©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction of Oct-1 with TFIIB
IMPLICATIONS FOR A NOVEL RESPONSE ELICITED THROUGH THE PROXIMAL OCTAMER SITE OF THE LIPOPROTEIN LIPASE PROMOTER (*)

(Received for publication, May 25, 1995; and in revised form, June 19, 1995)

Harikrishna Nakshatri Poornima Nakshatri R. Alexander Currie (§)

From the Laboratory of Gene Regulation, The Picower Institute for Medical Research, Manhasset, New York 11030

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The ubiquitous human POU domain protein, Oct-1, and the related B-cell protein, Oct-2, regulate transcription from a variety of eukaryotic genes by binding to a common cis-acting octamer element, 5`-ATTTGCAT-3`. The binding of Oct-1 and Oct-2 to the functionally important lipoprotein lipase (LPL) promoter octamer site was stimulated by the general transcription factor, TFIIB. Comparative analysis of the LPL, histone H2B (H2B), and herpes simplex virus ICPO gene promoter octamer sites revealed that nucleotide sequences within and flanking the octamer sequence determined the degree of TFIIB-mediated stimulation of Oct-1 DNA binding. TFIIB was found to decrease the rate of dissociation of Oct-1 from the LPL octamer site, whereas it increased the rate of association, as well as decreased the rate of dissociation, of Oct-1 from the H2B octamer site. A monoclonal antibody against TFIIB immunoprecipitated a ternary complex containing TFIIB, Oct-1, and the LPL and H2B octamer binding sites. TFIIB did not alter the DNase I footprints generated by Oct-1 on the LPL and H2B promoters. However, Oct-1 prevented TATA-binding protein and TFIIB from footprinting the perfect TATA box sequence located 5` of the LPL NF-Y binding site. In transfection experiments, transcription from reporters containing the LPL octamer, and either the SV40 or the yeast transcription factor GAL4-dependent enhancers, initiated at a precise position within the octamer sequence. Transcription from reporters containing the H2B octamer and the SV40 enhancer initiated at several positions within and flanking the octamer site, whereas transcription initiated at a precise position within the octamer from reporters with both the H2B octamer and the GAL4-dependent enhancer. These results suggest that octamers and their flanking sequences play an important role in positioning the site of transcription initiation, and that this could be a function of the interaction of Oct-1 with TFIIB.


INTRODUCTION

Accurate and efficient transcription initiation from RNA polymerase II promoters requires the combined action of basal transcription factors and transcription activators. Basal transcription factors, along with RNA polymerase II, form a preinitiation complex over the core promoter DNA region and this assembly process is stimulated by transcription activators (Choy and Green, 1993; for review, see Zawel and Reinberg(1993)).

Two types of cis-acting motifs, the TATA box located 25-30 base pairs upstream from the transcription initiation site, and an initiator element (INR) (^1)located at the transcription initiation site, have been identified as the genetic elements that can independently specify the location of the preinitiation complex formation on a promoter (for review, see Breathnach and Chambon(1981) and Weis and Reinberg(1992)). The process of functional preinitiation complex formation in vitro on TATA-containing promoters involves the initial binding of TFIID to the TATA box, and subsequent ordered assembly of TFIIB, TFIIF, RNA polymerase II, TFIIE, and TFIIH onto the TATAbulletTFIID complex (Buratowski, 1994). Transcription initiation from INR-containing promoters requires specific interaction of INR binding proteins with the INR sequence, and subsequent assembly of basal transcription factors over the INR element (Martinez et al., 1994; Roy et al., 1993a; for review, see Weis and Reinberg(1992)). There are a number of promoters which contain neither a canonical TATA box nor any known INR-type elements, suggesting that there may be additional classes of elements that specify the location of preinitiation complex assembly in these promoters.

Among the basal transcription factors, TFIID and TFIIB have been studied extensively. TFIID is a multisubunit complex comprising the TATA-binding protein (TBP), and several tightly associated proteins commonly referred to as the TBP-associated factors (TAFs). Interestingly, in a reconstituted in vitro transcription system with purified components, TBP along with TFIIB and RNA polymerase II can initiate transcription in the absence of TAFs (Parvin and Sharp, 1993; Pugh and Tjian, 1990). However, an effective response to activators in such a system requires TAFs and TBP as well as distinct components of the transcription activators called coactivators (Meisterernst et al., 1991; Chiang et al., 1993; Chen et al., 1994). TAFs associate with distinct activation domains of transcription activators, TFIIB, TBP, or among themselves in the TFIID complex (Chen et al., 1994; Goodrich et al., 1993). TBP can also interact directly with several transcription activators (Stringer et al., 1990; Hateboer et al., 1993; Kashanchi et al., 1994; Caron et al., 1993; Liu et al., 1993).

TFIIB, which by itself cannot bind to double-stranded DNA, acts as a bridging factor between DNA-bound TBP, RNA polymerase II, and several different activators (Ha et al., 1991; Hisatake et al., 1993). Recent reports have indicated that TFIIB, in association with RNA polymerase II, functions to select the transcription initiation site (Li et al., 1994). In Drosophila, TFIIB has been shown to repress nonspecific transcription initiation (Wampler and Kadonaga, 1992). Furthermore, TFIIB is required for efficient interaction of TFIID with the INR, but not to the TATA box sequences in vitro (Kaufmann and Smale, 1994). In addition, TFIIB has also been shown to interact with transactivation domains of various transcription factors (Colgan et al., 1993; Lin and Green et al., 1991; Roberts et al., 1993; Ing et al., 1992; Kerr et al., 1993).

The octamer sequence motif, 5`-ATGCAAAT-3` (or its complement 5`-ATTTGCAT-3`), the GC box, and the CCAAT box sequence are the three most common elements found close to the TATA box/INR of cell type-specific and ubiquitously expressed genes. Several transcription factors are known to interact with the octamer and the CCAAT box. For example, the ubiquitous transcription factor Oct-1, and lymphoid-enriched transcription factor Oct-2, bind to the octamer sequence, whereas CTF/NF1, C/EBP, and NF-Y bind to the CCAAT box sequence (Muller et al., 1988; Scheidereit et al., 1988; Sturm et al., 1988; Hooft van Huisduijnen et al., 1990; Jones et al., 1987; Johnson et al., 1987). Sp1 is the most common factor that interacts with the GC box (Kadonaga et al., 1986). Specific TAFs which interact with the activation domains of CTF/NF1 and Sp1 have also been identified (Gill et al., 1994; Tanese et al., 1991; Chiang and Roeder, 1995). These factors are essential for efficient assembly of the preinitiation complex over a TATA box/INR (Arnosti et al., 1993; Mantovani et al., 1992; Seto et al., 1993).

Oct-1 and Oct-2 belong to the POU family of transcription factors which bind to their target sequences through their bipartite POU DNA-binding domains (Sturm et al., 1988; Scheidereit et al., 1988; Muller et al., 1988). Both proteins contain distinct transcription activation domains, located N-terminal and C-terminal to the POU domain, that are responsible for promoter-specific activity (Tanaka and Herr, 1990, 1994; Tanaka et al., 1992, 1994). Since Oct-1 is essential for the transcription of both mRNA-type as well as snRNA-type RNA polymerase II promoters, it is suggested that the promoter-specific activation domains of Oct-1 serve in conjunction with specific coactivators to activate gene expression (Yang et al., 1991; Tanaka et al., 1992; Pfisterer et al., 1994; Strubin et al., 1995; Gstaiger et al., 1995).

Lipoprotein lipase is a hydrolytic enzyme which is synthesized in a number of animal tissues, but functions at the luminal surface of the vascular endothelium. LPL plays an important role in lipoprotein metabolism by hydrolyzing triglycerides contained in chylomicron and VLDL particles and providing fatty acids to tissues for storage or combustion. The proximal 730-base pair LPL promoter region upstream of the transcription start site contains several potential binding sites for known transcription factors. They include a putative TATA box at position -27, three octamer sites at positions -580, -186, and -46, two CCAAT boxes at -65 and -506, sequences with partial homology to glucocorticoid response element at -644, a cyclic AMP responsive element at -306, and HNF-3/fork head family transcription factor binding sites at positions -702 and -468 (Deeb and Peng, 1989; Enerback et al., 1992; Previato et al., 1991). Among these sites, only the octamer at -46, and the CCAAT box at -65 have been shown to specifically bind the known transcription factors Oct-1 and NF-Y, respectively (Currie and Eckel, 1992; Previato et al., 1991). An element that functions as a silencer of LPL transcription in Chinese hamster ovary and HeLa cells has also been identified recently (Tanuma et al., 1995).

In this study, the interaction of basal transcription factors and transcription activators with the LPL promoter was investigated. Mutagenesis experiments revealed that the octamer motif (position -46 to -39), and the NF-Y CCAAT box (position -65 to -61), but not the putative TATA box (position -27 to -22), are essential for promoter activity. The binding of Oct-1 and Oct-2 to the LPL octamer element was investigated using purified Escherichia coli-derived recombinant proteins. We observed that recombinant Oct-1 and Oct-2 bound to this element only weakly on their own and that binding was stimulated 12-fold by TFIIB. Comparative analysis of TFIIB stimulation of Oct-1 binding to the LPL octamer versus the histone H2B promoter octamer and the herpes simplex virus ICPO octamer, suggested that the mechanism and the extent of stimulation depends upon nucleotide sequences both within and flanking the octamer. In a transfection assay, the LPL octamer element functioned as an INR element, raising the possibility that like other INR elements, TFIIB may recruit TFIID to the octamer site in vivo and Oct-1 may influence this process by recruiting TFIIB to the octamer site.


