Collagen Gene Expression - Molecular and Cellular Biology

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Jun 15, 1988 - Cell 44:261-272. 14. Laimins, L., M. Holmgren-Konig, and G. Khoury. 1986. ... Muglia, L., and L. B. Rothman-Denes. 1986. Cell type-specific.
MOLECULAR AND CELLULAR BIOLOGY, Nov. 1988, P. 4851-4857

Vol. 8, No. 11

0270-7306/88/114851-07$02.00/0 Copyright © 1988, American Society for Microbiology

Interactions between the Promoter and First Intron Are Involved in Transcriptional Control of al(I) Collagen Gene Expression BORNSTEIN,l.2* JOHN McKAY,1 DEANN J. LISKA,1 STEPHEN APONE,' AND SREELEKHA DEVARAYALU' Department of Biochemistry1 and Department of Medicine,2 University of Washington, Seattle, Washington 98195 PAUL

Received 15 June 1988/Accepted 16 August 1988

The first intron of the human collagen al(l) gene contains several positively and negatively acting elements. We have studied the transcription of collagen-human growth hormone fusion genes, containing deletions and rearrangements of collagen intronic sequences, by transient transfection of chick tendon fibroblasts and NIH 3T3 cells. In chick tendon fibroblasts, but not in 3T3 cells, inversion of intronic sequences containing a previously studied 274-base-pair segment, A274, resulted in markedly reduced human growth hormone mRNA levels as determined by an RNase protection assay. This inhibitory effect was largely alleviated when deletions were introduced in the collagen promoter of plasmids containing negatively oriented intronic sequences. Evidence for interaction of the promoter with the intronic segment, A274, was obtained by gel mobility shift assays. We suggest that promoter-intron interactions, mediated by DNA-binding proteins, regulate collagen gene transcription. Inversion of intronic segments containing critical interactive elements might then lead to an altered geometry and reduced activity of a transcriptional complex in those cells with sufficiently high levels of appropriate transcription factors. We further suggest that the deleted promoter segment plays a key role in directing DNA interactions involved in transcriptional control.

Regulatory elements have recently been identified in the first intron of several collagen genes (1, 2, 10, 19, 20; P. Savagner, T. Miyashita, and Y. Yamada, FASEB J. 2:A359, 1988). None of these elements has been characterized at the nucleotide level, but evidence for functionality using chloramphenicol acetyltransferase (1, 2, 10, 19), globin (20), and human growth hormone (hGH) (1) reporter genes has been reported. In the human al(I) gene, several sites for interaction of DNA-binding proteins have been deduced by DNase I protection mapping of an intronic segment (2). Rossouw et al. (20) inserted a 782-base-pair intronic fragment of the human al(I) gene into the first intron of the al-globin gene, driven by an al(I) collagen promoter, and demonstrated enhanced transcription when the chimeric gene was introduced either into Xenopus oocytes by microinjection or into NIH 3T3 cells by transfection. The same intronic fragment was also stimulatory in oocytes, but only in the negative orientation, when placed upstream of a longer collagen promoter. In this case, as well as in the case of the mouse x2(I) gene (19), orientation specificity of enhancerlike elements was attributed largely to proximity effects. In our own studies of regulatory elements in the first intron of the human al(I) gene, we noted that a 274-bp sequence (A274), in its appropriate 3' location relative to the start of transcription, was highly inhibitory to transcription of a collagen-hGH fusion gene. This inhibition occurred only when the sequence was oriented negatively, that is, opposite to its orientation in the gene (1). When oriented positively in the same location, the A274 sequence was essentially neutral or, at best, only slightly inhibitory (1). This specificity of orientation was puzzling since one would have expected the natural orientation that exists in the gene to be most effective. We now find that the transcription of collagen-hGH plas*

mids, in which other segments of the first intron have been inverted, is also reduced in comparison to transcription of plasmids containing the same intronic sequences in their correct orientation. Furthermore, when segments of the 5' flanking collagen sequence are deleted, the ability of the A274 sequence to inhibit transcription is markedly reduced, even though the deletions themselves are not inhibitory. We suggest that transcriptional activation of the al(I) collagen gene is regulated by a complex set of interactions, mediated by DNA-binding proteins, between intronic and 5'-flanking promoter sequences. When a segment of the intron is inverted, the correct organization of the transcription initiation complex is disrupted, resulting in reduced transcription. We propose that 5'-flanking deletions interfere with such interactions at a distance, thereby neutralizing the effect of the intronic inversion and reducing transcriptional activity to the basal level observed in the absence of the intron. MATERIALS AND METHODS

