©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Promoter Sequences in the RI Subunit Gene of cAMP-dependent Protein Kinase Required for Transgene Expression in Mouse Brain (*)

(Received for publication, August 18, 1995; and in revised form, October 20, 1995)

Christopher H. Clegg (§) Harald S. Haugen Landin F. Boring (¶)

From the Bristol Myers-Squibb Pharmaceutical Research Institute, Seattle, Washington 98121

ABSTRACT
INTRODUCTION
MATERIALS and METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Neural-specific expression of the mouse regulatory type-Ibeta (RIbeta) subunit gene of cAMP-dependent protein kinase is controlled by a fragment of genomic DNA comprised of a TATA-less promoter flanked by 1.5 kilobases of 5`-upstream sequence and a 1.8-kilobase intron. This DNA contains a complex arrangement of transcription factor binding motifs, and previous experiments have shown that many of these are recognized by proteins found in brain nuclear extract. To identify sequences critical for RIbeta expression in functional neurons, we performed a deletion analysis in transgenic mice. Evidence is presented that the GC-rich proximal promoter is responsible for cell type-specific expression in vivo because RIbeta DNA containing as little as 17 base pairs (bp) of 5`-upstream sequence was functional in mouse brain. One likely regulatory element coincides with the start of transcription and includes an EGR-1 motif and 3 consecutive SP1 sites within a 21-bp interval. Maximal RIbeta promoter activity required the adjacent 663 bp of 5`-upstream DNA where most, but not all, of the regulatory activity was localized between position -663 and -333. A 37-bp direct repeat lies within this region that contains 2 basic helix-loop-helix binding sites, each of which are overlapped by two steroid hormone receptor half-sites, and a shared AP1 consensus sequence. Intron I sequences were also tested, and deletion of a 388-bp region containing numerous Sp1-like sequences lowered transgene activity significantly. These results have identified specific regions of the RIbeta promoter that are required for the expression of this signal transduction protein in mouse neurons.


INTRODUCTION

The variety of neuronal cell types that comprise the mammalian nervous system is determined by highly refined spatial and temporal patterns of gene expression(1, 2) . This idea is supported by the restricted expression patterns of numerous transcription factors within the developing nervous system. Members of the homeobox(3, 4) , POU domain(5, 6) , and bHLH (^1)(7, 8) families of DNA binding proteins help initiate the cascades of gene expression that establish region-specific classes of neurons. The diversity of neuronal phenotypes is further enhanced during the life of the organism by stimulus-dependent modifications in differentiated neurons. This adaptive plasticity, controlled by various neurotransmitters and cytokines, elicits new patterns of gene expression and long-term changes in cell behavior(9, 10) .

A better understanding of how neuronal diversity and function is achieved will be facilitated by the identification of the mechanisms that regulate neural-specific gene expression. A mouse gene that we have focused on encodes the regulatory type Ibeta (RIbeta) subunit of cAMP-dependent protein kinase (PKA)(11) . Protein phosphorylation by PKA serves a pivotal role in neuronal function (12, 13, 14) . The R subunits of the holoenzyme, of which four genes have been identified, prevent catalysis in the absence of cAMP(15) . Each type of regulatory subunit contributes distinct qualities that broadens enzyme function and helps regulate the myriad of cellular responses controlled by this second messenger pathway. The RIbeta subunit gene is expressed primarily in neurons, and within the central nervous system RIbeta mRNA appears throughout the brain and within the spinal cord (16, 17) . Relative to the other R subunits, RIbeta makes the holoenzyme more sensitive to cAMP(18) , and recent gene disruption experiments indicate that mice lacking RIbeta show deficits in long-term depression and depotentiation in hippocampal neurons(19) .

