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(Received for publication, May 24, 1996, and in revised form, July 31, 1996)
From the ABSTRACT The promoter-distal half of the spacer separating
the tandem Xenopus laevis rRNA genes consists of ``0''
and ``1'' repetitive elements that have been considered unimportant
in polymerase I transcriptional activation. Utilizing oocyte
microinjection, we now demonstrate that the 0/1 region, as well as its
component 0 and 1 repeats, substantially stimulate transcription from a
ribosomal promoter in cis and inhibit transcription when
located in trans. Both the cis and
trans responses increase linearly with increasing numbers
of 0 or 1 repeats until saturation is approached. The 0/1 block and its
component elements stimulate transcription in both orientations, over
distances, and when placed downstream of the initiation site,
properties for which the 60/81-base pair (bp) repeats have been defined
as polymerase I enhancers. In their natural promoter-distal rDNA
location, the 0/1 repeats can stimulate transcription from the rRNA
gene promoter, above the level afforded by the intervening 60/81-bp
repeats and spacer promoter. In addition, as with the 60/81-bp
repeats, the 0/1 repeats bind a factor in common with the rDNA
promoter. Thus, the entire X. laevis rDNA intergenic spacer
(the 0 repeats, 1 repeats, spacer promoter repeats, and 60/81-bp
repeats) acts together to enhance ribosomal transcription.
INTRODUCTION Eukaryotic ribosomal RNA (rRNA) constitutes 75% of total cellular
RNA and is synthesized by RNA polymerase I (pol
I)1 as a precursor to the 18, 5.8, and 28 S
RNAs of the ribosome (1). The typical eukaryotic cell contains from
several hundred (in vertebrates) to several thousand (in plants) rRNA
genes organized in head-to-tail tandem arrays located at one or a few
chromosomal sites (2). Each rDNA repeating unit consists of the
transcribed pre-rRNA region and an intergenic spacer (IGS), whose
length varies considerably (e.g. from ~2.5 kb in
Saccharomyces cerevisiae (3) to ~30 kb in humans
(4)). Within a given species, and even within the individual rDNA
repeats of a single organism, the length of the IGS may be polymorphic
(e.g. ~3 kb to ~9 kb in Xenopus laevis (5)).
In all cases examined, this length polymorphism is due to differences
in the numbers of repetitive sequence elements (6, 7, 8, 9, 10). Indeed,
all metazoan organisms for which sequence information is available
have one or more types of reiterated sequence elements that constitute
a substantial portion of their promoter-proximal IGSs.
The IGS of X. laevis rDNA is composed almost entirely of
four types of repeated elements (Fig. 1) (11, 12). The
promoter-proximal portion of the IGS consists of blocks of 60/81-bp
repeats (6-12 copies of 60- or 81-bp elements) that alternate with a
pol I spacer promoter (SP). This unit is generally repeated two to
three times/spacer, but can be repeated up to eight times. The SP is
~90% identical to the gene promoter (
The promoter-proximal portion of the X. laevis IGS can
substantially affect transcription from the rRNA gene promoter (15,
16). The 60/81-bp repeats are pol I transcriptional enhancers because
they function in both orientations and over considerable distances to
stimulate transcription from an rDNA promoter located in
cis, relative to one on a separate DNA molecule (17, 18, 19).
The 60/81-bp repeats can also stimulate a promoter in cis in
the absence of a competitor template (20), and their
cis-stimulatory and trans-competitive effects can
each be ~10-fold (17, 20). Although the SP does not stimulate
transcription from the gene promoter by itself, it potentiates the
enhancement observed from the 60/81-bp repeats, in a process that is
not yet understood (21, 22).
In contrast to the transcriptional effects of the promoter-proximal
repetitive elements, previous studies have concluded that the
promoter-distal region of the X. laevis rDNA IGS containing
the 0 repeats and the 1 repeats had no appreciable effect on
transcription (19, 22, 23). However, the assays used in these
experiments were considerably less sensitive than those used to
demonstrate the effects of the promoter proximal elements, and they
might not have detected the effects of the 60/81-bp repeats either.
This report re-examines the transcriptional role of the 0/1 repetitive
elements.
Following the X. laevis paradigm, the promoter-proximal
repetitive elements of the rDNA IGS in mouse (24, 25),
Arabidopsis (26), and Acanthamoeba (27), the
promoter-proximal spacer promoter repeats in Drosophila (28,
29) and mouse (30), and the promoter-proximal spacer promoter and/or
repetitive elements in rat (31) have been found to stimulate
transcription from the respective cis-located gene
promoters, much as in frog. Thus, there are many examples of
promoter-proximal rDNA repeats acting to stimulate pol I
transcription.
Using oocyte microinjection assays under conditions that can separately
detect cis stimulation and trans competition, we
have directly examined the effects of the promoter-distal half of the
X. laevis IGS. We show that the 0 repeats, the 1 repeats,
and the combined 0/1 repeats can significantly influence transcription
from the rRNA gene promoter. In cis, these repeats serve as
potent enhancers of both spacer promoter and gene promoter
transcription, acting independent of orientation and over distances,
both upstream and downstream of the initiation site. When located in
trans, these promoter-distal repetitive elements instead act
as inhibitors of transcription. By footprint competition, the 0 and 1 repeats were found to specifically interact with a factor that binds to
the rDNA promoter; gel shift analysis indicates that the pol I
transcription factor UBF can bind to these sequences. Thus, all of the
criteria that establish the 60/81-bp repeats as pol I transcriptional
enhancers also apply to the promoter-distal 0/1 transcriptional
enhancers, indicating that virtually the entire X. laevis
IGS consists of repetitive elements that enhance ribosomal
transcription.
