Tyrosine hydroxylase (TH) ()converts L-tyrosine to 3,4-dihydroxy-L-phenylalanine and is the first and rate-limiting enzyme in catecholamine synthesis (Nagatsu et al., 1964; Levitt et al., 1965). TH is expressed in specific cell types in the peripheral and central nervous systems. Sympathetic ganglia and chromaffin cells of the adrenal medulla are major sites of peripheral TH expression. In the central nervous system, TH-expressing neurons are located in the diencephalon, midbrain, brainstem, olfactory bulb, and retina (Bjorklund and Lindvall, 1984).

TH activity is regulated at the protein level and the RNA level. Activation at the protein level is short term with a time course of less than 1 h and mainly occurs via phosphorylation of preexisting protein molecules, which increases TH activity (reviewed by Zigmond et al.(1989)). Induction of TH at the mRNA level is long term, resulting in increased mRNA levels for hours to days. Long term increases of TH activity are induced by various physiological stimuli including cyclic AMP (cAMP), epidermal growth factor, glucocorticoids, nerve growth factor, transsynaptic neuronal activity, and depolarization. These inducers have been shown to increase TH mRNA levels, and several studies indicate that they increase mRNA levels by inducing transcription: cAMP (Lewis et al., 1983, 1987; Tank et al., 1986, 1990; Fader and Lewis, 1990; Carroll et al., 1991; Fossom et al., 1992; Fung et al., 1992; Kim et al., 1993a), epidermal growth factor (Lewis and Chikaraishi, 1987), glucocorticoids (Lewis et al., 1983, 1987; Tank et al., 1986, 1990), nerve growth factor (Gizang-Ginsberg and Ziff, 1990), and transsynaptic neuronal activity and depolarization (Hefti et al., 1982; Black et al., 1985; Tank et al., 1985; Faucon-Biquet et al., 1989; Erlich et al., 1990; Kilbourne and Sabban 1990, 1992; Banerjee et al., 1992). Investigation of the DNA regulatory elements that direct basal and induced TH transcription has been of interest because of the role TH has in the long term regulation of catecholamine biosynthesis.

The 5`-flanking sequence of the rat TH gene contains several sequences that bear homology to cis-acting regulatory elements, including the AP2, AP1, E box (E2A/Myo D), octamer/POU, heptamer, Sp1, cAMP response element (CRE), and TATA box found in other genes. The AP1, octamer, Sp1, and CRE sites and their relative positions from the RNA initiation site are highly conserved in the rat, human, bovine, and mouse TH genes, suggesting that they may be important for transcriptional regulation (Harrington et al., 1987; Coker et al., 1988; Kobayashi et al., 1988; Cambi et al., 1989; D'Mello et al., 1989; Iwata et al., 1992). In peripheral nervous system-derived cell lines that express TH (PC12, PC8b, and SK-N-BE(2)C), the CRE confers cAMP responsiveness (Fader and Lewis, 1990; Fung et al., 1992; Kim et al., 1993a); the CRE also mediates induction by depolarization in PC12 cells (Kilbourne et al., 1992). The AP1 site partially mediates nerve growth factor induction in PC12 cells (Gizang-Ginsberg and Ziff, 1990) and may confer cAMP responsiveness in PC8b cells (Fung et al., 1992). Previous studies of basal and induced TH transcription have been limited to these peripheral nervous system-derived cells because of the lack of TH-expressing central nervous system cell lines. However, it is also of interest to examine how central nervous system catecholaminergic cells regulate TH transcription.

Recently, a mouse catecholaminergic central nervous system cell line, CATH.a, was derived from a TH-expressing brainstem tumor in a transgenic mouse carrying the SV40 T antigen oncogene under the transcriptional control of rat TH 5`-flanking DNA (Suri et al., 1993). CATH.a cells are morphologically undifferentiated under normal culture conditions; they grow in clumps, most are round, and most lack significant processes. Nevertheless, they exhibit differentiated neuronal characteristics. They express neurofilament proteins, the intermediate filaments specifically expressed in neurons, but do not express glial fibrillary acidic protein, characteristic of glial cells. CATH.a cells express synaptophysin, an integral membrane protein of synaptic vesicles, dopamine -hydroxylase, and TH. They synthesize, secrete, and accumulate high levels of dopamine and norepinephrine (Suri et al., 1993). CATH.a cells express voltage-gated tetrodotoxin-sensitive sodium currents and high voltage activated calcium currents similar to those reported in other neurons (Lazaroff, et al., 1992). In total, these data suggest that CATH.a cells are immortalized derivatives of central nervous system noradrenergic neurons. Two other TH-expressing cell lines, CATH.b and PATH.2, were derived from brainstem and adrenal tumors, respectively, from different mice. CATH.b arose from the same transgenic lineage as CATH.a, while PATH.2 was derived from an independent lineage. PATH.2 cells express neurofilament protein, synaptophysin, and TH, and they synthesize dopamine and norepinephrine (Suri et al., 1993). CATH.b cells express TH ()but otherwise have not yet been characterized.