MATERIALS AND METHODS

Construction of Recombinant Plasmids

To construct the plasmid pLPLCAT, the human LPL fragment (-480 to +118 base pairs) from pGEM-7zf+LPL (Currie and Eckel, 1992) was first cloned into the EcoRI-BamHI site of SK- (Stratagene), isolated subsequently as a BamHI-XhoI fragment and cloned into BamHI-XhoI sites of pBLCAT3 (Luckow and Schultz, 1987). Point mutations in pLPLCATm1 to pLPLCATm5 were created by a two-step polymerase chain reaction procedure as described by Loh et al.(1989) using pLPLCAT as a template. In pLPLCATm1, the wild-type sequence 5`-CATAAG-3` at position -27 to -22 of the LPL promoter was changed to 5`-TATAAA-3`. pLPLCATm2 is similar to pLPLCATm1 except that the sequence, 5`-CATAAGCA-3`, was changed to 5`-GAGACCTG-3`. In pLPLCATm3, the octamer sequence, 5`-ATTTGCAT-3`, at position -46 to -39 was changed to 5`-TGTTCAGA-3`. In pLPLCATm4, the CCAAT box sequence, 5`-GCCAATAGGTGA-3`, at position -66 to -55 was changed to 5`-CTGCAGGAATTC-3`. In pLPLCATm5, the wild-type sequence, 5`-ATTTTATA-3`, at position -74 to -67 was changed to 5`-CAGGTCTC-3`.

To generate plasmids containing the SV40 enhancer, the 198-base pair EcoRI SV40 enhancer fragment derived from pOVEC-S (Westin et al., 1987) was cloned into the EcoRI site of SK- in both orientations and compatible flanking restriction sites of SK- were used to clone the enhancer into pBLCAT3. For example, to generate the plasmid pS1, the SV40 enhancer was cloned into the BamHI-HindIII site of pBLCAT3, such that the orientation of the SV40 enhancer in relation to the CAT gene was the same as in the SV40 early gene promoter (Tooze, 1981). In pS2, the SV40 enhancer had an orientation identical to the SV40 late gene promoter. The plasmid pS3 contains the SV40 enhancer downstream of the CAT gene-SV40 polyadenylation signal. The GAL4 binding site containing reporter, pG17, was created by two-step plasmid construction. First, a HindIII-EcoRI fragment from 17-mer thymidine kinase-CAT (Webster et al., 1988), which contains the 17-mer GAL4 binding site, a GC box, and the CCAAT box from the thymidine kinase promoter, was cloned into the HindIII-EcoRI site of SK-. Subsequently, a GAL4-binding site containing fragment from SK- was isolated as a BamHI-HindIII fragment, and cloned into the BamHI-HindIII site of pBLCAT3.

The synthetic oligonucleotide, 5`-GATCAGGTGATGAGGTTTATTTGCATATTTCC-3` (complementary strand: 5`-GATCGGAAATATGCAAATAAACCTCATCACCT-3`), corresponding to the nucleotides -59 to -33 of the LPL promoter, was cloned into the BglII site of pBLCAT3, pS1, pS2, pS3, and pG17, which gave rise to plasmids pLO+, pLO+S1, pLO+S2, pLO+S3, and pLO+G17, respectively. In these plasmids, the orientation of the inserted oligonucleotide was such that the octamer sequence, 5`-ATTTGCAT-3`, was located in the coding strand, as in the LPL gene. Plasmids pLO-, pLO-S1, pLO-S2, and pLO-G17, are similar to pLO+, pLO+S1, pLO+S2, and pLO+G17, respectively, except that the orientation of the LPL octamer-containing oligonucleotides was reversed. The plasmid pLOm+S1 is similar to pLO+S1 except that the octamer sequence 5`-ATTTGCAT-3` was changed to 5`-TGTTCAGA-3`.

The synthetic oligonucleotide, 5`-GATCCAACTCTTCACCTTATTTGCATAAGCGA-3` (complementary strand: 5`-GATCTCGCTTATGCAAATAAGGTGAAGAGTTG-3`), corresponding to nucleotides -63 to -36 of the H2B promoter (LaBella et al., 1988), was inserted into the BglII site of pBLCAT3, pS1, pS2, pS3, and pG17, which gave rise to plasmids pHO+, pHO+S1, pHO+S2, pHO+S3, and pHO+G17, respectively. In these plasmids the orientation of the inserted oligonucleotide was such that the octamer sequence 5`-ATTTGCAT-3` was located in the coding strand, as in the H2B gene. Plasmids pHO-, pHO-S1, pHO-S2, and pHO-G17 are similar to pHO+, pHO+S1, pHO+S2, and pHO+G17, respectively, except that the octamer sequence 5`-ATGCAAAT-3` was in the coding strand.

The plasmid pET15bOct-1 was constructed by blunt-end ligation of the HindIII-BamHI fragment of pBSOct1+ (Strum et al., 1988) into the BamHI site of pET15b (Novagen). OctDeltaN contains the Oct-1 cDNA sequence from nucleotides 918 to 2525 (amino acids 268 to 743) and was created by two-step plasmid construction. First, the HincII-HindIII fragment of pBSOct1+ was cloned into SK-, Oct-1 sequences were isolated as HincII-BamHI fragment and cloned into the pET15b vector that had been linearized with XhoI, blunt ended by Klenow treatment, and digested with BamHI. OctDeltaC contains the Oct-1 cDNA sequence from nucleotides 1 to 1774 (amino acids 1-590). This plasmid was created by digesting pET15bOct-1 with NcoI and ligation of the NcoI fragment containing the Oct-1 sequence into the NcoI site of pET15b. pET15bOct-2 was created by cloning Oct-2 cDNA (LeBowitz et al., 1988) into the XhoI-BamHI sites of pET15b. Plasmid vectors expressing protein A:Oct-1 POU domain fusion proteins and GST-TFIIB were a kind gift from Dr. P. A. Sharp (MIT, Cambridge, MA), and Dr. Inder Verma (The Salk Institute, San Diego, CA), respectively.

Recombinant Proteins

To obtain Oct-1, OctDeltaN, OctDeltaC, and Oct-2 recombinant proteins, expression vectors were transformed into E. coli strain BL21. Expression of recombinant proteins was induced by the addition of isopropyl-1-thio-beta-D-galactosidase. Three h after induction, the cells were collected, washed in phosphate-buffered saline, and resuspended in buffer D (20 mM HEPES, pH 7.9, 20% glycerol, 300 mM KCl, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin). Resuspended cells were lysed by sonication, centrifuged at 13,000 g for 20 min, and the supernatant was subjected to DEAE-cellulose chromatography. The flow-through fraction from DEAE-cellulose was applied to a Ni-NTA-agarose column (Novagen). This column was washed extensively with buffer D containing 10 mM imidazole. The column-bound Oct protein was eluted by a 50 mM to 1 M gradient of imidazole in buffer D. Fractions containing higher DNA binding activity as measured by EMSAs, and very few contaminating proteins as identified in a Coomassie Blue-stained SDS-PAGE gel, were dialyzed in buffer D containing 100 mM KCl and applied to a heparin-agarose column. Oct proteins, bound to the column were eluted using a step KCl gradient of 0.4, 0.6, and 1.0 M. Oct-1 protein containing only the POU domain was prepared as described previously (Kristie and Sharp, 1990). GST-TFIIB was prepared as described by Kerr et al.(1993), cleaved with thrombin (Novagen), and purified using glutathione-agarose beads. TBP was purchased from Promega.

Electrophoretic Mobility Shift Assays (EMSAs)

EMSAs were performed in a 10-µl reaction volume in the DNA binding buffer: 12 mM Tris, pH 7.9, 20 mM HEPES-KOH, pH 8.4, 100 mM KCl, 0.12 mM EDTA, 6 mM beta-mercaptoethanol, 20 mM dithiothreitol, 120 µg/ml BSA, and 2 µg of poly(dG-dC)bulletpoly(dG-dC). Incubations were at 30 °C for 40 min and the reactions were analyzed on a 5% polyacrylamide (60:1), 1 TBE gels at room temperature. In competition-dissociation assays, cold competitor DNA was added after 40 min of incubation. LPL and H2B probes used in the assays are as described in Currie and Eckel(1992). ICPO oligonucleotide probe is as described by Stern et al.(1989).

DNA Coimmunoprecipitation Assay

DNA binding reactions as described above were incubated with 0.2 µg of mouse anti-TFIIB antibody (Promega, Madison, WI) for 30 min on ice. Subsequently, rabbit anti-mouse IgG (0.2 µg, Zymed, San Francisco, CA) was added and the incubation continued for 30 min on ice. Protein A-Sepharose (20 µl, bead volume) (Pharmacia Biotech Inc.) was added to the reaction and incubated at 4 °C for 1 h with gentle rocking. Protein A-Sepharose was pelleted at 4 °C and washed five times with DNA binding buffer. SDS-PAGE sample buffer was added to the pellet and the samples were electrophoresed over 10% acrylamide gels.

DNase I Footprinting

DNase I footprinting was performed with a 3` end-labeled LPL probe (nucleotides -110 to +18; Currie and Eckel(1992)) or H2B probe (-90 to -20; LaBella et al.(1988)). DNA-protein binding reactions were performed in a buffer containing 25 mM Tris, pH 8.0, 0.5 mM EDTA, 10% glycerol, 0.5 mM dithiothreitol, and 0.01% Nonidet P-40, in a reaction volume of 50 µl for 40 min and digested with 0.03 units of DNase I (Boehringer Mannheim) for 1 min. The reaction was stopped by the addition of 0.4% SDS in 150 mM NaCl and 25 mM EDTA, phenol-chloroform extracted, and analyzed on an 8% sequencing gel.

Cell Transfection and CAT Assays

3T3-L1 cells were maintained and differentiated as described previously (Currie and Eckel, 1992). Undifferentiated cells were cotransfected with 20 µg of the indicated LPLCAT constructs and 0.5 µg of pcDNA3 (Invitrogen) by a calcium phosphate procedure (Kumar et al., 1987). Fourty-eight h after transfection, G418 (800 µg/ml) was added. Medium was changed every 4 days and G418-resistant colonies obtained after 20 days were pooled and replated across two plates. One plate was treated with differentiating agents, and harvested at day 7. Cell extracts were prepared, and heated to 65 °C for 5 min; CAT assays were performed as described previously (Kumar et al., 1987).