Plasmid construction. The construction of pCol-hGH, pCol-hGHA292-1440, pCol-hGHAA274(+), and pCol-hGH A&A274(-) has been described (1). The latter two plasmids were constructed by blunt-end ligation of a 274-bp intronic sequence (bases 820 to 1093; see reference 2); flanked by AvaI sites, into a modified SstII site created by deletion of the intronic sequences between bases 292 and 1440 (see Fig. 1). The remaining plasmids, shown schematically in Fig. 1, were constructed by reinserting intronic sequences, flanked by SstII sites and fractionated by agarose gel electrophoresis, into pCol-hGHA292-1440. Thus, pCol(671-1440)hGH and pCol(1440-671)hGH contain the sequences indicated by the bases in parentheses in either the positive or negative orientation, respectively, as do pCol(292-671)hGH and pCol (671-292)hGH. Deletions in the 5'-flanking sequence of the al(I) collagen gene were initially introduced into a collagen-bovine growth

Corresponding author. 4851

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Plasmid

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24+t2.3 (13) 26 ±2.8(6) 2 ±0.6 (7) 61

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FIG. 1. Diagrammatic representation of deletions and rearrangements created in pCol-hGH and tabulation of transcriptional activity of the resulting plasmids. The start of transcription at base 1, which initiates the collagen exon (open rectangle), is indicated by the angled arrow. The hGH minigene cassette (heavy line), extending from the 3' portion of the third intron to the 3'-flanking sequence, was fused to the collagen first intron at a HindlIl site (base 1447; see references 4 and 5). Unique SstII (S) sites in the collagen intron are indicated. Deletions and reinsertions were performed by SstII cleavage followed by ligation of the intronic fragment in the desired orientation. The 274-bp intronic sequence flanked by Aval sites (A274, open arrow) was reinserted into the deleted intron by blunt-end ligation (see Materials and Methods and reference 5). The orientation of the intronic segment in a plasmid is indicated either by (+) or (-) in the case of AA274 or by the sequence of the base numbers flanking the intronic segment, e.g., (671-1440) and (1440671). The orientation of the shorter intronic segment (292-671) is also indicated by a solid arrow. The transcriptional activity of each plasmid standard error, relative to pCol-hGH which has been set at 100%, is tabulated. The number of independent determinations is given in parentheses.

hormone (bGH) plasmid described by Bornstein et al. (P. Bornstein, J. McKay, S. Devarayalu, and S. C. Cook, Nucleic Acids Res., in press). The plasmid, pCol-bGH, contains the same collagen promoter sequence as does pCol-hGH (to -804) but was constructed by fusing collagen exon 1 in frame to bGH exon 2. pCol-bGH, when transfected into a variety of cells, directs the production of secreted, immunologically reactive bGH (Bornstein et al., in press). A sequence flanked by two unique EcoRV sites in the collagen promoter (at -625 and -443) was deleted by EcoRV digestion followed by religation to produce pCol(A -625 to -443)bGH. Larger deletions in the collagen promoter were produced by exonuclease III digestion of EcoRV-cleaved plasmids. Exonuclease III digestion was followed by S1 nuclease treatment, blunt-ending with Klenow fragment, addition of SstI linkers, SstI digestion, and ligation. The resulting deleted clones were mapped preliminarily by restriction enzyme mapping. To create a deletion series with a constant upstream endpoint, an SstI linker was introduced into the EcoRV site of pCol(A180-362)bGH and a vector was prepared by digestion with SstI and SstII (SstII cleaves pCol-bGH uniquely in the third intron of the bGH gene). DNA fragments extending from SstI to SstII were then transferred from deletion clones to this vector. The resulting deletion series was sized carefully by comparison of radiolabeled restriction fragments with known collagen promoter restriction fragment, as well as with an MspI digest of pBR322, and three deletions extending from bases -625 to -380, -625 to -293, and -625 to -161 were chosen for further study. The accuracy of determination of the 3' endpoint of each deletion was considered to be +5 bp. Each deletion, as well as the base-line deletion A -625 to -443,

