©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Far-upstream Enhancer of the Carbamoyl-phosphate Synthetase I Gene Is Responsible for the Tissue Specificity and Hormone Inducibility of Its Expression (*)

(Received for publication, July 5, 1995)

Vincent M. Christoffels Maurice J. B. van den Hoff Antoon F. M. Moorman Wouter H. Lamers (§)

From the University of Amsterdam, Department of Anatomy and Embryology, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The role of the proximal promoter and the far-upstream enhancer in the hepatocyte-specific and hormonal regulation of the carbamoyl-phosphate synthetase I (CPS) gene was investigated in transient transfection assays using primary rat hepatocytes, hepatoma cells, and fibroblasts. These experiments revealed that the activity of the promoter is comparable in all cells tested and is, therefore, not responsible for tissue-specific expression. The 5`-untranslated region of the mRNA is a major, non-tissue specific stimulator of expression in FTO-2B hepatoma cells, acting at the post-transcriptional level. A 469-base pair DNA fragment, 6 kilobase pairs upstream of the transcription start-site in the CPS gene, confers strong hormone-dependent tissue specific expression, both in combination with the CPS promoter and a minimized viral thymidine kinase promoter. Sequences similar to a cyclic AMP-responsive element and a glucocorticosteroid-responsive element were found in the isolated enhancer. Substitutional mutations in these sites strongly affected hormone-induced expression. Analysis of the interaction between the enhancer and parts of the CPS promoter revealed that, in addition to the TATA box, the GAG box, a motif similar to the GC box near the TATA motif, is instrumental in conferring the enhancer activity.


INTRODUCTION

Carbamoyl-phosphate synthetase (CPS) (^1)is the first enzyme of the ornithine cycle. CPS expression can be detected from the 15th embryonic day onward in the liver of the rat. Expression is initially found in a few hepatocytes only, but toward the end of the fetal period all hepatocytes have been recruited to express CPS(1, 2, 3) . After birth, the expression gradually becomes confined to the hepatocytes surrounding the portal veins(3, 4) . The only other cells producing appreciable levels of CPS mRNA and protein are the enterocytes of the small intestine(3, 5) . After birth, CPS enzyme and mRNA levels change in parallel under all experimental conditions(5, 8, 10, 11) , suggesting that hormonal regulation, tissue specificity, and zonal restriction of expression of CPS is regulated at the level of transcription. Accordingly, it was shown that glucocorticosteroids and cyclic AMP enhance transcription of the CPS gene in adult rat hepatocytes(6, 7, 8, 9, 12) .

The CPS gene is a single-copy, 110 gene that contains 38 exons and is surrounded by matrix attachment regions(13, 14, 15, 16) . The mRNA is 5546 nucleotides in length (excluding the poly(A) tract) and consists of a 140-nt 5`-untranslated region, an open reading frame of 4500 nt, and a 3`-UTR of 906 nt. Functional analysis of the 5`-part of the gene showed that the minimal, fully active promoter is located within the 161 nt upstream of the transcription initiation site(15) . DNase I footprint analysis of this region revealed three protected sites (sites I-III (17, 18) ), but the actual identity of the factors occupying these sites is not known(19) . In between the TATA motif at position -21 and protected site I, a so called ``GAG'' element was identified (19) that resembles the element recognized by the TFIIIA-like Cys(2)/His(2) zinc finger class of transcription factors, including Sp1, but is not a target of the Sp1 protein itself.

Two MspI restriction sites upstream of the transcription start site were found to be differentially methylated(15) . The site at -6.3 kbp becomes demethylated shortly after birth in liver and intestine. The other site, at -4.0 kbp relative to the transcription start site is fully methylated in liver and partly demethylated in postnatal small intestine. A 4-kbp fragment containing the -6.3-kbp MspI site responds to cyclic AMP and dexamethasone in transient expression assays, giving a 10-fold rise in reporter gene expression in FTO-2B hepatoma cells(15) .

The regulatory regions of the CPS gene were analyzed with respect to their role in tissue-specific expression and hormone sensitivity. A 469-bp far-upstream enhancer fragment was found to be responsible for hormone-dependent tissue-specific expression of the CPS gene. It was also investigated whether the proximal promoter and the 5`-UTR of the mRNA contributed to tissue-specific expression. Exploration of the interaction of the enhancer fragment with parts of the CPS promoter revealed an important role for the GAG element in the transduction of the hormonally induced activation signal from the enhancer.


EXPERIMENTAL PROCEDURES

Cell Culture

FTO-2B rat hepatoma cells(20) , Rat-1 fibroblasts(21, 22) , Chinese hamster ovary cells (CHO-K1), and Caco-2 cells (23) were cultured in Dulbecco's modified Eagle's medium/F-12 (Life Technologies, Inc.), supplemented with 10% fetal calf serum (FCS; Life Technologies, Inc.) and MH1C1 hepatoma cells were cultured in Ham's F-10 medium (Life Technologies, Inc.), supplemented with 17.5% FCS. The FCS was selected for its inability to induce CPS expression in cultured hepatocytes. All cells were cultured in a 5% CO(2)/air atmosphere at 37 °C. FTO-2B and MH1C1 cells express CPS, but Rat-1, CHO-K1, and Caco-2 cells do not (see ``Results''). Cell lines were tested monthly for the presence of mycoplasms.

DNA Transfection

Exponentially growing cells were transfected by electroporation(24) . The capacity of CPS gene sequences to direct expression of a reporter gene was analyzed by cloning into the vector pLT1 or pT81luc. The pLT1 vector, based on pBluescript SK+ (Stratagene), contains the firefly luciferase gene (25) as a reporter in conjunction with SV40 small t-antigen intron and polyadenylation signal optimized for expression(26) . The vector pT81luc (27) contains the firefly luciferase reporter gene driven by the first 81 base pairs of the viral thymidine kinase promoter (28) and is referred to as ``minimized TK promoter.'' To transfect equimolar amounts, 3 µg/kbp of CsCl-purified supercoiled plasmid was used. As a control the vector pLT1 without inserted promoter sequences was used. This vector produces 70 times 10^3 relative light units/mg of protein, which is 1.4 ± 0.4% (S.E., n = 6) of the activity of pLT1 in which the minimal, fully active CPS promoter (-161 to +138 nt with respect to the transcription start site) is inserted (background: 0.1 times 10^3 relative light units/mg of protein). Differences in transfection efficiency were corrected for by co-transfecting 3 µg of vector pRSVcat (RSV LTR(29) ). After each transfection the cell suspension was divided into equal parts, one being grown in culture medium and the other(s) in culture medium supplemented with 100 nM dexamethasone or with 100 nM dexamethasone, 1 mM dibutyryl cyclic AMP (Bt(2)cAMP; Boehringer) and 0.1 mM 3-isobutyl-1-methyl-xanthine (IBMX, Sigma) (7) . 44 hours after transfection cells were harvested, lysed in 100 mM KH(2)PO(4)/K(2)HPO(4), pH 7.6, 0.1% Triton X-100, and tested for CAT activity(30) , luciferase activity(31) , and protein concentration (bicinchoninic acid reagents, Pierce).

