Vol. 10, No. 1

0270-7306/90/010165-11$02.00/0 Copyright C) 1990, American Society for Microbiology

Factor Substitution in a Human HSP70 Gene Promoter: TATADependent and TATA-Independent Interactions IAN C. A. TAYLOR 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 18 July 1989/Accepted 2 October 1989

To investigate interactions between transcription factors on mammalian promoters, we constructed a set of 24 variations of the human HSP70 gene promoter in which six upstream sequence motifs are paired in every possible combination with four TATA motifs. These promoters were analyzed for in vivo expression, and selected constructs were examined by in vitro template commitment studies. Activation transcription factor (ATF) and CP1 showed dramatically different interactions with the factor(s) bound to the TATA region. CP1 functioned in vivo regardless of the TATA motif that it was paired with and was not capable of sequestering the core promoter complex in a template commitment assay. ATF activity was dramatically altered by changing the TATA motif, and ATF was able to sequester the core promoter complex. These data suggest that CP1 and ATF function by distinct mechanisms that differ with respect to interaction with the factor(s) at the TATA box. Factor Spl also appeared to function by a TATA-independent mechanism. These data imply that the ability of a factor to function is determined not only by the intrinsic properties of the factor but also by promoter context.

Elucidating the mechanism by which protein factors activate transcription is basic to understanding gene regulation. Many cis-acting DNA sequence elements have been identified as being important in eucaryotic promoter activity: core promoter elements (i.e., that TATA box and cap site [3]), upstream sequence elements (for reviews, see references 16 and 40), and enhancer elements (55). Nuclear proteins that act at these sites have been identified. For example, the development of in vitro transcription systems has led to identification of general transcription factors (TFIIA, TFIIB, TFIID, TFIIE, and RNA polymerase II) that are required for appropriate transcription from the core promoter elements (17, 41, 49-51). Of these, only TFIID is known to bind directly to the promoter at the TATA box and cap site (45,

qualitatively extend contact of TFIID with the promoter (27, 28). This change coincides with increased transcription from the adenovirus EIV promoter (22, 37). The discovery that a yeast TATA-binding factor can function in a mammalian system lends credence to the view that this protein is a common target (6, 7). However, not all eucaryotic upstream transcription factors contain acidic activation domains. For example, the activation domain of Spl is glutamine rich, raising the possibility that different upstream factors function by distinct mechanisms (10). This hypothesis has a precedent in procaryotic systems, as kinetic experiments have shown that activators such as AcI and catabolite gene activator protein affect separate rate-limiting steps in the regulatory pathway of initiation (24, 39; reviewed in reference 42). Similarly, some upstream factors in mammals may act via the TATA complex, whereas others may act directly on RNA polymerase II. To address the question of whether all mammalian upstream factors function similarly, we focused on their ability to interact with TATA complexes. While biochemical evidence is lacking, genetic evidence argues that both yeast and human cells utilize several distinct TATA motifs (56, 57). We reasoned that if all mammalian upstream factors functioned via the TATA complex, as proposed for ATF (22, 28), then each would show a similar pattern of interaction with different TATA motifs. Alternatively, if upstream factors differ with respect to interactions with TATA complexes, exchanging TATA motifs might reveal distinct effects on the transcriptional activity of the upstream factors. We therefore created a set of promoters in which six different upstream motifs were paired in all possible combinations with four TATA motifs. We used these promoters as tools in in vivo and in vitro transcription studies. Our results indicate that there exist at least two classes of upstream transcription factors: one class that functions independently of the TATA complex to increase transcription, and a second class that activates transcription only when matched with a particular set of TATA elements.