RNA Extraction, RNase Protection, and Primer Extension

Cells were transfected by a calcium phosphate procedure. RNA was prepared by RNAzol (Tel-Test Inc) 48 h after transfection. RNA was treated with RNase-free DNase I before primer extension. Primer extension with the 5` end-labeled primer 5`-CGATGCCATTGGGATATATC-3` (corresponding to nucleotides 521 to 540 of pBLCAT3, Luckow and Schultz(1987)) was performed as described previously (Sambrook et al., 1992). RNase protection with the human glyceraldehyde-3-phosphate dehydrogenase probe was performed as described previously (Ausubel et al., 1989).


RESULTS

Functional Analyses of the Octamer, the CCAAT Box, and the Putative TATA Box Region of the LPL Promoter

To investigate the functional importance of the octamer, the CCAAT box, and the putative TATA box region, the human proximal LPL promoter fragment spanning nucleotides -480 to +115 was cloned into the enhancer- and promoter-less chloramphenicol acetyltransferase (CAT) reporter vector pBLCAT3 (Luckow and Schultz, 1987). Several mutants were generated from this vector (Fig. 1A). In pLPLCATm1, the wild-type putative TATA box sequence, CATAAG, was changed to the canonical TATA box, TATAAA. In other mutants, the wild-type motifs were changed such that the corresponding transcription factor did not bind efficiently in an EMSA. The effect of various mutations on LPL promoter activity was determined by measuring the CAT enzyme activity in 3T3-L1 preadipocytes that had stably integrated CAT reporters. The CAT activity of these cells after their differentiation into adipocytes was also measured to determine the effect of mutations on the LPL promoter activity in mature adipocyte cultures (Fig. 1B). The wild-type LPL promoter displayed 7-fold more activity than the parental vector, pBLCAT3 (Fig. 1B, compare pBLCAT3 with pLPLCAT). Mutating the putative TATA box sequence to TATAAA increased the activity 2-fold (compare pLPLCAT with pLPLCATm1). Surprisingly, mutating the putative TATA to GAGACC, which does not bind TBP in vitro, further increased CAT activity, suggesting that the CATAAG sequence may not function as a TATA box (compare pLPLCAT with pLPLCATm2). The inverse TATA-like sequence located at position -74 to -67 of the LPL promoter was also determined not to be essential for promoter activity, since mutation of this sequence (pLPLCATm5) did not reduce CAT activity. In contrast, mutating the octamer sequence (pLPLCATm3), or the CCAAT box (pLPLCATm4) completely abolished LPL promoter activity. These results suggest that the transcription factors Oct-1 and NF-Y which bind to these elements in vitro (Currie and Eckel, 1992; see below) are required for LPL gene regulation. Our results differ from those reported by Enerback et al.(1992) with respect to cell type-specific activity of this promoter. Those investigators observed a 6-fold induction in promoter activity upon differentiation of 3T3L1-F22A preadipocytes containing a stably transfected construct similar to our wild-type construct; this was not detected in our experiments. This difference could be due to variation in the cells or differentiation conditions.


Figure 1: Activity of the LPL promoter in 3T3-L1 cells. A, partial sequence of the wild-type and mutated human LPL promoter in CAT reporters. Binding sites for the transcription factors, Oct-1 and NF-Y, and two putative TATA boxes are underlined. The mutations introduced to these elements are also indicated. B, CAT activity after transfection with reporter constructs. Transfection conditions are outlined under ``Materials and Methods.'' CAT activity obtained with extracts from cells transfected with the parental vector, pBLCAT3, was set arbitrarily as 1 unit. CAT activity in differentiated cells and undifferentiated cells are represented by open and striped bars, respectively. Activity of a CAT reporter under the control of cytomegalovirus enhancer-promoter (pCMVCAT) is also shown as a positive control. Results are average of two independent experiments.



Binding of Recombinant Oct-1 to LPL Octamer Sequence Is Stimulated by TFIIB

Previously, it was shown that Oct-1 from HeLa and 3T3-L1 cells binds to the proximal LPL promoter octamer site much more efficiently than to a similar element from the H2B promoter. This effect was mainly attributed to flanking A/T-rich sequences in the LPL promoter octamer site (Currie and Eckel, 1992). Since the putative LPL TATA box was shown to be nonfunctional, and the proximal octamer is a high affinity Oct-1 binding site, we have hypothesized that this octamer site might function as a surrogate TATA box to nucleate assembly of TFIID and other basic transcription factors. To explore this possibility, binding of TBP, TFIIB, and Oct-1 to the octamer element was investigated in EMSAs. In parallel, binding of these factors to the H2B octamer and the ICPO octamer elements was also tested (Fig. 2A). Neither TBP nor TFIIB alone, or in combination, bound specifically to any of the above elements (Fig. 2A, lanes 1-3, 10-12, and 19-21). Oct-1 bound to the ICPO element much more strongly than to the LPL and the H2B octamer elements (Fig. 2A compare lanes 4, 13, and 22). Addition of TFIIB, but not TBP, enhanced the binding of Oct-1 to all three elements (Fig. 2A, lanes 6, 15, and 24). Among these elements, TFIIB-dependent stimulation of Oct-1 binding to the LPL probe was the strongest. TFIIB also stimulated the DNA binding of HeLa cell Oct-1 that had been partially purified by wheat germ agglutinin chromatography (Segil et al., 1991) approximately 3-fold (data not shown). No stimulation was observed with Oct-1 synthesized in rabbit reticulocyte lysates, possibly due to the presence of other stimulatory factors, such as HMG2, in reticulocyte lysate (Zwilling et al., 1995; data not shown). TFIIB-dependent stimulation of Oct-1 binding was not attributable to nonspecific protein effects since neither BSA (lanes 5, 14, and 23), nor the basic peptide poly-L-lysine, nor glutathione S-transferase (GST) protein stimulated Oct-1 DNA binding (Bannister and Kouzarides, 1992; data not shown). The observed increase in Oct-1 binding was unlikely due to improved recovery of the Oct-1bulletDNA complex from Eppendorf tubes in the presence of TFIIB, since experiments in siliconized tubes gave identical results (data not shown). Highly purified recombinant TFIIB obtained from a different source (Promega), also stimulated Oct-1 DNA binding (data not shown). The complex obtained in the presence of Oct-1 and TFIIB corresponds to a bona fide Oct-1bulletDNA complex, since this complex could be supershifted by Oct-1 specific antibody, and was competed by Oct-1 specific unlabeled oligonucleotides (data not shown). DNA binding of recombinant Oct-1 was efficient when the binding reactions were performed under reducing conditions (6 mM beta-mercaptoethanol and 20 mM dithiothreitol), and in the absence of Mg. Mg appears to stimulate ``exonuclease-like'' activity of recombinant Oct-1 (or a copurifying protein) as the DNA probe size was reduced by two nucleotides accompanied with the loss of P-labeled nucleotides (data not shown).


Figure 2: DNA binding of recombinant Oct-1. EMSAs of recombinant Oct-1 either alone (lanes 4, 13, and 22), with BSA (lanes 5, 14, and 23), TFIIB (lanes 6, 15, and 24), TBP (lanes 7, 16, and 25), TBP and BSA (lanes 8, 17, and 26), or with TFIIB and TBP (lanes 9, 18 and 27) are shown. DNA binding of TBP (lanes 1, 10, and 19), TFIIB (lanes 2, 11, and 20), or both together (lanes 3, 12, and 21) is also shown. DNA probes used were the LPL octamer (lanes 1-9), the H2B octamer (lanes 10-18), and the ICPO octamer (lanes 19-27). In experiments described here and in subsequent EMSA figures, the concentration of Oct-1 was 1 ng/reaction, whereas TFIIB and TBP were 5 ng/reaction.



Deletion Analysis of Oct-1 Protein

The Oct-1 protein can be subdivided into three functional domains: an N-terminal glutamine-rich activation domain, a central DNA-binding POU domain, and serine/threonine-rich C-terminal activation domain (Tanaka et al., 1992). To investigate which of these domains is involved in TFIIB-mediated stimulation of Oct-1 binding, recombinant proteins containing either the Oct-1 POU domain fused to Staphylococcus aureus-protein A (Kristie and Sharp, 1990), the POU domain with N-terminal domain (OctDeltaC), or POU domain with C-terminal domain (OctDeltaN) (see ``Materials and Methods'') were tested for binding to the LPL octamer (Fig. 3A), and the H2B octamer (Fig. 3B), with or without TFIIB. Although TFIIB stimulated DNA binding of all the three mutant Oct-1 proteins, the extent of stimulation decreased significantly with deletion of the N- and C-terminal transactivation domains suggesting that maximum stimulation requires the full-length Oct-1. Two DNA-protein complexes were observed in the DNA binding reactions of Oct-1 proteins containing only the POU domain (Fig. 3, A and B, lanes 13-18). This observation could result from binding of Oct-POU as monomers (bottom band) and dimers (top band, Kemler et al.(1989), Poellinger and Roeder (1989)), or due to degradation of protein during purification. In general, the Oct-1 protein is highly susceptible to proteolysis during purification, and this was enhanced with OctDeltaN (data not shown).


Figure 3: Oct-1 domains required for stimulation by TFIIB. EMSAs of indicated deletion mutant Oct-1 proteins (OctDeltaN, amino acids 268-743; OctDeltaC, amino acids 1-590; Oct-POU, amino acids 270-441) were performed with the LPL probe (A) or the H2B probe (B). DNA binding of Oct-1 mutants either alone (lanes 1, 7, and 13), with BSA (lanes 2, 8, and 14), TFIIB (lanes 3, 9, and 15), TBP (lanes 4, 10, and 16), TBP and BSA (lanes 5, 11, and 17), or TBP and TFIIB (lanes 6, 12, and 18) is shown.