Determination of Transfection Efficiency

FTO-2B hepatoma cells and Rat-1 fibroblasts were transiently transfected as described (24) with 31 µg of luciferase construct and 25 µg of pRSV-n-LacZ, encoding beta-galactosidase carrying a nuclear translocation signal. 44 hours after transfection, luciferase activity and protein concentration were determined in one well. Cells in the other well were stained for galactosidase activity(32) . The specific luciferase activity driven by the promoter of interest (well 1), divided by the transfection efficiency (well 2), is a measure for the activity per transfected cell (24) .

Isolation and Transfection of Primary Rat Hepatocytes

Adult Wistar rats were obtained from the Broekman Institute, B.V., Someren, The Netherlands. Hepatocytes were freshly prepared from 17-day-old fetuses as described(33) , except that DNase was omitted to prevent degradation of plasmid DNA during transfection. Cells were transfected as described, and plated in rat tail collagen-coated dishes containing Dulbecco's modified Eagle's medium/F-12 and 10% FCS. Non-adhering cells were removed after 5 h. The cultures were incubated in Dulbecco's modified Eagle's medium/F-12 and 2.5% FCS in the absence or presence of added hormones, and harvested after 20 h.

ExoIII Deletions

ExoIII-deletion analysis of the 4-kbp fragment that contains the upstream enhancer (15) (Fig. 5) was performed as described in Promega Protocols and Applications Guide. Constructs B, C, D, and E are derived from construct A, in which the 4-kbp enhancer fragment is cloned in positive direction upstream of the 299-bp CPS promoter-containing fragment (-161 to +138). Constructs F and G are derived from a plasmid in which the 4-kbp enhancer fragment was cloned in the opposite direction upstream of the CPS promoter. Constructs H, I, and J are subclones derived from constructs D and F, and are cloned in positive direction upstream of the 299-bp CPS promoter-containing fragment.


Figure 5: Delineation of the far-upstream enhancer. Transient-transfection assays of constructs containing sequences derived from an exonuclease III library of the 4-kbp enhancer fragment, cloned upstream of the CPS promoter-containing fragment (construct 1 in Fig. 1). The open box at position A indicates the original 4-kbp enhancer fragment(15) . The ``RI'' indicates a landmark EcoRI site in the fragment, which is positioned 6 kbp upstream (-6 kbp) of the transcription start site in the CPS gene. On the left, size and position of the test sequences are represented relative to the 4-kbp fragment (fragment A). The middle and right panels represent the activities of the fragments shown on the left after transfection into FTO-2B hepatoma cells and into Rat-1 fibroblasts, respectively. The bars indicate the normalized specific luciferase activity (``Experimental Procedures'') relative to the activity obtained from the construct containing only the CPS promoter (-, construct 1 in Fig. 1), which is arbitrarily set at 1. The black bars indicate activities of cells grown in the absence of added hormones, gray bars in the presence of dexamethasone, and hatched bars, activities in the presence of dexamethasone, Bt(2)cAMP, and IBMX (see ``Experimental Procedures''). Error bars indicate the S.E. of at least three independent transfections. The hatched bars showing the activity of enhancer fragments A, B, C, D, and F in FTO-2B cells represent the mean of two independent transfections, with the error bar showing the variance. ND indicates values not determined.




Figure 1: Functional analysis of the CPS promoter. Transient transfection assays of CPS promoter deletion constructs in FTO-2B hepatoma cells and Rat-1 fibroblasts. Regions of the CPS promoter that were cloned upstream of the luciferase reporter gene are shown to the left. The TATA box (black box), GAG box, and sites III, II, and I of the CPS promoter (19) are indicated. The bottom-most structure, shown in gray, indicates the minimized TK promoter. The TATA box (rectangle) and the GC box of the TK promoter are given. The bars indicate the normalized specific luciferase activity (luciferase activity/CAT activity) relative to the activity obtained from the construct harboring the 161-bp CPS promoter, which was arbitrarily set at 1. The black bars indicate activities of cells grown in the absence of added hormones, while the hatched bars show activities in the presence of dexamethasone, Bt(2)cAMP, and IBMX (``Experimental Procedures''). Error bars indicate the S.E. of at least three independent transfections.



Site-directed Mutagenesis and Construction of Expression Plasmids

pRc/RSV-CREB (expression vector expressing CREB) and pRc/RSV-CBP (expression vector encoding full-length mouse CBP) were gifts from R. H. Goodman, 6RGR (expression vector encoding glucocorticoid receptor) was a gift from K. R. Yamamoto, and MT-CEValpha (expression vector encoding Calpha) was a gift from G. S. McKnight. Site-directed mutagenesis was performed using the ``megaprimer'' method (34) with modifications(35) .

All CPS promoter constructs are derived from the 299-bp PstIHindIII promoter-containing fragment (position -161 to +138, Fig. 1, HindIII site derived from polylinker of pBluescript SK+) described in (15) . To delete sites III and II from the CPS promoter, a 238-bp AflIII-HindIII fragment (position -100 to +138, Fig. 1) was cloned into pLT1. For deletion of sites III, II, and I, primer CAGCCCCTCCTCCCTCTAGAATGTCCAGAGATG (complementary to sense-strand) was used to create a XbaI site (underlined) at position -74 (Fig. 1). The resulting 212-bp XbaI-HindIII fragment was cloned into pLT1. To delete sites III, II, I and the GAG box, a 176-bp AccI-HindIII fragment (position -38 to +138, Fig. 1) was cloned into pLT1.

Deletions in the 5`-UTR of the mRNA were made by creating HindIII sites in the 5`-UTR at 50, 89, and 115 nt downstream of the transcription start site of construct 2 (see Fig. 1, construct 2) with oligonucleotides GGGAAGGAAAGCTTTGTGGAGAC, CATGAAAGCTTGTTGTCCAATTTGC and GTGACTAAGCTTAAATCACAAATATCTC, respectively (all complementary to the sense strand; HindIII sites are underlined). The T7 primer of pBluescript was used as complementary primer. All PCR products were sequenced. The putative CRE and GRE element in the 469-bp enhancer fragment (see Fig. 5, construct J) was mutated after subcloning into pBluescript SK+. Primer TTACTTTAGAATCATATTGAGGACTTATTA was used to disrupt the putative CRE and CATCAGAGAAGTTTGATCTGCTCAGCACAT to disrupt the putative GRE. Underlined nucleotides were substituted (see Fig. 6). The T3 and T7 primers were used to fill in both sides flanking the mutagenesis primer. The PCR products were sequenced in both directions.