In this study we investigated transcriptional regulation of the rat TH gene in the CATH.a, CATH.b, and PATH.2 cell lines by examining expression of a transiently transfected chloramphenicol acetyltransferase (CAT) reporter gene under the transcriptional control of TH 5`-flanking DNA. Deletion analysis suggests that upstream regions between -4.8 and -0.272 kilobase pairs (kbp) are not necessary for expression in these cells. Mutational analysis of various conserved sites within 0.272 kbp indicates that the CRE is critical for TH transcription in both the central nervous system-derived and the peripheral nervous system-derived cells. Expression of a dominant negative mutant CREB protein, KCREB, decreased CAT expression, indicating that a CRE-binding protein (CREB, ATF-1, and/or CREM) promotes transcription. The AP1 (A) and an overlapping dyad (D) symmetry element whose core is an E box site, both of which are also located within 0.272 kbp, appear to have a less important role in CATH and PATH cells. The primacy of the CRE is further supported by experiments in which minimal enhancer constructs containing the CRE, AP1, and/or dyad/E box elements directed reporter expression. The CRE supported full expression, comparable to that of the intact -0.272-kbp region. These findings contrast our previous work in PC8b cells (a TH-expressing PC12 pheochromocytoma subclone; Tank et al. (1990)), where the AP1 and dyad/E box sites are primarily responsible for TH transcription and the CRE has a less important role (Cambi et al., 1989; Fung et al., 1992; Yoon and Chikaraishi, 1992). Therefore, TH transcription may require the CRE, AP1, and dyad/E box elements to varying extents in different cells.

MATERIALS AND METHODS

Cell Culture

DNA Constructs

Figure 1: DNA elements critical for TH transcription in CATH.a, CATH.b, and PATH.2 cells reside within 0.272 kbp of TH 5`-flanking DNA. A, diagram of the deletion constructs from the rat TH 5`-flanking region. Cells were transiently transfected with constructs containing the CAT reporter gene under the transcriptional control of various lengths of TH 5`-flanking DNA. Normalized CAT activities are expressed as a percentage of that obtained with -0.272 THCAT from the same experiment in CATH.a cells (B), CATH.b cells (C), and PATH.2 cells (D). The number of transfected plates (n) is indicated above each bar. Normalized percent conversion values for -0.272 THCAT in CATH.a cells were 27.1 ± 2.8 (n = 8), 56.5 ± 1.0 (n = 4), and 106.7 ± 8.9 (n = 2) in three different experiments; in CATH.b cells, 11.1 ± 1.2 (n = 4) and 2.8 ± 0.3 (n = 6) in two different experiments; and in PATH 2 cells, 8.1 ± 0.2 (n = 2) and 8.6 ± 0.7 (n = 6) in two different experiments.

Figure 2: The CRE is critical for TH transcription in CATH.a, CATH.b, and PATH.2 cells. A, schematic diagram showing the upstream region of the rat TH gene and the positions of enhancer motifs. Site-specific mutants of AP1, dyad/E box, octamer, heptamer, Sp1, and CRE sites within the -0.272 THCAT construct were generated previously (Yoon and Chikaraishi, 1992). The wild type and mutated sequences (underlined, bold) are indicated. Normalized CAT activity in transfected CATH.a cells (B), CATH.b cells (C), and PATH.2 cells (D) is expressed as described in Fig. 1. E, normalized CAT activity in CATH.a cells transfected with wild type -0.272 THCAT and -4.8 THCAT and their corresponding mutant CRE constructs, -0.272 CRE and -4.8 CRE. For all experiments, each DNA precipitate was tested in duplicate plates. For CATH.a cells, at least two different DNA preparations of each construct were tested except Sp1 and -4.8 CRE; different DNA preparations of any one construct gave similar results. The number of transfected plates (n) is indicated above each bar. The normalized values for -0.272 THCAT were as follows: CATH.a cells in panelB, 23.9 ± 0.9 (n = 6), 30.4 ± 2.9 (n = 6), 69.4 ± 0.8 (n = 2), 59.0 ± 4.0 (n = 4), and 43.4 ± 3.2 (n = 4) in five different experiments; CATH.a cells in panelE, 55.0 ± 0.3 (n = 2), 69.4 ± 0.8 (n = 2), 59.0 ± 4.0 (n = 4), and 43.4 ± 3.2 (n = 4) in four different experiments; CATH.b cells in panelC, 5.8 ± 1.2 (n = 6) and 6.0 ± 2.0 (n = 4) in two different experiments; and PATH.2 cells in panelD, 0.6 ± 0.1 (n = 4) and 5.4 ± 1.2 (n = 4) in two different experiments.