EXPERIMENTAL PROCEDURES All subclones of the 0/1 region
derive from pXlrs5, a subclone of pXlr14B (13) in which a 1.7-kb
fragment starting at the HindIII site at the end of the 28 S
coding region and extending to 92 nucleotides before the first
BamHI (an exonuclease III/S1 deletion to remove all residues
of the spacer promoter) was cloned between the HindIII and
SmaI sites of pUC18. This construct includes the eight
copies of the 34-bp 0 elements (2 of which are imperfect) and the six
copies of the 100-bp 1 elements of this IGS (Ref. 14, and data not
shown).
The 0.3-kb AvaII fragment of pXlrs5, containing the 0 repeats was S1-blunted and subcloned into the SmaI site of a
pUC18 derivative in which a BglII site replaced the
EcoRI site (pUCEco-Bgl), yielding pXlrs10.
Oriented polymers (32) of one to four copies of the 0 region from
pXlrs10, excised by BamHI-BglII digestion, were
inserted into the BamHI site of a pUC18 derivative in which
an EcoRI site replaced the HindIII site
(pUCHind-Eco) yielding subcloned 0, 02,
03, and 04 repeats
(pXlrs121-4).
The 0.85-kb HphI (S1-blunted)-SstI (polylinker
site) fragment of pXlrs5 containing the subcloned 1 repeats was cloned
into SmaI-SstI-digested pUCEco-Bgl,
yielding pXlrs13. Oriented monomer and dimer constructs of the
BamHI-BglII-excised 1 repeat of pXrs13 were
constructed as above, yielding 1 and 12 clones
(pXlrs141-2).
A subcloned 0/1 repeat, pXlrs16, was made by first cloning the
~1.3-kb BssHII (S1-blunted)-SstI (polylinker
site) fragment of pXlrs5 into SmaI-SstI digested
pUCEco-Bgl (yielding pXlrs15) and then excising this insert
at the flanking polylinker BamHI and BglII sites
and inserting it into the BamHI site of
pUCHind-Eco.
The A and B plasmids are pUC-based subclones of the X. laevis rRNA gene promoter (residues The E plasmid contains a subcloned block of 10 X. laevis
60/81-bp enhancer repeats (20). The E:A and E:B plasmids have the
60/81-bp block from E inserted upstream of the gene promoter in the A
and B plasmids (20). A:E and e2:A (containing one 60- and
one 81-bp element) were made by cloning into pUC18 the 1.1-kb
SalI or the 0.47-kb HindIII-SalI
fragment from A:E(pBR) or e2:A(pBR), respectively (20).
Template SP was made by subcloning the leftmost spacer promoter from
pXlr14 (14) (SmaI fragment, residues
About 30 stage V-VI X. borealis oocytes from mature frogs were each
injected in the nucleus with ~30 nl of plasmid DNA, to deliver: 0.6 fmol of template plasmid plus 1 fmol of the 5 S gene control plasmid in
the cis stimulation assay, 0.3 fmol each of the template and
the competitor plasmid in the trans competition assay, and
0.3 fmol of each template plasmid plus 1 fmol of the 5 S gene control
plasmid in the cis/trans assays. The total DNA injected was brought to
1.5-3 ng with pUC18. Injections were performed as described (20, 34);
the oocytes were centrifuged for 5 min at 30 × g
immediately preceding injection and 10 µg/ml The 25-µl binding reaction
was 20 mM HEPES, pH 7.9, 105 mM KCl, 5 mM MgCl2, 10% glycerol, 2 mM DTT,
0.14 mM EDTA and contained 5 µl of mouse S-100 extract
(~10 mg of protein/ml), 200 ng of single-stranded calf thymus DNA,
400 ng of HhaI-digested competitor construct, and 1 ng of a
HindIII-EcoRI fragment of the rDNA promoter
(plasmid pUC 230: a 230-bp HhaI fragment of rDNA, residues
Recombinant xUBF protein was synthesized in
vitro from the expression plasmid pxUBF-CITE and purified (38).
The mRNA transcription reaction (1.2 ml) contained 960 units of
RNasin, 30 µg of plasmid template (XbaI-linearized), and
1500 units of T7 RNA polymerase, as recommended by the supplier (Life
Technologies, Inc.). After 1 h at room temperature, an additional
750 units of T7 RNA polymerase was added for another 1-h incubation,
then 80 µg of DNase I was added for 15 min at 37 °C, and the RNA
(750 µg) was phenol/CHCl3-extracted and
ethanol-precipitated. The 10-ml in vitro translation
reaction was 20 mM HEPES, pH 7.4, 100 mM KCl,
0.5 mM spermidine-(HCl)3, 2 mM DTT,
8 mM creatine phosphate, 25 µM amino acids
and contained 400 µg of the xUBF transcript and 4 ml of micrococcal
nuclease-treated (39) reticulocyte lysate (Green Hectares, Oregon, WI).