Figure 6: Nucleotide sequence of fragment I (Fig. 5), containing the 469-bp enhancer fragment (fragment J). The landmark EcoRI site depicted in Fig. 5and the differentially methylated MspI/HpaII site (CCGG) at -6.3 kbp (Fig. 9) are underlined. The imperfect CRE (TGACGTCA, 148-155) and the three GRE half-sites (TGTTCT, 382-401) are double underlined. On top of these sites the substitutional mutations to inactivate the putative CRE and GRE are shown. The arrow indicates the position of the most 5` nt of fragment E of Fig. 5. Sites with similarity to binding sites of liver-enriched factors HNF3 (Ca/tAAa/gTCAATA), HNF4 (GGGCCANNNa/ga/gGTCCA), and HNF5 (Ta/gTTTGc/t) are given in italics.




Figure 9: Enhancer analysis in enterocytes. Transient-transfection assays of constructs containing regions upstream of the transcription start site of the CPS gene in Caco-2 cells. CPS-A and TK-A represent constructs in which a 4-kbp fragment (fragment A in Fig. 5) was cloned upstream of the CPS promoter (fragment 1 in Fig. 1) or the minimized TK promoter (-81 to +52), respectively. CPS-B and TK-B represent constructs in which a 4-kbp fragment containing the differentially methylated site at position -4.0 kbp (15) was cloned upstream of the 161-bp CPS promoter or the minimized TK promoter. Bars indicate the normalized CAT activity (Panel A) or luciferase activity (Panel B). Black bars show the activities of cells grown in the absence of added hormones and hatched bars the activities of cells grown in the presence of dexamethasone, Bt(2)cAMP, and IBMX. The values were normalized either to the activity of construct CPS-A in the absence of added hormones (Panel A) or to the activity of construct TK-A in the absence of added hormones (Panel B). Values are the mean of two independent transfections, error bars indicating the variance.



RNA Isolation and Quantification

5 times 10^6 FTO-2B hepatoma cells were transiently transfected with 30 µg of construct UTR 50, UTR 115, or UTR 138 as described and divided into equal parts. After 48 h the luciferase activity and protein content was determined in the cells of one well, while the cells of the other well were lysed in guanidinium isothiocyanate and loaded onto CsCl cushions for total RNA isolation(36) . In addition, the RNA samples were treated with 3 units of RNase-free DNase I (RQ1, Promega) for 1 h at 37 °C in the presence of 56 units of RNasin (Promega), extracted with phenol and chloroform, and precipitated two times in 2 M LiCl. The concentration of the RNA was determined at 260 nm and the integrity by formaldehyde-agarose gels. The concentration of luciferase-SV40 mRNA hybrids was determined by reverse transcriptase-PCR (avian myeloblastosis virus-reverse transcriptase, Promega), using a mimic DNA construct as internal standard(37) . The mimic construct contains identical SV40 sequences, but, in addition, a 129-bp unrelated fragment. The primers (SV40+, CTGTGGTGTGACATAATTGG; and SV40-, TACTAAACACAGCATGACTCA) recognize sites flanking the intron, leading to amplification of a 263-bp fragment from the mRNA, a 328-bp fragment from the unspliced RNA or from the plasmid DNA, and a 465-bp fragment from the mimic construct. In each reaction 1 µg of total RNA was used. The products of the RT-PCR reaction were separated on 3% Metaphor-agarose (FMC).

General Methods

Plasmid DNA isolation, PCR, subcloning, restriction analysis, and Western blot analysis were performed as described(32) . Oligonucleotides were obtained from Eurogentec, Seraing, Belgium. Double-stranded DNA was sequenced using the dideoxy procedure(38) .


RESULTS

Three regions in the CPS gene appeared to be involved in the regulation of expression, viz. the 161-bp proximal promoter, the 138-bp 5`-UTR of the mRNA, and the 4-kbp far-upstream enhancer fragment(15) . These regions were systematically investigated for their contribution to tissue specificity and response to physiologically relevant hormones.

The Proximal Promoter of the CPS Gene

Two approaches were used to investigate whether the 299-bp CPS promoter-containing fragment (from -161 to +138 relative to the transcription start site(15) ) confers tissue-specific expression. As a first approach, 5` sequences of the CPS promoter were deleted, and the resulting derivatives were tested in FTO-2B hepatoma cells and Rat-1 fibroblasts (Fig. 1). Elements upstream of the GAG box did not substantially alter reporter gene expression in either FTO-2B or Rat-1 cells. Deletion of the GAG box, however, decreased activity to 30-40%. In both cell lines the CPS promoter appeared to be sensitive to added hormones, possibly due to enhanced expression of general transcription factors. The minimal fully active CPS promoter was 50 times more active in FTO-2B cells than the minimized TK promoter.

Since the activity of the CPS promoter was similar in hepatoma and fibroblast cells (Fig. 1), this sequence probably does not confer tissue specificity. To establish this conclusion more firmly, luciferase activity per transfected cell was determined. This approach (Fig. 2) clearly demonstrated that the activity of the CPS promoter was quantitatively comparable in FTO-2B and Rat-1 cells and, hence, does not confer tissue specificity. The minimized TK promoter, containing only the proximal GC box and a TATA box, was less active in the hepatoma cell lines than in the fibroblasts. The GC box, target of the Sp1 protein and the main determinant of the strength of this promoter (28, 39) may therefore be hardly functional in hepatoma cell lines, in accordance with the relatively low concentrations of Sp1 in hepatocytes(40) .


Figure 2: Lack of tissue specificity of the CPS promoter. Comparison of the activity of the CPS promoter (left panel) and the minimized TK promoter (right panel) in FTO-2B hepatoma cells and Rat-1 fibroblasts. Transfection efficiency was determined by cotransfecting the expression vector pRSV-n-LacZ (``Experimental Procedures''). Bars indicate normalized luciferase activity (relative light units/mg of protein times fraction of blue cells times 10^6). Error bars indicate the S.E. of at least four independent transfections. Black bars represent the activities of FTO-2B cells, gray bars the activities of Rat-1 cells. *, p (two-tail) = 0.39, **, p (two-tail) = 0.03, using the Student's t test for independent samples.