Figure 4: The CRE minimal enhancer construct supports full CAT expression. A, the enhancerless THCAT construct is shown at the top. The TATA box is boxed. Underlined nucleotides comprise the partial CRE. At the bottom, the linker is shown. Nucleotides shown in bold are authentic TH sequences, and those shown in regulartype are linker sequences. Minimal enhancer constructs containing synthetic oligonucleotides corresponding to the CRE, AP1, and dyad/E box in various combinations in front of the TH promoter in the THCAT construct are shown. The synthetic CRE, AP1, and dyad/E box elements were inserted into restriction sites of the polylinker of the THCAT construct. B, normalized CAT activity in CATH.a cells transiently transfected with the constructs shown in A is expressed as in previous figures. C, normalized CAT activity in CATH.a cells transiently transfected with THCAT and minimal enhancer constructs lacking the partial CRE. For all of the constructs in C, the partial CRE was mutated to 5`-GACAATT-3`, as shown in parentheses under the partial CRE sequence to give -38 THCAT. HIII is HindIII. Normalized values for -0.272 THCAT for CATH.a cells in B were 17.7 ± 1.4 (n = 4), 55.0 ± 0.2 (n = 2), and 43.4 ± 3.2 (n = 4) in three different experiments and for CATH.a cells in C were 55.0 ± 0.2 (n = 2) and 58.9 ± 4.0 (n = 4) in two different experiments. For all experiments, each DNA precipitate was tested in duplicate plates. At least two different DNA preparations of each construct were tested; different DNA preparations of any one construct gave similar results.

Figure 5: The AP1 and dyad/E box can direct THCAT transcription when located close to the TH promoter but not when located at -205/-182 bp; the CRE can direct THCAT transcription when located close to the promoter and when it is moved to the AP1 site. A, diagram of the AD THCAT minimal enhancer construct, -0.095/-0.072 AD THCAT, -0.272 CRE (CRE mutant -0.272 THCAT), -0.272 A>C, and wild type -0.272 THCAT constructs. Distances from the TH promoter of the AP1, dyad/E box, and CRE sites are indicated underneath in parentheses. Boxed regions represent authentic TH sequences, and dashedlines represent polylinker sequences. B, normalized CAT activity in CATH.a cells transiently transfected with constructs shown in A is expressed as in previous figures. -0.272 CRE data is taken from Fig. 2B. The normalized value for -0.272 THCAT in CATH.a cells was 125.9 ± 5.3 (n = 4).

Transfections

CAT Assays and -Galactosidase Assays

-Galactosidase activity was determined by incubating 50 µg of protein lysate in 0.1 M sodium phosphate (pH 7.0), 10 mM KCl, 1 mM MgSO, 83 mM -mercaptoethanol, and 2.2 mMo-nitrophenyl--D-galactopyranoside (Sigma) at 22 °C. The colorimetric reaction was measured (A) after 1.5-5 h for CATH.a cells and 6-24 h for CATH.b and PATH.2 cells when RSVgal served as the internal control for transfection efficiency. The colorimetric reaction was measured after 20-24 h for CATH.a cells when SV2gal served as the internal control for transfection efficiency.

For each transfected plate of cells, CAT activity was expressed as the percentage of [C]chloramphenicol converted to acetylated forms per µg of protein lysate per hour, divided by the measure of -galactosidase enzyme activity (A) per µg of protein lysate per hour to normalize for differences in transfection efficiency. CAT activities have been corrected for the molarity of the given construct, since the length of the 5` region used varied. In all figures normalized CAT activities are expressed as a percentage of that obtained with -0.272 THCAT from the same experiment except for Fig. 7, where normalization was to -4.8 THCAT. For all experiments, each DNA precipitate was tested in duplicate plates. At least two different DNA preparations of each construct were tested except where noted; different DNA preparations of any one construct gave similar results.

Figure 7: KCREB reduces basal and cAMP-induced transcription. CATH.a cells were transiently transfected with 0, 3, or 6 µg of RSV-KCREB (as indicated on y axis), 2 µg of -4.8 THCAT, 2 µg of SV2gal, and 6, 3, or 0 µg of pGEM-1 to bring the total DNA amount to 10 µg/plate. Cells were treated with or without 1 mM dibutyryl cAMP for 18-24 h. Normalized CAT expression is expressed as in previous figures. The normalized values for basal -4.8 THCAT transfected without RSV-KCREB were 209.3 ± 29.3 (n = 2) and 66.2 ± 7.1 (n = 8) in two different experiments.