52). Many promoter-specific transcription factors that bind to the upstream and enhancer elements have been purified, and in some cases the genes that encode them have been cloned (for a review, see reference 30). Examples include Spl (4, 15, 32), activation transcription factor (ATF; also called CREB [20, 26, 29, 35, 43]), API (2, 36), octamer transcription factor 1 (OTF-1) (18, 54; called Oct-1 in reference 59), and CP1 (9, 23; called NF-Y in reference 12). While it is clear that these gene-specific factors increase the strength of a promoter, their functions in the formation of a preinitiation complex or in the subsequent initiation of transcription remain unclear. Recent experiments in both procaryotes and eucaryotes suggest that many upstream transcription factors contain stretches of acidic amino acids that function as activating domains. Presumably, acidic domains all utilize similar activation mechanisms. One model states that the acidic domains of promoter-bound factors interact with a component of the transcriptional machinery (i.e., general transcription factors or RNA polymerase II; for a review, see reference 47). TFIID is a potential target, for in vitro DNA binding and transcription assays have shown that both a DNA-bound mammalian (ATF) and yeast (GAL4) factor *

Corresponding author. 165




























TAGGCAGT UPSTREAM NONSENSE FIG. 1. Human HSP70 substitution mutations. The wild-type (WT) CCAAT and TATA sequences are highlighted. The consensus sequences of the various substituted elements are shown in the position in which they were substituted. Mixing and matching the wild-type and substitution elements gave 24 separate promoters. Spacing mutants were created by inserting the four bases shown at -57. The start site of transcription is marked by the arrow. Base changes resulting from the cloning procedures are shown above the wild-type sequence in small letters and are described in Materials and Methods.

MATERUILS AND METHODS Plasmids. All plasmids were purified by banding twice with ethidium bromide-CsCl centrifugation. DNA concentrations were determined spectrophotometrically and verified by agarose gel electrophoresis. Construction of the deletion mutation plasmids pAHS-34 and pAHS-16, as well as pA84, has been described previously (21). Construction of the single upstream substitution mutants containing the HSP70 gene TATA was as follows. A Sal-Xmn fragment from pAHS-34 was combined with an Xmn-Sac fragment from pA84 and with an annealed synthetic oligonucleotide linker designed to fill the gap between the Sac and Sal sites and to give the desired substitution in a three-part ligation. As a result of this cloning procedure, 3 base pairs at -36 of the HSP70 gene promoter (hereafter referred to as the HSP70 promoter) were altered (Fig. 1). For mutants substituting a TATA element and for the spacing mutants, a single oligonucleotide containing the desired substitutions was self-hybridized, filled in with deoxynucleotides by using Klenow fragment to give a doublestranded product, cut with Sal and Sac to give the appropriate cloning ends, and isolated as described previously (1). This fragment was then combined with the Xmn-Sac fragment from pA84 and the Sal-Xmn fragment from pAHS-16 in a three-part ligation. As part of this cloning procedure, a Sal site was inserted at -17 of the HSP70 promoter. As a result, 3 base pairs were altered and an additional 2 base pairs were inserted between the TATA site and the initiation site (Fig. 1). All plasmids were sequenced by the dideoxy-chain termination method to confirm their structures. The nomenclature used throughout the text in referring to these promoters is as follows: upstream sequence element/TATA element (e.g., ATF/EIIa; octamer [OCTA]/simian virus 40 [SV40] early TATA [SV40E]; Spl/hsp7O). Plasmid -53MLP, which drives the shorter G-less cassette, has been described elsewhere (60). For construction of the remaining G-less cassette-bearing plasmids, a HindIllRsrII (-84 to -7) fragment of the selected substitution mutant HSP70 promoters was ligated with the HindIII and RsrII vector fragment of plasmid pHC2AT. pHC2AT was constructed by ligating an oligonucleotide containing the HSP70 cap site (from the RsrII site at -7 to +5) to the G-less cassette (from plasmid pC2AT [51]), which was then inserted between the Sall and BamHI sites in pUC13. In vitro binding assays. Labeled promoter fragments containing bases -84 to +7 were analyzed in gel mobility shift