The 5`-Untranslated Region

A promoter fragment lacking the 5`-untranslated region of the mRNA, showed a dramatically lower expression in transfection studies(15) . Several promoter fragments lacking parts of the 5`-UTR were therefore transfected into FTO-2B cells. Fig. 3A shows that the entire UTR was needed for high promoter activity. If only 23 nt were deleted (construct U 115), 80% of the activity was lost. In primary hepatocytes the activity of U 115 was 47 ± 5% (S.E., n = 5) of that of construct U 138, showing that the observation does not result from using established cell lines. The ratio between hormone-stimulated expression and basal expression is comparable for all constructs, indicating that the 5`-UTR is not involved in the hormonal sensitivity of promoter. The contribution of the 5`-UTR to tissue specificity was tested by transfection to Rat-1 fibroblasts and MH1C1 hepatoma cells (Table 1). Although promoter activity was most affected by the deletions of the 5`-UTR in FTO-2B, the effects were similar in MH1C1 and Rat-1, indicating that it plays no role in tissue specificity. To test whether the 5`-UTR is an independent stimulator of expression, it was cloned downstream of the minimized TK promoter. Fig. 3B shows that this fragment (+3 to +138) is not able to enhance expression in combination with this promoter. To discriminate between transcriptional and post-transcriptional effects on expression, the levels of RNA obtained from the constructs containing the full-length UTR and two deletion constructs were determined (Fig. 4). Whereas the relative concentrations of luciferase-SV40 mRNA hybrids were found to be similar, the full-length UTR gave a 4-5-fold higher expression than both deletion constructs. This result shows that the 5`-UTR affects expression at the post-transcriptional, and probably at the translational level.


Figure 3: Functional analysis of the 5`-UTR. A, deletion analysis of the stimulation of reporter gene expression by the 138-nt 5`-UTR. Constructs harboring parts of the 5`-UTR were transiently transfected in FTO-2B hepatoma cells. The bars indicate the normalized specific luciferase activity (``Experimental Procedures'') relative to the activity obtained from the construct containing the full-length 5`-UTR and 100 bp of the CPS promoter (UTR 138 = construct 2, Fig. 1), which is set to 1. Other constructs harbor the same upstream promoter sequences, but lack 5`-UTR regions +115 to +138 (U 115), +89 to +138 (U 89), +50 to +138 (U 50), and +7 to +138 (U 7) relative to the transcription start site. The small ORF (position 97-108) is present in constructs U 115 and U 138, and absent in the other constructs. Black bars indicate the activity of FTO-2B cells incubated in the absence of added hormones, hatched bars in the presence of dexamethasone, Bt(2)cAMP, and IBMX (``Experimental Procedures''). Error bars indicate the S.E. of at least three independent transfections. B, activity of the 5`-UTR of the CPS mRNA in the context of the minimized TK promoter (-81 to +52). The 5`-UTR (+3 to +138, black box) was cloned into the BglII site (position +52) of the TK promoter (gray) in both directions (arrows). The activity of the TK promoter was set to 1. Black bars indicate the activity in the absence of added hormones, hatched bars in the presence of dexamethasone, Bt(2)cAMP, and IBMX. Error bars indicate the S.E. of at least three independent transfections.






Figure 4: Luciferase mRNA levels produced by full-length and truncated 5`-UTR constructs in FTO-2B cells. Constructs U 138, U 115, and U 50 (Fig. 3) were transiently expressed in FTO-2B hepatoma cells and assayed by determination of luciferase-SV40 hybrid mRNA concentration (``Experimental Procedures''). Lanes 1, 4, 7, 13, and 19, U 138; lanes 2, 5, 8, 14, and 20, U 115; lanes 3, 6, 9, 15, and 21, U 50; lanes 11, 12, 17, and 18, rat liver total RNA; lanes 10 and 16, 10 fg of mimic DNA. Lanes 1, 2, and 3, 200 fg of mimic DNA; lanes 4, 5, and 6: 50 fg of mimic DNA; lanes 7, 8, and 9, 10 fg of mimic DNA. Lanes 16-21, without reverse transcriptase (RT).



Delineation of the Element That Confers Tissue Specificity and Hormone Responsiveness in the Far-upstream CPS-enhancer Fragment

A 4-kbp DNA fragment harboring sequences from -8 to -4 kbp relative to the transcription start site of the CPS gene was shown to be transcriptionally active in transient-transfection assays(15) . To delineate these cis-acting elements more precisely, the 4-kbp fragment was subjected to an ExoIII-deletion analysis. The fragments from the ExoIII library were tested in combination with the 161-bp CPS promoter (Fig. 5). None of the fragments caused stimulation of basal expression, but the response to both dexamethasone and the combination of dexamethasone and Bt(2)cAMP was found to be delimited by the 5` and 3` borders of constructs D and F, respectively (Fig. 5). This region alone (construct H in Fig. 5) was as active as either construct D or F. Further deletions confined the enhancer activity to a 469-bp fragment (construct J in Fig. 5). The differentially methylated CCGG (15) site is located within this fragment. A number of sites resembling known liver-enriched factor binding-sites (HNF3, -4, and -5) and hormone-responsive elements (CRE and GRE) were identified (Fig. 6).

Tissue Specificity of the Far-upstream Enhancer

The same enhancer fragments that caused hormonal induction of reporter gene expression in FTO-2B hepatomas, also stimulated hormone-dependent expression in Rat-1 fibroblasts (Fig. 5), but 6-fold less. This cell-specific difference in activation properties of the enhancer became more pronounced when the 469-bp enhancer fragment was tested in combination with the minimized TK promoter (Fig. 7). The expression obtained with this fragment in FTO-2B cells was 340-fold induced, compared to the TK promoter itself, while only a 12-fold induction was seen in Rat-1 cells, i.e. an almost 30-fold difference. These results show that the tissue specificity conferred by the enhancer is independent of the promoter. The strength of the 470-bp enhancer fragment was also tested in two hepatoma cell lines (FTO-2B and MH1C1) and two fibroblast lines (Rat-1 and CHO-K1) and compared to the endogenous CPS expression in these cell lines. A clear relation between the strength of the enhancer (Fig. 8A) and the level of expression of CPS protein in the different cell lines (Fig. 8B) was observed. The difference in the fold stimulation of the luciferase reporter gene and the endogenous CPS gene probably results from the difference in stability of both gene products.