35S-Labeled xUBF tracer was synthesized in a 1/40 size
reaction using 300 µCi of [35S]methionine. After 90 min
at 30 °C, the reactions were combined, diluted to 40 ml with CB100
(CBn (where n equals KCl concentration) is 25 mM HEPES, pH 7.4, n mM KCl, 5 mM MgCl2, 20% glycerol, 1 mM DTT,
0.1 mM EDTA, and 0.5 mM PMSF), and loaded onto
a 5-ml Q-Sepharose column in CB100. The column was washed with 30 ml
each of CB100 and CB250, and then eluted with 20 ml of CB600. The xUBF
fractions, identified by 35S tracer, were combined diluted
to 100 mM KCl, applied to a 5-ml Bio-Rex 70 column in
CB100, washed with 25 ml each of CB100 and CB300, and eluted with a
30-ml linear gradient from CB300 to CB1400. The peak xUBF-containing
fractions were combined, diluted to 100 mM KCl, applied to
a 1-ml Mono-Q FPLC column in CB100, washed with 6 ml each of CB100 and
CB350, and eluted with a 10-ml linear gradient from CB350 to CB600.
Peak xUBF-containing fractions were dialyzed against 50 mM
HEPES, pH 7.9, 200 mM KCl, 40% glycerol, 2 mM
DTT, 0.2 mM EDTA, and 0.5 mM PMSF, yielding
~500 ng of purified xUBF.
UBF gel shift analysis was conducted as described (40). The 10-µl
shift reaction was 10 mM HEPES, pH 7.9, 30 mM
KCl, 10% glycerol, 0.3 mM DTT, 0.03 mM EDTA,
and contained 3 µg of bovine serum albumin, 6 fmol of probe (a
5 RESULTS To
examine whether the 0 and 1 repeats that constitute the promoter-distal
portion of the X. laevis rDNA IGS (Fig. 1)
could affect rDNA transcription, we initially utilized a template
(0/1:SP; Fig. 2A) that contains the
promoter-distal 0/1 repeats and their natural adjacent pol I initiation
signal, the 3
The RNA preparations were also assayed for the transcript of the
co-injected control 5 S maxi gene to verify that the injections were
reproducible (Fig. 3A, lower panel). (This
analysis was routinely performed but is omitted from the later
figures.) Reproducibility was also demonstrated by routinely performing
each injection in duplicate. The duplicate results were usually within
~20% of each other; only one set of injections is shown in the
figures. Furthermore, each experiment was repeated by microinjecting
into the oocytes from more than one frog. Although, as expected, the
quantitative level of transcription varied between different
Xenopus individuals, the qualitative results were highly
reproducible between individuals.
To determine if the 0/1 repeats could stimulate the rRNA gene promoter
as well as the spacer promoter, transcription from a plasmid containing
the A gene (a marked X. laevis rRNA gene promoter segment;
Ref. 20) to which the 0/1 region had been joined upstream in
cis (0/1:A, Fig. 2B), was similarly assessed by
oocyte microinjection (Fig. 3B). The 0/1 repeats markedly
stimulate transcription from the rRNA gene promoter (lanes 1 and 2). This level of stimulation (~20-fold) is slightly
higher than that exerted by the 60/81-bp enhancer block (E:A, Fig.
3B, lane 8) in almost all frogs examined.
To determine whether the separated 0 and 1 repeats stimulate
transcription, the templates 0:A and 1:A (Fig. 2B) were
examined in cis stimulation assays (Fig. 3B).
Individually, both the 0 repeats (eight 34-bp repeats) and the 1 repeats (six 100-bp repeats) also stimulate transcription from the gene
promoter (lanes 1, 3, and 4). Their
levels of stimulation are somewhat lower than for the combined 0/1
repeats (lane 2), with the relative order of
cis-stimulatory potential being 0/1 > 60/81 The cis-stimulatory effect of the 0, 1, and 0/1 repeats is
not dependent on the sequence of the transcribed reporter region.
Templates analogous to those of Fig. 3B but transcribing the
rDNA B gene (20) give analogous results (data not shown). Furthermore,
the observed cis stimulation is not attributable to an
inhibitory sequence having been inadvertently introduced upstream of
the basal promoter whose action is shielded by the inserted stimulatory
elements, since the A and B genes have been cloned into pUC and pBR322
vectors at numerous sites and in both orientations without any
appreciable effect on the level of their transcription (Ref. 20 and
data not shown).
One of the distinguishing characteristics of the 60/81-bp pol I
enhancers, like pol II enhancers, is that they are capable of
stimulating their respective promoters in both orientations. To assess
the orientation dependence of the 0/1, 0, and 1 repeats, the templates
0/1r:A, 0r:A, and 1r:A (Fig.
2B) were examined in cis stimulation assays (Fig.
3B). In the reverse orientation these repeats, both together
and separately, also stimulate transcription from the gene promoter
(lanes 5-7). Thus the 0/1, 0, and 1 repeats share with
recognized enhancers the ability to function in both orientations.
The second
characteristic feature of the 60/81-bp enhancer repeats is their
ability to stimulate a promoter over several hundred bp distance
(although quantitatively, their stimulation diminishes at increasing
distance; Ref. 20). To assess whether the 0 and 1 repeats also
stimulate over a distance, a 0.2-kb segment of prokaryotic DNA was
inserted to generate the templates 0:200:A, 1:200:A, and 0/1:200:A
(Fig. 2B) in which the stimulatory elements are separated
from the rRNA gene initiation site by 0.45 kb. These displaced 0, 1, and 0/1 repeats also stimulate transcription from a downstream gene
promoter, to approximately the same extent (± ~25%) as in the
parental 0:A, 1:A, and 0/1:A constructs (Fig.
