Organization of Multiple Regulatory Elements in the Control Region of ...

Vol. 8, No. 3

MOLECULAR AND CELLULAR BIOLOGY, Mar. 1988, p. 1147-1159

0270-7306/88/031147-13$02.00/0 Copyright © 1988, American Society for Microbiology

Organization of Multiple Regulatory Elements in the Control Region of the Adenovirus Type 2-Specific VARNAl Gene: Fine Mapping with Linker-Scanning Mutants JOHNNY F. RAILEY Ilt AND GUANG-JER WU* Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322 Received 14 August 1987/Accepted 15 December 1987

The adenovirus type 2-specific virus-associated RNA 1 (VARNAl) gene is transcribed by eucaryotic RNA polymerase III. Previous studies using deletion mutants for transcription have shown that the VARNAl gene has a large control region which is composed of several regulatory elements. Twenty-five exact linker-scanning mutations in the control region, from -33 to +77, of this gene were used for definition of the number and boundaries of these elements. The effects of these mutations on transcription and competition for transcription factors in human KB cell extracts revealed five positive regulatory elements. The essential element, which coincided with the B block, was absolutely required for both transcription and formation of stable complexes. A second element, which included the A block, was also required for both transcription and formation of stable complexes. Although this element is not as essential as the B-block element, together with the B-block element it may be necessary for formation of the most basal form of transcription machinery. Therefore, these two elements are the promoter elements in this gene. In addition, one possible element in the interblock region and two elements in the 5' flanking region were also required for efficient transcription, but they were moderately required for formation of stable complexes. Transcription of these mutants and the wild-type gene using an extract of 293 cells was stimulated at least threefold over that with the KB cell extract, as expected. Similar regulatory elements of this gene were revealed, however, when the 293 cell extract was used for transcription of these mutants, suggesting that the ElA-mediated specific transcription factors act on the transcription machinery in a sequence-nonspecific manner.

Genes transcribed by eucaryotic RNA polymerase III have been categorized into two groups with respect to differences in their essential regulatory sequences in the intragenic control region (for reviews, see reference 10 and E. P. Geiduschek and G. P. Tocchini-Valentini, Annu. Rev. Biochem., in press). One group includes the genes encoding somatic and oocyte 5S rRNAs (for a review, see reference 23). This group of genes is characterized by the presence of an internal transcriptional control region consisting of two essential regulatory elements, the anterior A block and the posterior C block (10, 32). Transcription of these genes is dependent upon RNA polymerase III plus three fractions of factors, TFIIIA, TFIIIB, and TFIIIC (28, 43). The TFIIIA is a single protein, specific and necessary only for transcription of the 5S rRNA genes (13, 20, 35). This transcription factor makes its tightest contacts with the posterior C block element (41). The other group consists of the genes encoding for all tRNAs (16, 19, 44; Geiduschek and Tocchini-Valentini, in press), U6 RNA (27), human Alu-family RNAs (12, 22, 45), rodent Bi (24, 26), B2 (25, 47), 4.5S (40), and 7S RNAs (29, 49), adenovirus-specific VARNAs (1, 9, 46), and EpsteinBarr virus-specific EBERs (39). This group of genes is characterized by the presence of an internal transcriptional control region consisting of two essential regulatory elements, the anterior A block and the posterior B block (14). Transcription of these genes is dependent upon RNA polymerase III plus two fractions of factors, TFIIIB and TFIIIC (8, 15, 28).

The VARNAl gene has been an excellent model system for studies on the regulatory mechanisms of RNA polymerase III-mediated genes (14, 18, 53-55, 57-59). Systematic dissection of this gene has revealed a region larger than the internal transcriptional control region required for efficient transcription (59). We have evidence to suggest that this large control region may consist of multiple regulatory elements (59). To define further the number of regulatory elements and their boundaries, we have constructed 25 exact linker-scanning (LS) mutants, covering the entire region from -33 to +77 which contains most of the transcriptionally important regulatory sequences, and used them for transcriptional studies. We have quantitatively determined the effect of LS mutations on the transcription efficiency of the gene and on the ability of the gene to form stable complexes. Furthermore, we have determined the effect of the LS mutations on the use of start and termination sites. From the above results, we have clearly defined the number and the boundaries of these regulatory elements and suggested their functions in the control region of the VARNAl gene. Furthermore, we have compared the effect of LS mutations on the transcription efficiency of the gene using an extract of line 293 cells with like results obtained using a human KB cell extract for determination of regulatory elements affected by the ElA-mediated transcriptional augmentation. Some of the results have been presented (J. F. Railey II and G.-J. Wu, Abstr. Annu. Meet. Am. Soc. Microbiol. 1987, S32, p. 313).