Figure 7: Tissue specificity of the far-upstream enhancer. Transient-transfection assays of the construct containing the 469-bp enhancer fragment coupled to the minimized TK promoter in FTO-2B hepatoma cells and Rat-1 fibroblasts. Black bars represent the activity of cells incubated in the absence of added hormones, gray bars the activity in the presence of dexamethasone, and hatched bars the activity in the presence of dexamethasone, Bt(2)cAMP, and IBMX. The activities indicate the normalized specific luciferase activity relative to the activity obtained from the minimized TK promoter and are corrected for the effect of hormonal stimulation on the TK promoter without enhancer. Bars indicate the S.E. of four independent transfections.




Figure 8: Relation between expression of the endogenous CPS gene and the far-upstream enhancer activity. Panel A, transient transfection of the 469-bp far-upstream element (fragment J in Fig. 5) in combination with the CPS promoter (fragment 1 in Fig. 1) in Chinese hamster ovary cells, Rat-1 fibroblasts, and FTO-2B- and MH1C1-hepatoma cells. The bars indicate the normalized luciferase activity relative to the activity obtained from the basal CPS promoter without enhancer. The activities are corrected for the effect of added hormones on the basal CPS promoter without enhancer. Black bars indicate the activity of cells cultured without added hormones, hatched bars the activity of cells to which dexamethasone, Bt(2)cAMP, and IBMX were added. Error bars indicate the S.E. of at least three independent transfections. Panel B, Western blot analysis of CPS protein content in the respective cell lines in the absence(-) and the presence of dexamethasone, Bt(2)cAMP, and IBMX (+). In each lane 50 µg of total protein was applied.



To verify the validity of the use of hepatoma cells as a model for assessing tissue-specific expression, the enhancer was also tested in freshly isolated hepatocytes. In these cells the CPS promoter was stimulated 4.6-5.0-fold (n = 2) by dexamethasone, Bt(2)cAMP, and IBMX. When combined with the 469-bp enhancer, the expression was reduced to 27 ± 3% (n = 5) of that of the promoter alone in the absence of added hormones. In the presence of hormones, however, the expression was stimulated 63-79-fold (n = 2) compared to the promoter, the hormonal induction being more than 250-fold.

Expression in Intestinal Cells

The enhancer element at -6.3 kbp is selectively demethylated in CPS-expressing cells. To test whether sequences surrounding the site at -4.0 kbp which is partially demethylated in the small intestine only(15) , have enhancer properties in intestinal cell lines, they were tested in Caco-2 cells (Fig. 9). Only the -6.3 kbp site-containing fragment conferred enhanced expression in the presence of dexamethasone, Bt(2)cAMP, and IBMX, both in combination with the CPS promoter and with the minimized viral TK promoter fragment. Combined with the data from other cell lines, we conclude that only the fragment harboring the MspI site at -6.3 kbp has the capacity to enhance CPS gene expression.

Interaction between the Enhancer and the Promoter

In the presence of added hormones, the enhancer stimulates expression via the proximal promoter. To investigate which elements of the promoter mediate this bridging function, a series of CPS promoter deletions was tested in combination with the 1003-bp enhancer fragment (fragment H in Fig. 5) in the FTO-2B hepatoma cell line. Fig. 10shows that both the TATA box and the GAG box were needed for an optimal transactivation by enhancer elements. When the GAG box was deleted, transactivation decreased to 50% of that of the fully active promoter. Neither sites I-III nor the 5`-UTR are involved in the interaction between the promoter and the enhancer fragment. These effects became even more pronounced when the 469-bp fragment (fragment J in Fig. 5) was used. When tested in combination with the TK promoter (Fig. 10), the hormonal stimulation decreased to 25% of that of the fully active CPS promoter. Obviously, the GC box is not able to functionally replace the GAG box.


Figure 10: Delineation of promoter elements involved in relaying the activation signal of the enhancer. Hormonal inducibility of reporter gene expression by the far-upstream enhancer in combination with CPS promoter deletion constructs in FTO-2B hepatoma cells. The promoter fragments, shown in the left panel were coupled to the 1003-bp enhancer fragment (fragment H in Fig. 5) or the 469-bp enhancer fragment (fragment J in Fig. 5). The TATA box (black rectangle), GAG box, and sites I-III of the CPS promoter (19) are indicated. In gray the minimized TK promoter is indicated, with the TATA box (rectangle) and the GC box. Hormonal inducibility is expressed as the ratio of the activity in the presence of added hormones over the activity in absence of added hormones after correction for the effect of hormones on the promoter-deletion construct.



When the distance between the enhancer and the minimized TK promoter was decreased, luciferase expression levels increased strongly (Fig. 11). Such distance effects were virtually absent in combination with the CPS promoter ( Fig. 5and Fig. 11). Whether or not sites upstream of the TATA box of the CPS promoter are functioning as coupling elements between the enhancer and the promoter was tested. A promoter fragment in which all sites upstream of the TATA box were deleted, leaving only the TATA box and 5`-UTR (Fig. 1, construct 4) was coupled to either the 1003-bp enhancer fragment (H in Fig. 5) or the 469-bp fragment (J in Fig. 5) and transfected to FTO-2B cells (Fig. 11). The truncated promoter was more sensitive to increasing distance than the CPS promoter, but not as sensitive as the TK promoter. These results underline the role of the GAG box.


Figure 11: Effects of distance between promoter and enhancer on the effectiveness of the enhancer. The distance (bp) between the TATA box and the center of the 469-bp enhancer fragment is depicted on the horizontal axis and reporter gene activity on the vertical axis. FTO-2B hepatoma cells were incubated in the presence of the hormones dexamethasone, Bt(2)cAMP, and IBMX. All activities were normalized to the activity of the CPS promoter fragment which was set to 1. Circles (bullet) represent the activity of the CPS promoter containing elements I-III, the GAG box, and the TATA box (construct 1, Fig. 1). Triangles () represent the activity of the CPS promoter containing only the TATA box (construct 4 of Fig. 1) and squares () represent the activities of the minimized TK promoter (construct TK in Fig. 1). Promoters were combined with the 469-, 547-, 1003-bp, and the 2-kbp enhancer fragments (Fig. 5, fragments J, I, H, and D, respectively). The solid lines are curves proportional to (bp)^q. Theoretically, the exponent q is a measure for the probability of a DNA site to be in the vicinity of the second. Mathematically, for random distribution in three dimensions, q = -1.5 (the Gaussian limit(55) ). q (CPS) = -0.2, q (truncated CPS) = -1, q (TK) = -3.5.



