif El) was purified as described previously . Briefly, the purification procedure consisted of fractionation of whole cell extract derived from rat mammary adenocarcinoma. Ascites cells on DEAE-sephadex, heparin-sepharose, CM-sepharose and 37 bp oligo columns. The final preparation was subjected to polyacrylamide gel electrophoresis under denaturing conditions .
Electrophoretic mobility shift assay The synthetic oligonucleotides containing 43 bp spacer region ( - 2.220 kb to - 2.262 kb) and 37 bp spacer region ( - 2.183 kb to - 2.219 kb) of the rat ribosomal gene were labelled with 32p