The neural-specific component of RIbeta gene expression was localized to 3.5 kb of genomic DNA that includes 1.5 kb upstream of a GC-rich (TATA-less) promoter, exon I, and a 1.8-kb intron. A fusion gene containing this promoter fragment and the bacterial lac z coding region mimicked the expression pattern of the endogenous RIbeta gene in the central nervous system of transgenic mice(20) . Analysis of sequence and protein binding activity of this DNA (21) has identified a complex array of recognition sequences for general transcriptional activators like Sp1(22) , developmental regulators like bHLH and POU-domain proteins(6, 23) , and physiological regulators such as immediate-early gene products (9) and steriod hormone receptors (SHR) (24, 25) . To address the functional role of these various binding sites in vivo, we performed a deletion analysis in transgenic mice. The results indicate that the sequences required for neuronal expression lie within the GC-rich proximal promoter and that flanking regions upstream of transcription start and within intron I are required for full transgene promoter activity.


MATERIALS and METHODS

DNA Plasmids

All RIbetalac constructs were derived from RIbetalac 3.5(20) , which is referred to here as RIbetalac-1515. This plasmid contains a 3.5-kb genomic fragment of the RIbeta promoter fused to the bacterial lac z coding sequence and a 3`-intron and poly(A) addition site from the mouse protamine-1 gene(26) . Deletions in the RIbeta promoter sequence were generated using the restriction sites shown in Fig. 1. The numbering system is relative to the 5`-most transcription initiation site(20) , which is designated +1. RIbetalac-7500 was made by cloning a 6.4-kb fragment of genomic DNA into the XbaI site at position -1063 bp of RIbetalac-1515.


Figure 1: DNA sequence of the RIbeta promoter and flanking regions. Exons I and II are boxed, and the location of intron I is indicated. Transcription initiation sites are identified by the ˆ , and the 5`-start site is designated +1. The translation initiation codon at +1993 is labeled MET. The coding region of the lac z gene was fused in frame at this ATG. Consensus binding motifs for known transcription factors are shown in bold and underlined. Closed circles indicate SHR superfamily half-sites. Cleavage sites for the restriction enzymes used in the deletion analysis are indicated by the black arrowheads.



Transient Transfections and beta-Galactosidase Assay

Neuro-2a (NB2a) cells (American Type Culture Collection) were derived from the murine C1300 neuroblastoma line(27) . Cells (10^6 cells per 10-cm plate) were cultured for 24 h in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Hyclone) and then transfected by calcium phosphate precipitation using 10 µg of DNA/plate of pBSRIbetalac or the promoterless lac z gene plac F(26) . As an internal control for transfection efficiency, we included 0.1 µg of RSVluc(28) . The DNA precipitate was added to cells for 20 min at room temperature. Cultures were then fed 10 ml of complete media and incubated overnight (16 h). The precipitate was removed, and the cells were treated with 10% Me(2)SO in Dulbecco's modified Eagle's medium for 4 min at 37 °C, washed twice, and cultured in complete media for an additional 28-30 h. Cells were harvested and lysed in buffer containing 100 mM KPO(4), pH 7.8, 1 mM dithiothreitol, and 0.1 mM phenylmethanesulfonyl fluoride. Protein concentration was determined using the Bio-Rad protein assay reagent. beta-Galactosidase (beta-gal) activity was measured spectrophotometrically using O-nitrophenyl-beta-D-galactopyranoside (Sigma) as the substrate(20) . 20-50 µg of protein from each plate was assayed with 1.1 mMO-nitrophenyl-beta-D-galactopyranoside in buffer containing 100 mM NaHPO(4), 10 mM KCl, 1 mM MgCl(2), and 54 mM beta-mercaptoethanol. Reactions were carried out in 1-ml plastic cuvettes at 37 °C for 180 min. Absorbance was measured at 420 nm. Luciferase activity was determined using a Monolight 2010 luminometer (Analytical Luminescence Laboratory) in buffer containing 1 mM luciferin, 100 mM KPO(4), 40 µM ATP, and 15 mM MgSO(4). Luciferase activities measured in 1 µg of transfected cell protein typically ranged from 800 to 1200 relative fluorescence unit/s.