4A).
The position dependence of the promoter-distal stimulatory elements was
further examined using the templates A:0, A:1, and A:0/1 (Fig.
2B) in which the repeats were positioned 0.15-0.2 kb
downstream of the rRNA gene initiation site. The 0, 1, and 0/1 repeats
also stimulate when 3 The
extent of cis-enhancement by the 60/81-bp repeats increases
with the number of copies (Fig. 2B) (22, 42); in some frogs,
this additive effect does not plateau until more than two blocks of the
60/81-bp repeats are present, while in other frogs it plateaus at one
of these 0.7-kb blocks (Ref. 20 and data not shown; see also Ref. 19,
where the regions examined included variable numbers of inactive or
active spacer promoters). To determine if multimers of the 0 and 1 repeats can further stimulate transcription over the level obtained
from a single block, these elements were polymerized to >1 kb of
repeat sequence and similarly inserted upstream of the A gene (Fig.
2B). Because we anticipated variation between individuals
(19), oocytes from several frogs were separately microinjected and
compared using the cis-enhancement assay (Fig.
5). Increasing numbers of 0 repeats afforded increasing
levels of stimulation over the basal promoter. Under conditions of weak
basal promoter activity, the 0 polymer series can give an almost linear
increase in transcription up to four copies of the 0 block (Fig.
5A, lanes 1-5), while in oocytes from other
frogs, saturation was reached at about two copies (Fig. 5A,
lanes 8-12). On a per length basis, the 0 repeat block
(~0.3 kb with eight copies of the 34-bp 0 element) is about as
efficient at stimulating transcription as the 60/81-bp enhancer repeats
(0.5-2 times as efficient in different individuals). A similar
analysis with a polymer of the 1 repeats revealed that in oocytes from
some individuals the cis-stimulatory effect is already
saturated with one copy of the 1 block (Fig. 5B), while in
others additional repeats can afford more stimulation (data not
shown).
Like the subcloned 60/81-bp enhancer block that
inhibits transcription from an rDNA promoter when coinjected in
trans (17), the subcloned 0/1 repeats (Fig. 2C)
strongly inhibit rDNA promoter activity in trans (Fig.
6A, lanes 1, 5, and
6). Additionally, both the separated 0 and 1 repeats inhibit
transcription in trans, and this effect increases with
increasing numbers of repeats (1 repeats in Fig. 6A,
lanes 1, 3, and 4; 0 repeats in Fig.
6B). These elements exhibit a trans-competitive
effect of comparable magnitude to that of the 60/81-bp enhancer repeats
on a per length basis. As expected, the trans-competitive
strength of these repeats varies somewhat between individuals
(e.g. Fig. 6, A and B, lanes
2), but in all cases these promoter-distal repeats have a
substantial inhibitory effect on transcription of a pol I promoter in
trans.
Analysis of the 60/81-bp enhancers
frequently has utilized a cis/trans assay in which two
different marked promoter-containing plasmids are coinjected, at least
one of which bears the enhancer repeats (e.g. Ref. 19), as
in Fig. 7A (lanes 1-3). Using the
cis/trans assay, the 0/1 repeats (lane 4) exert
an effect similar to the 60/81-bp repeats (lane 3),
enhancing transcription of the promoter carrying the repeats (B gene)
and inhibiting transcription of the coinjected promoter lacking the
repeats (A gene). Thus, the effect of the 0/1 repeats is also highly
obvious in the cis/trans enhancer assay.
We next examined whether the 0/1 repeats could function in their
natural, promoter-distal spacer arrangement (see Figs. 1 and
2A), through the intervening promoter-proximal 60/81-bp
enhancer and spacer promoter, to further stimulate transcription from
the downstream gene promoter. When examined in frogs whose
cis-stimulatory response is not saturated by the 60/81-bp
repeats and the spacer promoter (Fig. 7B, lanes
1-3; see also Ref. 21), the additional presence of the 0/1
repeats directs still greater enhancement of gene promoter
transcription (lane 4). In the individual of Fig.
7B, the presence of each of these additional elements
increases transcription from the downstream gene promoter by about
2-fold.
The trans competition results (Fig. 6)
suggest that the 0/1 repeats bind a factor that is required for
transcription at the ribosomal promoter. To more directly examine
whether the 0/1 repeats interact with a rDNA promoter-binding factor,
we used footprint competition analysis. When the X. laevis
rDNA promoter, 5
Because the 0 and 1 repeats and 60/81-bp repeats all inhibit formation of the promoter
footprint similarly (Fig. 8), they might all bind the same rDNA
transcription factor. UBF is a polymerase I transcription factor that
interacts within the upstream rDNA promoter domain in a functionally
relevant manner (38, 45). UBF is also the only protein thus far
reported to bind the 60/81-bp enhancer, based on a characteristic DNase
I footprint (25, 46) and mobility shift (40) created by isolated or
recombinant UBF on those repeats. On the basis of these results, UBF
has been thought to be important for enhancer function (40, 46), even
though its binding exhibits limited sequence specificity (see
``Discussion''). To examine whether UBF binds to the 0 and the 1 enhancer repeats like it binds to the 60/81-bp enhancer repeats,
X. laevis UBF was synthesized by in vitro
transcription/translation and was purified to homogeneity (Fig.
