(Received for publication, January 4, 1995)
From the
A method for preparation of transcriptionally active nuclear extracts from the ciliated protozoan Tetrahymena thermophila is described. Cells were lysed in the presence of gum arabic, and nuclei were further purified in the presence of Ficoll 400. Highly concentrated nuclear extracts were prepared by ultracentrifugation of nuclei in a buffer containing potassium glutamate and spermidine. These extracts supported accurate transcription initiation of T. thermophila class II and III genes. Using the histone H3-II gene as a template, we demonstrated that physiologically induced changes in transcriptional activity in vivo were reflected in the transcriptional activity of the nuclear extract in vitro. By electrophoretic mobility shift assays, five conserved sequence elements in the upstream region of the histone H3-II gene were shown specifically to bind proteins in extracts from exponentially growing as well as from starved cells, and by UV cross-linking we further characterized the specific binding of two proteins to an oligonucleotide containing a conserved CCAAT box motif. Transcription competition experiments showed that addition of this oligonucleotide decreased transcription significantly. Competition with oligonucleotides corresponding to the two proximal conserved sequence elements almost completely abolished transcription of the H3-II gene suggesting that binding of transacting factors to these elements is crucial for initiation of transcription.
In higher eukaryotes mRNA levels are controlled by a complex interplay of transcriptional and post-transcriptional mechanisms. In lower eukaryotes examples of post-transcriptional regulation of mRNA abundance have been described (Warner et al., 1993), but generally, transcriptional control of mRNA levels appears to prevail. The ciliated protozoans represent one of the earliest divergent branches of the eukaryotic lineage (Sogin et al., 1986) and have as model organisms been of considerable importance for studies of fundamental molecular mechanisms such as RNA self-splicing (Zaug et al., 1986) and telomere formation (Greider and Blackburn, 1987). A considerable number of protein-encoding genes from ciliates have been cloned and characterized, but no functional analyses of promoter regions have been reported. To date, analyses of promoter structures in Tetrahymena have been limited to mapping of DNase hypersensitive regions in the promoters of the L1 and S25 ribosomal protein genes (Nørgaard et al., 1992) and in the promoter of the histone H4-I gene (Pederson et al., 1986). Apart from these studies, identification of putative promoter elements in ciliates has been restricted to computer-assisted searches for sequence elements exhibiting similarity to known eukaryotic promoter elements (Brunk and Sadler, 1990).
Recently, transformation of Tetrahymena thermophila with protein-encoding genes was reported (Yao and Yao, 1991; Kahn et al., 1993; Gaertig et al., 1994), but transformation was in all cases accomplished by homologous recombination making this approach unsuitable as a general procedure for functional analyses of promoters in ciliates. An alternative approach would be to develop an in vitro transcription system specific for ciliate genes. Systems capable of accurate in vitro transcription are well established for higher eukaryotes (Manley et al., 1980; Dignam et al., 1983; Parker and Topol, 1984), but it has consistently been found difficult to achieve in vitro transcription of class II genes in extracts from lower eukaryotes (Lue and Kornberg, 1987).
Tetrahymena extracts capable of accurate transcription of rDNA by polymerase I have been described (Sutiphong et al., 1984; Matsuura et al., 1986), but transcription in vitro of ciliate class II genes has not been reported. In this report we describe the preparation of a nuclear extract from T. thermophila that supports accurate transcriptional initiation of exogenously added ciliate class II and class III genes. Extracts prepared from exponentially growing and starved cells, respectively, differed in their ability to transcribe the histone H3-II gene. By competition with double-stranded oligonucleotides, we demonstrated that four conserved sequence elements in the intergenic region between the divergently transcribed histone H3-II and H4-II genes are involved in the regulation of transcription of the histone H3-II gene. Interestingly, electrophoretic mobility shift assays revealed that these oligonucleotides bound different complements of proteins in extracts prepared from exponentially growing and starved cells, respectively.