was transferred to an hGH plasmid series that included pCol-hGH, pCol-hGHA292-1440, pCol-hGHAA274(+), and pCol-hGHAA274(-), using standard recombinant DNA techniques. The net result of these manipulations was to create a matrix in which any of five configurations of the promoter (the intact sequence or deletions from -625 to -443, -625 to -380, -625 to -293, or -625 to -161) could be matched with any of four configurations of the intron [the intact sequence, A292-1440, AA274(+), or AA274(-)]. Transient transfection. Chick tendon fibroblasts (CTF) and NIH 3T3 cells were cultured in 100-mm dishes. Cells were subcultured at a 1:3 split ratio. Results with CTF were only reproducible when early (less than lOth)-passage cells were used. Plasmid DNA was introduced into cells by the calcium phosphate coprecipitation procedure as described previously (1, 2). Efficiency of uptake of DNA was monitored by cotransfection of a plasmid bearing the ,-galactosidase gene under control of the Rous sarcoma virus long terminal repeat. Cell lysates were assayed for 3-galactosidase activity by fluorescence using 4-methylumbelliferyl ,-D-galactoside as a substrate. Analysis for hGH mRNA. RNA was extracted from cells using a single-step acid guanidinium thiocyanate-phenolchloroform method (4) and purified by isopropanol precipitation. Possible contamination with residual transfected plasmid DNA was avoided by brief digestion with RNasefree fast-protein liquid chromatography-purified DNase I (Pharmacia). hGH mRNA was analyzed by an RNase protection assay using 32P-labeled antisense RNA synthesized with T7 RNA polymerase as previously described (1, 15). DNA mobility shift assays. Nuclear extracts were prepared from CTF as described by Shapiro et al. (23). These extracts were functional in an in vitro transcription assay utilizing the collagen al(I) promoter (unpublished observations). A274 was 5'-end labeled with 32P using T4 polynucleotide kinase. Binding reactions were performed in 20 mM HEPES (N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.9)20% glycerol-100 mM KCI-0.2 mM EDTA-0.2 mM EGTA

[ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid]-2 mM dithiothreitol in a volume of 20 ,ul. After preincubation of nuclear extract (20 ,ug) with 4 ,ug of poly(dIdC) at 4°C for 10 min, labeled probe (1 ng; 15,000 cpm) with or without unlabeled DNA (competitor) fragment was added, and incubation was continued at 20°C for 20 min. Competitor DNA fragments were prepared by restriction enzyme digestion and purified by electroelution from agarose gels. Samples were loaded directly on 5% preelectrophoresed polyacrylamide gels (30:1 polyacrylamide-bisacrylamide in 25 mM Tris-188 mM glycine-1 mM EDTA), and electrophoresis was performed in the same buffer at 20°C and 12 V/cm. Gel bands were visualized by radioautography. RESULTS We have previously shown that a 274-bp intronic sequence, flanked by AvaI sites (A274), contained several regions that were contacted by DNA-binding proteins (2) and acted to inhibit transcription when cloned 5' to the simian virus 40 (SV40) early promoter (2) or either 5' or 3' to collagen promoters driving chloramphenicol acetyltransferase or hGH genes (1, 2). A puzzling feature of this inhibitory activity was that the A274 element, when separated from adjacent intronic sequences, was strongly inhibitory of transcription only when placed in a negative orientation, i.e., opposite to the natural orientation, 3' to collagen promoter sequences. In this work we have examined this aspect of the

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TRANSCRIPTIONAL CONTROL OF otl(I) COLLAGEN GENE