and methylation interference assays as described previously (21). Half-times of dissociation of CP1 and ATF from their cognate sites in the various promoter contexts were determined by gel shift analysis. Labeled promoter fragments were incubated with HeLa nuclear extract (4 jig) for 30 min, after which time an excess of cold competitor fragment was added for various lengths of time (0 to 30 min). Gels (21) were loaded running and quantitated by using a Betascope 603 blot analyzer (Betagen). Percent bound versus time of competition was plotted, and the t412 of dissociation was determined from the graph. The competitor fragment used was either CCAAT/TATA nonsense or ATF/TATA nonsense. DNase protection assays were performed with a labeled promoter fragment from -266 to +7 and containing either the wild-type CCAAT sequence or the substituted AP1 site. Partial DNase I (Promega Biotec) digestion and urea sequencing gel analysis were performed as described previously (34). Transfection protocol. HeLa cells were passaged in Dulbecco minimal essential medium containing 10% fetal bovine serum. The calcium phosphate coprecipitation method was used as described previously (31). Transfections for chloramphenicol acetyltransferase (CAT) assays contained 20 ,ug of the test plasmid and 1 or 2 jig of pXGH5, which expresses human growth hormone and was used as a control for transfection efficiency. Transfections for RNA contained 10 ,ug of the test plasmid, 10 ,ug of pIR17-84, and 1 or 2 ,ug of pXGH5. Human growth hormone and CAT assays were performed as described previously (21). RNA preparation and analysis. Total cellular RNA was obtained by the guanidinium-CsCl method (1). Preparation of the single-stranded human HSP70 probe (containing bases +229 [5' end labeled] to -133 of the HSP70-CAT fusion gene) and Si nuclease digestion analysis have been described elsewhere (34). Gels were quantitated by film densitometry with a Kratos model SD3000 (Schoeffel Instrument Co.) spectrodensitometer. Template commitment assays. Transcription reaction mixtures (25 RI) contained 40 mM N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid (HEPES; pH 8.4), 12 mM Tris hydrochloride (pH 7.3), 60 mM KCI, 12% glycerol, 0.12 mM EDTA, 7 mM MgCl2, 0.6 mM CTP and ATP, 25 jiM UTP, 5 ,uCi of [c-32P]UTP (800 Ci/mmol), 15 U of RNase T1 (Pharmacia, Inc.), 0.1 mM 3'-O-methyl-GTP, 135 jig of HeLa nuclear extract (15 ,ul; 9 mg/ml), and 800 ng each of the test plasmid and -53MLP. Transcription reactions were per-


VOL. 10, 1990




R; F



SP. 8 F D _2










WT F _











x, s_ _% 4v^


-~~~~~~~~~~~~~~~~~~~~~~i1% il







S. 4 4%






6 7



ts ti u4e d I


Region --

3 4

FIG. 2. In vitro DNA-binding assays of HSP70 substitution mutants. (A) Methylation interference assay of proteins binding to the upstream substituted regions. A 5'-labeled DNA fragment containing bases -84 to +7 (2 ng) was treated with dimethyl sulfate under limiting conditions and incubated with HeLa cell nuclear extract (25 Fg). Bound (B) and free (F) DNAs were separated by mobility shift gel electrophoresis, the desired bands were excised, and the cleavage reaction was performed (21). Unincubated probe DNA (D) was also subjected to the chemical reactions and run as a sequencing ladder (lanes 3 and 6). The wild-type (WT) HSP70 promoter was treated and run as a reference for the substituted region (lane 9). The substituted sites (matched with the HSP70 TATA) are shown above the brackets. Solid arrows mark underrepresented G residues in the substituted region; broken arrows show some underrepresentation in the GC box of the Spl/hsp70 construct. (B) DNase I footprinting analysis of a factor binding to the substituted AP1 site. A labeled DNA fragment (-266 to +7; 7.5 ng) containing either the wild-type CCAAT (lanes 1 to 5) or substituted AP1 site (lanes 6 to 9) matched with the HSP70 TATA sequence was incubated with various amounts (indicated in microliters at the top) of a HeLa cell nuclear extract (4 mg/ml) before partial DNase I digestion. Open boxes mark regions of protection; the dotted box extends to cover hypersensitive sites. Lane M, a labeled pBR322 MspI

digest used


size marker.

formed at 30°C for 60 min. Transcripts were isolated as previously described (51) and resolved on a 4% polyacrylamide (acrylamide/bisacrylamide ratio, 30:0.8)-7 M urea gel. For template commitment assays, each construct (800 ng) was preincubated in 135 ,ug of HeLa nuclear extract (15 pAl; 9 mg/ml) with or without -53MLP (800 ng) for 60 min. -53MLP was added to the latter reaction mixture, and incubation of both continued for an additional 20 min. Nucleotides (in 1 ,ul; final concentrations same as noted above) were added, and transcription (final reaction volume, 25 pl) was allowed to continue for 30 min, after which time the transcripts were processed as described above. Gels were quantitated by using a Betascope 603 blot analyzer (Betagen).

