MOLECULAR AND CELLULAR BIOLOGY, Apr. 1990, p. 1319-1328 0270-7306/90/041319-10$02.00/0 Copyright © 1990, American Society for Microbiology

Vol. 10, No. 4

TATA-Dependent and TATA-Independent Function of the Basal and Heat Shock Elements of a Human hsp7O Promoter JOHN M. GREENEt AND ROBERT E. KINGSTON*

Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115 Received 12 October 1989/Accepted 14 December 1989

We have characterized the interactions between the TATA element and other sequence elements of a human heat shock protein 70 (hsp7O) promoter by a mutational approach. Expression of a distal element of this promoter requires an intact TATA element in human cell lines. The hsp7O TATA element can be functionally replaced for this interaction by TATA elements from the simian virus 40 early and adenovirus EIla promoters. The TATA element in this promoter therefore both determines the appropriate start site and determines strength by allowing function of the distal element. In contrast, three proximal upstream elements necessary for basal and heat-regulated transcription have no requirement either for a TATA element or for any other proximal element. The behavior of promoters multiply mutant in these proximal elements implies that these elements function independently. We examined the interaction between the heat shock element (HSE) and the TATA element as the distance between the two factor-binding sites was increased. It was necessary to create a mutant HSE with an extended consensus sequence in order for the HSE to function at a distance. Moving this extended HSE 500 bases upstream did not increase its dependence on the TATA element, suggesting that the TATA independence of this element is intrinsic to its function and is not determined by distance from the promoter.

Mammalian promoters transcribed by RNA polymerase II contain multiple sequence elements, each of which is capable of binding specific protein factors (for reviews, see references 8 and 16). These elements can be spread out over several hundred to several thousand base pairs. Whereas in vivo mutational analysis has shown that multiple elements can contribute to the expression of a gene, little is known about how the respective factors interact to regulate transcription. Biochemical and genetic evidence suggests that upstream factors can have important interactions with the factor(s) that binds to the TATA element. In yeast cells, the TATA element appears to be necessary for function of certain upstream activation sequences (28). In mammals, introduction of whole or half turns between the elements of the simian virus 40 (SV40) early or adenovirus Elb promoter varies transcription levels in a way that suggests an interaction between factors bound to the TATA element and upstream elements (30, 38), although in the Elb promoter biochemical studies do not support such an interaction (25). Two upstream mammalian factors have been shown either to alter the size of the TATA-associated factor TFIID footprint (ATF [12]) or to bind synergistically with TFIID (USF/ MLTF [24]). Upstream factors may also interact with each other. The presence or absence of the octamer sequence can alter the ability of enhancers to increase transcription from the thymidine kinase gene promoter (21). Similarly, the ability of either the SV40 72-base-pair (bp) repeat or multiple Xenopus heat shock elements (HSEs) to efficiently enhance P-globin transcription is altered by deletion of proximal elements in the ,-globin gene promoter (4, 31). In vitro, the yeast GAL4 factor requires a site for any one of a number of upstream

mammalian transcription activators to be present near TATA for function from a distance, although no such binding site is necessary if GAL4 is bound next to the TATA element (15). One interpretation of these studies is that proximal factors can anchor more distal factors to a proximal location that then allows the distal factors to interact with the transcription machinery. Finally, certain factors may not require any interaction with other DNA-bound factors. These factors could directly interact with RNA polymerase II or associated, soluble factors to stimulate transcription (25). These considerations suggest that mammalian upstream factors could fall into three (not necessarily mutually exclusive) categories in terms of factor interaction. One group could function by altering the ability of a second DNA-bound factor (e.g., TFIID) to function. A second group could require a physical interaction to anchor the factor in an appropriate position for function. A final group could be able to function directly, without any other factor present on the promoter. Determining these types of interactions is a necessary first step to defining the mechanism by which factors activate transcription. We and others have been characterizing a human hsp70 promoter as a model system for understanding gene regulation in humans. This promoter is expressed at a basal level in human cells and is induced 10- to 20-fold by heat shock (35). At least five different transcription elements play a role in this regulation (9, 18, 19, 33, 36). In this study, we used a mutational approach to disrupt transcription factor-binding sites in order to examine interactions between such factors. We demonstrate that these promoter elements can be divided into two classes on the basis of their dependence on interactions with the TATA element. MATERIALS AND METHODS Plasmids. Plasmid DNA was grown in M9 minimal medium and purified by banding twice by ethidium bromideCsCl gradient centrifugation. DNA concentrations were

Corresponding author. t Present address: Laboratory of Molecular Genetics, National Institute of Child Health and Development, Bethesda, MD 20892. *