Transgenic Analysis

Transgene DNA was separated from plasmid sequences by restriction digestion and gel electrophoresis and injected (10 ng/µl) into the pronuclei of fertilized embryos of C57 Bl/6 times C3H F1 mice. After incubation overnight, two-cell embryos were transferred to the oviducts of pseudopregnant foster mothers. Transgenic animals were identified by dot hybridization using tail DNA and P-labeled lac z DNA as a probe. Gene copy number was measured by dot hybridization employing a standard curve equivalent to 0-300 copies per cells. Adult mice were killed by cervical dislocation. Brains were divided at the midline; one half was frozen on dry ice and subsequently used to quantitate enzyme activity, and the other half was fixed for histochemistry in phosphate-buffered saline containing 2% formaldehyde and 0.02% glutaraldehyde at 4 °C. Coronal brain sections (approximately 0.5 mm) were cut on a series 1000 Vibratome (Technical Products International), and incubated for 6 h at 37 °C in buffer containing 5-bromo-4-chloro-3-indolyl-beta-galactoside (X-gal). Sections from transgenic lineages were scored for relative X-gal staining in ten separate regions of the brain, using a subjective scale from 0-5. The values for each region were averaged for all the lineages expressing a given transgene. Enzyme activity was measured in brain extracts as described previously(20) ; 100-200 µg of protein was incubated in plastic cuvettes containing 1.1 mMO-nitrophenyl-beta-D-galactopyranoside in the buffer described above at 37 °C for 120-180 min. Each sample was measured in triplicate. Average units of beta-gal were calculated for each lineage after subtracting activities obtained from non-transgenic brain extracts followed by normalization to a beta-galactosidase enzyme standard. Statistical comparisons were measured using the Wilcoxon Rank Sum test, which is applied for data sets that contain large internal variation(29) . This test examines the relative position of each data point rather than the specific value. For any two transgenes being compared, we ranked the reporter gene activities in descending order and then summed the assigned numbers for each set. The sum from one data set was compared to a distribution of values that are calculated for specific sample sizes(29) . Values that lie outside this distribution are considered different, and a p value is assigned for determining the stringency at which the null hypothesis is rejected. For these studies, p < 0.05 was used as a criterion for statistical significance.


RESULTS

Previous results demonstrated that 3.5 kb of DNA encompassing the RIbeta promoter was sufficient to direct lac z gene expression in the central nervous system of transgenic mice(20) . Fig. 1presents the sequence of this DNA and highlights the complex array of transcription factor binding sites used by various tissue-specific regulators, immediate-early genes, and mediators of hormone action. There are, for instance, 12 SP1 binding sites clustered within a 350-bp region surrounding the GC-rich proximal promoter. An additional six SP1 motifs are located within a 200-bp region in intron I. Most of these consensus sequences (14/18) are the ``GT'' box version, which has been reported to be the preferred binding sequence of a brain-specific member of the SP1 family(30) . The proximal promoter region also contains two EGR-1 (31, 32) and two AP2 motifs (33) . 12 out of 15 E-boxes are located upstream of the transcription initiation sites. This sequence, CANNTG, is recognized by the bHLH family of proteins(34) , which includes regulators of nerve cell differentiation(8, 23) . Another family of proteins implicated in neural development recognizes the POU consensus sequence(35) ; five out of six POU motifs are clustered in a 250-bp region of intron I. The most prevalent consensus sequence is recognized by the superfamily of SHRs. At least 40 half-sites comprised mostly of GGTCA, GGTGA, and AGGACA were identified. Employing gel mobility shift assays, we established that many of these consensus sequences bind proteins found in brain nuclear extracts as well as purified SP1, AP2, and MyoD/E47 dimers(21) .