RESULTS Experimental design. Our goal was to examine the transcriptional activity of a given upstream transcription factor in a variety of promoter contexts as a means of determining

the molecular mechanism of transcription factor action. In designing these experiments, we were faced with a dilemma. We could replace sites in a natural promoter, putting them in the context of all of the other functional sites found in that promoter. Alternatively, we could create promoters de novo, mixing only defined upstream sequences with defined TATA sequences while creating artificial sequences to fill out the rest of the promoter. We reasoned that not enough was known about mammalian promoter function to confidently construct artificial promoters that were functional and that did not contain fortuitous factor-binding sites. We therefore chose to use the human HSP70 promoter (62) as a template on which we created different promoter contexts. Detailed mutagenesis and in vivo and in vitro expression studies of the human HSP70 promoter have revealed three critical proximal promoter elements: an inverted CCAAT site at -65, a Spl-binding site at -45, and a TATAAA sequence at -28, with the CCAAT and TATA elements contributing most to the basal level of expression (21, 61,



59a, 63). A factor with binding properties similar to those of ATF binds to a sequence at -37 in vitro, and factor AP2 may bind to sequences at -17 and +15 in vitro (21, 61; H. Prentice and R. Kingston, unpublished observations). Mutation of the latter three binding sites does not alter function of the promoter under any condition tested to date. We targeted the CCAAT and TATA sites for mutagenesis, as they contributed most to basal function in vivo. The consensus binding sites for ATF, Spl, AP1, and OTF-1 were substituted for the CCAAT site of the wild-type HSP70 promoter and matched with the wild-type HSP70 TATA sequence (Fig. 1). An 8-base-pair upstream nonsense sequence served as a control. The six upstream elements (including the wild-type CCAAT) were also matched with the SV40 early TATA and adenovirus EIIa TATA sequences as well as with a TATA nonsense control sequence. These substitutions create a set of 24 promoters that we used as tools to examine the effects of the various promoter contexts on transcription factor activity. Binding of upstream factors to chimeric promoters. To determine whether the upstream transcription factors were able to bind to their consensus binding sites when placed in the context of the human HSP70 promoter, in vitro binding assays were performed. By using a gel mobility shift assay, retarded bands specific to each substituted element were identified by competition experiments, using unlabeled fragments either with or without the element under investigation (data not shown). No specific retarded bands were identified for the substituted AP1 element or the upstream nonsense control. To ensure that the specific bands identified by mobility shift in fact were due to factor binding to the substituted upstream element, methylation interference assays were performed on the retarded bands. Methylated G residues were underrepresented in the appropriate substituted region of the promoter for the ATF, OCTA, and Spl elements (Fig. 2A), as was previously shown for the CCAAT element (21). Because the ATF site showed dramatically different transcriptional activities in the various promoter contexts (see below), we analyzed binding of ATF to each construct in more detail. The off-rate of ATF for each construct was found to be identical in competition studies, implying that the TATA mutations did not significantly alter the ability of ATF to bind to the promoter (data not shown). We used DNase footprinting to assay for AP1 binding to the substituted AP1 consensus site. The HeLa nuclear extract contained both CCAAT- and APl-binding activities (Fig. 2B). DNase footprinting performed with a purified AP1 fraction (generous gift of Michael Comb) confirmed that the AP1 protein itself elicited this binding activity (data not shown). Upstream factor function in vivo. To determine the level of transcription from each of the 24 promoters, we transfected the constructs into HeLa cells by the calcium phosphate precipitation method. All of the promoters were truncated at -84 to remove the heat shock element to ensure that the resulting expression levels were not due to stressing of the cells during transfection. Since each of the promoters was positioned upstream of the bacterial CAT gene, the product of this gene was used initially to assay expression levels. To verify that these expression levels were due to appropriate initiation of transcription, we isolated total cellular RNA in separate transfections and assayed transcript levels and initiation sites by S1 nuclease digestion. Transfections analyzed directly for RNA also contained the pseudo-wild-type plasmid pIR17-84 as an internal reference. In many cases,