9A) (38). Incubation of recombinant UBF
protein with a radiolabeled fragment containing two of the 60-bp and
two of the 81-bp sequences results in a characteristic (40)
UBF-mediated electrophoretic mobility shift (Fig. 9B,
lane 1) that is inhibited by co-incubation with unlabeled
60/81-bp repeats (lanes 2-4) to a greater extent than by
co-incubation with a comparable amount of nonspecific dA-dT DNA
(lanes 11-13). Notably, both the isolated 0 repeats and
isolated 1 repeats also inhibit this mobility shift (lanes
5-10) to an extent comparable to the 60/81-bp repeats. In
separate experiments, we found that incubation with UBF also causes a
mobility shift and discrete DNase I and exonuclease III footprint
patterns on the isolated 0 elements and on the isolated 1 elements
(data not shown). Thus, by all the criteria used to infer that the pol
I transcription factor UBF binds to the 60/81-bp enhancer repeats, it
binds in a similar manner to the 0 and the 1 enhancer repeats.
DISCUSSION The X. laevis 0 and 1 spacer repeats are enhancers of
pol I transcription. The X. laevis rDNA intergenic spacer
consists almost entirely of four kinds of repeated elements. The
promoter-proximal portion contains the 60/81-bp enhancer repeats and
spacer promoters, both of which serve to stimulate pol I transcription,
while the promoter-distal portion contains two other types of repeats,
the 0 and 1 repeats (11, 23, 47). We report here that the 0/1 repeats,
as well as the individual 0 and 1 repeats, are enhancers of pol I
transcription and exhibit all of the known properties of the 60/81-bp
enhancers (17, 19, 20). The 0, 1, and 0/1 repeats stimulate
transcription from a promoter when located in cis (Fig. 3,
A and B), when in the reverse orientation
relative to the promoter (Fig. 3B), when moved hundreds of
bp upstream from the promoter (Fig. 4A), and when located
within the transcribed region downstream from the promoter (Fig.
4B). They also stimulate transcription from a promoter in
cis relative to that from a coinjected promoter in
trans (Fig. 7A). Thus, virtually the entire
multikilobase X. laevis IGS is made up of pol I enhancer
repeats.
The extent of cis stimulation afforded a pol I promoter by
the 0 and 1 repeats increases with an increasing number of repeats
until the saturation level is approached (Fig. 5). This saturation
point varies between individual frogs, a phenomenon also observed with
the 60/81-bp enhancers (20, 42). Notably, the level of cis
stimulation afforded by the 0 and 1 repeats is comparable to and in
most frogs somewhat greater than that afforded by similarly positioned
60/81-bp enhancer repeats, on a per length basis.
In oocytes from individuals in which the maximal level of enhanced
transcription is very high relative to the level from the promoter
alone, we also observe that the 0/1 repeats in their natural
promoter-distal location in the IGS stimulate transcription from the
downstream gene promoter, above the level directed by the intervening
promoter-proximal 60/81-bp enhancers and spacer promoter (Fig.
7B). In the mature oocytes of other frogs, the level of gene
promoter transcription appears maximal with a single 60/81-bp block and
no additional stimulation from the more distal spacer promoter and 0/1
repeats is observed (data not shown). This presumably explains why
previous studies concluded that the 0 and 1 repetitive elements do not
exert a significant transcriptional effect (19, 22, 23). These
conclusions were based entirely on the type of experiment of Fig.
7B, where detection of stimulation by the distal 0/1 repeats
is frog-dependent; in the oocytes examined, the maximal
level of transcription may have been reached in the absence of the 0/1
region. These early studies did not specifically examine the
promoter-distal repeats in experiments like those of Fig. 3-7.
In trans
competition assays, the subcloned 0, 1, and 0/1 enhancer repeats
inhibit transcription from a coinjected ribosomal promoter on a
separate plasmid molecule (Fig. 6), as do the 60/81-bp enhancer repeats
(17, 20), suggesting that both types of enhancers may bind a pol I
transcription factor (see also Refs. 23 and 48). This hypothesis was
strengthened by the finding that formation of a footprint on the rDNA
promoter that is diagnostic for a stable transcription complex is
specifically inhibited by the 0, 1, and 0/1 repeats (Fig. 8), as well
as by the 60/81-bp repeats, but not by various nonspecific DNAs.
Additionally, the 1 repeats exhibit a strong sequence identity with the
upstream domain of the rRNA gene promoter (Fig.