Figure 1: The histone H3-II containing plasmids used in this study. Transcribed regions are shown in black. Polylinker regions are shaded. Closed arrows indicate the T3 or T7 RNA polymerase initiation sites in the pBluescript vectors. Open arrows indicate the H3-II and H4-II transcription initiation sites.
p5S rDNA SS256 contains a SalI fragment of a 5 S rRNA gene (subcloned from pBS-A4 obtained from R. Hallberg, Department of Biology, Syracuse University) with 145 bp of upstream sequences and the entire transcribed region except for the last 4 bp in the 3`-end of the gene. This fragment was cloned into the SalI site of pBluescript KS+.
The oligonucleotides were annealed
pairwise by mixing 50 µl each of oligo(A) and (B) (100 pmol/µl)
with 6.8 µl of 1 M Tris-HCl, pH 7.5, and 1 µl of 100
mM EDTA. This mixture was boiled for 3 min and then cooled
slowly to the annealing temperature (23 °C for oligonucleotides 1
and 2, 20 °C for oligonucleotides 3 and 5 and 33 °C for
oligonucleotide 4). After annealing for 1 h, 1.7 µl of 1 M MgCl, 1.7 µl of 100 mM DTE, 17 µl of
1 mg/ml bovine serum albumin, 42.5 µl of dATP, dGTP, dCTP, and dTTP
(2 mM each), and 16 units of T7 DNA polymerase were added. The
fill-in reaction was allowed to proceed for 30 min at the annealing
temperature. Five µl of 0.5 M EDTA and 16 µl of 3 M sodium acetate were added, and the reaction mixture was
extracted with phenol/chloroform. For preparation of labeled
oligonucleotides, 4 pmol were annealed in a volume of 10 µl and
filled-in in the presence of [
-
P]dCTP to a
specific activity of 3000 Ci/mmol. The double-stranded oligonucleotides
were precipitated by addition of 3 volumes of 99% ethanol and were
subjected to electrophoresis in a native 12% polyacrylamide gel.
Following electrophoresis the part of the gel containing the
double-stranded oligonucleotide was detected by UV shadowing or
autoradiography and isolated. The oligonucleotides were eluted by the
addition of 200 µl of elution buffer (0.5% SDS, 250 mM sodium acetate, pH 8.0, 1 mM EDTA) followed by gentle
shaking overnight at 37 °C. The supernatant was isolated, and 200
µl of elution buffer was added to the gel pieces followed by an
additional incubation for 2 h. The supernatants were combined and
extracted with phenol/chloroform. The oligonucleotides were
precipitated with 3 volumes of ethanol, washed twice with 75% ethanol,
dried, and dissolved in a solution of 25 mM sodium acetate, pH
8.0, 1 mM EDTA. The concentrations of the double-stranded
oligonucleotides were determined by UV spectroscopy.
By using this procedure and by adding Ficoll during the preparation of nuclei, we were able to obtain nuclear extracts containing 20-80 mg of protein/ml. As described below, these extracts supported in vitro transcription of cloned genes from T. thermophila.
Figure 6: The intergenic region of the divergently transcribed H3-II-H4-II histone genes. Thirty sequences in the EMBL data base were aligned using the PILEUP program of the GCG package (Deveraux et al., 1984). The consensus was displayed with the program PRETTY counting 20 conserved nucleotides as a consensus. The sequence of the intergenic region of T. thermophila is shown with the nucleotides identical to the consensus shown in uppercase. The ATG translation start codons are underlined. The oligonucleotides used for electrophoretic mobility shift assays and competition experiments are shaded, and the sites of transcription initiation are shown with open arrows. The sequence is numbered counting the A in the ATG start codon of the H3-II gene as +1.