TFIIB Stimulates Oct-2 Binding to DNA

To determine if functional differences between Oct-1 and Oct-2 (Tanaka and Herr, 1990; Tanaka et al., 1992) could be correlated with differences in TFIIB-stimulated DNA binding, EMSAs were performed with recombinant Oct-2. Recombinant Oct-2 bound the LPL and H2B probes only weakly, and their binding was not improved by addition of either BSA or TBP (Fig. 4, compare lanes 1, 2, and 4 and 7, 8, and 10). Oct-2 binding, however, was stimulated by TFIIB (compare lanes 1 and 3 and 7 and 9) in a manner similar to Oct-1. These results suggest that DNA binding of both Oct-1 and Oct-2 can be stimulated by TFIIB and, correspondingly, that the TFIIB interaction does not contribute to the functional difference between these proteins.


Figure 4: DNA binding of recombinant Oct-2. DNA binding of Oct-2 either alone (lanes 1 and 7), with BSA (lanes 2 and 8), TFIIB (lanes 3 and 9), TBP (lanes 4 and 10), TBP and BSA (lanes 5 and 11), or TBP and TFIIB (lanes 6 and 12) is shown. Probes were the LPL octamer in lanes 1-6, and the H2B octamer in lanes 7-12.



Mechanism of Stimulation of Oct-1 Binding by TFIIB

To determine the mechanism by which TFIIB enhances the DNA binding activity of Oct-1, the rate of association of Oct-1 with the LPL and H2B octamers was examined in EMSAs. With the LPL probe, maximum DNA binding of Oct-1 alone occurred after a 1-min incubation and remained stable for up to 5 min, after which the DNA-protein complex started to dissociate and less than half of the initial complex remained after 2 h of incubation (Fig. 5A, upper panel). In the presence of TFIIB, however, the Oct-1bulletDNA complex was stable through 2 h of incubation. With the H2B octamer, the rate at which Oct-1 associated with DNA was slower than with the LPL probe, as maximum binding was observed at 40 min and from this point on, the amount of complex decreased (Fig. 5A, lower panel). In the presence of TFIIB, however, maximum binding was observed at 10 min, and the complex was stable throughout the incubation period. These results indicate that TFIIB-mediated stimulation of Oct-1 DNA binding can involve both an increase in the rate of association, and a decrease in the rate of dissociation, depending on the specific octamer element. Note in this respect that the LPL and H2B octamer probes used in these assays differ only in their flanking sequences, not in their core octamer sequence.


Figure 5: Kinetics of Oct-1 DNA binding with or without TFIIB. A, rate of Oct-1 association with the LPL octamer probe (top) and with the H2B octamer probe (bottom). Reaction mixtures containing Oct-1 and the probe were incubated for various length of time (in minutes) in the presence of BSA or TFIIB as indicated and analyzed on EMSA. Only DNA-protein complexes are shown. B, rate of Oct-1 dissociation from the LPL octamer probe (top) and the H2B octamer probe (bottom). Reaction mixtures were incubated for 40 min followed by addition of 50-fold excess unlabeled LPL (top) and the H2B probe (bottom) octamer oligonucleotides. Aliquots at the indicated times were analyzed by EMSA.



To further explore the effect of TFIIB on the dissociation rates of Oct-1bulletDNA complexes, competition experiments were performed. First, labeled probes were incubated with Oct-1 alone, or together with TFIIB for 40 min. After complex formation, 50-fold excess specific unlabeled DNA was added to minimize rebinding of the dissociated Oct-1 with labeled probe. Aliquots of the binding reactions were assayed in EMSAs at different time points (Fig. 5B). It is apparent that TFIIB reduces the rate of dissociation of Oct-1bulletLPL octamer and the Oct-1bulletH2B octamer complexes. Similar results were obtained when the initial binding reaction was less than 40 min, and challenged with unlabeled probe DNA (data not shown).

The equilibrium dissociation constant (K) was determined by binding saturation experiments, in which the amount of labeled DNA was increased in the presence of a fixed amount of protein, followed by Scatchard analysis. Oct-1 alone gave a K value of 1.65 nM with the LPL octamer probe, and a value of 0.14 nM with TFIIB. For unknown reasons, the K value of Oct-1 alone with the H2B probe varied in different experiments, but in the presence of TFIIB, the K value for this probe was reproducibly calculated as 0.13 nM. These results suggest TFIIB decreases the rate of Oct-1 dissociation from the LPL probe 12-fold. Note that in these experiments incubation of probe with proteins was for 40 min. With different incubation times, the fold stimulation by TFIIB may differ, depending on the element tested. To our knowledge, this is the first report of Oct-1 DNA binding affinity measurement using E. coli-derived full-length Oct-1. Previously, Verrijzer et al.(1990) reported a K value of 0.07 nM for the Oct-1 POU domain derived from a vaccinia virus expression system using an adenovirus 4 octamer site as a DNA probe.

TFIIB, Oct-1, and Octamer DNA Form a Ternary Complex

Surprisingly, the presence of TFIIB did not appear to affect the mobility of the Oct-1bulletDNA complex in EMSAs; and therefore TFIIB might not be a stable component of this complex. In this respect, TFIIB stimulation of Oct-1 DNA binding is similar to Phox1 stimulation of serum response factor binding to DNA (Grueneberg et al., 1992), human T-cell leukemia virus type I tax stimulation of bZIP proteins binding to DNA (Wagner and Green, 1993), and TFIIB stimulation of COUP-TF transcription factor binding to DNA (Tsai et al., 1987; Ing et al., 1992). As observed in human T-cell leukemia virus type I tax stimulation of activating transcription factor-DNA interaction, stimulation of Oct-1 DNA binding was dependent upon the concentration of Oct-1 in the assay (data not shown). These results indicate that TFIIB overcomes a protein concentration-dependent step that normally limits the extent of DNA binding by Oct-1. Since the mobility of the Oct-1bulletDNA complex in EMSAs was not altered by TFIIB, it was necessary to determine whether TFIIB is incorporated into Oct-1bulletDNA complexes at any stage of Oct-1 DNA binding. For this purpose, the DNA coimmunoprecipitation assay (Wagner and Green, 1993) was employed. In this assay, DNA binding reactions were performed with either TFIIB alone, or TFIIB with Oct-1 in the presence of labeled, octamer-containing target DNA. TFIIB in the reaction was immunoprecipitated with anti-TFIIB antibody and the immunoprecipitate was analyzed by SDS-PAGE and autoradiography. Since TFIIB alone binds DNA very poorly (Hisatake et al., 1993), in reactions containing only TFIIB and the probe, anti-TFIIB antibody should precipitate very little or no labeled probe. However, if TFIIB, Oct-1, and octamer probe form a ternary complex, then anti-TFIIB antibody should coprecipitate TFIIB, Oct-1, and the DNA probe. As shown in Fig. 6, very little of the octamer-containing LPL and H2B probes, a mutant LPL octamer site probe, or a control nonspecific probe which contains a MHC class II promoter CCAAT box sequence (Ealpha), was precipitated by anti-TFIIB antibody from DNA binding reactions containing either TFIIB alone or TFIIB with nonspecific GST protein (Fig. 6, lanes 1, 2, 4, 5, 7, 8, 10, and 11). In contrast, reactions containing TFIIB and Oct-1, anti-TFIIB antibody immunoprecipitated LPL, as well as the H2B probe, but not the Ealpha probe (Fig. 6, lanes 3, 9, and 12). In addition, the quantity of the LPL mutant probe precipitated was considerably lower than the LPL wild-type probe (Fig. 6, lane 6). These results suggest that TFIIB associates with Oct-1 during the DNA binding reaction and interaction survives immunoprecipitation conditions, but not EMSA conditions. Unlike, TRbeta-TFIIB interactions (Baniahmad et al., 1993), interaction of TFIIB with Oct-1 in the absence of octamer-containing DNA was unstable (data not shown).


Figure 6: DNA coimmunoprecipitation assay. DNA binding reactions were performed with indicated proteins and either the LPL octamer probe (lanes 1-3, 13, and 14), LPL mutated octamer probe (lanes 4-6), H2B octamer probe (lanes 7-9), or a probe corresponding to the CCAAT box of the MHC class II mouse Ealpha gene (lanes 10-12). Protein complexes formed on the P-labeled probes were immunoprecipitated with an anti-TFIIB antibody (except lane 14), and analyzed by SDS-PAGE. The reaction in lane 14 contained Oct-1 and the LPL probe DNA, and was precipitated with protein A-agarose without the addition of antibodies (Ab).



DNase I Footprinting of LPL and H2B Promoter with Recombinant Oct-1

Since the above experiments indicated that Oct-1 may physically associate with TFIIB when bound to an octamer sequence, DNase I footprinting was performed to determine whether Oct-1 together with TFIIB provides extended protection over the octamer site. For this purpose, the LPL promoter and H2B promoter fragments were incubated with increasing concentrations of Oct-1 alone, or with TFIIB, and subjected to partial DNase I digestion (Fig. 7A). Oct-1 alone protected the region -59 to -33 of the LPL promoter, and -63 to -36 of the H2B promoter (Fig. 7A, compare lanes 2-5 and 11-14). Overall, the pattern of protection observed with Oct-1 and TFIIB together was similar to that of Oct-1 alone (Fig. 7A compare lanes 3 and 6, 4 and 7, 5 and 8, 12 and 15, 13 and 16, and 14 and 17). Only a minor difference at the center of the protected region was observed with the LPL probe (Fig. 7A, compare lanes 3 and 6). These results suggest that in the ternary complex of Oct-1, TFIIB, and DNA, TFIIB might not contact DNA directly. However, we cannot rule out the possibility that TFIIB interacts with the minor groove of the octamer site which remains exposed when Oct-1 is bound to the octamer (Klemm et al., 1994). It is interesting to note that Walker et al.(1994) observed no difference between the footprint patterns protected by the octamer-POU versus octamer-POU-VP16 complex.