10A; a 63-bp segment of >75% identity),
suggesting that a common factor may bind to the promoter and to the
enhancers. The 60/81-bp repeats show a comparable extent of sequence
homology with the central region of the rRNA gene promoter (a 43-bp
segment of ~80% identity (
The pol I transcription factor UBF that acts at the rDNA promoter (see
Refs. 38, 45, and 50, and references therein) is the only protein
reported to bind to the 60/81-bp repeats and has been proposed to be
important in mediating enhancement by the 60/81-bp repeats (25, 40,
46). Since analogous assays show that UBF can bind similarly to the 0 and the 1 repeats (Fig. 9), UBF is also a candidate for mediating the
action of the 0 and 1 enhancers. However, competition studies using
footprinting, gel shift, and UV cross-linking do not reveal the extent
of binding specificity for UBF to the promoter or to any of the
enhancers, as is typical for pol II transcription factors (Ref. 51;
Fig. 9)3 and mutations of the 60/81-bp
sequence that inhibit enhancer function can have minimal effect on UBF
binding (42). Furthermore, UBF appears to recognize structural features
of DNA rather than a certain nucleotide sequence (51, 52, 53, 54, 55). From such
considerations (see also Refs. 49 and 56), it appears that UBF alone
may not mediate transcriptional enhancement, and it remains to be
proven whether UBF is indeed directly relevant to the in
vivo action of pol I enhancers, both the 60/81-bp repeats and the
0 and 1 repeats.
The
promoter-distal intergenic spacer of Xenopus borealis rDNA
contains extensive repetitive regions that have a high degree of
sequence identity to the 0 and 1 repeats of X. laevis. There
is >70% identity over a 70-bp region of a sequence that is repeated
nine times in the X. borealis IGS region 2 with the X. laevis 1 repeat (Fig. 10B, bottom). There is
also 100% identity between a 13-bp sequence that is repeated three
times in the more promoter-distal X. borealis IGS and
sequences of the X. laevis 0 repeat (Fig. 10B,
top). Furthermore, there is 70% identity between a 28-bp
sequence that is repeated four times in the promoter-distal X. clivii IGS (57) and sequences of the X. laevis 0 repeat
(Fig. 10B, top). These repetitive elements in the
spacers of other Xenopus species that have sequence
identities to the 0 and 1 repeats may also be promoter-distal enhancers
of pol I transcription. Furthermore, in mouse, Drosophila,
Arabidopsis, and Acanthamoeba where
promoter-proximal repetitive enhancers have been demonstrated, there
are also more promoter-distal repetitive elements (26, 27, 58, 59, 60, 61);
these might also function as promoter-distal enhancers of pol I
transcription.
FOOTNOTES REFERENCES
Volume 271, Number 43,
Issue of October 25, 1996
pp. 27138-27145
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
,§ and
Department of Biological Chemistry, Johns
Hopkins University, School of Medicine, Baltimore, Maryland 21205
140 to 1) and the 60/81-bp
repeats have ~80% identity to 50 bp of the gene promoter (121 to
72) (11, 13). The promoter-distal portion of the X. laevis
IGS consists of ``0'' repeats, 34-bp elements reiterated ~2-10
times/spacer, and ``1'' repeats, 100-bp elements reiterated ~six to
nine times/spacer (11, 12, 14).
Fig. 1.
The organization of X. laevis
rDNA. Slightly more than one repeating unit of X. laevis rDNA is represented. The transcribed region is
boxed. The intergenic spacer, represented by a solid
line, is expanded below. Darkly shaded objects
represent enhancers, lightly shaded boxes represent the gene
promoter (GP) or spacer promoter (SP), and the
arrows indicate sites of transcription initiation.
T3 is a promoter-proximal terminator.
[View Larger Version of this Image (10K GIF file)]
245 to +13), transcribing
two different reporter sequences (see Ref. 20, and references therein).
The monomer and oligomers of the 0 repeats, 1 repeats, and 0/1 repeats
were then inserted in the forward and reverse (r) direction, upstream
of the gene promoter, by cloning the EcoRI fragment
containing these repeats from pXlrs121-4,
pXlrs141-2, and pXlrs16 into the EcoRI site
upstream of the promoter in the A or B constructs. This created the
plasmids 01-4:A, 0r1-4:A, 0:B,
0r:B, 11-2:A, 1r1-2:A, 1:B,
1r:B, 0/1:A, 0/1r:A, 0/1:B, and
0/1r:B. Transcription in all these templates proceeds away
from the HindIII end of the polylinker. A:0, A:1, and A:0/1
were constructed by cloning the same 0-, 1-, or 0/1-containing
fragments from pXlrs12, pXlrs14, or pXlrs16 (EcoRI-excised,
S1-blunted, and joined to HindIII linkers) into the
HindIII site of the A construct. 0:200:A, 1:200:A, and
0/1:200:A were constructed by replacing a 40-bp AccI
polylinker fragment of 0:A, 1:A, or 0/1:A with a 238-bp
HpaII fragment of pBR322 (residues 1020-1258).
252 to +50 relative
to this promoter, joined to ClaI linkers) into the
AccI site of pUC18 and then inserting, between the upstream
XbaI and EcoRI polylinker sites, the X. laevis promoter-proximal terminator (residues 243 to 188
relative to the gene promoter, originally cloned into the
SmaI site of pUC18 as pXlT32 and
excised with XbaI and EcoRI). E:SP contains the
60/81-bp E block joined upstream of residue 252 of the spacer
promoter at an introduced SalI site. 0/1:SP contains the 0/1
region from pXlr14B (1.5-kb BssHII (S1 blunted and joined to
ClaI linkers)-BamHI fragment) cloned between the
BamHI site of SP and the AccI site of the vector
recreating a native spacer segment. SP:E:A and 0/1:SP:E:A contain the
rDNA inserts from SP and 0/1:SP (excised using the HindIII
(Klenow-blunted) and EcoRI (polylinker) sites) inserted into
E:A (cleaved with XbaI (S1-blunted) and EcoRI).
