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The Dual Role of Helix ± Loop ± Helix-Zipper Protein USF in Ribosomal RNA Gene Transcription in Vivo

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The Dual Role of Helix ± Loop ± Helix-Zipper Protein USF in Ribosomal RNA Gene Transcription in Vivo Oncogene (1997) 14, 589 ± 594 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 The dual role of helix ± loop ± helix-zipper protein USF in ribosomal RNA gene transcription in vivo Asish K Ghosh, Prasun K Datta and Samson T Jacob* Department of Pharmacology and Molecular Biology, The Chicago Medical School, 3333 Green Bay Road, North Chicago, Illinois 60064, USA We have previously demonstrated that the core poliovirus infection, in response to dierentiation, heat promoter of rat ribosomal RNA gene (rDNA) contains shock or inhibition of protein synthesis, and by an E-box-like sequence to which the core promoter glucocorticoid treatment of lymphosarcoma cells binding factor CPBF binds and that the 44 kDa subunit (Jacob, 1995). of this protein is immunologically related to USF1, the In general, rDNA transcription is species-speci®c helix ± loop ± helix-zipper DNA binding protein. Further, (Mishima et al., 1982; Grummt et al., 1982; Ishikawa et we showed that RNA polymerase I (pol I) transcription al., 1991) although a strict species speci®city can be in vitro is competed by oligonucleotides containing altered under certain conditions (Pape et al., 1990; USF-binding site, which suggested a key role for USF Ghosh et al., 1996). Three key cis-acting elements in rDNA transcription. To prove the potential role of namely core promoter, enhancers and terminators are USF in pol I transcription in vivo, USF1 and USF2 involved in the initiation, stimulation and termination homodimers and USF1/USF2 heterodimer were over- of rDNA transcription. Similarly, trans-acting factors expressed in CHO cells by transfection of the respective that speci®cally interact with the cis-acting elements are cDNAs. Co-transfection of a plasmid containing rDNA required for the initiation and/or regulation of rDNA followed by primer extension analysis showed that transcription. A complex called SL1 that consists of overexpression of USF1 and USF2 as homodimers TATA box-binding protein TBP and pol I-speci®c resulted in inhibition of rDNA transcription by as much TBP-associated proteins (TAFs) constitutes one of the as 85 ± 90% whereas overexpression of USF1/USF2 in essential trans-acting factors involved in the initiation the heterodimeric form activated transcription approxi- of rDNA transcription (Comai et al., 1992). Recent mately 3.5-fold. Transfection of mutant USF2 cDNA study has suggested the potential involvement of other that is devoid of the basic DNA-binding domain factors in the initiation of rDNA transcription (Joost et produced only minimal inhibition of rDNA transcription. al., 1994). Two other factors, E1BF/Ku and CPBF These data show that USF can modulate transcription (core promoter-binding factor), are involved in the of rRNA gene in vivo by functioning as a repressor basal or initiation of rDNA transcription (Zhang and (homodimer) or activator (heterodimer) of pol I Jacob, 1990; Ghosh et al., 1993; Ho and Jacob, 1993; transcription in vivo and suggest that inhibition of Ho et al., 1994; Liu and Jacob, 1994). Antibodies rDNA transcription may be responsible for the against the Ku protein can inhibit initiation of rDNA antiproliferative action of USF homodimers. transcription and this inhibition is signi®cantly restored following addition of puri®ed E1BF/Ku protein (Ho Keywords: ribosomal RNA gene; upstream stimulatory et al., 1994). The factor CPBF is required for rDNA factor; repressor; activator; pol I transcription transcription in a reconstituted system (Liu and Jacob, 1994). Further study showed that CPBF and E1BF/Ku interact physically and functionally to promote rDNA transcription (Niu et al., 1995). It is not known Introduction whether CPBF and E1BF/Ku directly interact with SL1 complex. It is also unclear how the various factors Ribosomal RNA (rRNA) is synthesized in the involved in the initiation of transcription confer species nucleolus by RNA polymerase I (pol I) as a large speci®city. precursor RNA (pre rRNA) which is processed to Recent study in our laboratory showed that mature rRNAs (28S, 18S and 5.8S) by a series of mammalian rRNA gene promoter contain an E-box- speci®c cleavage reactions (Paule, 1993; Jacob, 1995; like sequence (CACGcTG) to which the basic helix ± Moss and Stefanowsky, 1995). rRNA synthesis can be loop ± helix-zipper DNA binding protein USF binds regulated by a variety of physiological, pathological (Datta et al., 1995). Antibodies against USF1 cross- and nutritional conditions. The up-regulation of rRNA reacted with the 44 kDa polypeptide of rat CPBF. gene (rDNA) transcription is achieved by glucocorti- Further, oligonucleotide probes corresponding to coid in non-lymphoidal cells/tissues in response to USF-binding site and rDNA core promoter inhibited growth or cell proliferation and SV40-induced infec- both pol I and pol II transcriptions. Only tion. It is down-regulated by nutrient deprivation, oligonucleotides that contain the USF binding sequence competed eectively in