Figure 7: Effect of TFIIB and TBP on DNase I footprints generated by Oct-1. A, DNase I footprinting of the LPL and H2B promoters with Oct-1 and TFIIB. DNase I footprinting of the LPL probe (lanes 1-9), and the H2B probe (lanes 10-18) incubated with increasing concentrations of Oct-1, either alone (lanes 3-5 and 12-14) or in combination with 5 ng of TFIIB (lanes 6-8 and 15-17) is shown. DNase I digestion pattern of the probe alone is shown in lanes 2 and 9 (LPL probe), lanes 11 and 18 (H2B probe). A+G sequencing reaction for the LPL probe (lane 1), and the H2B probe (lane 10) is also shown. DNase I-protected regions in LPL and H2B probes are indicated by a vertical line on the left and the right side, respectively. 1 Oct-1 is equivalent to 1 ng. B, DNase I footprinting of the LPL probe with Oct-1, TBP, and TFIIB. The LPL probe was incubated with the indicated proteins and subjected to DNase I footprint analysis. Proteins used were as follows: TBP (one footprint unit, Promega; lane 3), TFIIB (lane 4), TBP and TFIIB (lane 5), Oct-1 (lane 6), Oct-1 with TBP (lane 7), Oct-1 with TFIIB (lane 8), and Oct-1 with TBP and TFIIB (lane 9). DNase I digestion pattern of the probe alone (lanes 2 and 10) and A+G sequencing reaction (lane 1) are also shown. The major DNase I protected region when the probe was incubated with TBP is indicated by a vertical line on the right and designated I, whereas protection by Oct-1 alone or in combination with other proteins is indicated by a vertical line on the right and designated II.



Since EMSAs indicated that TBP does not interact with the LPL octamer, the possibility of TBP interacting with other regions of the LPL promoter was investigated by DNase I footprinting. Similar assays were also performed to determine the effect of TFIIB and Oct-1 on TBP binding to the LPL promoter. TBP alone partially protected a region containing the TATA box-like sequence (-80 to -60 of Enerback et al.(1993), Fig. 7B, lane 3, protected region I). This interaction might not be physiologically relevant since mutation of this sequence did not affect promoter activity (Fig. 1B). TFIIB alone did not provide any protection (Fig. 7B, lane 4), however, along with TBP it partially protected a long stretch of DNA extending from the octamer site to the TATA-like region (-80 to -30). Interestingly, in the presence of Oct-1, either alone, or together with TBP and TFIIB, only the region containing the octamer sequence was protected (Fig. 7B, compare lane 5 with lanes 6-9). These results indicate that when Oct-1 is present, TBP and TFIIB cannot bind to the TATA box-like sequence, either due to steric hindrance, or due to association of TBP, TFIIB, and Oct-1 as a heterotrimeric complex contacting only the octamer sequence. In DNA coimmunoprecipitation assays, we did not detect such a heterotrimeric complex (data not shown). This could be due to technical difficulties in preserving the complex during TBP antibody precipitation.

Octamer of the LPL Promoter Can Function as an INR in Vivo

The inability of TBP to interact with the TATA-like sequence in the presence of Oct-1 (Fig. 7B), the dispensability of the TATA-like element in in vivo LPL promoter assays (Fig. 1B), and the interaction of TFIIB with DNA-bound Oct-1 (Fig. 6), taken in conjunction with the known activity of TFIIB in transcription initiation (see Introduction), raised the possibility that the octamer element by itself may function as a basal promoter element on which a functional preinitiation complex can assemble in vivo. To test this possibility, an oligonucleotide corresponding to the Oct-1-footprinted region of the LPL promoter (-59 to -33) was cloned in either orientation into pBLCAT3 (Fig. 8A, pLO+, pLO-). Similar constructs containing a sequence corresponding to the Oct-1-protected region of the H2B promoter were also prepared (Fig. 8A, pHO+, pHO-). The SV40 enhancer sequence, or a binding site for the yeast transcription factor GAL4, was inserted into these octamer-containing vectors to generate enhancer-containing CAT reporters (Fig. 8A, see ``Materials and Methods'' for details). These reporters were transfected into Cos-1 cells, RNA was prepared and the transcription start site was identified by primer extension analysis (Fig. 8, B, C, and D). Among various SV40 enhancer-LPL octamer containing reporters, specific initiation of CAT gene transcription was observed only with pLO+S1 which contains the SV40 enhancer in the ``early gene'' orientation (in relation to the CAT gene coding sequence) located upstream from the LPL octamer site (Fig. 8B, lane 5, indicated by an arrow). Note that the orientation of the LPL octamer sequence in relation to the CAT gene is identical to the wild-type gene promoter. The initiation site maps to the CA dyad within the octamer sequence 5`-ATTTGCAT-3`. With other SV40 enhancer-containing reporters, transcription initiated at numerous sites, including the site obtained with pLO+S1 (Fig. 8B). Of note is the construct pLO+S2 which, in addition to the LPL octamer sequence, contains the SV40 promoter with the ``late gene'' orientation relative to the downstream CAT gene. Approximately 50% of the transcripts from this reporter initiated at the LPL octamer site while the remaining transcripts initiated within the late gene promoter (Fig. 8B, lane 8). Also, transcription from a reporter containing a mutated LPL octamer sequence (5`-TGTTCAGA-3`), in the context of wild-type flanking sequences, and the SV40 enhancer in the early gene orientation, initiated at several sites including the CA nucleotide of the mutated octamer sequence (Fig. 8B, pLOm+S1, lane 12).


Figure 8: LPL octamer functions as an INR. A, schematic representation of reporter constructs. The construction of various reporters are described in detail under ``Materials and Methods.'' Arrows indicate the orientation of the enhancers and octamer sequence with respect to the CAT coding sequence. Plasmids containing LPL octamers are abbreviated (LO), whereas those with H2B octamers as (HO). Similarly, plasmids with SV40 enhancer are abbreviated (S), while those with GAL4 binding site as (G17). , CAT gene; ▪, mutant octamer of the LPL gene; &cjs2090;, octamer of the H2B gene; &cjs2099;, GAL4 binding site; &cjs2110;, octamer of the LPL gene; , SV40 enhancer. B, Primer extension analysis. 10 µg of the indicated CAT reporters were transfected into Cos-1 cells, and RNA was analyzed by primer extension. The most prominent transcription initiation site is indicated by an arrow. RNA were also subjected to RNase protection with an antisense glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe to measure the quantity and quality of the RNA; the corresponding control is shown below each lane. C, primer extension analyses of RNA from cells transfected with CAT reporters, with or without GAL4 and GALVP16. Cos-1 cells were transfected with 10 µg of the indicated reporters either alone (lanes 1, 4, 7, 10, 13, and 16), with 2 µg of GAL4 (lanes 2, 5, 8, 11, 14, and 17), or with 2 µg of GALVP16 (lanes 3, 6, 9, 12, 15, and 18) and RNA was analyzed by primer extension. The major transcription site is indicated by an arrow and the levels of glyceraldehyde-3-phosphate dehydrogenase RNA is shown below each lane. D, primer extension analysis of RNA from cells transfected with the H2B octamer-containing CAT reporters. Cos-1 cells were transfected with 10 µg of the indicated reporters either alone (lanes 1-8 and 11), with 2 µg of GAL4 (lanes 9 and 12), or with 2 µg of GALVP16 (lanes 10 and 13). The major transcription site is indicated by an arrow, and the glyceraldehyde-3-phosphate dehydrogenase RNA level is shown below each lane.



To determine whether the specific start site utilized by the reporter pLO+S1 is specified by the SV40 enhancer or the LPL octamer sequence, similar analyses were performed with reporters containing the LPL octamer sequences in the context of a binding site for GAL4 (Fig. 8A, pLO+G17 and pLO-G17). In these experiments, reporters were transfected either alone, with the GAL4 expression vector, or with a vector which codes for a protein containing the GAL4 DNA-binding domain fused to the acidic activation domain of VP16 (Webster et al., 1988). As shown in Fig. 8B, specific initiation of CAT gene transcription was observed only in cells transfected with pLO+G17 and either GAL4 or GALVP16. The transcription initiation site in pLO+G17 was the same as that of the pLO+S1 vector (Fig. 8C, lanes 13-15, indicated by an arrow). Note that specific initiation of transcription from pLO-G17 vector was also observed, although the level was relatively lower than the pLO+G17 vector. The transcription initiation from this vector occurred at a ``CA'' dyad located at the same distance from the CAT primer (Fig. 8C, lanes 16-18). These results suggest that the octamer sequence is primarily responsible for the transcription start site selection. Also, depending on the enhancer context in which it appears, the LPL octamer can function as an INR in either orientation. Similar results were obtained in HeLa cells, suggesting that the INR-like function of octamer is not cell type-specific (data not shown).

We next investigated whether transcription initiation takes place at the same site in reporters containing the H2B octamer sequence (Fig. 8D). In contrast to reporters containing the LPL octamer site, transcription initiation from the H2B octamer-containing reporters, particularly those with the SV40 enhancer (pHO+S1, pHO-S1, pHO+S2, and pHO+S3), occurred at numerous sites (Fig. 8D, lanes 3-8). For example, transcription from pHO+S1 initiated at a minimum of six different locations within the H2B sequence (Fig. 8D, lane 3), whereas transcription from pHO-S1 initiated at sequences both within and outside the H2B sequence (Fig. 8D, lane 4). Unlike pHO+S1, transcription from the GAL4 binding site and H2B octamer-containing reporters (pHO+G17 and pHO-G17) also initiated mostly within the H2B octamer sequence (Fig. 8D, lanes 8-13). Taken together, these results suggest that octamer sequences, like transcription factor YY1 and E2F binding sites, can function as INRs (Weis and Reinberg, 1992).