Figure 2:
In vitro transcription of the
histone H3-II gene. The transcription products were detected with T3
RNA polymerase generated antisense transcripts of XbaI
linearized pH3-II RB449. Lane 1, 0.5 µg pH3-II RB449
transcribed with 5 µl (165 µg) of nuclear extract; lane
2, as lane 1, but with 2 µg/ml -amanitin; lane 3, 0.5 µg of pBluescript KS+ transcribed with 5
µl of nuclear extract; lane 4, as lane 1 but
stopped immediately after the addition of nuclear extract; lane
5, 0.5 µg of pH3-II RB449 transcribed with 5 µl of nuclear
extract from starved cells; lane 6, 0.5 µg of pH3-II RB449
transcribed with 2.5 µl of nuclear extract from starved cells and
2.5 µl of nuclear extract from exponentially growing cells; lane t, RNase protection with 40 µg of wheat germ tRNA; lane T, RNase protection with 40 µg of T. thermophila total RNA. Open and closed arrows indicate in vitro and in vivo transcribed RNA,
respectively.
To demonstrate that the
nuclear extract supported accurate transcription initiation,
transcripts from a number of different templates were analyzed (Fig. 3). Analysis of total RNA isolated from exponentially
growing T. thermophila showed that transcription of the H3-II
gene was initiated at two sites mapping to position -60 (major
transcription start point) and -46 (minor transcription start
point) relative to the A in the translation start codon ATG (Fig. 3, lane T). The in vitro transcription
with pH3-II RH3.6 and pH3-II RB449 as templates revealed that the same
transcription start points were used in vitro (Fig. 3, lanes 1 and 2). Transcripts generated from pH3-II
KB351 contain 14 nucleotides of the polylinker region in common with
the labeled antisense RNA. Accordingly, transcription of pH3-II KB351
gave rise to protected products that were extended by 14 nucleotides (Fig. 3, lane 3). Addition of -amanitin to 2
µg/ml abolished transcription (Fig. 3, lane 4).
Thus, the in vitro transcription system displayed the expected
-amanitin sensitivity and utilized transcription start sites
identical to those used in vivo. However, it is noteworthy
that transcripts initiated at position -60 dominated in
vivo, whereas transcripts initiated at position -46 were
most abundant in vitro.
Figure 3:
In vitro transcription of the
histone H3-II gene. The transcription products were detected with T3
RNA polymerase generated antisense transcripts of XbaI
linearized pH3-II KB351. Templates: lane 1, 0.5 µg of
pH3-II RB449; lane 2, 0.5 µg of pH3-II RH3.6; lane
3, 0.5 µg of pH3-II KB351; lane 4, 0.5 µg of
pH3-II KB351 + 2 µg/ml -amanitin; lane 5, 0.5
µg of pH3-II KB351 linearized with XbaI; lane t,
RNase protection with 40 µg of wheat germ tRNA; lane T,
RNase protection with 40 µg of T. thermophila total RNA. Open and hatched arrows indicate in vitro transcribed RNA with no protected polylinker and in vitro transcribed RNA with a protected polylinker, respectively. Closed arrows indicate in vivo transcribed
RNA.
We examined the influence of DNA topology by comparing in vitro transcription of a supercoiled template with a linearized template (Fig. 3, lanes 3 and 5). In this particular experiment, the linearized template was more efficiently transcribed than the supercoiled one, but other experiments showed no difference between supercoiled and linearized templates.
Figure 4:
The
effect of magnesium acetate and spermidine on in vitro transcription. Each in vitro transcription reaction
contained 0.5 µg of pH3-II RB449 as template, and the transcription
products were detected using T3 RNA polymerase generated antisense
transcripts of XbaI linearized pH3-II RB449. To the reactions
marked 0 mM MgAc, EDTA was added to a concentration of 10
mM to chelate Mg present in the extract.
(For these reactions, magnesium acetate was added to a total
concentration of 16 mM prior to the addition of DNase.) The
lanes containing reactions with 0, 0.25 and 1.0 mM magnesium
acetate were exposed for 14 h without an intensifying screen; the lanes
containing reactions with 2.5 and 10 mM magnesium acetate were
exposed for 50 h with an intensifying screen. Sper. spermidine.