interaction between intronic and promoter sequences in greater detail and conclude that this inhibition results from a requirement for extensive, highly specific, promoter-intron interactions which we believe underlie the regulation of this gene. In accord with our earlier findings (2), deletion of the majority of the collagen intronic sequence from pCol-hGH reduced transcription by a factor of about 4 (Fig. 1). Replacement of the isolated A274 sequence in the positive (natural) orientation had little or no effect, but replacement of A274 in the negative orientation reduced transcription by a factor of at least 10 (Fig. 1). We have now found that inclusion of a longer collagen intronic fragment, containing A274, augmented transcription when the sequence (6711440) was placed in the positive orientation but that reversal (1440-671) again inhibited transcription by a factor of about 10 (Fig. 1). The finding that transcriptional activity of pCol(1440-671)hGH is almost as low as that of pColhGHAA274(-) argues against the possibility that a negatively acting element in A274 is highly sensitive to distance. Finally, the reintroduction of a shorter intronic sequence which lacks A274 (292-671) also stimulated transcription, but reversal (671-292) abolished this stimulation (Fig. 1). In the last case the negative orientation of the intronic sequence produced little if any inhibition of transcription relative to the level observed with the intron-deleted clone (A292-1440). We have reanalyzed some of the RNAs from the experiments described in Fig. 1 with a riboprobe to collagen exon 1 that extends 5' to the start of transcription and have obtained essentially the same results (not shown). This finding argues against an attenuation mechanism that might account for the differential effects of orientation of the intronic segments and indicates that significant new transcription start sites are not generated in some of these plasmids. We interpret the findings in Fig. 1 in the following way. Productive interactions between a series of DNA-binding proteins that recognize elements in the 5'-flanking promoter region of the al(I) collagen gene and another series in the intron require the correct spatial orientation of these proteins, as might occur in a DNA looping model for such interactions (17, 18; see Discussion). Reversal of a segment of the intron that contained sequences crucial for these interactions might lead to a disruption of the normal spatial geometry of the transcriptional complex. If this assumption were true, it might be possible to prevent the inhibition created by the presence of the A274 sequence in the negative orientation by deleting a region of the 5'-flanking sequence with which the intronic segment interacted. To interpret such reversal of inhibition easily, it would be preferable if this 5'-flanking sequence were not essential, per se, for transcriptional activity of the collagen promoter. The experiments described in Tables 1 and 2 support both this requirement and the postulate. Deletions extending from -625 to -443, -625 to -293, and -625 to -161 were introduced into the collagen promoter, and the resulting promoters were fused to the hGH gene as described in Materials and Methods. There was no major reduction in expression of the hGH gene when these plasmids were introduced into CTF by transient transfection, and levels of hGH mRNA were compared to those resulting from transfection of pCol-hGH (Table 1). Indeed, the deletion in pCol(A -625 to -443)hGH consistently increased expression by a factor of 2, suggesting the presence of negatively acting sequences in this region. A similar

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TABLE 1. Transcriptional activity of pCol-hGH plasmids containing promoter deletions Plasmida

Activityb

pCol-hGH .................. pCol(A -625 to -443)hGH .................. pCol(A -625 to -293)hGH .................. pCol(A -625 to -161)hGH ..................

100 204 ± 10 (7) 92 ± 15 (4) 101 ± 14 (5)

a Promoter deletions were constructed by a combination of restriction enzyme and exonuclease III digestion from a common EcoRV site at -625 as described in Materials and Methods. b Transcriptional activity was determined by an RNase protection assay of hGH mRNA in transiently transfected CTF. The level for hGH was arbitrarily set at 100, and values are given + standard error with the number of independent determinations in parentheses.

effect of deletion was noted in a more promoter-proximal segment of the mouse a2(I) gene (22). We then transferred the promoter deletions to plasmids containing various deletions of the intron. The data in Table 2 summarize the results of experiments in which the ratio of hGH mRNA levels obtained with plasmids containing the A274 sequence, reinserted in the deleted intron in either the positive or negative orientation [AA274(+)/AA274(-)], was expressed as a function of the absence or presence of deletions in the promoter. As determined previously (see Fig. 1), this ratio was about 13 when an intact promoter was used but was reduced to about 2 when deletions extending from -625 to -293 and -625 to -161 were examined. Deletions extending from -625 to -443 gave intermediate results. An example of an experiment in which a larger promoter deletion (-625 to -161) was compared with a smaller deletion (-625 to -443) is shown in Fig. 2. It can be seen that the inhibition associated with the inversion of the intronic sequence in the shorter promoter deletion (Fig. 2, compare lanes 7 and 8) was largely relieved when the promoter deletion was extended (compare lanes 3 and 4). The greater orientation independence of the effect of intronic sequences with increasing extent of promoter deletion results in part from a reduced effectiveness of A274(+), but mainly from an increased transcriptional activity of A274(-). Figure 2 also demonstrates that the promoter deletions, per se, do not inhibit transcription (compare lanes 1, 5, and 9). In contrast to CTF, NIH 3T3 cells showed little or no ability to distinguish the inversion of intronic segments, regardless of whether the promoter was intact or not (Fig. 3). Similarly, with increasing doubling number CTF were erratically less able to distinguish inversion of intronic sequences, and in BHK cells negatively oriented intronic sequences did TABLE 2. Transcriptional activity as a function of collagen promoter and intron structurea Promoter deletion

None .................................. -625 to -443 ................... -625 to -293 ................... -625 to -161 ....................