Efforts to identify the functional domains of the RIbeta promoter were initiated using standard transient transfection protocols and cell lines that expressed RIbeta protein. It was determined that the RIbeta promoter retained tissue-specific regulation in vitro since the RIbetalac reporter plasmid was active in NB2a and HT-22 neuroblastoma cell lines but inactive in cells of non-neuronal origin such as Chinese hamster ovary and JEG cells (data not shown). To localize the regulatory sequences important for this expression pattern, we introduced a series of deletion constructs into N2Ba cells. It was discovered, however, that plasmid sequences in the expression vector influenced promoter activity. As indicated in Fig. 2A, cells transfected with 1.5 kb of 5`-upstream DNA (pBSRIbetalac-1515) produced less beta-gal activity than cells given a plasmid containing just 17 bp of 5`-upstream sequence (pBSRIbetalac-17). This result might suggest that sequences between -1515 and -17 had an inhibitory effect on promoter function. Alternatively, this ``induction'' was caused by the juxtaposition of plasmid sequences closer to the RIbeta proximal promoter. To distinguish between these possibilities, we repeated this transfection using plasmid-free RIbetalac DNA and discovered that the relative strengths of these two constructs were reversed (Fig. 2B). Thus, when assayed in the absence of plasmid DNA, it appears that deleting the majority of 5`-upstream DNA has a negative effect on promoter activity in vitro.


Figure 2: Transient transfection of NB2a neuroblastoma cells with RIbetalac DNA. A, RIbetalac plasmids with different lengths of 5`-upstream DNA (see Fig. 1) were transfected by calcium phosphate precipitation and assayed for beta-gal enzyme activity. B) beta-gal activity obtained with the same RIbetalac deletions as in A with the exception that all plasmid sequences were removed prior to transfection. The data are expressed as the fold increase in enzyme activity obtained in comparison to transfections using a promoterless lac z gene, plac F(26) . Each bar represents the average ± S.D. of two experiments performed in triplicate.



Because of the contradictory results obtained with transient transfection and because of the uncertain physiological relevance of testing neuroblastoma cells instead of neurons, we measured the activity of various RIbetalac constructs in transgenic mouse brain. Fig. 3A illustrates the transgenes used in this experiment and indicates the number of lineages that expressed lac z. Animals were considered positive if beta-gal staining was detected in sections of brain tissue. Note that almost all of the lines expressed the transgene even with as little as 17 bp of upstream sequence. As expected, no animals expressed transgenes containing deletions through the region of transcription initiation and exon I (RIbetalac+243). Upon inspection of beta-gal staining in brain slices, we noted that enzyme levels appeared to be quite variable within each group of transgenic mice. Fig. 4, for instance, shows the expression pattern of two lineages carrying RIbetalac-7500. beta-gal staining in line 349 was strong and broadly dispersed throughout all the major regions of the brain (Fig. 4, A and C), whereas expression in line 344 was completely absent or significantly reduced in many regions (Fig. 4, B and D). These patterns were heritable and observed in siblings of different gender and at different ages. Line 344 contained three times the number of integrated transgenes as line 349, so the reduced expression in 344 could not be attributed to gene copy number. In fact, no correlation between copy number and transgene expression was observed in 75 independent lineages. This suggests that the variability of lac z expression probably relates to the random nature of transgene integration and the domain effects caused by the flanking host chromatin(36) .


Figure 3: Expression of RIbetalac 5`-deletion constructs in the brains of adult mice. A, transgenes containing the indicated amounts of 5`-DNA are shown. The restriction sites used for this analysis were as follows: BII, BglII; X, XbaI; K, KpnI; S, StuI; SII, SstII; E, EagI; and n, NsiI. The number of expressing lineages out of the total number of lineages positive for transgene incorporation are shown on the right (Exp/TG). B, transgene expression in regions of mouse brain. Tissue slices were incubated with X-gal, and the relative intensity of staining in the indicated regions was scored using an arbitrary scale from 0 to 5. The number of lineages examined is shown above the graph. A minimum of three mice was assayed from each lineage. The graph indicates averaged values. C, beta-gal enzyme activity in whole brain extracts. The number of expressing lineages that were assayed is shown above the graph. A minimum of three mice was assayed from each lineage. The Wilcoxon Rank Sum test (29) was used to determine statistical significance (p < 0.05; see text).