Mutational Analysis of cis-Elements in the Enhancer

Expression of the CPS gene in vivo is regulated at the transcriptional level by both glucocorticosteroids and cyclic AMP(8, 12) . The activity of the upstream enhancer depends entirely on the presence of these hormones and was therefore investigated for the presence of hormone-responsive elements. The 469-bp enhancer contains sequences that qualify as a potential cAMP-responsive element (CRE, Fig. 6), the binding site for dimers of the CREB family, and a glucocorticosteroid-responsive element (GRE, Fig. 6). When 4 nt of the CRE were substituted (CREmut, Fig. 6), basal expression decreased 60% (Fig. 12A). By binding to the regulatory subunit of protein kinase A, cyclic AMP releases the active catalytic subunit (Calpha)(41) . Expression vectors for Calpha, CREB(51, 52) , and CBP (53, 54) were co-transfected with the wild-type enhancer or the CRE mutant. These additions resulted in a 2-fold increase of the expression signal of the wild-type enhancer, but the CRE mutant no longer conferred this effect (Fig. 12A). To functionally test the existence of a GRE, one nucleotide that has been shown to be essential for receptor binding (42) was substituted in each of the three putative GRE half-sites (Fig. 6). The activity of the mutated enhancer (GREmut) was decreased to 20% of that of the wild-type enhancer when tested in combination with an expression vector encoding the GR in the presence of dexamethasone (Fig. 12B). Interestingly, the fold-induction by dexamethasone of the CRE mutant was not affected.


Figure 12: Functional identification of hormone-responsive elements in the enhancer. Panel A, functional identification of putative CRE by transient transfection of the enhancer into FTO-2B hepatoma cells. Cells were cultured in the absence of added hormones. Black bars indicate the activities of the CPS promoter (construct 1, Fig. 1) combined with the 469-bp enhancer (wt) or the 469-bp enhancer containing a mutation in the putative CRE (CREmut). Hatched bars show activities in the presence of 4 µg each of the co-transfected expression vectors encoding Calpha of protein kinase A, CREB, and CBP. The activity of the CPS promoter was set to 1. Error bars represent the S.E. of at least three independent transfections. Panel B, functional identification of the putative GRE by transient transfections into FTO-2B hepatoma cells. Bars indicate the relative luciferase activity of the CPS promoter (construct 1, Fig. 1) in conjunction with the 469-bp enhancer (wt), carrying a mutation in the putative GRE site (GREmut) or the putative CRE site (CREmut), in the presence of 4 µg of a cotransfected expression vector encoding the glucocorticosteroid receptor. The black bars indicate the activity in the absence, and hatched bars in the presence of dexamethasone. The activity of the CPS promoter was set to 1. The activities of hormonally stimulated cells were corrected for the effect of added hormones on the CPS promoter without enhancer. The ratio of the activity in the presence of dexamethasone (hatched bars) over the activity in the absence of dexamethasone (black bars) is depicted above the bars. Error bars represent the S.E. of at least three independent transfections.




DISCUSSION

The aim of this study was the functional characterization of the regulatory regions of the CPS gene with respect to their role in tissue-specific expression and hormone sensitivity. Three regions were functionally analyzed. The CPS promoter, comprising a TATA box, a GAG box, and elements I-III, associated with the binding of presently unknown proteins(19) , the 138-bp 5`-untranslated region of the mRNA, and the far-upstream enhancer at -6.3 kbp.

Our data show that, in vitro, the CPS promoter has comparable strength in hepatoma cells, which do express CPS, and in fibroblasts, which do not express CPS, demonstrating that it is not involved in tissue-specific regulation of transcription. Furthermore, our findings clearly show that sites I, II, and III (-150 to -79 nt) are not essential for basal promoter activity or transduction of the hormonally induced activation signal from the enhancer. The GAG box, however, is quantitatively important because its absence reduces both the promoter activity and the effect of the enhancer on the promoter 2-3-fold. These data suggest that the protein(s) binding to the GAG box are involved in transducing the activation signal from the enhancer to the promoter as well as in the functioning of the promoter itself.

A promoter fragment lacking the 5`-UTR of CPS mRNA (+7 to +138) is hardly active in FTO-2B cells ( (15) and Fig. 3). Such a finding can be due to an incorrect mapping of the transcription start site, or to the involvement of downstream sequences in the basal transcription complex(15) . These possibilities can now be virtually ruled out because addition of up to 115 nt of the 5`-UTR downstream of the transcription start site does not restore expression levels to those obtained with the full-length 5`-UTR (Fig. 3A). When combined with the minimized TK promoter (Fig. 3B), the full-length 5`-leader of CPS mRNA inhibits, rather than stimulates expression. This result makes the presence of cis-elements in the 5`-UTR DNA, which, in combination with a promoter, are able to stimulate transcription, rather unlikely. Quantification of RNA levels (Fig. 4) shows that the 5`-UTR acts at the translational level. The 5`-UTR contains a small upstream open reading frame (uORF) of four codons (Met-Arg-Tyr-Leu) starting with the initiation codon at position 97 (position relative to the transcription start site) in a suboptimal context compared to the more downstream initiation codon of the CPS reading frame (50 and 70% similarity to the consensus sequence(43) , respectively), followed by three stop codons. uORFs are thought to be able to suppress translation of downstream cistrons(44, 45, 46) . Strikingly, other eukaryotic CPS genes of which the 5`-UTR sequences are known, the human CPS I gene(47) , the shark CPS III gene(48) , and the yeast CPA1 gene(46) , also contain one or more uORFs. The uORF of the CPA1 gene is known to suppress translation in a regulated manner. Constructs U 115 and U 138 (Fig. 3) both contain the uORF, but differ markedly in luciferase activity. Deletion of the 23 nt between the uORF and the luciferase start codon affects inter-cistronic length, the sequence context at the uORF stop codon, and secondary structure, parameters which were found to be important for translational control (49) .

Scanning sequences up to 12 kbp upstream and 4 kbp downstream of the promoter fragment, only one fragment located at 6 kbp from the transcription start site could be found that had the capacity to stimulate reporter gene expression in FTO-2B cells and Rat-1 fibroblasts(15) . This enhancer element has now been confined to a 469-bp fragment (Fig. 5). Transgenic mice, harboring the CPS promoter and 12-kbp upstream DNA in combination with the CAT reporter gene, give rise to hepatocyte-specific expression of CAT mRNA, which co-localizes with the endogenous CPS mRNA in the liver. Mice harboring only the proximal promoter show extremely weak CAT activity, which is not tissue-specific. (^2)Combination of in vivo and in vitro results suggests that the proximal promoter and the 469-bp far-upstream enhancer are both necessary and sufficient for tissue-specific CPS expression.