AA274(+)/AA274(-)

13b ............... ............... ..............

8.3 ± 1.8 (5) 1.9 0.6 (4) 2.4 ± 0.4 (7)

a The ratio of the transcriptional activity of plasmids containing the A274 sequence in the positive orientation, AA274(+), to that of plasmids with A274 in the negative orientation, A274(-), is tabulated as a function of the structure of the collagen promoter. Transcriptional activity was determined by an RNase protection assay of hGH mRNA in total RNA of transiently transfected CTF. Values are given + standard error with the number of independent determinations in parentheses. b Ratio obtained from data in Fig. 1.

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BORNSTEIN ET AL. Competitor Ce)

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FIG. 2. Autoradiogram of hGH mRNA analyzed by an RNase protection assay. Total RNA (4 to 8 ,ug) from CTF, transiently transfected with collagen-hGH fusion gene plasmids, was hybridized with a 32P-labeled antisense hGH riboprobe. After RNase digestion, ethanol-precipitable material was analyzed by electrophoresis in a 6% acrylamide denaturing gel. Lanes 1 through 4, pCol(A -625 to -161)hGH: lane 1, intact intron; lane 2, A292-1440; lane 3, AA274(+); lane 4, AA274(-). Lanes S through 8, pCol(A -625 to -443)hGH: lane 5, intact intron; lane 6, A292-1440; lane 7, AA274(+); lane 8, AA274(-). Lane 9, pCol-hGH; lane 10, pColhGHA292-1440; lane 11, RNA from mock-transfected CTF; lane 12, RNA from transgenic mouse liver containing the hGH gene; lane 13, hGH riboprobe. The relative hGH mRNA levels in this experiment, corrected for RNA concentration and efficiency of transfection, are as follows: lane 1, 2.3; lane 2, 0.36; lane 3, 0.24; lane 4, 0.16; lane 5, 2.1; lane 6, 0.29; lane 7, 0.25; lane 8, 0.02; lane 9, 1.0; lane 10, 0.50.

not reduce transcription below the level observed with intron deletion (data not shown). These negative findings provide important controls since they tend to exclude factors intrinsic to the plasmid DNA, such as differences in topology, that might otherwise account for differences in transcriptional efficiency. We conclude that the ability of negatively oriented collagen intronic sequences to inhibit transcription results from the presence of a cell type-specific DNA-binding protein(s) that facilitates promoter-intronic interactions.


3' hGH

hGH

FIG. 5. A model that accounts for orientation specificity in collagen promoter-intronic interactions. (Top) A transcriptional complex is shown involving TATA(T) and CCAAT(C) motifs and 5' flanking and intronic elements. The latter two regions of the DNA interact by looping of the DNA, and these interactions are stabilized by several types of protein-DNA interactions (symbols). The magnitude of the transcriptional response of the collagen-hGH fusion gene is indicated by the width of the arrow that marks the transcription start site (base 1). (Middle) Inversion of the intronic segment from base 671 to base 1440 leads to an altered geometry of the transcriptional complex, although promoter interactions still occur. A reduced transcription rate is indicated by the narrow horizontal arrow. (Bottom) Deletion of the promoter sequence from -625 to -293 disrupts promoter-intron interactions. Despite inversion of the intronic segment, transcription proceeds at a higher rate than in the fusion gene depicted in the middle panel. The promoter deletion does not, per se, reduce transcriptional activity (see Table 1).