Different DNA preparations of the same construct gave essentially the same percent conversion in a given experiment. However, there were differences between the absolute values of normalized CAT activities from different experiments, which probably reflect differences in the state of the cells at the time of transfections. For example, in one particular experiment with CATH.a cells, four different preparations of -0.272 THCAT DNA gave CAT activities of 27.1 ± 2.8%. In a second experiment, two of the same DNA preparations were transfected and gave CAT activities of 56.5 ± 1%. The reason for such variability is unclear but may be due to unidentified microenvironmental cues and/or variable culture conditions. Despite this variability, the relative ratios of CAT expression among the various THCAT constructs and the enhancerless negative control THCAT construct were the same in all experiments.

RNase Protection Assay

RESULTS

Deletion Analysis

The amount of CAT activity obtained from CATH.a cells was usually about 5-50-fold higher than from CATH.b and PATH.2 cells. These differences correlate with higher endogenous TH levels expressed in CATH.a cells compared to CATH.b and PATH.2 cells. Western blots and immunohistochemistry indicate that CATH.a cells express much more TH than do CATH.b and PATH.2 cells. In addition, TH activity is 5-fold higher in CATH.a cells than in PATH.2 cells, and Northern analysis of TH mRNA indicates that CATH.a cells express more TH than do PATH.2 cells (Suri et al., 1993).

Site-directed Mutation Analysis

-0.272 THCAT constructs bearing mutations at the AP1, dyad/E box (includes the E box and the 20-bp dyad symmetry element in which it lies), octamer/POU, heptamer, Sp1, and CRE sites (Fig. 2A) were transfected into CATH.a, CATH.b, and PATH.2 cells. In all three lines, mutation of the CRE diminished transcriptional activity 80-90% (Fig. 2, B-D). In addition, a -4.8 THCAT construct in which the CRE was mutated, -4.8 CRE, diminished CAT activity to near background levels in all three lines (CATH.a data shown in Fig. 2E; CATH.b and PATH.2 data not shown). These results demonstrate that the CRE is the crucial site mediating expression in the CATH.a, CATH.b, and PATH.2 cell lines. In contrast, mutations of the AP1 and dyad 4/E box (right half of the dyad/E box) only modestly reduced CAT activity (20-40%), while dyad 3/E box mutations (left half of the dyad/E box) had no effect (Fig. 2, B-D). Therefore, the AP1 and dyad/E box sites have significantly less enhancer activity than the CRE under basal conditions (with no inducers in the culture medium). This is in contrast to the situation in PC8b cells where the AP1 and dyad/E box are critical for expression and the CRE is less important; CAT expression was reduced 95% by mutation of the AP1 and 65-85% by mutation of the dyad, whereas CAT expression was reduced 50% by mutation of the CRE in PC8b cells (Yoon and Chikaraishi, 1992).

Mutations of the octamer and Sp1 sites in the -0.272 THCAT construct gave 35-45% less CAT expression than did wild type -0.272 THCAT in the CATH.b and PATH.2 cells (Fig. 2, C and D). These decreases in expression were not further investigated. It is possible that these sites, as well as the AP1 and dyad/E box sites, have a more prominent role than seen here in regulating TH expression in response to various physiological stimuli or during certain stages of development.

In CATH.b and PATH.2 cells, mutation of the heptamer site in the -0.272 THCAT construct gave more CAT expression than did the wild-type -0.272 THCAT (Fig. 2, C and D), suggesting that in some neuronal cells, the heptamer may repress expression, perhaps via Oct-2 (see Dawson et al.(1994)). A similar result was obtained in PC8b cells (Yoon and Chikaraishi, 1992).

RNase protections confirmed that transcription in CATH.a cells was initiated at the correct start site for the wild type, mutant AP1, and mutant dyad/E box -0.272 THCAT constructs. Consistent with CAT activity measurements, no correctly initiated CAT RNA could be detected from the mutant CRE -0.272 THCAT construct (Fig. 3, A and B). RNA from transfected cells was hybridized to a 397-nt probe corresponding to -109 to +274 nt of THCAT plus 14 nt of vector sequence. Hybridization of correctly initiated transcripts generates a 274-nt fragment, which was observed with RNA from cells transfected with wild type, mutant AP1, and mutant dyad/E box -0.272 THCAT constructs. In contrast, a 274-nt protected fragment was not obtained with RNA from cells transfected with the mutant CRE construct (even at longer exposure times; data not shown), indicating that with this construct, transcription was not correctly initiated. These results support the contention that the CRE is critical for TH transcription, but the AP1 and dyad/E box sites are not.