Corresponding author. t Present address: Department of Pathology, University of California at San Francisco, San Francisco, CA 94143.

[a-32P]UTP (350 to 410 Ci/mmol) and [o-thio35S]dATP (650 Ci/mmol) were obtained from Amersham. [y-32P]ATP (4,500 Ci/mmol) and [a-32P]dGTP (2,903 Ci/

MATERIALS AND METHODS Materials.

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MOL. CELL. BIOL.

RAILEY AND WU

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FIG. 1. Construction of LS mutations of the VARNAl gene. pAdHinfC(ABB)R was used for generation of two deletion mutant libraries that contained a KpnI linker at the deletion junction. The heavy line stands for the inserted Hinfl fragment (from 10,069 to 10,685 bp) of the adenovirus type 2 genomic DNA (57, 59). The entire coding region of the VARNAl gene and the 5' coding region of the VARNA2 gene are, respectively, indicated as open bars with and without an arrowhead, which indicates the direction of transcription. LS mutants were constructed as described in Materials and Methods. The KpnI linker in each mutant was located at different locations between the XbaI and BamHI sites, as reconfirmed by DNA sequencing after the final construction. Restriction sites: S, SalI; E, EcoRI; H, HindIlI; Hf, Hinfl; X, XbaI; Bm, BamHI; Ba, BAL 1.

mmol) were from ICN. BAL 31 nuclease, T4 phage DNA ligase, Klenow fragment of Escherichia coli DNA polymerase 1, phage M13 primer (dTCCCAGTCACGACGT), pBR322 EcoRI site primer (dGTATCACGAGGCCCT), and pBR322 HindIII site primer (dGCAATTTAACTGTGAT) for DNA sequencing and restriction enzymes were from New England BioLabs. T4 phage polynucleotide kinase and some restriction enzymes were from Bethesda Research Laboratories. The restriction enzyme ASP718 was from Boehringer-Mannheim. Dideoxyribonucleoside triphosphates, deoxyribonucleoside triphosphates, and bacteriophage M13 mplO were from P-L Biochemicals. KpnI linker (8-mer, dCGGTACCG) was from Collaborative Research. Formamide (FX420) from Matheson, Coleman and Bell was deionized and stored at -70°C until used. Ultrapure urea was from Schwarz/Mann. Acrylamide, bis-acrylamide, and TEMED (N,N,N',N'-tetramethylethylenediamine) were from Eastman Kodak. General chemicals were from Fisher, American Scientific Products, or Sigma Chemical Co. Growth of human KB cells and 293 cells. Human KB cells were maintained' in exponential growth as a suspension culture (53-55, 58). Human 293 cells, obtained from Arnold