DISCUSSION

This study provides additional evidence for the emerging concept that the differential transcription regulation by Oct-1 involves selective recruitment of other transcription factors to the octamer site. Recruited cofactors may either increase the transcriptional activation potential of Oct-1, or suppress nonspecific transcription initiation by promoting the assembly of a preinitiation complex at a particular location. Our results suggest that TFIIB is one cofactor that is recruited to the octamer motif by Oct-1. We speculate that depending on the sequences flanking the octamer site, and the specific set of enhancer-binding factors, recruited TFIIB directs the assembly of a functional preinitiation complex. Octamer sites with unique flanking AT-rich sequences, like the proximal LPL site, may form a distinct group of octamers that have evolved for this particular function.

On-rate and Off-rate of Oct-1-DNA Interaction Were Altered by TFIIB

We have shown here that Oct-1 and Oct-2 associate with TFIIB in the presence of the octamer sequence, and this association stabilizes the Oct-octamer interaction. With the LPL promoter octamer, TFIIB appears to decrease the off-rate of DNA binding, whereas with the H2B promoter octamer it appears to both increase the on-rate and decrease the off-rate (Fig. 5). We did not observe a stable association of TFIIB with Oct-1 in solution (data not shown). In this respect, TFIIB association with Oct-1 is different from its reported association in solution with c-Rel, TRbeta, and vitamin D(3) receptor (Kerr et al., 1993; Baniahmad et al., 1993; MacDonald et al., 1995). There are some similarities in TFIIB stimulation of Oct-1 DNA binding to Phox1 stimulation of serum response factor DNA binding, human T-cell leukemia virus type I tax stimulation of activating transcription factor DNA binding, reciprocal stimulation of NF-kappaB and C/EBP DNA binding and Fos/Jun stimulation of NF-kappaB DNA binding (Grueneberg et al., 1992; Wagner and Green, 1993; Stein et al., 1993a, 1993b). For example, in EMSAs, none of the DNA binding proteins displayed any shift in mobility in the presence of stimulatory proteins. However, the mechanism of stimulation may be different in this case, since TFIIB did not induce homodimerization of Oct-1 as observed with Tax-activating transcription factor interaction. Also, unlike Phox1-serum response factor interaction, TFIIB appears to decrease the off-rate of Oct-octamer interaction. Since DNA binding assays were performed in a reaction containing 20 mM dithiothreitol and 6 mM beta-mercaptoethanol, the mechanism of TFIIB stimulation of Oct-1 binding does not resemble the redox sensitive stimulation of the Jun/Fos DNA binding by Ref-1 (Abate et al., 1990).

Two models can be envisioned for the TFIIB-mediated stimulation of Oct-1 binding. One model would suggest that TFIIB stabilizes Oct-1- and Oct-2-induced DNA bending (Verrijzer et al., 1991). It is thought that induced DNA bending stores elastic energy which can cause local unwinding of double-stranded DNA. Since TFIIB has greater affinity for single-stranded DNA than for double-stranded DNA, TFIIB may interact with Oct-1-bound DNA at the unwound region, and stabilize the DNA bending angle which in turn stabilizes the Oct-1-DNA interaction. However, since the present study was not performed with probes that are suitable to test this model, additional experiments with additional probes are required. The second model would predict TFIIB-mediated conformational changes in Oct-1 which would enable Oct-POU domains to interact more efficiently with DNA. It is possible that the activators themselves undergo a conformational change when they induce similar changes in TFIIB (Roberts and Green, 1994). Although preliminary results using a chymotrypsin protease clipping assay (Leid, 1994) did not reveal specific TFIIB-induced conformational changes in Oct-1, quantitative differences in protease-resistant fragments were observed (data not shown). Further experiments using different proteases are required to test this possibility.

The LPL Promoter Is a TATA-less Promoter and the Octamer Element May Assume TATA Box Functions

Mutational analysis of the LPL promoter suggested that Oct-1 and NF-Y transcription factors are essential for LPL promoter activity (Fig. 1). However, previous deletion analysis experiments suggested that these elements alone are not sufficient for LPL promoter activity (Previato et al., 1991; Enerback et al., 1992). Elements that bind to the HNF-3/fork head family of transcription factors are also necessary for LPL promoter activity (Enerback et al., 1992). Taken together, the cell type-specific expression of LPL may require coordinate interaction of cell type-specific transcription factors that bind to the enhancer region and ubiquitously expressed factors, such as Oct-1 and NF-Y, that bind to the promoter and the enhancer regions.

Our results also revealed that the putative TATA box at position -26 to -22 could be mutated without affecting LPL promoter activity. In fact, replacing the putative TATA box with a sequence which does not bind TBP in vitro increases promoter activity (Fig. 1). Furthermore, the activity of the promoter with this mutation was almost equivalent to that of the promoter in which the wild-type sequence was mutated to a perfect TATA box. These results suggest that the wild-type sequence at position -26 to -22 is not a functional TATA box. Since mutation to a perfect TATA box, or to a sequence that does not bind TBP, both enhance the activity of the promoter, it can be argued that the observed effect of mutation is not due to generation of a new transcription factor binding site. The slight increase in promoter activity of these mutants suggest that the wild-type sequence may function weakly as a negative regulatory element. Interestingly, of these proximal elements only the LPL CCAAT box and the octamer, but not the putative TATA box, are conserved between human and mouse genes (Hua et al., 1991).

The results presented here indicate that the LPL promoter cannot be considered a classical TATA-containing promoter. A number of recent studies have demonstrated that transcription from such TATA-less promoters is regulated differentially by certain oncogenes and tumor suppressor genes (Roy et al., 1993b; Shrivastava et al., 1993; Mack et al., 1993). As with several other TATA-less promoters, this might allow the c-myc oncogene and p53 tumor suppressor gene to regulate LPL promoter activity. It is interesting to note that c-myc has been shown to suppress adipocyte-specific gene expression in preadipocytes (Freytag and Geddes, 1992).

At this point, it is appropriate to speculate that the octamer site of LPL functionally replaces the TATA box, since TFIIB can be recruited to this site by Oct-1 in vitro. Experiments presented in Fig. 8demonstrated that the LPL octamer can direct the assembly of a preinitiation complex in vivo. However, it was unexpected that transcription initiated from sequences within the octamer. One possible explanation for these results is that the LPL octamer is flanked by INR-like sequences which, due to the presence of an Sp1-binding site in the SV40 enhancer, are able to function as INRs independently of Oct-1. This seems unlikely since the CA dyad with its flanking sequences has been shown to account for only 10% of the canonical INR function in an in vivo INR functional assay (Javahery et al., 1994). Moreover, the H2B octamer, which contains the sequence 5`-CCTTATTT-3`, that has been shown in an in vivo INR assay to have 62% of wild-type INR activity (Javahery et al., 1994), did not function as an INR in our experimental system. Similarly, several of the INR-like sequences present in the plasmid vectors (both SV40 enhancer and GAL4-binding site enhancer) failed to function as INRs. Furthermore, transcription initiated at several sites from the reporter pLO+mS1, which has a mutated LPL octamer with wild-type flanking sequences (Fig. 8B). Taken together, these results strongly suggest that both the LPL octamer and its flanking sequence are essential for INR activity. Also, octamer INRs can be classified as ``strong INRs'' since sequences between -33 and -14 in our CAT reporters are GC-rich, which is not compatible with ``weak INR'' activity in vivo (Javahery et al., 1994).

Our attempts to identify the site of initiation from LPL CAT reporters (Fig. 1) by primer extension were not successful, possibly due to the low level of CAT mRNA (data not shown). However, reverse transcription-polymerase chain reaction analysis using different pairs of primers revealed that transcription from all CAT reporters, except pLPLCATm3, initiated at a position close to the previously identified LPL promoter transcription start site (Deeb and Peng, 1989). The residual CAT transcripts from pLPLCATm3 initiated at upstream sites, suggesting that the mutation of the octamer sequence results in nonspecific transcription initiation (data not shown). Although the transcription initiation site of the natural LPL promoter and LPL CAT reporters are different from that of the pLO+S1 and pLO+G17 reporters, possibly due to the influence of enhancers, the results clearly demonstrate that the LPL octamer has a specific role in transcription initiation site selection.

With currently available experimental systems, it is diffi-cult to provide direct evidence for Oct-1 interaction with TFIIB in vivo, and to define the role of such an interaction in INR element-like function of the LPL octamer site. For example, cotransfection of TFIIB with Oct-1 did not increase the activity of octamer-containing reporters (data not shown). A similar experimental approach was not useful in determining the interaction of glutamine-rich activation domains with TFIIB in vivo (Colgan et al., 1993). However, considering the fact that TFIIB is required for the transcription start site selection and for recruitment of TBP to INR-containing promoters (see Introduction), it is conceivable that in vivo, the RNA polymerase II-TFIIB complex is recruited to the octamer site by Oct-1, allowing transcription initiation to occur within the octamer. Additionally, TBP may also be recruited to the same site by its dual ability to interact with Oct-1 and TFIIB (Zwilling et al., 1994; Yamashita et al., 1993). Mechanistically, such an interaction on an octamer site may be possible since the POU domains of Oct-1 dock only to the major groove of the octamer sequence (Klemm et al., 1994), whereas TBP generally binds within the minor groove (Starr and Howley, 1991; Lee et al., 1991). Interestingly, the CA dinucleotide of the octamer from which transcription is initiated faces the minor groove (Klemm et al., 1994). Since the LPL octamer behaved as a strong INR and compared to the H2B octamer, is flanked by AT-rich sequences, it is possible these AT-rich flanking sequences stabilize the TBPbulletTFIIBbullet Oct-1bulletoctamer complex and make them available for multiple rounds of transcription initiation (Arnosti et al., 1993).