Figure 5: In vitro transcription of a 5 S rRNA gene. The transcription products were detected with T7 RNA polymerase-generated antisense transcripts of XhoI linearized p5SrRNA SS256. Lane 1, transcription of 0.5 µg of p5SrRNA SS256 (supercoiled); lane 2, control reaction with 0.5 µg of pBluescript KS+. Open and closed arrows indicate in vitro transcribed and endogenous RNA, respectively. Asterisks indicate the positions of correctly trimmed antisense probes that were complementary to the protecting endogenous 5 S rRNA and in vitro transcribed 5 S rRNA, respectively.
To analyze the functional importance of the conserved elements, double-stranded oligonucleotides (Fig. 6) corresponding to these regions were prepared. Oligonucleotide 1 encompasses the transcription initiation site, oligonucleotide 2 the HiNF-A homology, oligonucleotide 3 one of the CCAAT boxes, oligonucleotide 4 the H4TF-1 consensus, and oligonucleotide 5 the octamer consensus site.
Electrophoretic mobility shift assays (Staudt et al., 1986) showed that all oligonucleotides bound protein(s) in the extracts prepared from exponentially growing and starved cells, respectively, and furthermore, reciprocal competition experiments demonstrated that the binding was specific for each oligonucleotide (Fig. 7). An unrelated oligonucleotide did not bind any protein in the nuclear extract (results not shown). The complexes formed with nuclear extracts from exponentially growing cells were clearly distinct from those formed with nuclear extracts prepared from starved cells (Fig. 7, and results not shown).
Figure 7: Electrophoretic mobility shift assay of oligonucleotides 1-5 with nuclear extract prepared from exponentially growing cells (A) or from cells starved for 24 h (B). Each individual oligonucleotide was subjected to competition with oligonucleotide 1-5 as indicated. Nuclear extract was preincubated with or without 5 nmol of competitor followed by the addition of 10 fmol of labeled oligonucleotide. Free probe and complexes were separated on native gels as described under ``Materials and Methods.'' C and O, indicate competitor and labeled oligonucleotide, respectively.
Figure 8: UV cross-linking of oligonucleotide 3 to proteins in nuclear extract prepared from exponentially growing cells. Lane 1, standard cross-linking reaction; lane 2, cross-linking in the presence of a 500-fold molar excess of oligonucleotide 1; lane 3, cross-linking in the presence of a 500-fold molar excess of oligonucleotide 3; lane 4, cross-linking followed by treatment with proteinase K; lane 5, cross-linking reaction with no nuclear extract. The migration of prestained markers is indicated to the right.
Figure 9: Oligonucleotide mediated competition of in vitro transcription. Transcription of 0.5 µg of pG78 RH3.6 in the presence of 12 pmol (100-fold molar excess) of oligonucleotide 1-5 as indicated. The transcription products were detected with T3 RNA polymerase generated antisense transcripts of XbaI linearized pH3-II RB449. Lane 1, in vitro transcription in the absence of competitor; lanes 2-6, in vitro transcription in the presence of a 100-fold molar excess of the double-stranded oligonucleotides 1-5 as indicated.