Sites homologous to an imperfect CRE, with the structural characteristics of a so called ``low affinity site'' (50) and a GRE were found to be essential elements in the minimal enhancer fragment (Fig. 12); in the absense of added hormones, the enhancer was inactive, while mutations of the CRE and GRE significantly decreased hormone-dependent enhancer activity. Expression of the construct carrying the CRE mutation was still responsive to glucocorticoids (Fig. 12B), indicating that the CRE and GRE are not functionally linked. On the other hand, when sequences upstream of position 339, including the CRE (Fig. 6) are deleted (construct E of Fig. 5), the enhancer looses all activity. The deleted area contains nearly perfect consensus sequences for HNF3 and, to a lesser extent, for HNF4.

In our transfection analysis the hormonal stimulation by the CPS enhancer was found to be independent of the distance between the CPS promoter and enhancer, whereas this distance was important when combining TK promoter and enhancer (Fig. 11). This promoter-specific effect is probably highly relevant in vivo, because the CPS promoter and the enhancer are approximately 6-kbp apart. One way to explain the differences is to hypothesize that transcriptional activity is related to the probability of the enhancer to contact the promoter. The curves, proportional to (bp)^q, represent this probability and exponent q the slope in a double logarithmic plot (Fig. 11). Mathematically, when the promoter and the enhancer are randomly distributed in space, q = -1.5 (the Gaussian limit(55) ). q approx 0 (CPS promoter) means that the probability is 1, i.e. that the promoter and the enhancer are connected independently of distance. q = -1 (truncated CPS promoter) approaches a random distribution and q = -3.5 (TK promoter) indicates hindrance of enhancer-promoter interaction. The main difference between the CPS and the truncated CPS promoter is the GAG box (see also Fig. 10), indicating that this motif is instrumental in conferring the hormonal activation of the enhancer to the transcriptional complex. The main difference between the minimized TK promoter and the truncated CPS promoter is the GC box. This GC box might be the actual cause of the distance dependence of the minimized TK promoter in conjunction with the CPS enhancer, possibly by preventing interaction of enhancer and promoter through steric hindrance.

In summary, our data support a model in which the tissue-specific expression of CPS is determined by the far-upstream enhancer. The activity of this enhancer is strictly dependent on the presence of glucocorticoids and cyclic AMP. The activated enhancer complex will stimulate transcription through interaction with factors bound to the GAG box and the TATA region.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X90476[GenBank].

§
To whom correspondence should be addressed. Tel.: 31-20-5664927; Fax: 31-20-6976177.

(^1)
The abbreviations used are: CPS, carbamoyl-phosphate synthetase I; kbp, kilobase pair(s); nt, nucleotides; FCS, fetal calf serum; UTR, untranslated region; TK, thymidine kinase; Bt(2)cAMP, dibutyryl-cyclic AMP; IBMX, 3-isobutyl-1-methylxanthine; (u)ORF, (upstream) open reading frame; CRE, cyclic AMP-responsive element; GRE, glucocorticoid-responsive element; bp, base pair(s); RSV, Rous sarcoma virus; CREB, cAMP response element-binding protein; RT-PCR, reverse transcriptase-polymerase chain reaction.

(^2)
V. M. Christoffels, P. A. J. de Boer, M. C. Lamers, A. F. M. Moorman, and W. H. Lamers, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. R. P. S. Kwok and Dr. R. H. Goodman for kindly providing the pRc/RSV-CREB and pRc/RSV-CBP expression vectors, Dr. K. R. Yamamoto for 6RGR and Dr. G. S. McKnight for MT-CEValpha. We also thank E. H. van Beers for the gift of Caco-2 cells. We are grateful to Carol Pocock-Verhoek for taking care of the cell lines and to Jitske Weegenaar for her contribution to the data presented.