TFIIB May Be One of Several Cofactors Responsible for Promoter-specific Activity of Octamer Transcription Factors

A number of studies have indicated that the promoter and the cell type-specific functions of Oct-1 and Oct-2 are due to their ability to recruit cofactors into the preinitiation complex in an octamer-site dependent manner (Tanaka and Herr, 1990, 1994; Tanaka et al., 1992, 1994; Pfisterer et al., 1994; Strubin et al., 1995; Gstaiger et al., 1995). Examples include the recruitment of Jun protein to the octamer site of the interleukin-2 promoter upon antigenic stimulation (Ullman et al., 1993), VP16 and host cell factor to the octamer sites in the herpes simplex virus genome (Gerster and Roeder, 1988; Greaves and O'Hare, 1990; Kristie and Sharp, 1990; Stern et al., 1989; Wilson et al., 1993; Xiao and Capone, 1990), viral precursor terminal protein-DNA polymerase complex to the adenovirus replication origin (Coenjaerts et al., 1994), and the OBF-1 coactivator to the immunoglobulin promoter octamer sites (Strubin et al., 1995). In this report, we have presented evidence for a unique feature of the LPL octamer site, in that it can function as an INR and that the sequences flanking the octamer are partially responsible for this property. Whether Oct-1 bound to the LPL octamer site undergoes a distinct conformational change induced by the flanking sequences similar to the conformational change in Oct-1 bound to an octamer flanked by the GARAT sequence (Walker et al., 1994) remains to be established. Considering the diversity in Oct-1 functions specified by its binding site, and the influence of sequence surrounding the octamer site, it is likely that distinct conformational changes in Oct-1 serve as a major regulatory event for site-specific cofactor interaction and overall promoter regulation. Site-specific conformational changes and co-recruitment of additional transcription factors have been described as a key regulatory phenomenon in transcription regulation by the steroid hormone superfamily of transcription factors for instance (Saatcioglu et al., 1993; Pearce and Yamamoto, 1993). Thus, specific effects, particularly in terms of protein-protein interactions, subsequent to the conformational change may be responsible for the diverse transcription regulatory potential of Oct-1.


FOOTNOTES

*
This work was supported by the Picower Institute for Medical Research, by grants from the Weight Watchers Foundation, Inc.(0892N), the Coronary Heart Division of the American Health Assistance Foundation, and United States Public Health Service Grant DK-47272, from the National Institute of Diabetes and Digestive and Kidney Diseases (to R. A. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 516-562-9432; Fax: 516-365-5090.

(^1)
The abbreviations used are: INR, initiator element; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; LPL, lipoprotein lipase; SV40, simian virus 40; TAFs, TBP-associated factors; TBP, TATA-binding protein; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; VLDL, very low density lipoprotein.


ACKNOWLEDGEMENTS

We are grateful to Drs. P. Chambon, W. Herr, W. Schaffner, P. A. Sharp, and I. Verma for various constructs and to Dr. Kirk Manogue for a critical reading of the manuscript.