For 5 S maxi, the 0.35-kb HindIII fragment containing the 5 S maxi gene of pXbs115/77 (33) was cloned into pUC18. The rDNA
constructs are diagrammed in Fig. 2.
Fig. 2.
X. laevis rDNA constructs.
Schematic representation of templates containing the spacer promoter
(panel A) or gene promoter (panel B), and of
constructs used in trans competition experiments
(panel C).
[View Larger Version of this Image (17K GIF file)]
-amanitin was used to
allow transcription of the 5 S control template. At 6 h
post-injection, the nucleic acid was isolated (35, 36) and RNA from two
oocyte equivalents was analyzed by S1 nuclease mapping (34, 36). The
probes for the A and B gene transcripts were
AvaI-HindIII fragments (residues 60 to +97 or
60 to +60) from the A or B plasmids, respectively, 5
-end-labeled,
denatured in 95% formamide, and separated on a nondenaturing 6%
polyacrylamide gel. The probe for the X. laevis spacer
promoter transcript was a BglI-HindIII fragment
(residues 25 to +71) of the SP plasmid, 5-end-labeled and strand
separated on a 4% polyacrylamide, 9 M urea gel, recovering
the longer antisense strand. The 5 S maxi gene probe was a
BamHI fragment of the template (residues 77 to +115),
prepared similarly to the A and B gene probes except using a 5%
polyacrylamide gel. The transcription signals were quantitated by
densitometry or phosphorimaging.
210 to +20, blunted and cloned into the SmaI site of
pUC9),2 5-end-labeled at +20 (EcoRI). After 30 min at 30 °C, 800 units of exonuclease III was added in 25 µl of
binding buffer and incubation was continued for 10 min at 30 °C. The
DNA products were resolved on a 6% sequencing gel and visualized by
autoradiography. Factors of the mouse cell S-100 extract can bind to
and transcribe frog and mouse rDNA promoters with approximately equal
efficacy (37). Similar exonuclease III footprints are observed at the
3 border of the stable complex region on both frog and mouse rDNA
promoters,2 but the most distinctive footprint is at the
upstream border of the frog rDNA promoter, as used in this
analysis.
-end-labeled 282-bp enhancer fragment containing a tetramer of the
60/81-bp element), ~2 ng of purified recombinant xUBF, and the
indicated amounts of isolated competitor fragment. Reactions were
incubated 15 min at room temperature.
spacer promoter, as well as a terminator element (16,
41) added to prevent read-through inhibition. The level of
transcription from this construct was compared to that of an otherwise
identical construct lacking the 0/1 repeats (SP; Fig. 2A) by
separately coinjecting X. borealis oocytes with either of
the rDNA templates plus a control 5 S maxi gene and then quantitating
the resultant transcripts by S1 nuclease analysis (see ``Experimental
Procedures''). The 0/1 repeats are indeed capable of stimulating
transcription from the adjacent pol I promoter, by up to 30-fold (Fig.
3A, upper panel, lanes
1 and 2). This is the largest cis
stimulation reported for a pol I transcriptional enhancer, reports of
which have ranged from ~3-fold (mouse rRNA gene promoter by mouse
rDNA enhancer; Ref. 25) to ~10-fold (X. laevis rRNA gene
promoter by the 60/81-bp rDNA enhancer; Ref. 20). A comparable extent
of stimulation was also observed for this promoter by a similarly
located block of 60/81-bp enhancer repeats (Fig. 3A,
upper panel, lane 3).
Fig. 3.
Stimulation of pol I transcription by
cis-located 0 and 1 repeats. The indicated rDNA
template containing the spacer promoter (panel A) or the
gene promoter (panel B) plus elements of the X. laevis IGS (see Fig. 2) was co-injected with the maxi-5 S control
template into nuclei of X. borealis oocytes. The transcripts
from the spacer promoter (SP), 5 S control gene
(control), and the rDNA gene promoter (A) were
quantitated by S1 nuclease analysis and densitometry. Relative
intensities are given below the lanes in this and the subsequent
figures. In most frogs used in these studies, the 0/1 repeats and the
60/81-bp repeats caused a ~10-30 fold transcriptional
cis-enhancement; however, as observed previously (20), some frogs
exhibit minimal cis-enhancement (e.g. see frog 2 of Fig.
5A).
[View Larger Version of this Image (30K GIF file)]
1 0.
Fig. 4.
Transcriptional stimulation by the 0 and 1 repeats is not dependent on their precise positions. The indicated
templates (see Fig. 2B) were co-injected with the maxi-5 S
control template and analyzed as in Fig. 3.
[View Larger Version of this Image (42K GIF file)]
of the promoter, and with nearly the same
efficacy as when upstream (Fig. 4B, lanes 1-7).
An analogous result is observed with a similarly positioned 60/81-bp
enhancer block (Fig. 4B, lanes 8 and
9). The ability of the 60/81-bp repeats to function 3 of
the promoter was also suggested earlier using a different kind of assay
(18); an apparently contradictory result (20) was likely due to that
particular clone. Thus, the 0 and 1 repeats exhibit the same
stimulatory properties as the 60/81-bp enhancer repeats; therefore, the
0 and 1 repeats are likewise pol I enhancers.
Fig. 5.
Stimulation by polymers of the 0 and 1 repeats. The indicated templates were co-injected with the maxi-5
S control template and analyzed as in Fig. 3. Panel A shows
the results for two different frogs whose cis response was
not saturated with a single 0 block.