Figure 4: Variability of transgene expression in adult mouse brain. A and C, Lineage 349, transgenic for RIbetalac-7500, expressed beta-gal activity in most regions of the brain, including the neocortex (N), caudate-putamen (Cp), septum (S), hippocampus (H), thalamus (T), hypothalamus (Hy), and piriform cortex (P). B and D, lineage 344, also transgenic for RIbetalac -7500, showed intense staining in the cortex but little to no enzyme activity in other anatomical regions. E, lineage 874, transgenic for RIbetalac -17, expressed only in the neocortex (arrows show approximate lateral extent of labeled cells). F, lineage 867, also transgenic for RIbetalac -17, showed scattered expression in the cingulate region of the neocortex, the dorsal hypothalamic nucleus, and the amygdala (A). Positive cells are also scattered throughout the hypothalamus and piriform cortex.



To quantitate the expression of each transgenic lineage, we scored the relative level of lac z staining in specific regions of the brain (Fig. 4B), and as a second method we measured lac z activity in whole brain extracts (Fig. 4C). The results of these assays showed a similar trend; the construct with the most 5`-upstream DNA, RIbetalac-7500, was equivalent to RIbetalac -663 in activity. Removal of DNA down to position -333 reduced promoter activity, and deletion to -17 diminished transgene expression significantly. Although RIbetalac -17 was rather weak, beta-gal expression was always restricted to the central nervous system and undetectable in non-neuronal tissue. To calculate whether the differences observed between these deletion constructs were statistically significant, we employed the Wilcoxon Rank Sum test ((29) , see ``Materials and Methods''), which is used for data sets with large variances. Using this test, it was determined that the differences between -663, -333, and -17 were statistically significant (p < 0.01). Two-way comparisons involving -7500, -1456, -1063, and -663 showed no statistical differences (p > 0.10). We conclude from this series of deletions that sequences positioned between intervals -663/-333 and -333/-17 contribute to high level expression of the RIb promoter. Moreover, the residual activity of RIbetalac -17 ( Fig. 3and Fig. 4) indicates that neural-specific expression is controlled by additional sequences downstream of -17. We suggest that these sequences encompass the RIbeta proximal promoter and transcription initiation (see ``Discussion'').

Intron I of the RIbeta gene also contains a variety of protein binding motifs (Fig. 1). To test the functional requirement for this DNA, we made transgenic mice with the constructs shown in Fig. 5. The largest deletion removed 1900 bp from the end of exon I to the beginning of the initiator MET of the lac z gene. 6 out of 10 founders demonstrated only very weak staining in brain sections (Fig. 5B). No expression was detected in whole brain extracts (Fig. 5C). Another deletion removed most of the intron but left intact the 5`- and 3`-splice sites (Delta+243/+1661). Again, the loss of activity was dramatic: 4 of 10 founders expressed only low levels of beta-gal. Finally, a relatively minor deletion that removed 388 bp of intron sequence (Delta+243/+643) also had a marked effect when compared to the intact intron. These results demonstrate that intron I is required for meaningful RIbeta expression in vivo, although its absence does not eliminate brain-specific expression.


Figure 5: Expression of RIbetalac intron deletions in the brains of adult mice. A, various amounts of intron I were removed from RIbetalac-1515. The number of transgenic mice established for each construct is indicated with the number that expressed lac z. RIbetalac -1456, which has an intact intron, served as the positive control. The restriction sites used were RI, EcoRI; BII, BglII; A, AvaI; SII, SstII; X, XbaI; and n, NsiI. B, transgene expression in different brain regions, as measured by relative X-gal staining. C, beta-gal activity in whole brain extracts. The number of expressing lineages that were assayed is shown above the graph. A minimum of three mice was assayed from each lineage.