REFERENCES

  1. Lamers, W. H., Janzen, J. W. G., te Kortschot, A., Charles, R., and Moorman, A. F. M. (1987) Differentiation 35,228-235 [Medline] [Order article via Infotrieve]
  2. Gaasbeek Janzen, J. W., Westenend, P. J., Charles, R., Lamers, W. H., and Moorman, A. F. M. (1988) J. Histochem. Cytochem. 36,1223-1230 [Abstract]
  3. Moorman, A. F. M., de Boer, P. A. J., Das, A. T., Labruyère, W. T., Charles, R., and Lamers, W. H. (1990) Histochem. J. 22,457-468 [Medline] [Order article via Infotrieve]
  4. Gaasbeek Janzen, J. W., Moorman, A. F. M., Lamers, W. H., and Charles, R. (1985) J. Histochem. Cytochem. 339,1205-1211
  5. Ryall, J. C., Quantz, M. A., and Shore, G. C. (1986) Eur. J. Biochem. 156,453-458 [Abstract]
  6. Lamers, W. H., and Mooren, P. G. (1981) Mech. Aging Dev. 15,93-118 [Medline] [Order article via Infotrieve]
  7. Lamers, W. H., Zonneveld, D., and Charles, R. (1984) Dev. Biol. 105,500-508 [Medline] [Order article via Infotrieve]
  8. Morris, S. M., Jr., Moncman, C. L., Rand, K. D., Dizikes, G. J., Cederbaum, S. D., and O'Brien, W. E. (1987) Arch. Biochem. Biophys. 256,343-353 [Medline] [Order article via Infotrieve]
  9. van Roon, M. A., Eier, W., Charles, R., and Lamers, W. H. (1989) Differentiation 41,139-147 [Medline] [Order article via Infotrieve]
  10. de Groot, C. J., Zonneveld, D., de Laaf, R. T. M., Dingemanse, M. A., Mooren, P. G., Moorman, A. F. M., Lamers, W. H., and Charles, R. (1986) Biochim. Biophys. Acta 866,61-67 [Medline] [Order article via Infotrieve]
  11. Moorman, A. F. M., de Boer, P. A. J., Charles, R., and Lamers, W. H. (1990) FEBS Lett. 276,9-13 [CrossRef][Medline] [Order article via Infotrieve]
  12. Morris, S. M. (1992) Ann. Rev. Nutr. 12,81-101 [CrossRef][Medline] [Order article via Infotrieve]
  13. Nyunoya, H., Broglie, K. E., Widgren, E. E., and Lusty, C. J. (1985) J. Biol. Chem. 260,9346-9356 [Abstract/Free Full Text]
  14. Lagacé, M., Howell, B. W., Burak, R., Lusty, C. J., and Shore, G. C. (1987) J. Biol. Chem. 262,10415-10418 [Abstract/Free Full Text]
  15. van den Hoff, M. J. B., van de Zande, L. P. W. G., Dingemanse, M. A., Das, A. T., Labruyere, W., Moorman, A. F. M., Charles, R., and Lamers, W. H. (1995) Eur. J. Biochem. 228,351-361 [Abstract]
  16. Adcock, M. W., and O'Brien, W. E. (1984) J. Biol. Chem. 259,13471-13476 [Abstract/Free Full Text]
  17. Lagacé, M., Goping, I. S., Müller, C. R., Lazzaro, M., and Shore, G. C. (1992) Gene (Amst.) 118,231-238 [Medline] [Order article via Infotrieve]
  18. Goping, I. S., Lagacé, M., and Shore, G. C. (1992) Gene (Amst.) 118,283-287 [Medline] [Order article via Infotrieve]
  19. Goping, I. S., and Shore, G. C. (1994) J. Biol. Chem. 269,3891-3896 [Abstract/Free Full Text]
  20. Killary, A. M., and Fournier, R. E. K. (1984) Cell 38,523-534 [Medline] [Order article via Infotrieve]
  21. Botchan, M., Topp, W., and Sambrook, J. (1976) Cell 9,269-287 [Medline] [Order article via Infotrieve]
  22. Topp, W. C. (1981) Virology 113,408-411 [CrossRef][Medline] [Order article via Infotrieve]
  23. Pinto, M., Robine-Leon, S., Appay, M. D., Kedinger, M., Triadou, N., Dussaulx, E., Lacroix, B., Simon-Assmann, P., Haffen, K., Fogh, J., and Zweibaum, A. (1983) Biol. Cell 47,323-330
  24. van den Hoff, M. J. B., Christoffels, V. M., Labruy è re, W. T., Moorman, A. F. M., and Lamers, W. H. (1995) in Methods in Molecular Biology (Nickoloff, J. A., ed) Humana Press Inc., Totowa, NJ
  25. de Wet, J. R., Wood, K. V., DeLuca, M., Helinski, D. R., and Subramani, S. (1987) Mol. Cell. Biol. 7,725-737 [Medline] [Order article via Infotrieve]
  26. van den Hoff, M. J. B., and Lamers, W. H. (1993) Nucleic Acids Res. 21,4987-4988 [Medline] [Order article via Infotrieve]
  27. Nordeen, S. K. (1988) BioTechniques 6,454-457 [Medline] [Order article via Infotrieve]
  28. McKnight, S. L., and Kingsbury, R. (1982) Science 217,316-324 [Medline] [Order article via Infotrieve]
  29. Gorman, C. M., Merlino, G. T., Willingham, M. C., Pastan, I., and Howard, B. H. (1982) Proc. Natl. Acad. Sci. U. S. A. 79,6777-6781 [Abstract]
  30. Seed, B., and Sheen, J.Y. (1988) Gene (Amst.) 67,271-277 [CrossRef][Medline] [Order article via Infotrieve]
  31. Brasier, A. R., Tate, J. E., and Habener, J. F. (1989) BioTechniques 7,1116-1122 [Medline] [Order article via Infotrieve]
  32. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  33. Blommaart, P. J. E., Zonneveld, D., Meijer, A. J., and Lamers, W. H. (1993) J. Biol. Chem. 268,1610-1617 [Abstract/Free Full Text]
  34. Sarkar, G., and Sommer, S. S. (1990) BioTechniques 8,404-407 [Medline] [Order article via Infotrieve]
  35. Kuipers, O. P., Boot, H. J., and de Vos, W. M. (1991) Nucleic Acids Res. 19,4558 [Medline] [Order article via Infotrieve]
  36. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18,5294-5299 [Medline] [Order article via Infotrieve]
  37. Cottrez, F., Auriault, C., Capron, A., and Groux, H. (1994) Nucleic Acids Res. 22,2712-2713 [Medline] [Order article via Infotrieve]
  38. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,5463-5467 [Abstract]
  39. Majumder, S., Miranda, M., and DePamphilis, M. L. (1993) EMBO J. 12,1131-1140 [Abstract]
  40. Saffer, J. D., Jackson, S. P., and Annarella, M. B. (1991) Mol. Cell. Biol. 11,2189-2199 [Medline] [Order article via Infotrieve]
  41. Lalli, E. and Sassone-Corsi, P. (1994) J. Biol. Chem. 269,17359-17362 [Free Full Text]
  42. Truss, M., and Beato, M. (1993) Endocr. Rev. 14,459-479 [Abstract]
  43. Kozak, M. (1987) Nucleic Acids Res. 15,8125-8148 [Abstract]
  44. Geballe, A. P., and Morris, D. R. (1994) Trends Biochem. Sci. 19,159-164 [CrossRef][Medline] [Order article via Infotrieve]
  45. Kozak, M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92,2662-2666 [Abstract]
  46. Werner, M., Feller, A., Messenguy, F., and Piérard, A. (1987) Cell 49,805-813 [Medline] [Order article via Infotrieve]
  47. Haraguchi, Y., Uchino, T., Takiguchi, M., Endo, F., Mori, M., and Matsuda, I. (1991) Gene (Amst.) 107,335-340 [Medline] [Order article via Infotrieve]
  48. Hong, J., Salo, W. L., Lusty, C. J., and Anderson, P. M. (1994) J. Mol. Biol. 243,131-140 [CrossRef][Medline] [Order article via Infotrieve]
  49. Kozak, M. (1992) Annu. Rev. Cell Biol. 8,197-225 [CrossRef]
  50. Nichols, M., Weih, F., Schmid, W., DeVack, C., Kowenz-Leutz, E., Luckow, B., Boshart, M., and Schütz, G. (1992) EMBO J. 11,3337-3346 [Abstract]
  51. Yamamoto, K. K., Gonzalez, G. A., Biggs, W. H., III, and Montminy, M. R. (1988) Nature 334,494-498 [CrossRef][Medline] [Order article via Infotrieve]
  52. Gonzalez, G. A., and Montminy, M. R. (1989) Cell 59,675-680 [Medline] [Order article via Infotrieve]
  53. Kwok, R. P. S., Lundblad, J. R., Chrivia, J. C., Richards, J. P., Bächinger, H. P., Brennan, R. G., Roberts, S. G. E., Green, M. R., and Goodman, R. H. (1994) Nature 370,223-226 [CrossRef][Medline] [Order article via Infotrieve]
  54. Arias, J., Alberts, A. S., Brindle, P., Claret, F. X., Smeal, T., Karin, M., Feramisco, J., and Montminy, M. (1994) Nature 370,226-229 [CrossRef][Medline] [Order article via Infotrieve]
  55. Mossing, M. C., and Record, M. T. (1986) Science 233,889-892 [Medline] [Order article via Infotrieve]

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