[View Larger Version of this Image (32K GIF file)]
Fig. 6.
Competition of transcription by the 0 and 1 repeats in trans. Equimolar amounts of the A gene promoter
template and the indicated competitor construct (see Fig.
2C) were co-injected, and their transcripts were analyzed as
in Fig. 3.
[View Larger Version of this Image (35K GIF file)]
Fig. 7.
The 0/1 repeat can provide a selective
advantage to a cis-located gene promoter in competition
against another gene promoter in trans and with an
intervening 60/81-bp enhancer block and spacer promoter. Panel
A, the A gene and the indicated B gene promoter constructs were
co-injected along with the maxi-5 S control template, and their
transcripts were analyzed as in Fig. 3. The B gene templates are
identical to their A gene counterparts, except that they contain a
different reporter region. Panel B, gene promoter templates
containing the indicated elements of the X. laevis IGS in
cis were co-injected with the maxi-5 S control template, and
their transcripts were analyzed as in Fig. 3.
[View Larger Version of this Image (18K GIF file)]
-end-labeled downstream from the initiation site, is
preincubated in transcriptionally competent cell extract, a complex is
formed that blocks the progression of exonuclease III at the borders of
the region required for stable transcription complex formation (43,
44), resulting in a strong exonuclease stop at residue 141 (Fig.
8).2 The band that results from the blockage
of exonuclease progression indeed represents a specific complex,
because its formation is not inhibited by the presence of unlabeled
plasmid vector alone (lane 4) or by several other subcloned
nonspecific DNA sequences (data not shown), but is inhibited by rDNA
promoter-containing plasmid (lane 9) and by 60/81-bp
enhancer-containing plasmid (lane 8).2 When used as
unlabeled competitors, the plasmids containing the subcloned 0, 1, and
0/1 repeats similarly inhibit formation of the exonuclease III
footprint on the promoter (lanes 5-7). This suggests that
the 0 and 1 repeats in addition to the 60/81-bp repeats may both bind a
factor in common with the rDNA promoter.
Fig. 8.
The 0 and 1 repeats specifically inhibit
complex formation on the rDNA gene promoter. A X. laevis promoter probe (residues 210 to +20,
32P-labeled at the +20 end) was incubated in mouse S-100
cell extract in the presence of the indicated HhaI-digested
competitor DNAs (HhaI does not digest within the repetitive
elements). Following treatment with exonuclease III, the digestion
products were analyzed on a sequencing gel and the autoradiogram is
shown. The arrow indicates the strong exonuclease stop at
the upstream border of the promoter. M,
HpaII-digested pBR322 markers; A+G, purine
cleavage marker of the probe DNA; Extract, no S-100
extract in the binding reaction; pUC,
HhaI-digested pUC18 competitor DNA.
[View Larger Version of this Image (63K GIF file)]
Fig. 9.
UBF can bind to the 0 and 1 repeats.
Panel A, SDS-PAGE of the purified xUBF; left
lane, autoradiogram; right lane, silver stain. Markers:
215,000-Da myosin H-chain, 105,100-Da phosphorylase b,
69,800-Da bovine serum albumin, 43,000-Da ovalbumin, and 28,300-Da
carbonic anhydrase. Panel B, a 282-bp
32P-end-labeled 60/81-bp probe was incubated with ~2 ng
of purified xUBF in the presence of the indicated isolated competitor
fragments or nonspecific DNA competitor at the indicated mass ratio of
competitor to probe. The nucleoprotein complexes were subject to
electrophoretic mobility shift analysis and autoradiography.
[View Larger Version of this Image (42K GIF file)]
114 to 72); Refs. 11, 12, 13); and an
artificially polymerized version of the central region of the rRNA
gene promoter can function as a pol I enhancer (49).
Fig. 10.
The 0 and 1 repeats have sequence identity
to regions of the gene promoter and to elements in the IGSs of other
Xenopus species. Panel A, the program BESTFIT of
the GCG package was used to identify a >75% identity between residues
167 to 105 of the X. laevis rDNA promoter (13) and the 1 repeats (12). Panel B, the X. borealis rDNA
repeat of pXbr101A (62) is schematically represented. Regions of
identity between the 0 and 1 repetitive elements and repetitive
elements in the spacers of X. borealis (62) and X. clivii (57) were identified as in A. BESTFIT analysis
showed the X. borealis region designated 0 to have 100%
identity within the 0 repeats of X. laevis, and the X. borealis region 2 to have a >70% identity within the X. laevis 1 repeats. Identities between the E region of X. borealis and the 60/81-bp repeats of X. laevis, as well
as between the spacer and gene promoters of X. borealis and
X. laevis have been noted (47). Region ga
contains the simple repeat GA, and all other labels are as in Fig. 1.
The identity between the X. laevis 0 repeats and the
X. clivii 1 repeats has been noted (47).
[View Larger Version of this Image (26K GIF file)]
§
Present address: Dept. of Chemistry, New York University, New York,
NY 10003.
1
The abbreviations used are: pol I, polymerase I;
IGS, intergenic spacer; kb, kilobase(s); bp, base pair(s); DTT,
dithiothreitol; SP, spacer promoter.
2
L. K. Pape, unpublished data.
3
E. B. Mougey, unpublished observation.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT