MOLECULAR AND CELLULAR BIOLOGY, Dec. 1990, p. 6533-6543

Vol. 10, No. 12

0270-7306/90/126533-11$02.00/0 Copyright © 1990, American Society for Microbiology

Identification of cis-Acting Regulatory Elements in the Promoter Region of the Rat Brain Creatine Kinase Gene GRACE M. HOBSON,' GEORGE R. MOLLOY,' AND PAMELA A. BENFIELD2* Experimental Station, E. I. du Pont de Nemours and Company, Wilmington, Delaware 198800328,2 and School of Life and Health Sciences, University of Delaware, Newark, Delaware 197161 Received 4 May 1990/Accepted 5 September 1990

The functional organization of the rat brain creatine kinase (ckb) promoter was analyzed by deletion, linker scanning, and substitution mutagenesis. Mutations were introduced into the ckb promoter of hybrid ckblneo (neomycin resistance gene) genes, and the mutant genes were expressed transiently in HeLa cells. Expression was assayed by primer extension analysis of neo RNA, which allowed the transcription start sites and the amount of transcription to be determined. Transfections and primer extension reactions were internally controlled by simultaneous analysis of transcription from the adenovirus VA gene located on the same plasmid as the hybrid ckblneo gene. We demonstrate that 195 bp of the ckb promoter is sufficient for efficient in vivo expression in HeLa cells. A nonconsensus TTAA element at -28 bp appears to provide the TATA box function for the ckb promoter in vivo. Two CCAAT elements, one at -84 bp and the other at -54 bp, and a TATAAA TA element (a consensus TATA box sequence) at -66 bp are required for efficient transcription from the TTAA element. In addition, we present evidence that the consensus j8-globin TATA box responds to the TATAAATA element in the same way as the ckb nonconsensus TTAA element.

Initiation of transcription by RNA polymerase II is a major point at which regulation of gene expression occurs (15). The unique pattern of transcription characteristic of each gene is largely due to the interactions among all the various regulatory factors that bind the gene in its promoter and enhancer regions and to the further interaction of these factors with the transcriptional machinery. The transcriptional machinery is thus able to detect and integrate the regulatory information imposed on the gene. We have been studying transcriptional regulation of the creatine kinase genes. Creatine kinase (adenosine 5'-triphosphate-creatine phosphotransferase; EC 2.7.3.2) is an enzyme that catalyzes the reversible transfer of a phosphoryl group between ATP and creatine (for reviews, see references 35 and 59). Two cytosolic isoforms of creatine kinase, the brain (B) isoform and the muscle (M) isoform, which randomly associate to form three isozymes, MM, MB, and BB, have been described. Separate genes for the two cytosolic isoforms have been cloned from several species and appear to be the products of single-copy, unlinked genes (3, 32, 39, 45, 58). There is also a mitochondrial isoform that self-associates into octameric structures (47). A human mitochondrial gene has been isolated and characterized (25). The variety of modes of expression for the ck genes that must exist to accommodate the widely varying energy needs of cells make this gene family an interesting system in which to examine the molecular mechanisms involved in differential gene expression. The two cytosolic isoforms of creatine kinase, the muscle isoform and the brain isoform, have different tissue-specific patterns of expression (56). The muscle isoform is expressed predominantly in skeletal and cardiac muscle. The brain isoform, while being expressed at highest levels in brain, has a much broader range of expression than the muscle isoform has. Regulatory phenomena associated with brain isoform expression include up-regulation during differentiation of monocytes into macrophages *

(37), induction by peptide and steroid hormones (22 and references therein), high levels of expression in transformed cells (20), and stimulation by viral infection (11). Differential expression of the ckb gene in liver and brain (42) and up-regulation of the ckm gene during skeletal muscle differentiation (E. Arpaia and T. Smith, unpublished results; 32) have been shown to be regulated at least in part at the level of transcription. We previously reported evidence that a nonconsensus TTAA sequence at -28 bp appears to provide the principal TATA box function for the ckb promoter in vivo, even though there is a consensus TATAAATA sequence at -66 bp relative to the transcription start site (28). Under certain conditions in vitro, however, the upstream consensus TATA AATA sequence can function as a TATA box to mediate transcription from an upstream start site (28, 42). In addition, we previously identified a region of the ckb promoter between -104 and -43 bp that is important for efficient in vitro transcription of the rat ckb gene from the downstream TTAA sequence. This region contains the TATAAATA sequence at -66 bp flanked by two CCAAT sequences at -84 and -54 bp. These sequences were identified as binding sites for potential trans-acting regulatory proteins. The TA TAAATA sequence binds a protein that we have called TARP. Recently, we showed that TARP-binding sites are also present in the ckm enhancer and that TARP is present in both muscle and nonmuscle sources (31a). In this study, we have refined our analysis of the rat ckb promoter by synthesizing deletion, linker scanning, and substitution mutations in the ckb promoter and studying the effects of these mutations on transient expression in HeLa cells. The expression results are discussed with respect to our previous description of potential regulatory factors that bind the ckb promoter. MATERIALS AND METHODS Gene transfer. HeLa cells were grown and gene transfer experiments were performed as previously described (Horlick et al., in press).

Corresponding author. 6533

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RNA isolation and analysis. RNA was isolated from transfected cells by the procedure of Chirgwin et al. (10) as described elsewhere (Horlick et al., in press). Primer extension analysis was carried out essentially as described elsewhere (Horlick et al., in press). The oligodeoxynucleotides used as probes were 5'-CGTGCAATCCATCTTG-3' (bases +26 to +41 from the start of translation in the neo gene) (a kind gift from Tatjana Loh) and 5'-CGCCATGATACCCTT GCG-3' (bases +32 to +49 from the start of transcription in the VA gene). Primer extension products were quantitated with an AmBis radioanalytic imaging system (AmBis systems, San Diego, Calif.). The relative transcription level is defined as the ratio of mutant gene transcripts to control VA transcripts divided by the ratio of wild-type transcripts to control VA transcripts. Construction of test plasmids. The pNVD cloning and expression vector (see Fig. 1) was constructed from restriction fragments derived from pVA, (a kind gift from R. Kovelman) (29), pRSVneo (23), and pUCPLcat (6). Synthetic and/or restriction fragment promoter region inserts were ligated between the HindlIl site and the BgIII site. A strategy based on that used by Karlsson et al. (33 and M. D. Walker, personal communication) was used for systematic linker scanning mutagenesis of the ckb promoter. A set of wild-type oligodeoxynucleotides of 16 to 27 bases was prepared in addition to a set of mutant oligodeoxynucleotides with the linker sequence GCATCGATAC, which contains a ClaI restriction site. The oligodeoxynucleotides were phosphorylated in individual reactions with T4 polynucleotide kinase. Terminal oligodeoxynucleotides containing the HindIII and BgIII restriction site overhangs were not phosphorylated. A wild-type promoter region and 18 linker scanning mutant promoter regions were assembled individually by mixing 20 appropriate wild-type and mutant oligodeoxynucleotides, hybridizing them, and ligating them into 218-bp synthetic promoter regions. Multimers did not form because the end oligodeoxynucleotides were not phosphorylated. The synthetic inserts contained ckb promoter sequences from -195 to +5 bp. The synthetic promoter regions for the wild-type ckb promoter and LS series of mutants with Hindlll and BglII overhangs were ligated into the pNVD HindIII-BglII vector fragment without purification of the correctly sized promoter inserts, creating pwt and the LS mutants (LS1 to LS18). LPwt was made by ligating a 2.8-kb ckb promoter fragment into pwt. 5' and 3' deletion mutants were made from LS mutants by taking advantage of a ClaI restriction site in the linkers and the Hindlll and BglII sites at either end of the promoter sequence. The DC series of mutants and the 3-globin constructs were created with restriction fragments of LS mutants and the following oligodeoxynucleotides:

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RESULTS Functional analysis of the ckb promoter. Calcium phosgene transfer of wild-type and mutant ckb promoter constructs into HeLa cells was used to determine the regions of the ckb promoter that are important for expression in vivo. HeLa cells were chosen for several reasons. We previously demonstrated that HeLa cells express their endogenous ckb gene (28) and would therefore be an appropriate cell line to use to determine minimal sequence requirements for ckb expression. In addition, the HeLa cell in vitro transcription system is well characterized, and we showed that ckb is transcribed efficiently in HeLa cell extracts (28). In the pNVD expression vector series that was created for these experiments (Fig. 1), the ckb promoter regions drive expression of the neomycin resistance gene (neo). Primer extension analysis of neo RNA allows detection of RNA transcripts with different 5' ends without interference from endogenous ckb RNA. The ability to detect RNAs with different 5' ends is important because of the potential for transcription from both the upstream and downstream start sites in the ckb promoter. In addition, primer extension analysis allows differentiation between initiation at the ckb start sites and initiation within the reporter gene. The pNVD expression vector also contains the polymerase III adenovirus VA gene to serve as an internal control. Godbout et al. (21) have shown that transcription from an internal polymerase III gene can be used as an effective internal control in transient gene transfer experiments. It has been reported that expression from some cotransfected genes can be stimulated by VA, RNA (1, 34) by regulating the stability of ribosome-bound RNAs (53). We cannot rule out the possibility that the VA internal control RNA in our experiments affected the stability of the neo transcripts. However, VA RNA signals were similar within experiments. In addition, results similar to those for the in vivo experiments presented in this report have been obtained with the same constructs in in vitro transcription experiments (M. T. Mitchell, unpublished observations) in which RNA was not ribosome bound. We therefore believe that VA serves as an effective internal control for these in vivo experiments. Analysis of the ckb promoter function by 5' deletion mutagenesis. An initial experiment (Fig. 2a) was performed to compare LPwt containing 2.9 kb of ckb 5'-flanking sequence with pwt containing 200 bp of ckb 5'-flanking sequence. Transcription with these constructs was initiated from the downstream start site, the same site that is used in rat brain and all in vivo sources previously tested (28, 42). LPwt and pwt have similar transcriptional efficiencies. Thus, the 200-bp promoter contains sufficient information for efficient ckb transcription in vivo in HeLa cells. The slightly higher

phate-mediated

5'-CGATACTGAATGGGCTATAAATAGCCGCAT-3' 3'-TATGACTTACCCGATATTTATCGGCGTAGC-5' 5'-GGCAGGGCAGAGCATATAAGGTGAGGTAGGATCAGTTGCTCCTCACATTTGCTTCTGACA-3' 3'-CCGTCCCGTCTCGTATATTCCACTCCATCCTAGTCAACGAGGAGTGTAAACGAAGACTGTCTAG-5'

Mutant constructs were identified and checked by restriction digest analysis. Mutants created with synthetic oligodeoxynucleotides were also checked by sequence analysis with a Sequenase kit (United States Biochemical Corp.). Compressions were resolved with dlTP reactions.

level of expression seen with LPwt (1.4-fold higher than that seen with pwt) may have been due to positive cis-acting elements upstream of the 200-bp promoter region. Also, the results do not rule out the possibility of a combination of positive and negative elements operating in this region.

cis-ACTING REGULATORY ELEMENTS IN ckb GENE

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TATA box and a CAP site alone were previously shown to require additional elements for efficient transcription in vivo (60). These results are consistent with in vitro transcription data obtained previously with a series of 5' deletion mutants and showing that there is a dramatic decrease in transcriptional efficiency from the downstream start site when the 61-bp region between -104 and -43 bp and containing the

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(Hind III)/Nhe I, 2310 Pst I, 2299

Sal I, 2293 Xba I, 2287

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\.. BamH I, 2022 Sma I, 2027 Kpn I, 2031 EcoR1, 2043 I.,Sst I,2049

FIG. 1. Structure of the pNVD cloning and exprression vector. Test promoter and enhancer inserts can be ligates d between the unique Hindll and BgIII sites. Arrows indicate thte direction of transcription of the bacterial neo (reporter) gene, the E gene. intemal control gene, and the bacterial amp (P-lactan of is the origin of replication, and an arrow indicates tl replication. The vector also contains the splicing an(d polyadenylation signals derived from simian virus 40 (SV40O ). Parentheses indicate that the site was destroyed during cloning. R'estriction sites shown in boldface type are unique sites.

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A series of 5' deletions into the 200-bp 5'-flainking region similarly tested to localize cis-acting elerments in this region (Fig. 2b and c). All transcription was iinitiated from the downstream start site. Little transcriptiornal efficiency was lost with 115A', in which sequences betwe en -185 and -75 bp and containing the CCACCC seque :nce and the upstream CCAAT sequence are deleted. Hov vever, a dramatic decrease in transcriptional efficiency of greater than 10-fold relative to the transcriptional efficienc3y of the wild type was observed with 13A5', in which al dditional sequences between -75 and -56 bp and contai ining the upstream TATAAATA sequence are deleted. Figiure 2c shows a longer autoradiographic exposure of the neo F )ortion of the gel shown in Fig. 2b, indicating that with 13A5' transcription initiated from the same start sites as with I pwt. Correct initiation therefore occurs independently of t he upstream consensus TATAAATA box and is probably n ot due to the upstream consensus TATAAATA box acting as ,a TATA box at an unprecedented distance. Faint bands wer e seen in the longer exposure (Fig. 2c) with 14A5' and 16A5'; however, no characteristic lower band was present. These faint bands may have been the result of some minimal level of transcription either due to TTAA or the surrounding seq luence or due to sequences at or near the site of initiation (511). However, it is more likely that the faint bands represent background, since they were seen with constructs that wvould not be expected to give transcripts of this size (data n ot shown). A was

two CCAAT sequences, the upstream TATAAATA sequence, and the CCACCC sequence is deleted (28, 42). Analysis of the ckb promoter function by linker scanning mutagenesis. A series of linker scanning mutants, LS1 to LS18 (Fig. 3b), was tested in HeLa cells for a more detailed analysis of functional elements within the 200-bp ckb promoter. Mutated bases in the linker scanning constructs are indicated by white letters on a black background in Fig. 3b. All transcription with the LS mutants initiated from the downstream start site. Results of a typical experiment (Fig. 3) indicated that a reduction in transcriptional efficiency occurred with LS13, LS14, LS16, and LS17 and that there was a slight reduction with LS11. The results for LS11, in which a linker replaces the upstream CCAAT sequence, were consistent with the results for 115A', the 5' deletion mutant in which sequences between -185 and -75 bp and including the upstream CCAAT sequence are deleted. A greater reduction in transcriptional efficiency, from 4- to 10-fold, was observed with LS14, which has a linker in place of the downstream CCAAT sequence. LS13, which has a linker in place of the upstream TATAAATA sequence, led to a two- to fivefold reduction in transcriptional efficiency. LS12, which has a linker altering the first T of TATAAATA and the sequence immediately 5' of TATAAATA, had no effect on transcription, suggesting that these 5' sequences are not required for efficient transcription. A reduction in

accurately initiated transcripts of threefold

was observed with LS16, which has an alteration of the TA of the TTAA sequence. A reduction of three- to fivefold was observed with LS17, which has an alteration of the final A. LS16 has an alteration of additional sequences 5' of TTAA, and LS17 has an alteration of sequences 3' of TTAA, so sequences flanking TTAA may be important for transcriptional efficiency. However, LS15 and LS18, which have mutated flanking sequences further away from TTAA, had no effect on transcriptional efficiency. Faint bands were observed below those due to the major neo transcripts in Fig. 3a. These bands correspond to "aberrant" initiation in the region of several restriction sites created between the ckb promoter region and the neo gene during construction of these plasmids. These transcripts respond to linker scanning mutagenesis of the promoter region in a manner similar to that observed for initiation at the correct start sites. An exception occurs with LS16 and LS17, in which the region including TTAA is mutated. With these, a decrease in aberrant transcripts is not observed. In fact, there is a tendency for these transcripts to increase (LS16) and change in relative intensity (LS17). Simnilar effects have been seen more predominantly in vitro (M. T. Mitchell, unpublished observations). These transcripts presumably represent TTAA-independent initiations that are still able to respond to upstream sequences in the ckb promoter. The results of the linker scanning experiments indicate that the ckb promoter elements that are important for ckb expression in HeLa cells are the two CCAAT sequences, the upstream TATAAATA sequence, and the downstream TTAA sequence. The two CCAAT sequences and the TATAAATA sequence are required for efficient transcrip-

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Identification of cis-acting regulatory elements in the promoter region of the rat brain creatine kinase gene.

The functional organization of the rat brain creatine kinase (ckb) promoter was analyzed by deletion, linker scanning, and substitution mutagenesis. M...
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