Figure 3: Correctly initiated transcription is directed by wild type, mutant AP1, and mutant dyad/E box -0.272 THCAT constructs but not the mutant CRE -0.272 THCAT construct. RNA from transfected CATH.a cells was hybridized to a 397-nt probe corresponding to -109 to +274 of THCAT plus 14 nt of vector sequence followed by treatment with RNase A and RNase T1. A, schematic diagram of the probe, correctly initiated RNA, and read-through RNA. Thinlines below represent the size of the RNA probe protected by hybridization and correspond to the bands seen on the gel. B, RNase protections with RNA from CATH.a cells transfected with the wild type and mutant AP1, dyad/E box, and CRE -0.272 THCAT constructs. The 274-nt protected band marked by an arrow indicates correctly initiated RNA. The other bands marked by asterisks represent predicted read-through transcription shown in A. Negative control lanes include RNA from CATH.a cells not transfected and E. coli tRNA lanes. This experiment was repeated twice with different preparations of transfected cell RNA.

Additional protected fragments are likely due to read-through transcripts that originated from incorrect transcription start sites, some of which may be in the vector, as suggested by Gizang-Ginsberg and Ziff(1990) and Fung et al.,(1992). This is supported by the fact that protected fragment *1* is approximately 383 nt (Fig. 3B), the size predicted from hybridization of the 397-nt probe to a transcript that initiates from an incorrect transcription start site located upstream of -109. Since the 397-nt probe contains 14 nt of vector sequence that do not hybridize to the transcript, this leaves 383 nt of THCAT sequence in the 383-nt protected fragment.

Protected fragment *2* is 311 nt (Fig. 3B); this is the size predicted to result from hybridization of the probe to a transcript that initiates from an incorrect transcription start site located upstream of -109 and that contains a mutated CRE. Since the CRE located at -45 to -38 nt is mutated and does not hybridize to the probe, this region is digested and two smaller read-through fragments (311 and 63 nt) are generated rather than the larger 383-nt protected fragment. Only the 311-nt protected fragment is detected in Fig. 3B, because the 63-nt protected fragment was run off the gel. The origin of the 290-300-nt band is uncertain; it may be due to incomplete RNase digest of correctly initiated transcripts hybridized to the probe.

Similar read-through transcripts were detected by Fung et al.(1992) in PC8b cells transfected with various THCAT constructs. It is likely that the larger read-through transcripts are not translated into functional CAT protein because of translational stop signals upstream of the +1 site. Evidence for this is provided by Fung et al.(1992), where it was shown that the enhancerless -44 THCAT construct generates similar read-through products that do not give high levels of CAT activity in transfected PC8b cells.

Minimal Enhancer Construct Analysis

The synthetic enhancer constructs used in Fig. 4B contained a partial CRE at -44 to -38 as part of the -44 to +27 THCAT promoter region. The partial CRE, 5`-GACGTCA-3`, is shown underlined in Fig. 4A; it contains 7 out of 8 bp of the consensus CRE, 5`-TGACGTCA-3`. Given the low level of CAT expressed by the enhancerless THCAT construct, the partial CRE contributes little enhancing activity by itself under basal conditions (Fig. 4B). In fact, CAT activity obtained with THCAT was less than that obtained with pUCCAT, an enhancerless and promoterless construct (data not shown). In addition, the THCAT construct containing the partial CRE is not cAMP-responsive (Fig. 6, A and B). Nevertheless, it was of concern that this partial CRE might interact or synergize with synthetic enhancer sites. Hence a second set of minimal enhancer constructs was prepared in which the partial CRE was mutated to give -38 THCAT (see base pairs in parentheses in Fig. 4A). CATH.a cells transfected with these constructs gave similar results (Fig. 4C) to those obtained with constructs containing the partial CRE (Fig. 4B). The CRE increased expression to 77% of -0.272 THCAT. Interestingly, AD THCAT, lacking the partial CRE, gave about 65% activity, which was statistically similar to the C THCAT value. As in PC8b cells, the AP1 and dyad/E box sites work synergistically to direct transcription, since the AP1 and dyad/E box alone had little or no enhancing activity.