In this report we describe the preparation of a nuclear extract from the ciliated protozoan T. thermophila that supports accurate transcription initiation in vitro by RNA polymerase II and III. Previous attempts to achieve in vitro transcription of ciliate class II and III genes have been unsuccessful. The reasons for these failures are probably related to the preparation of the nuclear extract itself as well as to problems concerning the choice of suitable templates for in vitro transcription. Since no functional analyses of ciliate promoters have been performed so far, rational delimitation of a functional promoter has in most cases been impossible. Therefore, we decided to use clones of the histone H3-II gene as model templates for the development of a Tetrahymena based in vitro transcription system. In ciliates the genes encoding histones H3-II and H4-II are clustered and oriented in a head to head fashion with an intergenic region of approximately 345 bp (Brunk and Sadler, 1990). No introns are present in the two genes, and consequently, the intergenic region can be expected to contain the sequences necessary for at least basal transcription. Furthermore, the organization of the two histone genes has been shown to be conserved in a large number of ciliates making it possible to identify conserved sequence elements of possible regulatory importance (Brunk and Sadler, 1990; this report). For preparation of active nuclear extracts, minimization of nuclear leakage during isolation of nuclei was clearly of importance. Several ways of preparing nuclei were investigated, and the most successful combination was found to be disruption of the cells in a Potter-Elverhjem homogenizer in the presence of 4% gum arabic (Gorovsky, 1975), followed by a final purification of nuclei in a buffer containing 18% Ficoll 400 (Lue and Kornberg, 1987). Cell lysis in buffers containing Nonidet P-40 resulted in inactive extracts with very low concentrations of protein. Furthermore, the use of a potassium glutamate and spermidine containing buffer and centrifugation for the preparation of a nuclear extract with high concentration of protein according to the procedure of Kamakaka et al.(1991) were instrumental in obtaining active extracts. Finally, a number of observations has suggested that chloride ions inhibit the transcriptional activity of nuclear extracts (Lue and Kornberg, 1987; Shapiro et al., 1988; Verdier et al., 1990). Consequently, we substituted acetate for chloride, although we did not perform a systematic comparison of the performance of extracts prepared with chloride or acetate.
The transcriptional efficiency of the nuclear extract from T. thermophila was lower than those reported for metazoanderived systems (Shapiro et al., 1988; Kamakaka et al., 1991), but comparable to those reported for extracts prepared from yeast (Lue et al., 1989; Verdier et al., 1990) and Neurospora (Tyler and Giles, 1985). This relatively low transcriptional activity may be characteristic for extracts from lower eukaryotes, but the promoter organization and transcription start site patterns of the genes used as templates for in vitro transcriptions may well add to the low transcriptional efficiency. Thus, the genes from lower eukaryotes which have been used as templates for in vitro transcription initiate transcription from multiple start sites in vivo (Lue and Kornberg, 1987; Lue et al., 1989; Verdier et al., 1990; Tyler and Giles, 1985), and it is a general observation that such genes even in the more efficient extracts from mammalian cells normally are poorly transcribed in vitro (Farnham and Schimke, 1986; Osborne et al., 1987; Kageyama et al., 1988). Most if not all ciliate class II genes also seem to initiate transcription in vivo from multiple transcription start sites (Nielsen et al., 1986; Rosendahl et al., 1991; Hansen et al., 1991). In higher eukaryotes a substantial number of genes that initiate transcription from multiple start sites encode housekeeping proteins (Dynan, 1986). To this class of genes belongs the ribosomal protein genes which have been notoriously difficult to transcribe in vitro (Zahradka and Sells, 1988; Yoganathan et al., 1992; Chung and Perry; 1991). Like other genes encoding housekeeping proteins, these genes lack canonical TATA boxes in the promoter region. Interestingly, mutating an AT-rich region in the promoter of the S16 ribosomal protein gene to a canonical TATA box increased in vitro transcription dramatically (Chung and Perry, 1991). It is also noteworthy that in vitro transcription of other TATA-less mammalian genes from which transcription in vivo is initiated at multiple start sites failed to reflect the in vivo utilization of the individual start sites. Thus, minor in vivo start sites became the most prominent start site in vitro, whereas transcription from major in vivo transcription start sites became barely detectable in vitro (Farnham and Schimke, 1986; Osborne et al., 1987; Kageyama et al., 1988). The same phenomenon was observed by in vitro transcription of the T. thermophila histone H3-II gene, where the most abundant transcript was initiated at position -46, and the less abundant transcript at position -60, whereas the reverse was observed in vivo. Similarly, preferential utilization in vitro of minor in vivo transcription start sites was also observed with yeast extracts (Lue and Kornberg, 1987) and Neurospora extracts (Tyler and Giles, 1985). The reason for this difference in transcription start site utilization remains to be established, but it is obviously not a peculiarity of the Tetrahymena system.