transcription. This study revealed the functional relationship between Correspondence: ST Jacob CPBF and USF. *Present address: Medical Biochemistry, Ohio State University, 333 The present study was undertaken to determine Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210 The ®rst two authors contributed equally to this work whether USF can transactivate ribosomal RNA gene Received 22 July 1996; revised 2 October 1996; accepted 7 October promoter in vivo. Surprisingly, overexpression of 1996 USF1 and USF2 individually in cells inhibited pol I- rRNA gene regulation by USF AK Ghosh et al 590 mediated transcription whereas overexpression of kDa USF1/USF2 heterodimer activated the ribosomal 200.0 — L RNA gene promoter. This dual eect of USF on b-galactosidase RNA polymerase I-mediated transcription is speci®c, 97.4 — 69.0 — as overexpression of either subunits as homodimers or heterodimers did not aect a non E-box containing 46.0 — L USF2 promoter. L USF1 * M 1 2 3 4 5 Results Figure 2 Overexpression of USF1, USF2, DUSF2 homodimers and USF1/USF2 heterodimer in CHO cells. Whole cell extracts prepared from transfected CHO cells were subjected to 10% Overexpression of USF1, USF2 and DUSF2 in CHO SDS ± PAGE and transferred to nitrocellulose membrane. cells does not aect non E-box containing promoter Immunoblot analysis was done as described in Methods using antibodies against b-Galactosidase, USF1 and USF2. Lane M, Previous study showed that the 44 kDa subunit of the prestained molelcular weight standards (Amersham); lane 1, ribosomal RNA core promoter-binding factor CPBF is control extracts from pLTR and pSVb-gal DNA-transfected functionally and immunologically related to the basic cells; lane 2, pLTR, USF1 and pSVb-gal DNA-transfected cell extract; lane 3, pLTR USF2 and pSVb-gal DNA-transfected cell helix ± loop ± helix-zipper DNA binding protein USF1 extract; lane 4, pLTR DUSF2 (lacks the basic DNA binding (Datta et al., 1995). Oligonucleotides corresponding to domain) and pSVb-gal DNA DNA-transfected CHO cell extract, USF-binding site and rDNA core promoter inhibited and lane 5, USF1, USF2 and pSVb-gal DNA-co-transfected cell both pol I and pol II mediated transcription in vitro. extract. Arrows on the right indicate the position of 44 kDa (USF2) and 43 kDa (USF1) polypeptides and the 116 kDa b- This data suggested that USF trans-activated rDNA Galactosidase polypeptide. *denotes the endogenous cellular transcription by interacting with the USF-binding E- protein having similar immuno-reactivity with the anti-USF box-like sequence in the ribosomal RNA promoter antibodies in all transfected cell extracts element. To prove further the relevance of this ®nding under in vivo conditions, the 43 kDa USF1, 44 kDa USF2 and DUSF2 were overexpressed in CHO cells by devoid of the basic DNA-binding domain (see transfecting with the respective cDNA clones (see Materials and methods and Figure 1) was also Materials and methods and Figure 1 for plasmid overexpressed in the transfected CHO cells (lane 4). constructs). To monitor transfection eciency and to This study showed that the expressions of the demonstrate that overexpression of USF subunits does transfected cDNAs were signi®cantly greater than not in¯uence promoters lacking E-box sequence, a those of the endogenous USF genes (Figure 2, compare reporter construct pSVb-Galactosidase was used. To lane 1 with lanes 2 ± 5; also see the relatively lighter rule out the in¯uence of vector DNA on the expression bands of the endogenous 44 kDa USF2 and 43 kDa of reporter genes, pLTR vector DNA alone was USF1 in lanes 2 and 3, respectively). The immunoblot transfected. Forty-eight hours after transfection, whole analysis for the expression of b-galactosidase clearly cell extracts were made and subjected to Western blot demonstrates uniformity in the transfection assay and analysis using antibodies directed against b-Galactosi- that the overexpression of USF polypeptides either as dase, USF1 and USF2. The amounts of USF1 and homodimers or as heterodimers does not in¯uence the USF2 were indeed higher in the cells transfected with activity of the control promoter (SV40) and speci®cally USF1 and USF2 cDNAs separately than those a non E-box containing promoter. These data demon- transfected with the control (pLTR and pSVb-gal) strated that overexpression of USF polypeptides in plasmids (Figure 2, compare lanes 2 and 3 with lane 1). CHO cells does not aect the activity of a control Cells co-transfected with USF1 and USF2 cDNAs reporter plasmid (SV40-b-galactosidase) at the protein overexpressed both 43 kDa and 44 kDa polypeptides level. Subsequent series of experiments addressed the (Figure 2, lane 5). As an additional control for the in¯uence of the overexpression of USF subunits as functional studies described below, DUSF2 that is homodimers or as heterodimers on the ribosomal RNA gene promoter activity at the RNA level. Overexpression of 44 kDa USF2 protein inhibits pol I transcription in vivo Homodimer as well as heterodimer of both USF subunits (44 kDa and 43 kDa) are known to bind the E-box-like sequences with equal anity whereas monomers are unable to bind DNA (Sawadogo and Roeder, 1985; Sirito et al., 1992).
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