REFERENCES

  1. Abate, C., Patel, L., Raushcher, F. J., II, and Curran, T. (1990) Science 249,1157-1161 [Medline] [Order article via Infotrieve]
  2. Arnosti, D. N., Merino, A., Reinberg, D., and Schaffner, W. (1993) EMBO J. 12,157-166 [Abstract]
  3. Ausubel, F. M., Brunt, R., Kingston, R. E., Moore, D. D., Smith, J. A., Sridman, J. G., and Struhl, K. (1989) Current Protocols in Molecular Biology, 3rd Ed., Wiley and Sons, New York
  4. Baniahmad, A., Ha, I., Reinberg, D., Tsai, S., Tsai, M-J., and O'Malley, B. W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,8832-8836 [Abstract]
  5. Bannister, A. J., and Kouzarides, T. (1992) Nucleic Acids Res. 20,3523 [Medline] [Order article via Infotrieve]
  6. Breathnach, R., and Chambon, P. (1981) Annu. Rev. Biochem. 50,349-383 [CrossRef][Medline] [Order article via Infotrieve]
  7. Buratowski, S. (1994) Cell 77,1-3 [Medline] [Order article via Infotrieve]
  8. Caron, C., Rousset, R., Beraud, C., Mancollin, V., Egly, J. M., and Jalinot, P. (1993) EMBO J. 12,4269-4278, 2749-2762 [Abstract]
  9. Chen, J-L., Attardi, L. D., Verrijzer, C. P., Yokomori, K., and Tjian, R. (1994) Cell 79,93-105 [Medline] [Order article via Infotrieve]
  10. Chiang, C-M., and Roeder, R. G. (1995) Science 267,531-536 [Medline] [Order article via Infotrieve]
  11. Chiang, C-M., Ge, H., Wang, Z., Hoffmann, A., and Roeder, R. (1993) EMBO J. 12,2749-2762 [Abstract]
  12. Choy, B., and Green, M. R. (1993) Nature 366,531-536 [CrossRef][Medline] [Order article via Infotrieve]
  13. Coenjaerts, F. E. J., van Oosterhout, J. A. W. M., and van der Vliet, P. C. (1994) EMBO. J. 13,5401-5409 [Abstract]
  14. Colgan, J., Wampler, S., and Manley, J. L. (1993) Nature 362,549-553 [CrossRef][Medline] [Order article via Infotrieve]
  15. Currie, R. A., and Eckel, R. H. (1992) Arch. Biochem. Biophys. 298,630-639 [Medline] [Order article via Infotrieve]
  16. Deeb, S. S., and Peng, R. (1989) Biochemistry 28,4131-4135 [Medline] [Order article via Infotrieve]
  17. Enerback, S., Ohlsson, B. G., Samuelsson, L., and Bjursell, G. (1992) Mol. Cell. Biol. 12,4622-4633 [Abstract]
  18. Freytag, S. O., and Geddes, T. J. (1992) Science 256,379-382 [Medline] [Order article via Infotrieve]
  19. Gerster, T., and Roeder, R. G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,6347-6351 [Abstract]
  20. Gill, G., Pascal, E., Tseng, Z. H., and Tjian, R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,192-196 [Abstract]
  21. Goodrich, J. A., Hoey, T., Thut, C. J., Admon, A., and Tjian, R. (1993) Cell 75,519-530 [Medline] [Order article via Infotrieve]
  22. Greaves, R. F., and O'Hare, P. (1990) J. Virol. 64,2716-2724 [Medline] [Order article via Infotrieve]
  23. Grueneberg, D. A., Natesan, S., Alexandre, C., and Gilman, M. Z. (1992) Science 257,1089-1095 [Medline] [Order article via Infotrieve]
  24. Gstaiger, M., Knoepfel, L., Georgiev, O., Schaffner, W., and Hovens, C. M. (1995) Nature 373,360-362 [CrossRef][Medline] [Order article via Infotrieve]
  25. Ha, I., Lane, W. S., and Reinberg, D. (1991) Nature 352,689-695 [CrossRef][Medline] [Order article via Infotrieve]
  26. Hateboer, G., Marc Timmers, H. T., Rustgi, A. K., Billaud, M., van TVeer, L. J., and Bernards, R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,8489-8493 [Abstract/Free Full Text]
  27. Hisatake, K., Roeder, R. G., and Horikoshi, M. (1993) Nature 363,744-747 [CrossRef][Medline] [Order article via Infotrieve]
  28. Hooft van Huisduijnen, R., Li, X. Y., Black, D., Matthes, H., Benoist, C., and Mathis, D. (1990) EMBO J. 9,3119-3127 [Abstract]
  29. Hua, X., Enerback, S., Hudson, J., Youkhana, K., and Gimble, J. M. (1991) Gene (Amst.) 107,247-253 [CrossRef][Medline] [Order article via Infotrieve]
  30. Ing, N. H., Beekman, J. M., Tsai, S. Y., Tsai, M-J., and O'Malley, B. W. (1992) J. Biol. Chem. 267,17617-17623 [Abstract/Free Full Text]
  31. Javahery, R., Khachi, A., Lo, K., Zenzie-Gregory, B., and Small, S. T. (1994) Mol. Cell. Biol. 14,116-127 [Abstract]
  32. Johnson, P. F., Landschulz, W. H., Graves, B. J., and McKnight, S. L. (1987) Genes & Dev. 1,133-146
  33. Jones, K. A., Kadonaga, J. T., Rosenfeld, P. J., Kelly, T. J., and Tjian, R. (1987) Cell 48,79-89 [Medline] [Order article via Infotrieve]
  34. Kadonaga, J., Jones, K., and Tjian, R. (1986) Trends Biochem. Sci. 11,20-23 [CrossRef]
  35. Kashanchi, F., Piras, G., Radonovich, M. F., Duvall, J. F., Fattaey, A., Chiang, C-M., Roeder, R. G., and Brady, J. N. (1994) Nature 367,295-299 [CrossRef][Medline] [Order article via Infotrieve]
  36. Kaufmann, J., and Smale, S. T. (1994) Genes & Dev. 8,821-829
  37. Kemler, I., Schreiber, E., Muller, M., Matthias, P., and Schaffner, W. (1989) EMBO J. 8,2001-2008 [Abstract]
  38. Kerr, L. D., Ransone, L. J., Wamsley, P., Schmitt, M. J., Boyer, T. G., Zhou, Q., Berk, A. J., and Verma, I. M. (1993) Nature 365,412-419 [CrossRef][Medline] [Order article via Infotrieve]
  39. Klemm, J. D., Rould, M. A., Aurora, R., Herr, W., and Pabo, C. O. (1994) Cell 77,21-32 [Medline] [Order article via Infotrieve]
  40. Kristie, T. M., and Sharp, P. A. (1990) Genes & Dev. 4,2383-2396
  41. Kumar, V., Green, S., Stack, G., Berry, M., Jin, J., and Chambon, P. (1987) Cell 51,941-951 [Medline] [Order article via Infotrieve]
  42. LaBella, F., Sive, H. L., Roeder, R. G., and Heintz, N. (1988) Genes & Dev. 2,32-39
  43. LeBowitz, J. H., Kobayashi, T., Staudt, L., Baltimore, D., and Sharp, P. A. (1988) Genes & Dev. 2,1227-1237
  44. Lee, D. K., Horikoshi, M., and Roeder, R. G. (1991) Cell 67,1241-1250 [Medline] [Order article via Infotrieve]
  45. Leid, M. (1994) J. Biol. Chem. 269,14175-14181 [Abstract/Free Full Text]
  46. Li, Y., Flanagan, P. M., Tschochner, H., and Kornberg, R. D. (1994) Science 263,805-807 [Medline] [Order article via Infotrieve]
  47. Lin, Y. S., and Green, M. R. (1991) Cell 64,971-981 [Medline] [Order article via Infotrieve]
  48. Liu, X., Miller, C. W., Koeffler, P. H., and Berk, A. J. (1993) Mol. Cell. Biol. 13,3291-3300 [Abstract]
  49. Loh, E. Y., Elliott, J. F., Cwirla, S., Lanier, L. L., and Davis, M. M. (1989) Science 243,217-220 [Medline] [Order article via Infotrieve]
  50. Luckow, B., and Schultz, G. (1987) Nucleic Acids Res. 105,5490
  51. MacDonald, P. N., Sherman, D. R., Dowd, D. R., Jefcoat, S. C., Jr., and DeLisle, R. K. (1995) J. Biol. Chem. 270,4748-4752 [Abstract/Free Full Text]
  52. Mack, D. H., Vartikar, J., Pipas, J. M., and Laimins, L. A. (1993) Nature 363,281-283 [CrossRef][Medline] [Order article via Infotrieve]
  53. Mantovani, R., Pessara, U., Tronche, F., Li, X-Y., Knapp, A-M., Pasquali, J-L., Benoist, C., and Mathis, D. (1992) EMBO J. 11,3315-3322 [Abstract]
  54. Martinez, E., Chiang, C-M., Ge, H., and Roeder, R. G. (1994) EMBO J. 13,3115-3126 [Abstract]
  55. Meisterernst, M., Roy, A. L., Lieu, H. M., and Roeder, R. G. (1991) Cell 66,981-993 [Medline] [Order article via Infotrieve]
  56. Muller, M. M., Ruppert, S., Schaffner, W., and Matthias, P. (1988) Nature 336,544-551 [CrossRef][Medline] [Order article via Infotrieve]
  57. Parvin, J. D., and Sharp, P. A. (1993) Cell 73,533-540 [Medline] [Order article via Infotrieve]
  58. Pearce, D., and Yamamoto, K. R. (1993) Science 259,1161-1165 [Medline] [Order article via Infotrieve]
  59. Pfisterer, P., Annweiler, A., Ullmer, C., Corcoran, L. M., and Wirth, T. (1994) EMBO J. 13,1654-1663
  60. Poellinger, L., and Roeder, R. G. (1989) Mol. Cell. Biol. 9,747-756 [Medline] [Order article via Infotrieve]
  61. Previato, L., Parrott, C. L., Santamarina-Fojo, S., and Brewer, H. B., Jr. (1991) J. Biol. Chem. 266,18958-18963 [Abstract/Free Full Text]
  62. Pugh, B. F., and Tjian, R. (1990) Cell 61,1187-1197 [Medline] [Order article via Infotrieve]
  63. Roberts, S. G. E., and Green, M. R. (1994) Nature 371,717-720 [CrossRef][Medline] [Order article via Infotrieve]
  64. Roberts, S. G. E., Ha, I., Maldonado, E., Reinberg, D., and Green, M. R. (1993) Nature 363,741-744 [CrossRef][Medline] [Order article via Infotrieve]
  65. Roy, A. L., Malik, S., Meisterernst, M., and Roeder, R. G. (1993a) Nature 365,355-359 [CrossRef][Medline] [Order article via Infotrieve]
  66. Roy, A. L., Carruthers, C., Gutjahr, T., and Roeder, R. G. (1993b) Nature 365,359-361 [CrossRef][Medline] [Order article via Infotrieve]
  67. Saatcioglu, F., Deng, T., and Karin, M. (1993) Cell 75,1095-1105 [Medline] [Order article via Infotrieve]
  68. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  69. Scheidereit, C., Cromlish, J. A., Gerster, T., Kawakami, K., Balmaceda, C-G., Currie, R. A., and Roeder, R. G. (1988) Nature 336,551-557 [CrossRef][Medline] [Order article via Infotrieve]
  70. Segil, N., Roberts, S. B., and Heintz, N. (1991) Cold Spring Harbor Symp. Quant. Biol. LVI, 285-292
  71. Seto, E., Lewis, B., and Shenk, T. (1993) Nature 365,462-464 [CrossRef][Medline] [Order article via Infotrieve]
  72. Shrivastava, A., Saleque, S., Kalpana, G. V., Artandi, S., Goff, S. P., and Calame, K. (1993) Science 262,1889-1892 [Medline] [Order article via Infotrieve]
  73. Starr, B. B., and Hawley, D. K. (1991) Cell 67,1231-1240 [Medline] [Order article via Infotrieve]
  74. Stein, B., Baldwin, A. S., Jr., Ballard, D. W., Greene, W. C., Angel, P., and Herrlich, P. (1993a) EMBO J. 12,3879-3891 [Abstract]
  75. Stein, B., Cogswell, P. C., and Baldwin, A. S., Jr. (1993b) Mol. Cell. Biol. 13,3964-3974 [Abstract]
  76. Stern, S., Tanaka, M., and Herr, W. (1989) Nature 341,624-630 [CrossRef][Medline] [Order article via Infotrieve]
  77. Stringer, K. F., Ingles, C. J., and Greenblatt, J. (1990) Nature 345,783-786 [CrossRef][Medline] [Order article via Infotrieve]
  78. Strubin, M., Newell, J. W., and Matthias, P. (1995) Cell 80,497-506 [Medline] [Order article via Infotrieve]
  79. Sturm, R. A., Das, G., and Herr, W. (1988) Genes & Dev. 2,1582-1599
  80. Tanaka, M., and Herr, W. (1990) Cell 60,375-386 [Medline] [Order article via Infotrieve]
  81. Tanaka, M., and Herr, W. (1994) Mol. Cell. Biol. 14,6056-6067 [Abstract]
  82. Tanaka, M., Lai, J. S., and Herr, W. (1992) Cell 68,755-767 [Medline] [Order article via Infotrieve]
  83. Tanaka, M., Clouston, W. M., and Herr, W. (1994) Mol. Cell. Biol. 14,6046-6055 [Abstract]
  84. Tanese, N., Pugh, B. F., and Tjian, R. (1991) Genes & Dev. 5,2212-2224
  85. Tanuma, Y., Nakabayashi, H., Esumi, M., and Endo, H. (1995) Mol. Cell. Biol. 15,517-523 [Abstract]
  86. Tsai, S. Y., Sagami, I., Wang, H., Tsai, M-J., and O'Malley, B. W. (1987) Cell 50,701-709 [Medline] [Order article via Infotrieve]
  87. Tooze, J. (ed) (1981) DNA Tumor Viruses, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  88. Ullman, K. S., Northrop, J. P., Admon, A., and Crabtree, G. R. (1993) Genes & Dev. 7,188-196
  89. Verrijzer, C. P., Kal, A. J., and van der Vliet, P. C. (1990) Genes & Dev. 4,1964-1974
  90. Verrijzer, C. P., van Oosterhout, A. W. M., van Weperen, W. W., and van der Vliet, P. C. (1991) EMBO J. 10,3007-3014 [Abstract]
  91. Wagner, S., and Green, M. R. (1993) Science 262,395-399 [Medline] [Order article via Infotrieve]
  92. Walker, S., Hayes, S., and O'Hare, P. (1994) Cell 79,841-852 [Medline] [Order article via Infotrieve]
  93. Wampler, S. L., and Kadonaga, J. T. (1992) Genes & Dev. 6,1542-1552
  94. Webster, N., Jin, J. R., Green, S., Hollis, M., and Chambon, P. (1988) Cell 52,169-178 [Medline] [Order article via Infotrieve]
  95. Weis, L., and Reinberg, D. (1992) FASEB J. 6,3300-3309 [Abstract/Free Full Text]
  96. Westin, G., Gerster, T., Muller, M. M., Schaffner, G., and Schaffner, W. (1987) Nucleic Acids. Res. 15,6787-6798 [Abstract]
  97. Wilson, A. C., LaMarco, K., Peterson, M. G., and Herr, W. (1993) Cell 74,115-125 [Medline] [Order article via Infotrieve]
  98. Xiao, P., and Capone, J. P. (1990) Mol. Cell. Biol. 10,4974-4977 [Medline] [Order article via Infotrieve]
  99. Yamashita, S., Hisatake, K., Kokubo, T., Doi, K., Roeder, R. G., Horikoshi, M., and Nakatani, Y. (1993) Science 261,463-466 [Medline] [Order article via Infotrieve]
  100. Yang, J., Muller-Immergluek, M. M., Seipel, K., Janson, L., Westin, G., Schaffner, W., and Pattersson, U. (1991) EMBO. J. 10,2291-2296 [Abstract]
  101. Zawel, S., and Reinberg, D. (1992) Curr. Opin. Cell Biol. 4,488-495 [Medline] [Order article via Infotrieve]
  102. Zwilling, S., Annweiler, A., and Wirth, T. (1994) Nucleic Acids Res. 22,1655-1662 [Abstract]
  103. Zwilling, S., Konig, H., and Wirth, T. (1995) EMBO J. 14,1198-1208 [Abstract]

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