DISCUSSION

In contrast to many genes, the 3.5 kb of DNA that flank the RIbeta promoter contain a rather complex arrangement of transcription factor binding sites that includes 18 SP1 sites, 15 bHLH E-boxes, 40 steroid hormone receptor half-sites, and multiple EGR-1, AP2, AP1, and POU sequence motifs (Fig. 1, (21) ). The redundancy of these binding sites has presented an interesting challenge in identifying the sequences responsible for RIbeta gene expression, and rather than systematically mutating specific classes of binding sites, we prepared a series of constructs in which varying amounts of DNA were removed from the 5`-upstream region and from intron 1. Although a test of these deletions was initiated in neuroblastoma cells by transient transfection, we turned to transgenic mice to ascertain which mutations would cause a meaningful effect on gene expression in a functional nervous system.

Sequences primarily responsible for neuron-specific expression of the RIbeta gene most likely reside within the proximal promoter between positions -17 and +243. This conclusion is based on the activities of RIbetalac-17 and RIbetalac DeltaIV, both of which expressed beta-gal in a brain-specific manner, and RIbetalac+243, which failed to express in the absence of the proximal promoter. Consistent with this result was the observation that the -17/+243 promoter fragment stimulated lac z activity in NB2a neuroblastoma cells but did not function in non-neuronal cell types (data not shown). A likely regulatory element within the proximal promoter coincides with the start of transcription and includes an EGR-1 motif and three consecutive SP1 sites within a 21-bp interval (Fig. 1). Gel shift experiments confirmed that this region in RIbeta binds pure SP1 and additional proteins in brain nuclear extract(21) . Similar sequences are present in a number of genes expressed in neural tissue(37, 38) , including the promoter for the RIIbeta subunit of PKA(39) , and are required for expression of aldolase C in mouse brain(40) . Sequences such as these have also been shown to bind inhibitors of transcription(41) .

Although the proximal RIbeta promoter could function in a tissue-specific manner, its activity was enhanced considerably by 5`-upstream sequences. RIbetalac -663 provided maximum transgene expression, indicating that sequences between -663 and -17 function as positive regulators of the basal promoter. This region contains numerous transcription factor binding sites, possibly the most complex being a 37-bp direct repeat (located between -555 and -519) that contains two E-boxes, each of which are overlapped by two steroid hormone receptor half-sites, and a shared AP1 consensus sequence(21) . Again, gel mobility shift assays demonstrated that this multiple repeat sequence readily formed a complex with brain nuclear extract containing bHLH-, SHR-, and AP1-related proteins. Nuclear proteins isolated from liver failed to bind these oligonucleotides(21) . Sequences like these have been shown to function as enhancer elements in numerous genes (42, 43, 44) . Evidence that the 37-bp repeat may be required for high level expression is suggested by the significant reduction in transgene activity obtained with RIbetalac -333. This latter deletion construct, however, still enhanced basal promoter activity, suggesting that additional sequences, some of which are redundant with those in region -663/-333, are also involved in controlling promoter activity.

Two additional regulatory regions may also have been uncovered further upstream: a positive element between -7500 and -1456 whose removal lowered promoter activity and an inhibitory sequence between -1063 and -666 whose deletion increased expression. Interestingly, this latter segment contains a duplicated E-box (Fig. 1; near position -736), which in the low affinity nerve growth factor receptor gene acts as a negative regulatory element(45) . The significance of these deletions is hard to measure because of the large variability inherent to this experiment. However, if we use the Wilcoxon Rank Sum Test and combine the activities from RIbetalac -1456 and RIbetalac -1063 into a single data set and compare this to RIbetalac -663, then the differences between these become statistically significant (p < 0.05). We do not believe, however, that this inhibitory region contains the type of silencer elements observed in other neural-specific genes (46) because their elimination did not stimulate proximal promoter activity in non-neuronal Chinese hamster ovary or JEG cell lines nor did it induce expression in the non-neuronal tissues of these transgenic mice.