The physiological variation in the rate of transcription of the histone H3-II gene according to the nutritional status of the cells (Bannon et al., 1983) is clearly reflected in the transcriptional activity of extracts prepared from exponentially growing and starved cells, respectively. Thus, extracts from starved cells did not support detectable transcription of the H3-II gene. The results obtained by mixing extract prepared from exponentially growing and starved cells suggested that the extract from starved cells did not contain grossly inhibitory substances. Rather, the lack of detectable transcriptional activity could be attributed to a lack of positively acting factors.
A computer-assisted comparison of the intergenic region between the H3-II and the H4-II genes in 30 different ciliates identified five conserved sequence elements. By electrophoretic mobility shift assays, oligonucleotides corresponding to each of these elements were found specifically to bind proteins in extracts from exponentially growing as well as starved cells. Interestingly, each oligonucleotide bound a different complement of proteins in extracts from exponentially growing cells and starved cells. Oligonucleotide 3 harbors a canonical CCAAT motif. By UV cross-linking, we showed that two proteins in the extract from exponentially growing cells bound to this oligonucleotide. The molecular masses of these proteins were estimated to 23.5 and 31.5 kDa, respectively. In mammals as well as in yeast, CCAAT motifs appear to be recognized by a large family of transacting factors that bind in the form of heterodimers to the target sequences. This pattern appears to be followed in T. thermophila, and furthermore, the molecular masses of the two binding proteins are comparable with those of the Hap2 and Hap3 proteins in yeast for which predicted molecular masses of 16 and 30 kDa, respectively, have been reported (Chodosh et al., 1988). The functional importance of the conserved sequence elements was assessed in transcription competition experiments. These experiments revealed that oligonucleotides 1 and 2, in particular, exerted a profound effect on transcriptional activity of the histone H3-II gene leading to an almost complete abrogation of H3-II transcription implying that factors binding to these two proximal elements are critical for transcription. Oligonucleotides 3 and 4 also significantly decreased transcription, whereas oligonucleotide 5 only affected transcription marginally. Thus, the CCAAT box-binding proteins in T. thermophila appear to be bona fide transacting factors, although less decisive than the factors binding to the two proximal elements.
The performance of the T. thermophila transcription system was investigated using other class II and class III T. thermophila genes as templates. Accurate initiation of transcription was achieved with the histone H4-I gene and the actin gene. However, in analogy with results obtained with other in vitro transcription systems, we were unable to detect specific initiation of transcription from either of four ribosomal protein genes. Using a 5 S rRNA gene as template, we demonstrated that the nuclear extract also supported accurate and efficient transcription of a class III gene. Of interest, the 5 S rRNA gene utilized in this study did not contain a canonical TATA box in the proximal upstream region. In N. crassa (Tyler, 1987), Drosophila (Sharp and Garcia, 1988), and Bombyx mori (Morton and Sprague, 1984), the presence of a TATA box approximately 25 base pairs upstream from the transcription initiation site was shown to be essential for efficient and accurate initiation of 5 S rRNA transcription in vitro. Interestingly, another 5 S rRNA gene cloned from T. thermophila (Pederson et al., 1984) possesses a canonical TATA box at position -30 to -26. Thus, it is possible that a TATA box may be dispensable for 5 S rRNA transcription in the T. thermophila system as it is in Xenopus derived systems, where no conserved sequences upstream from the transcription start site are necessary for accurate initiation (Sakunjo et al., 1980; Morton and Sprague, 1984). Although no pseudogenes have been identified in T. thermophila to date, formally it can not be excluded that the 5 S rRNA gene used in this study is a pseudogene which, in analogy with a human pseudogene lacking upstream promoter elements (Nielsen et al., 1993), can be transcribed in vitro albeit with a low efficiency.