Berk, were first maintained as a monolayer culture in Dulbecco minimal essential medium (GIBCO, catalog no. 4301600) containing 5% fetal bovine serum. When a sufficient quantity of cells was grown, the 293 cells were maintained as a suspension culture in modified Eagle minimal essential medium (autoclavable for suspension culture) GIBCO, catalog no. 410-1800 containing first 10% fetal bovine serum and, later, 10% newborn calf serum. Preparation of cell extracts for in vitro transcription. Human KB cells, or 293 cells, were harvested, washed, and used for preparation of the cell-free extracts (S-100) by previously published procedures (7, 53-55, 57-59). Transcription in vitro. The reaction mixture (0.05 ml) contained 0.5 to 1 ,uCi of [a-32P]UTP, 0.45 u.g of pBR322 DNA, and 0.05 ,ug of template DNA (wild type or LS mutant genes) and other ingredients as described (53-55, 58). For the Si nuclease protection experiments, synthesis was carried out in the absence of [a-32P]UTP. The synthesis usually was carried out at 29°C for 2 h. For the studies on the stability of RNAs in vitro, the reaction mixture was scaled up to 0.4 ml. At each time point, before and after addition of 1/10 volume of the reaction mixture of a-amanitin (3 mg/ml), samples of 0.05 and 0.055 ml were withdrawn, and RNA was purified as described below. Preparation and gel electrophoresis of RNA. RNA synthesized in vitro as described above was treated with RNasefree DNase I, purified, and electrophoretically analyzed as described (7, 54, 55, 57, 59). Construction of LS mutants of the VARNAl. The wild-type VARNAl gene cloned into a plasmid, pAdHinfC(ABB)R, was used as the starting material for generation of two deletion libraries (7, 57, 59). Twenty exact LS mutants were constructed (33; Railey and Wu, Abstr. Annu. Meet. Am. Soc. Microbiol. 1987; J. F. Railey II, Ph.D. Thesis, Emory University, Atlanta, Ga., 1987) (Fig. 1). Five more mutants were generated by oligonucleotide-directed mutagenesis (62). The XbaI-HindIII fragment containing the VARNAl gene was excised from pAdHinfC(QBB)R, purified, and cloned into linearized phage M13mplO replicative-form DNA by a standard protocol (34). The sequences of the oligonucleotides corresponding to portions of the sequences on the coding strand of the gene, and containing the changes (the KpnI linker 8-mer sequence) underlined, are shown in Fig. 2. Oligonucleotides were purified (51). Ten picomoles of each of the five phosphorylated oligonucleotides was annealed to about 0.5 pmol (1.5 ,ug) of the single-stranded recombinant phage M13 DNA and polymerized with the Klenow fragment. After ligation, the double-stranded phage DNA was treated with 12.5 or 50 U of S1 nuclease to reduce the background of the single-stranded phage DNA. The S1 nuclease-treated phage DNA was used for transformation of E. coli JM71.18 cells. The positive phages were identified by the presence of the KpnI restriction site and further confirmed by sequence analysis, and the XbaI-HindIII fragment containing the mutated VARNAl gene was cloned back to a linearized plasmid pAdHinfC(ABB)R, deleted of the similar fragment containing the wild-type gene (Railey, thesis). The 5' CACCAGACCACGGAAGCGGTACCTTACAGGCTCTCCTT LSM(+5/+14) 5' CCACCAGACCACGGTACTGCCCGCTTAC

LSM(-2/+8)

LSM(+22/+3 1)5' TCCGCCATGATACCCTTGCGA_GTACACCAGACCACGGAAGAGTGCC LSM (+31/+40)5' GTCGTCCGCCATGATACGGTACGAATTTATCCACCAGAC LSM (+61/+70) 5 CGGACGGCCGGATCCGCGGTAGACCCCGGTCGTCCGCC

FIG. 2. Oligonucleotide sequences of some LS mutants. The KpnI linker 8-mer sequence is underlined.

VOL. 8, 1988

DNA sequence of each mutant plasmid was reconfirmed by DNA sequencing (Railey, thesis). Isolation of covalently closed circular plasmid DNA for transcription, mutagenesis, and DNA sequencing. For preparation of pure, covalently closed circular plasmid DNA, an economical large-scale method was used (56). The plasmid DNA was about 98% covalently closed circular DNA and free of proteins, transcriptionally inhibitory RNAs, and host DNA. Rapid DNA sequencing. DNA was sequenced by a method modified from the original method of Sanger et al. (42) and adapted for sequencing double-stranded plasmid DNA (6; Railey, thesis). [oa-thio-35S]dATP was used for labeling the DNA fragments. Determination of the 5' and 3' termini of RNAs by the Si nuclease method. Unlabeled RNA, synthesized from the wild-type gene or LS mutant genes prepared as described above, was used for hybridization with the [32P]DNA fragments as described below. 5'-End-labeled DNA from the wild-type gene and mutated genes was prepared as follows. The wild-type and LS mutant DNAs containing a BamHI site were linearized with the BamHI restriction enzyme, whereas the LS mutant DNAs without the BamHI site were linearized with the ASP718 restriction enzyme. The linearized DNA was dephosphorylated with calf intestine alkaline phosphatase (31), deproteinized, dialyzed, and labeled with [_y-32P]ATP and T4 phage polynucleotide kinase (31). 3'-Endlabeled DNAs from the wild-type gene and mutated genes were prepared as follows. The DNA, linearized with a proper restriction enzyme as described above, was labeled by filling in the first nucleotide in the 3'-terminal end with [cx-32P]dGTP and Klenow fragment (31). To determine the 5' and 3' termini of the RNA, a modified method of the Si nuclease mapping method was used (57).

Sequential transcription competition. Sequential transcrip-

tion competition was carried out in 0.05 ml of reaction mixture as described (59). In brief, 0.1 ,ug of the competing DNA, containing the wild-type or the LS mutant gene, and 0.1 ,ug of pBR322 DNA were incubated with the G75excluded KB cell extract S-100 plus all the necessary salts in the absence of exogenous nucleoside triphosphates (59) at 29°C for 15 min. DNA (0.05 ,ug) from the reference gene, JRB262, was added, and the reaction mixture was further incubated at the same temperature for an additional 15 min. At the end of the incubation, [a-32P]UTP (2 ,uCi) plus the other three cold nucleoside triphosphates and 1 mM potassium phosphoenolpyruvate were added, and the RNA synthesis was carried out at the same temperature for an additional 2 h of incubation. The RNA was purified and analyzed electrophoretically in a gel as described (57, 59). The relative competing strength of each DNA was calculated as described (59). RESULTS LS mutagenesis in the control region of the VARNA1 gene. Our previous studies defining the transcriptionally important regulatory sequences by using deletion mutants have suggested the possibility that multiple regulatory elements are present in the control region (from -30 to +70) of the VARNAl gene (14, 18, 59). To define the boundaries of each regulatory element in this control region and to eliminate possible artifacts introduced by bringing in distant DNA sequences characteristic of deletion mutant constructs, exact LS mutations with clustered-base changes were introduced to the region from -33 in the 5' flanking region

CONTROL REGION OF VARNAl GENE

1149

through +77 in the coding region of the gene and used for the studies here. Twenty exact LS mutant genes were constructed by joining together two appropriate members from the deletion libraries (Fig. 1). Since the digestion with BAL 31 nuclease tended to stop near sequences rich in GC base pairs, five more exact LS mutants were generated by oligonucleotide-directed mutagenesis (Railey, thesis). DNA sequences of the 25 exact LS mutants are shown in Fig. 3. The wild-type spacing is retained but now different clusters of mutations exist throughout the entire control region. The clustered-base mutations varied from 2- to 8-base-pair substitutions. Most of the LS mutants constructed in this report had more than four base-pair substitutions. Transcription of LS mutant genes in a homologous cell extract. Previously we have shown that the uninfected human KB cell extract, S-100, contains RNA polymerase III and necessary auxiliary transcription factors for faithful transcription of RNA polymerase III-mediated genes (53-55). Using this cell extract and suboptimal template DNA concentrations which had been shown to express the maximal phenotypes of the mutant genes (52, 59), we studied the effects of LS mutations on transcription of the VARNAl gene. Figure 4A shows an autoradiography of transcripts synthesized from the wild-type and LS mutant genes. The two LS mutations located in the B block, LSM(+57/+66) and LSM(+61/+70), and the two LS mutations located in the A block, LSM(+11/+20) and LSM(+14/+23), had the greatest negative effect on transcription (Fig. 4A). Furthermore, some of the LS mutations in the 5' flanking and the interblock spacing regions also had some negative effects on transcription (see Fig. 6 for quantitative results). Interestingly, some of the LS mutations in the 5' flanking region had some positive effects on transcription (see Fig. 6 for quantitative results). To test whether the apparent differences in transcription efficiency observed were owing to the introduced base substitutions or to some nonspecific inhibitors or stimulators in the plasmid preparations, transcription of each mutant gene was carried out in the presence of a reference gene, JRB262. JRB262 is a mutant of the wild-type VARNAl gene that has a deletion of a nonessential region, from +72 to +105. Therefore, it is transcribed as efficiently as the wildtype gene, but yields a smaller RNA product (59). Transcription of this reference gene in the presence of some of the LS mutant genes was slightly less efficient than in the presence of the wild-type gene (Fig. 4B). This was expected because these LS mutant genes were actually transcribed more efficiently than the wild-type gene. Therefore, the more efficient transcription of these LS mutant genes was not owing to the presence of stimulators in the DNA preparations. Transcription of the reference gene in the presence of most of the mutant genes, including the above four mutations in the A and B blocks, was equally or slightly more efficient than that in the presence of the wild-type gene (Fig. 4B). Therefore, the less efficient transcription of these LS mutant genes was not due to the presence of inhibitors in the DNA preparations. These results, therefore, support the notion that the transcription activities of the LS mutant genes are due to the effects of LS mutations introduced to the gene. Effect of LS mutations on stability of RNA in vitro. The low transcription activities of some of the LS mutant genes may not be due to a direct effect of LS mutations on the rate of transcription, but rather may be attributed to the mutation rendering the RNA unstable after synthesis. To test this

1150

RAILEY AND WU

MOL. CELL. BIOL.

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Kpn I LINKER LOCATION FIG. 7. Effects of LS mutations on the competing strength of the VARNAl gene. The relative competing strength of each mutant was determined as described in Materials and Methods. Each value was a mean of three duplicate determinations which varied less than 5%. Points represent mutants (standard deviation of the mean in parentheses) as follows: LSM(-34/-25) (10), LSM(-33/-24) (9), LSM(-30/-21) (7), LSM(-25/-16) (10), LSM(-24/-15) (11), LSM(-20/-11) (5), LSM(-11/-2) (10), LSM(-10/-1) (7), LSM(-9/+1) (8), LSM(-8/+2) (7), LSM(-2/+8) (7), LSM(+5/+14) (4), LSM(+11/+20) (16), LSM(+14/+23) (7), LSM(+22/+31) (6), LSM(+31/+40) (9.5), LSM(+39/+48) (8), LSM(+43/+52) (8), LSM(+49/+58) (9), LSM(+57/+66) (0), LSM(+61/+70) (18), LSM(+67/+76) (9), LSM(+68/+77) (19), LSM(+69/+78) (5), and LSM(+70/+79) (9). Standard deviation of the mean for the wild-type gene was 3. Horizontal lines indicate the location of the linker in each mutant gene.

flanking region having a positive effect on transcription (Table 1). Functions of multiple regulatory elements in the control region of the VARNAl gene. (i) The essential B-block element. We have shown in this and previous reports (18, 59) that the B-block element is essential for transcription and for soliciting factors for formation of stable complexes. This element is essential probably because it is where the first set of transcription factors, presumably TFIIIC, interact with the VARNAl gene. This stable DNA-protein complex subsequently commits the gene for further interaction with the TFIIIB and RNA polymerase III (8, 15, 28) before transcription initiation takes place. Therefore, we like to designate this element as the entering and commitment element for initiating transcription. (ii) The important A-block element. The A-block element, although not as essential as the B block, is very important for transcription, since the two LS mutations in this element respectively decreased the overall transcription efficiency of the gene about 33- and 200-fold. Furthermore, deletion mutant genes lacking all the sequences upstream of the A-block element (in which the mutated genes still have the intact A block and the rest of the gene downstream of it) are transcribed at about 2 to 3% of the rate of the wild-type gene (59). On the basis of these findings, we suggest that one of the functions of the A block is to act not as an enhancer, as suggested from the work of dissection of the internal transcriptional control region of a human Alu-family RNA gene (36), but rather as an important element required for interactions through transcription factors or RNA polymerase III with the B-block element to form the most basal form of transcription machinery. This notion seems to be consistent

with the recent work by Yoshinaga et al. (60), who showed that two fractions of the TFIIIC factors appeared to protect both the A- and B-block elements in a DNase I footprint experiment. Another function of the A-block element is to dictate the wild-type start site, as suggested from the 5'-end mapping TABLE 1. Regulatory elements in the control region of the VARNAl gene Location of regulatory element

B block

Boundaries

+55/+58 to +75

Possible functions

Promoter element;

entering and commitment A block

+ 11/+13 to +30

Interblock

+30 to +50

5' Flanking

Upstream of -25,

Promoter element promotion and

alignment; determination of start site Interblock spacing; efficient transcription Efficient transcription

perhaps to -36/-60 5' Flanking 5' Flanking

-17 to +2 -11 to -1

5' Flanking

-25 to -17

5' Flanking

+2 to +10

Efficient transcription Determination of start site Putative negative regulation Putative negative regulation

CONTROL REGION OF VARNAl GENE

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