The final region shown to have regulatory activity was intron I, which contains many binding motifs including Sp1, AP1, bHLH, POU, CarG, and SHR sites (Fig. 1, (21) ). We made two large deletions in intron I and saw a dramatic decrease in promoter activity. Interpretation of this effect is complicated by the observation that transgene expression is often dependent on 5`-intronic sequences(47) . While the loss of promoter activity may have resulted from a splicing requirement, we note that the RNA splice and donor sites were left intact in RIbetalacDelta418, and even RIbetalacDelta388, which removed one AP1, four SP1, and three SHR half-sites, had significantly lower promoter activity. It has recently been shown with transgenic mice that deletion of peripheral Sp1 sites causes de novo methylation and inactivation of the aprt gene(48) . Since the RIbeta promoter encompasses a GC island, a similar phenomenon may explain the loss of transgene expression following the removal of SP1 sites in RIbetalacDelta388.

Our experiments show some of the intricacies associated with analyzing promoter function. RIbetalac expression in mice, for instance, was subject to large variations in expression due to position effects at the site of transgene integration. The mechanism responsible for this inhibition is unknown but may involve methylation(48) . As a result of the variability in expression, a large number of transgenic mouse lineages were produced, and utilizing the Wilcoxon Rank Sum test as a statistical tool, we were able to delineate major regulatory regions in the RIbeta promoter. Using transgenic mice, however, to test the relative importance of the specific binding sites within each region may prove more difficult. The N2A neuroblastoma cell line was originally used for a deletion analysis because of the relative simplicity of transient transfections. The contradictory results obtained with these cells (Fig. 2) required that we first establish RIbeta promoter regulation in vivo. Now that a similar expression pattern has been established between transgenics and the transfection of RIbetalac genes (minus plasmid sequences) in vitro, it should be possible to use a cell culture system for detailed mapping of these regulatory regions.

Endogenous RIbeta mRNA is detected at varying levels in most regions of the brain and spinal cord(16) , and RIbeta transgene expression has been detected in peripheral nerves as well(20) . Our data suggest that expression of RIbeta reflects a constitutive regulation common to many types of neurons and that this occurs at the level of its GC-rich proximal promoter. The large number of transcription factor binding sites flanking this region may serve to integrate information from numerous signal transduction pathways, allowing expression of RIbeta mRNA to be finely regulated. The redundancy of these sequences may guarantee the continual expression of this gene. Given the major role PKA plays in the physiology of neurons, precise regulation of individual subunit gene expression may be necessary to provide appropriate cellular responses.


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) S72345[GenBank].

§
To whom correspondence should be addressed: Bristol Myers-Squibb Pharmaceutical Research Institute, 3005 1st Ave., Seattle WA 98121. Tel.: 206-727-3733; Fax: 206-727-3602.

Present address: The Gladstone Institute of Cardiovascular Disease, P. O. Box 419100, San Francisco, CA 94141-9100.

(^1)
The abbreviations used are: bHLH, basic helix-loop-helix; RIbeta, the beta regulatory subunit of type I cAMP-dependent protein kinase; PKA, cAMP-dependent protein kinase; SHR, steroid hormone receptors; X-gal, 5-bromo-4-chloro-3-indolyl-beta-galactoside; beta-gal, beta-galactosidase; kb, kilobase(s); bp, base pair(s).


ACKNOWLEDGEMENTS

-We are indebted to Nerville Koeiman and Teresa Wagner for expert technical assistance and to Dr. Bruce Bamber and Dr. Alan Wahl for helpful discussions.


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