Figure 6: The TH CRE is cAMP-responsive. CATH.a cells were transiently transfected with the indicated constructs and treated with or without 1 mM dibutyryl cAMP for 12-24 h. A, normalized CAT expression from various deletion and CRE minimal enhancer THCAT constructs. B, normalized CAT expression from mutants in -0.272 THCAT. Normalized values for basal -0.272 THCAT were 59.0 ± 6.2 (n = 4), 69.4 ± 7.9 (n = 2), and 43.3 ± 3.6 (n = 4) in three different experiments. Hatchedbox, basal; blackbox, 1 mM dibutyryl cAMP.

Distance Analysis

Alternatively, it is possible that in CATH.a cells, only enhancers that are relatively close to the TH promoter (e.g. within 100 bp) function well. In this case, the native CRE site at -45 bp would be fortuitously positioned within this region. To test this possibility, the AP1 site at -205 was mutated to a CRE site and the native CRE site at -45 was eliminated, essentially moving the CRE from -45 to -206 bp. This construct (-0.272 A>C) gave 90% the level of wild type -0.272 THCAT (Fig. 5, A and B), demonstrating that the CRE can effectively function at a more distant position, whereas the AP1 element cannot.

cAMP Induction

Dominant Negative Mutant CREB Analysis

To assess this, we transfected CATH.a cells with RSV-KCREB (killer CREB), which encodes a CREB point mutant that destroys DNA binding but not dimerization. Hence, KCREB functions as a dominant negative mutant by dimerizing with endogenous CREB, ATF-1, and CREM, preventing them from binding to DNA and thereby blocking their activation of transcription (Walton et al., 1992). If these proteins were responsible for activation, cotransfected KCREB should decrease -4.8 THCAT transcription. As shown in Fig. 7, KCREB decreased basal and cAMP-induced -4.8 THCAT expression 50-60%, suggesting that CREB, ATF-1, and/or CREM proteins contribute to basal and cAMP-induced TH transcription. KCREB reduced -0.272 THCAT expression similarly (data not shown).

DISCUSSION

We investigated transcriptional regulation of the rat TH gene in TH-expressing peripheral nervous system and central nervous system cell lines derived from transgenic mice bearing TH-expressing tumors. PATH.2 cells are peripheral nervous system-derived; CATH.a and CATH.b cells are central nervous system-derived, and are the first catecholaminergic central nervous system cell lines used to map TH regulatory elements. Deletional analysis performed with THCAT constructs containing various lengths of 5`-TH-flanking DNA suggests that regions within 0.272 kbp of the transcription start site are sufficient and necessary for TH expression (Fig. 1, A-D). Site-directed mutagenesis of the CRE (5`-TGACGTCA-3`), located at -45 to -38 bp, diminished CAT activity to near background levels. Site-directed mutagenesis of the AP1 (5`-TGATTCA-3`) and the partially overlapping dyad/E box sites (5`-TGATTCAGAGGCAGGTGCCTGTGA-3`), located at -205 to -182 bp, reduced CAT activity between 20 and 40% (Fig. 2, A-E). These results suggest that the CRE is critical for TH transcription in these cells, whereas the AP1 and dyad/E box sites have a less significant role. In CATH.a cells, a minimal enhancer construct, consisting of one copy of the CRE inserted in front of the TH promoter, gave 80-120% of -0.272 THCAT expression. The AP1 minimal enhancer construct increased CAT activity slightly (15-20%), but together with the dyad/E box increased CAT activity to 45-80% of wild type levels (Fig. 4, A-C, and 5, A and B). The dyad by itself had no enhancer activity. Therefore, the results of deletion analysis, site-directed mutations, and synthetic enhancer constructs demonstrate the prime importance of the CRE for TH transcription in these cells, whereas the AP1 and dyad play a lesser role.

Kim et al. (1993a), in a previous study using PC12 (a rat pheochromocytoma line) and SK-N-BE(2)C (a human peripheral nervous system neuroblastoma line), obtained similar results. They showed that mutation of the rat TH CRE site abolished expression, whereas deletion of 5` sequences containing the AP1 and dyad/E box sites reduced expression by 40%. Thus, it appears that the CRE is essential for TH expression in both central nervous system-derived CATH.a and CATH.b cells and peripheral nervous system-derived PATH.2, PC12, and SK-N-BE(2)C cells, whereas the AP1 and dyad/E box sites are less important. These findings contrast those obtained in PC8b cells, a subclone of PC12 cells, where mutations of the AP1 diminished expression by 95% and mutations at the dyad/E box reduced expression by 65-80%, suggesting that these sites were more critical than the CRE, whose mutation reduced expression by 50% (Yoon and Chikaraishi, 1992). As in the CATH and PATH lines, the AP1 and dyad elements had little or no enhancer activity by themselves and needed to work together to support transcription in PC8b cells. In summary, the same elements, AP1, dyad/E box, and CRE, seem to be important in a variety of TH+ cell lines including CATH.a, CATH.b, PATH.2, PC12, SK-N-BE(2)C, and PC8b. However, the relative contribution of each element to TH transcriptional activation varies among lines.