Table 2, may be required to demonstrate an optimal negative effect. Thus, in our studies a promoter deletion extending from -625 to -293 largely abrogates the inhibitory effect observed with pCol-hGHAA274(-). An alternative possibility is that Xenopus oocytes lack the appropriate transcription factor(s) necessary to mediate all of the DNA-protein interactions that are possible between the collagen promoter and intron. In this respect the transcriptional environment in Xenopus oocytes may be similar to that in BHK or NIH 3T3 cells, in which reversal of orientation of intronic sequences also has relatively little effect (Fig. 3). The notion that homologous cellular enhancer-promoter interactions differ from heterologous enhancer-promoter interactions is supported by the work of Garcia et al. (5), who showed that K light-chain and heavy-chain immunoglobulin

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enhancers stimulate their homologous enhancers far more effectively than they do either SV40 or metallothionein promoters. Additional evidence for the specificity of enhancer-promoter interactions comes from studies of the SV40 enhancer. The SV40 enhancer is far more effective in stimulating expression of the 3-globin and conalbumin genes than it is in stimulating a-globin gene expression (26). Robbins et al. (18) observed apparent cell specificity in the relative ability of the SV40 enhancer to stimulate herpesvirus tk gene expression when the gene was driven by either the tk or SV40 early promoter. This specificity was modulated by the addition of T antigen in trans. Robbins et al. (18) conclude that the SV40 enhancer probably does not possess cell specificity but rather that certain enhancer-gene interactions show cell-specific responses which depend on cellular variations in the elaboration of effective transcriptional protein complexes. We propose the model shown in Fig. 5 to account for our findings but stress that details of the model are speculative and may well require modification. The interaction of the intron with the promoter and 5'-flanking sequence in the collagen gene involves the participation of several DNAbinding proteins (Fig. 5, top). We postulate that one subset of these interactions predominates in securing the looping of the DNA necessary to make these interactions. When the orientation of a segment of the intron is reversed, promoterintronic interactions may still occur, but at the cost of disrupting the normal spatial geometry of the transcriptional complex (Fig. 5, middle). This could account for the low level of transcription seen with plasmids such as pColhGHAA274(-) and pCol (1440-671)hGH (see Fig. 1). If a crucial region of the promoter, which provides a major source of interactions with the intron, is deleted, the reversal of an intronic sequence affects transcription to a lesser extent, and as a consequence, the level of transcription reverts towards that seen with the deleted 5' promoter alone (Fig. 5, bottom). This postulate is supported by the data in Table 2 and Fig. 2, which show that clones in which promoter deletions extend 3' to base -443 are transcribed at a relatively high level in transfection experiments, despite the presence of an inverted sequence in the intron. It should be noted that the collagen promoter, together with possible upstream elements in the 5'-flanking sequence, is capable of relatively strong stimulation of transcription of the collagen gene in the absence of intronic elements. However, the existence of an intact intron further augments transcriptional activity by a factor of 3 or 4 in CTF (1; Fig. 1). This stimulation could well be greater in other cells or if some of the cis-acting intronic elements responded to an inductive stimulus. We therefore postulate that promoterintronic interactions may subserve regulatory functions necessary for appropriate expression of type I collagen genes. ACKNOWLEDGMENTS We thank Elizabeth Enari and Kathleen Doehring for excellent technical and secretarial assistance, respectively. This work was supported by Public Health Service grants AM 11248, DE 08229, and HL 18645 from the National Institutes of Health. LITERATURE CITED 1. Bornstein, P., and J. McKay. 1988. The first intron of the al(I) collagen gene contains several transcriptional regulatory elements. J. Biol. Chem. 263:1603-1606. 2. Bormstein, P., J. McKay, J. K. Morishima, S. Devarayalu, and

