(Received for publication, May 25, 1995; and in revised form, June 19, 1995)
From the
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.
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) (
)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 TATA
TFIID 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.
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). OctN 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. Oct
C 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.
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.
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.
Figure 3:
Oct-1 domains required for stimulation by
TFIIB. EMSAs of indicated deletion mutant Oct-1 proteins (OctN,
amino acids 268-743; Oct
C, 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.
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.
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-1DNA 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-1
LPL
octamer and the Oct-1
H2B 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.
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 E 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).
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.
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).
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.
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.
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 TBPTFIIB
Oct-1
octamer complex and make them
available for multiple rounds of transcription initiation (Arnosti et al., 1993).