A region between -503 and -578 bp has also been shown to direct rat TH expression in another PC12 line (Gandelman et al., 1990; Wong et al., 1994). Deletions that lacked the -503/-578 bp region but retained the CRE reduced expression by 66%. A deletion construct containing only the CRE gave very low expression, suggesting that the CRE is not important or only works in conjunction with the -503/-578 bp region. Differences between these data and those of Kim et al. (1993a) may reflect differences among the PC12 cells carried in different laboratories.

In CATH.a cells, the AD (AP1 and dyad/E box) element cannot support transcription when located at its native position (-205 bp) as demonstrated by the low CAT activity obtained from the -0.272 CRE and -4.8 CRE constructs (Fig. 2, A-D, and 5, B). However, restoration (45-80%) of CAT activity was obtained with three AD minimal enhancer constructs; two constructs positioned the AD element at -91 bp surrounded by linker sequences, and the other placed the AD at -95 bp surrounded by native TH sequences (Fig. 4, A-C, and 5, A and B). This suggests two possibilities. The first is that position itself is important such that only enhancers relatively close to the TH promoter function efficiently in CATH.a cells. The fact that a single copy of the CRE gives full expression when positioned at -206 bp (Fig. 5, A and B) would argue against this, although it is possible that only the AD and not the CRE is subject to position dependence. Alternatively, TH DNA sequences between -160 and -46 bp, which are present in the -0.272 CRE and -4.8 CRE but not in the AD THCAT minimal enhancer constructs, may contain repressor elements that prevent AD function. Since the AD is able to direct transcription in PC8b cells when positioned at -205 bp, as well as when located close to the TH promoter (Yoon and Chikaraishi, 1992), the putative position or repressor effect may be cell line-specific. Importantly, the finding that the AD can efficiently support reporter expression in CATH.a cells suggests that differences between various cell lines may not simply be due to the absence of factors that can activate at the AP1, dyad/E box, or CRE sites.

It is possible that, in vivo, different populations of TH-expressing cells or cells at certain stages of development differentially rely on the CRE, AP1, and dyad/E box, or other sites for TH transcription; this would allow for finer regulation among various TH-expressing populations. An analysis of the regulation of TH transcription in various cell groups and during development requires studies in transgenic mice, similar to those performed in cultured cells. At present, transgenic studies indicate that the regulation of TH transcription is complex and probably involves multiple positive and negative elements located further upstream of those elements required in cultured cells. Transgenic studies by Suri et al.(1993), Min et al.(1994), and Liu et al.(1994) suggest that crucial DNA elements reside between -9 and -0.773 kbp of 5`-flanking region of the rat TH gene. It is likely that upstream elements which direct expression in different groups of TH-expressing cells in vivo work in conjunction with proximal elements like the CRE to direct basal transcription and mediate responses to various physiological stimuli.

CRE motifs are of major importance for transcriptional regulation and cAMP induction of several other neuronally expressed genes including somatostatin (Montminy et al., 1986; Andrisani et al., 1987; Powers et al., 1989; Leonard et al., 1992), vasoactive intestinal peptide (Tsukada et al., 1987, Fink et al., 1988; Fink et al., 1991), proenkephalin (Comb et al., 1986, 1988), and dopamine -hydroxylase (Ishiguro et al., 1993; Lamouroux et al., 1993; Kim et al., 1994). Transcriptional control of these genes involves the binding of transcription factors to the CRE motif (for reviews see Goodman(1990), Habener et al.(1990), Meyer and Habener(1993), and Lee and Mason(1993)). These transcription factors include related families of CREBs (Montminy and Bilezikjian 1987; Yamamoto et al., 1988; Hoeffler at al., 1988, 1990; Yamamoto et al., 1990; Ruppert et al., 1992), ATFs (Hai et al., 1989; Gaire et al., 1990; Yoshimura et al., 1990), and CREMs (Foulkes et al., 1991a, 1992; Laoide et al., 1993), all of which have a basic DNA binding domain adjacent to a leucine zipper dimerization domain so that they bind to DNA as homodimers or heterodimers. ATFs are capable of selectively forming heterodimers with each other (Hai et al., 1989). In addition, ATF-1/CREB (Hurst et al., 1990; 1991; Rehfuss et al., 1991; Liu et al., 1993) and CREM/CREB (Foulkes et al., 1991a, 1991b; Laoide et al., 1993; Hummler et al., 1994) heterodimers bind to CREs.