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determined spectrophotometrically and were verified by restriction digest and agarose gel electrophoresis. All linker scan and spacing mutation plasmids were sequenced by the dideoxy method (5) to confirm their structures. Construction of the hsp7O linker scan mutations through the use of annealed synthetic oligonucleotides has been previously described (9). Double and triple linker scan mutations were constructed by this method, using appropriate oligonucleotides. Mutations were constructed in either plasmid pA1250 (containing hsp7O sequences to -1250) or plasmid pA84 (containing hsp7O sequences to -84). The linker scan mutations were also subcloned into pA120 (36) by first excising the Sca-Nhe fragment from -75 to +6 of the promoter region of each linker scan plasmid. This fragment was then joined with a 1.9-kilobase-pair (kb) Sac-Xmn fragment from pA120 and with a 3.4-kb Xmn-Nhe fragment from pA1250 in a three-part ligation. In all of these linker scan constructions, every base of the promoter sequence is in its proper position relative to the start site of transcription; there are no insertions or deletions of base pairs. For constructions in which promoter elements were moved from their normal loci, oligonucleotides containing the wild-type HSE, the extended HSE, and the distal CCAAT and GC boxes (9) and their complementary strands were designed so that as much hsp7O promoter sequence was retained as possible. These oligonucleotides contained a HindIll site at one end and a Sac site at the other end when annealed and were joined in a three-part ligation reaction as described above with a 5.1-kb HindIII-Nhe fragment derived from pA120 and an 80-bp Sac-Nhe fragment from either a wild-type or TATA linker scan mutant (LS22-26) hsp7O promoter. The resulting plasmids were subsequently digested with Xho at a site designed into the oligonucleotides immediately upstream of the Sac site and ligated to 21-, 42-, and 63-bp annealed complementary spacer oligonucleotides with Xho ends. For the experiments in which an HSE was moved 500 bases upstream, a 500-bp Sal fragment from bacteriophage lambda was ligated into the same Xho site. The internal reference plasmid cotransfected with the linker scan mutations was pIR17 (14), which contains wildtype human hsp7O promoter sequences to -1250. For experiments involving heat shock, the HindIII-Nhe fragment from -120 to +6 of the promoter region of pAl120 was substituted for the same fragment of pIR17 to create pIR17-120. This plasmid thus has hsp7O promoter sequences only to -120 instead of -1250 but retains the 33-bp deletion in the 5' untranslated region of the human hsp7O gene. A similar strategy was used to create pIR17-84, containing hsp7O promoter sequences to -84. Cells and transfections. BALB/c 3T3 and HeLa cells were cultured in Dulbecco modified Eagle medium containing 10% calf serum. A DNA mixture consisting of 10 p.g of each linker scan mutant plasmid, 10 ,ug of the internal reference plasmid, and 1 or 2 ,ug of pXGH5 (which encodes human growth hormone and can easily be used to monitor transfection efficiency [26]) was introduced into cells by calcium phosphate coprecipitation as described elsewhere (13). RNA preparation and analysis. Cells were harvested 45 to 50 h posttransfection for total cellular RNA by the guanidinium-CsCl method (2, 32). For heat shock experiments, one of two duplicate transfections was shifted to 43°C for 4 h immediately before harvest. To monitor transfection efficiency as noted above, human growth hormone levels in the medium at the time of harvest (or just before heat shock) were determined by using a solid-phase two-site radioimmu-

MOL. CELL. BIOL.

noassay kit under the conditions recommended by the manufacturer (Nichols Institute). Single-stranded end-labeled probes were prepared as described previously (2, 14) and contained bases +229 (labeled) to -133 of the hsp7O-chloramphenicol acetyltransferase fusion gene. Si nuclease analysis (2, 3) was performed as follows: 30 to 75 ,ug of total cellular RNA was mixed with 2 x 105 dpm of probe, ethanol precipitated, and suspended in 20 ,il of hybridization buffer [40 mM piperazine-N,N'-bis(2ethanesulfonic acid) (PIPES; pH 6.4), 1 mM EDTA, 0.4 M NaCl, 80% deionized formamide]. Hybridization took place at 30°C for 16 h. S1 nuclease digestion, analysis on 7% denaturing polyacrylamide gels, and autoradiography on preflashed film were carried out by standard procedures (2). Quantitation and determination of RTL. Gels were quantitated by scanning densitometry of exposures within the linear range of the film, using a Kratos SD3000 spectrodensitometer coupled to a Hewlett-Packard 3290A integrator. The relative transcription level (RTL) is a ratio that compares the level of transcription of a mutant promoter with the level of a wild-type promoter after normalization of both levels for variations in transfection efficiency by using an internal reference signal. This ratio is calculated by the formula (MUT/MUT REF)/(WT/WT REF), where MUT is the signal corresponding to the appropriately initiated RNA from each linker scan mutant promoter, MUT REF is the internal reference signal from that same lane of the gel, WT is the signal from the wild-type sequence hsp7O promoter in lane 1 of Fig. 2 to 5, and WT REF is the internal reference signal in that same lane. For heat-shocked lanes in experiments using pIR17-120 as the internal reference, the RTL was corrected to reflect the increase upon heat shock by multiplying it by the average fold increase seen upon heat shock induction obtained from all the pairs (plus and minus heat shock) of internal reference signals in that experiment. For Fig. 4 and 5, the ratio of the corrected RTL from the heat-shocked lane to the basal-lane RTL for each construct was then computed to determine the induction ratio upon heat shock. All RTLs represent the average of two or more independent determinations from separate transfections. Calculation of predicted promoter strength of multiply mutated promoters. The observed relative transcription levels underlined in Tables 1 and 2 were used to define the fold effect of mutation of each proximal element (or, in the case of the HSE, the absence of heat shock) on the level of appropriately initiated RNA. In BALB/c cells these effects were as follows: HSE, 16-fold; CCAAT element, 10-fold; G+C-rich element, 1.4-fold; and TATA box, 3.3-fold. In HeLa cells, these effects were as follows: HSE, 17-fold; CCAAT element, 7.1-fold; G+C-rich element, 2.3-fold; and TATA box, 8.3-fold. To predict the values for the seven remaining available mutant combinations, these factors were used as divisors after designating the RTL of the wild-type promoter after a 4-h heat shock as 1,600 (BALB/c; the 16-fold induction value is the average induction of 12 different determinations) or 1,700 (HeLa). These predictions were made by assuming that mutation of multiple independent promoter mutations will be equal to the product of the effects of each individual mutation. This means, for example, that a C/G (CCAAT element/G+C-rich element) mutant in the absence of heat shock should have an RTL 224-fold (16 x 10 x 1.4) lower than that of the wild-type promoter after heat shock. The statement that multiple independent mutations should effect promoter strength in a multiplicative instead of additive manner is based on the following argument. It is possible