R. E. Gelinas. 1987. Regulatory elements in the first intron contribute to transcriptional control of the human al(I) collagen gene. Proc. Natl. Acad. Sci. USA 84:8869-8873. 3. Boulet, A. M., C. R. Erwin, and W. J. Rutter. 1986. Cell-specific enhancers in the rat exocrine pancreas. Proc. Natl. Acad. Sci. USA 83:3599-3603. 4. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156-159. 5. Garcia, J. V., L. Bich-Thuy, S. Stafford, and C. Queen. 1986. Synergism between immunoglobulin enhancers and promoters. Nature (London) 322:383-385. 6. Godbout, R., R. Ingram, and S. M. Tilghman. 1986. Multiple regulatory elements in the intergenic region between the afetoprotein and albumin genes. Mol. Cell. Biol. 6:477-487. 7. Goodbourn, S., H. Burstein, and T. Maniatis. 1986. The human ,-interferon gene enhancer is under negative control. Cell 45: 601-610. 8. Halinger, A., and M. Karin. 1985. Upstream promoter element of the human metallothionein-IIA gene can act like an enhancer element. Proc. Natl. Acad. Sci. USA 82:8572-8576. 9. Hammer, R. E., G. H. Swift, D. M. Ornitz, C. J. Quaife, R. D. Palmiter, R. L. Brinster, and R. J. MacDonald. 1987. The rat elastase I regulatory element is an enhancer that directs correct cell specificity and developmental onset of expression in transgenic mice. Mol. Cell. Biol. 7:2956-2967. 10. Horton, W., T. Miyashita, K. Kohno, J. R. Hassell, and Y. Yamada. 1987. Identification of a phenotype-specific enhancer in the first intron of the rat collagen II gene. Proc. Natl. Acad. Sci. USA 84:8864-8868. 11. Jaynes, J. B., J. E. Johnson, J. N. Buskin, C. L. Gartside, and S. D. Hauschka. 1988. The muscle creatine kinase gene is regulated by multiple upstream elements, including a musclespecific enhancer. Mol. Cell. Biol. 8:62-70. 12. Kawamoto, T., K. Makino, H. Niwa, H. Sugiyama, S. Kimura, M. Amemura, A. Nakata, and T. Kakunaga. 1988. Identification of the human ,B-actin enhancer and its binding factor. Mol. Cell. Biol. 8:267-272. 13. Kimura, A., A. Israel, 0. LeBail, and P. Kourilsky. 1986. Detailed analysis of the mouse H2Kb promoter: enhancer-like sequences and their role in the regulation of class I gene expression. Cell 44:261-272. 14. Laimins, L., M. Holmgren-Konig, and G. Khoury. 1986. Transcriptional "silencer" element in rat repetitive sequences associated with the rat insulin 1 gene locus. Proc. Natl. Acad. Sci. USA 83:3151-3155. 15. Melton, D. A., P. A. Krieg, T. Rebagliati, T. Maniatis, and M. R. Green. 1984. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12:7035-7056. 16. Muglia, L., and L. B. Rothman-Denes. 1986. Cell type-specific negative regulatory element in the control region of the rat a-fetoprotein gene. Proc. Natl. Acad. Sci. USA 83:7653-7657. 17. Ptashne, M. 1986. Gene regulation by proteins acting nearby and at a distance. Nature (London) 322:697-701. 18. Robbins, P. D., D. C. Rio, and M. R. Botchan. 1986. trans activation of the simian virus 40 enhancer. Mol. Cell. Biol. 6:

1283-1295. 19. Rossi, P., and B. de Crombrugghe. 1987. Identification of a cell-specific transcriptional enhancer in the first intron of the mouse a2(type I) collagen gene. Proc. Natl. Acad. Sci. USA 84: 5590-5594. 20. Rossouw, C. M. S., W. P. Vergeer, S. J. du Plooy, M. P. Bernard, F. Ramirez, and W. J. de Wet. 1987. DNA sequences in the first intron of the human pro-al(I) collagen gene enhance transcription. J. Biol. Chem. 262:15151-15157. 21. Schlokat, U., and P. Gruss. 1986. Enhancers as control elements for tissue-specific transcription, p. 226-234. In P. Kahn and T. Graf (ed.), Oncogenes and growth control. Springer-Verlag, Berlin. 22. Schmidt, A., P. Rossi, and B. de Crombrugghe. 1986. Transcriptional control of the mouse a2(I) collagen gene: functional deletion analysis of the promoter and evidence for cell-specific

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expression. Mol. Cell. Biol. 6:347-354.

23. Shapiro, D. J., P. A. Sharp, W. W. Wahli, and M. J. Kelier. 1988. A high-efficiency HeLa cell nuclear transcription extract. DNA 7:47-55. 24. Theisen, M., A. Stief, and A. E. Sippel. 1986. The lysozyme enhancer: cell-specific activation of the chicken lysozyme gene by a far-upstream DNA element. EMBO J. 5:719-724. 25. Treisman, R. 1985. Transient accumulation of c-fos RNA fol-

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lowing serum stimulation requires a conserved 5' element and c-fos 3' sequences. Cell 42:889-902. 26. Treisman, R., M. R. Green, and T. Maniatis. 1983. cis and trans activation of globin gene transcription in transient assays. Proc. Natl. Acad. Sci. USA 80:7428-7432. 27. Watanabe, K., A. Saito, and T. Tamaoki. 1987. Cell-specific enhancer activity in a far upstream region of the human afetoprotein gene. J. Biol. Chem. 262:4812-4818.