Of the bZip proteins, a likely candidate for mediation of basal and cAMP-induced TH transcription is CREB-341, which has been purified from PC12 cells (Montminy and Bilezikjian, 1987) and rat brain (Yamamoto et al., 1988). CREB-341 has been shown to bind to the somatostatin CRE as a dimer (Montminy and Bilezikjian 1987; Yamamoto et al., 1988; Gonzalez et al., 1989). It becomes phosphorylated at serine 133 when cAMP-dependent protein kinase is activated by increased intracellular cAMP levels (Montminy and Bilezikjian, 1987; Gonzalez and Montminy, 1989); this phosphorylation increases somatostatin transcription through an associated co-activator, CBP (Yamamoto et al., 1988; Gonzalez and Montminy, 1989; Chrivia et al., 1993; Kwok et al., 1994). An alternatively spliced form of CREB-341, termed CREB-327, which is also an activator, has been described (Hoeffler et al., 1988, 1990; Yamamoto et al., 1990; Ruppert et al., 1992). It is also possible that CREB-327, ATFs, and/or CREM (a CREM activator) mediate basal and cAMP-induced TH transcription. Widnell et al.(1994) have shown that CATH.a cells contain CREB-327/341, but the presence of other factors has not been assayed.

To determine whether CREB-327/341, ATF-1, or CREM proteins direct -4.8 THCAT transcription, we compromised their function by co-expressing a dominant-negative mutant of CREB, KCREB. KCREB is identical to CREB-327 except for a point mutation which prevents binding to DNA. However, KCREB retains the ability to selectively dimerize with the endogenous activators CREB-327/341, ATF-1, and CREM. Hence, KCREB prevents these proteins from binding to the CRE and thereby prevents activation of transcription (Walton et al., 1992). KCREB decreased basal and cAMP-induced -4.8 THCAT and -0.272 THCAT expression 50-60%, suggesting that CREB-327/341, ATF-1, and/or CREM (or an unknown CREB dimerizer) contribute to basal and cAMP-induced TH transcription in CATH.a cells (Fig. 7).

The lack of complete inhibition may be due to our inability to express sufficient amounts of KCREB to sequester all endogenous CREB proteins within the time frame of the experiment. Alternatively, residual expression may be mediated by CRE-binding proteins that do not dimerize with CREB (or KCREB) such as CRE-BP1 (Maekawa et al., 1989; Kara et al., 1990; Benbrook and Jones, 1990; Macgregor et al., 1990; Matsuda et al., 1991) and CRE-BP2 (Ivashkiv et al., 1990); it is possible that TH transcription is in part mediated by such proteins. Finally, it is possible that when endogenous TH CRE-binding proteins are inactivated by KCREB, there is functional compensation by other proteins that do not normally direct TH transcription.

In summary, these results represent the first attempts to map specific DNA regulatory elements that direct TH expression in central nervous system cells. The finding that the proximal region (-0.272 kbp) and, in particular, the CRE site alone can support TH basal and cAMP-induced expression suggests that the CATH.a, CATH.b, and PATH.2 cell lines may be similar to some PC12 lines and SK-N-BE(2)C cells (Kim et al., 1993a) but different from PC8b cells (Cambi et al., 1989; Fung et al., 1992; Yoon and Chikaraishi, 1992) and other PC12 lines (Gandelman et al., 1990; Wong et al., 1994). These differences may be due to differences in the relative amounts or efficacy of various transcription factors in different cell lines; such differences may exist in vivo among different populations of TH-expressing cells or among TH-expressing cells at different stages of development.

FOOTNOTES

*

This work was supported by National Institutes of Health Grants NS22675 and NS29679. 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.

§

Current address: Dept. of Anatomy and Cell Biology, Cornell Medical College, 1300 York Ave., New York, NY 10021.

¶

To whom correspondence should be addressed: Neuroscience Program, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-6651; Fax: 617-636-7413; dchikaraishi{at}infonet.tufts.edu.

()

The abbreviations used are: TH, tyrosine hydroxylase; CRE, cAMP response element; CREB, CRE-binding protein; ATF, activating transcription factor; CREM, cAMP response element modulator; CAT, chloramphenicol acetyltransferase; nt, nucleotide(s); bp, base pair(s); kbp, kilobase pair(s); RSV, Rous sarcoma virus; PIPES, 1,4-piperazinediethanesulfonic acid; KCREB, killer CREB.

()

M. Lazaroff and D. M. Chikaraishi, unpublished observations.

()

S. Patankar, S. O. Yoon, and D. M. Chikarishi, unpublished observations.

ACKNOWLEDGEMENTS

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