VOL. 1,1O990

TATA FUNCTION IN A HUMAN hsp7O PROMOTER Ia

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to write the initiation reaction of a promoter as a twocomponent system involving RNA polymerase and the promoter (the latter includes bound factors). For example, if polymerase (PolIl) must bind to the promoter (Pro), open the DNA, and initiate, the reaction can be written: k2 k3 k, PolII(free) + Pro -- PolII(bound) -> PolII(open)

PolII(init) + Pro The rate of the reaction, and thus promoter strength, is therefore determined by the value [PolII(free)][Pro]kjk2k3. Assuming that the role of an upstream transcription factor is to facilitate some step in this reaction, the presence of an upstream factor should have an effect on k1, k2, or k3. These effects will therefore be multiplicative if they are kinetically independent. This argument makes several simplifying assumptions. We use it here as a formal agrument to explain the multiplicative effects of multiple mutations that we observe.

RESULTS The TATA element is necessary for function of a distal element in human cells. Basal expression of the human hsp7O promoter in human cells requires at least four elements: a TATA element (beginning at -28), a G+C-rich element (-49), a CCAAT element (-67), and a complex distal element (located upstream of -120). The effects of individual mutations in these elements have been defined through the use of linker scan mutations and deletions (Fig. 1; 9, 33, 36). The distal element, which contains a CCAAT element as well as other, as yet unspecified elements, confers a 5- to 10-fold increase in transcription of the human hsp7O promoter in human cells. We sought to determine whether any of the more proximal promoter elements were necessary for transmission of this effect. Analysis of previously published data (9) suggested that the distal element may not function in hsp7O promoters with a mutant TATA region. We therefore carried out experiments similar to those previously reported in a manner that allowed a more direct measure of the function of the distal element simultaneously in various mutant backgrounds.

Mutant promoters were transfected into HeLa cells with a control, pseudo-wild-type hsp7O promoter that contained flanking sequences to -1250. Transcript level from each promoter was determined by S1 nuclease analysis, which resulted in a 230-nucleotide band from the mutant promoter and a 130-nucleotide band from the internal reference promoter. The shorter transcript from the internal reference promoter was due to a deletion in the untranslated coding region of this construction. The requirement of the distal element for proximal elements was directly determined by analyzing transcription from plasmids that contained (pA1250) or lacked (pA84) the distal element and also contained various linker scan mutations in the proximal elements (Fig. 2). Such side-by-side comparisons of the effect of the presence of the distal element on transcription levels when combined with disrupted basal elements allowed us to determine whether distal element function was dependent on any proximal basal element. The distal element had an eightfold effect on basal transcription levels when the CCAAT element was mutant, a fivefold effect when the G+C-rich element was mutant, but no effect when the TATA element was mutant (Fig. 2, lanes 3 to 8). Function of the distal element therefore required an intact TATA element but did not require an intact G+C-rich or CCAAT element. We next mutated both the G+C-rich and CCAAT elements (Fig. 1). The distal element stimulated expression of this doubly mutant promoter eightfold (Fig. 2, lanes 9 and 10), suggesting that the TATA element is sufficient for transmission of the distal signal. A promoter mutant in both the CCAAT and TATA elements was not stimulated by the presence of the distal element, confirming the dependence on the TATA element (Fig. 2, lanes 11 and 12). Other TATA sequences can substitute for the hsp7O TATA motif to transmit the distal signal. We have tested two other TATA sequence motifs from other eucaryotic promoters to see whether they are also capable of transmitting the distal signal. The TATA motifs used were derived from the sequences of the adenovirus Ella and SV40 early promoters and used to replace the hsp7O TATA motif (Fig. 3) (30a). The resulting chimeric promoters were subcloned into constructs containing (-1250) or lacking (-84) the distal element.

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GREENE AND KINGSTON

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