CAT protein levels correlated closely with observed levels of appropriately initiated transcripts. However, in some instances CAT levels were significantly different from levels of appropriate transcripts, which may reflect contributions from starts in the vector in these constructs. We therefore report promoter strength as calculated from transcript level. To facilitate comparison, the expression level for each construct was related to the level of the wild-type HSP70 promoter (arbitrarily assigned the relative transcription level [RTL] value of 1.0; Fig. 3). Promoters are grouped by TATA motif in Fig. 3 and are presented in order of overall strength within that grouping. By comparing the strength of any given promoter with the strength of the upstream nonsense control within the appropriate TATA grouping, one can determine the ability of an upstream factor to function in the context of a particular TATA motif (Fig. 4). The first point of interest is that the factor that bound to the HSP70 CCAAT motif functioned efficiently in each of the promoter contexts. Both CP1 and CCAAT-box-binding factor have been reported to bind to this particular CCAAT motif in vitro (9, 44). It is not clear at this time which factor occupies the site in vivo, although in vitro avidity favors CP1 (M. Pelletier and R. Kingston, unpublished observations). Regardless, these data imply that the ability of this factor to stimulate transcription is independent of the TATA-binding factor(s). In contrast, the degree of stimulation by ATF varied dramatically upon changing the TATA motif. ATF worked extremely well when paired with the HSP70 TATA, resulting in a promoter as strong as the wild-type HSP70 promoter. ATF also worked when paired with the EIla TATA motif but displayed essentially no activity when paired with the SV40 early TATA motif or with a nonsense TATA. There was more than a 20-fold difference in the ability of ATF to function in the presence of the Ella TATA compared with its ability to function with the nonsense TATA. The ability of each upstream factor to function with a given TATA motif is summarized in Fig. 4. Vertical columns represent the set of promoters containing a given TATA sequence. We have arbitrarily classified upstream factors that stimulate less than twofold more than the nonsense upstream control as inactive, two- to fivefold activators as moderate, and greater than fivefold activators as efficient. Factor Spl functioned with at least moderate strength with every TATA motif. All promoters containing either the AP1 or OTF-1 sites were expressed weakly, appearing slightly active when paired with either the EIla or nonsense TATA motif. It is important to point out that accurate quantitation of these more weakly expressed promoters is difficult because of the low level of appropriately initiated transcripts. Therefore, the values given to these weak promoters are less precise, and small differences between such promoters (e.g., ATF/TATA nonsense versus upstream nonsense/TATA nonsense) should not be considered significant. The S1 analysis not only measures the transcript level from each construct but also characterizes the start site. The HSP70 TATA and EIla TATA motifs both specify one major start site at the normal HSP70 cap site. With both the SV40 early TATA motif and the nonsense TATA motif, a doublet start site approximately 25 base pairs downstream of the wild-type start site is used somewhat more than the normal start site. Effects of spacing on CP1 and ATF function. One interpretation of the data presented above is that CP1 and ATF differ in their requirements for an interaction with the TATA element. Alternatively, it was possible that the differences in CP1 and ATF function were an artifact of the spacing


VOL. 10, 1990






1 .0







0.9 |










0. 2

h5sp70! o hsp7 .

= ~NONS.






*~~0. .


.2 .2TA Q


ll .0 4 i~~~.0








i17 1.0 3


- 4 SPI









_.04 SI4E




-, NONS.





Factor substitution in a human HSP70 gene promoter: TATA-dependent and TATA-independent interactions.

To investigate interactions between transcription factors on mammalian promoters, we constructed a set of 24 variations of the human HSP70 gene promot...
3MB Sizes 0 Downloads 0 Views