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Analysis of the promoter sequence and the transcription initiation site of the mouse 5-HT1C serotonin receptor gene
Molecular Brain Research, 17 (1993) 194-200 © 1993 Elsevier Science Publishers B.V. All rights reserved 0169-328x/93/$06.00
194
BRESM 70556
Analysis of the promoter sequence and the transcription initiation site of the mouse 5-HT c serotonin receptor gene L a u r a J. B l o e m *, Y a n Chen, J i a n Liu, L e i g h a n S. Bye a n d Lei Y u Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202 (USA) ~'Accepted 15 September 1992)
Key words: Xenopus oocyte; Nuclear injection; Neurotransmitter receptor
The serotonin lc (5-HTIc) receptor is found in many brain regions, but is particularly enriched on the epithelial cells of the choroid plexus. A major challenge in neurobiology is to delineate the molecular processes that regulate the specific pattern of neuronal gene expression in the brain. As an initial step towards identi~ing cis-acting DNA sequences that control the expression of the 5-HTtc receptor, we have isolated the promoter sequence of its gene. Sequence analysis of a 1.8 kb fragment indicated that the 3' end of this fragment overlaps with the 5' untranslated region of the 5-HTtc receptor mRNA, and primer extension using mouse brain poly(A)+ RNA mapped the transcription initiation site within this fragment. There are a number of sequence elements upstream from the transcription initiation site that are homologous to regulatory elements found in other eucaryotic genes. To determine the promoter activity, a plasmid was constructed that contains this fragment as promoter region and the eDNA for the 5-HTtc receptor as the reporter. When injected into the nucleus of Xenopus oocytes, this construct resulted in functional expression of the reporter gene. Primer extension using the RNA extracted from the injected oocytes indicated a single transcription initiation site of the reporter mRNA. These results suggest that the 5-HTIc receptor was functionally expressed under the promoter activity of the 1.8 kb 5' sequence of its gene. This system will be useful for further analysis of the cis-acting elements in the promotor region of the 5-HTtc receptor gene and the trans.acting factors that regulate tissue-specific expression of the receptor.
INTRODUCTION
The diverse physiological effects of the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) are mediated by specific membrane receptors that have been classified into pharmacologically distinct subtypes~'2t,47. The 5-HTtc receptor is one of the major serotonin receptor subtypes in the brain. It is present at high density on the epithelial cells of the choroid plexus 22'46'5s'59. The eDNA for the receptor has been cloned in mouse 37,6~, rat 28 and human 5~. Using the cDNA as probe, in situ hybridization studies have shown that the 5-HT~c receptor is expressed in a variety of cortical and sub. :tica'. neurons in addition to the choroid plexus ~- 40,43 A major goal of neurobiology is to define in molecular terms the processes that regulate the regional ex-
pression pattern of neurotransmitter receptors. Regulation of receptor gene expression can be at the transcriptional level, at the translational level, and by posttranslational modifications 49. Many eucaryotic genes have been studied that are expressed only in certain cells and tissues and are regulated at the level of transcription 2't6'~9'26'56'62. Studies have suggested that c/s-acting sequences consisting of both positive and negative upstream regulatory sequences as well as enhancer elements are responsible for tissue-specific expression of a gene. Presumably, tissue-specific transcription factors interact with cis-acting DNA sequences to control the level of RNA synthesis in different cell typos 29'42. Little is known about the molecular processes that regulate the regional expression pattern of the 5-HTmc receptor. We report here the isolation of the 5' se-
Correspondence: L. Yu, Department of Medical and Molecular Genetics, Indiana University School of Medicine, 975 West Walnut Street, Indianapolis, IN 46202, USA. Fax: (1) (317) 274-2387 or (317) 274-1069. * Present address: Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
195 q u e n c e o f the m o u s e 5-HT~c r e c e p t o r gene a n d the analysis o f its p r o m o t e r activity. This work has b e e n p r e s e n t e d in abstract form 4'5. MATERIALS A N D METHODS
Library screening and recombinant DNA A mouse genomic library in EMBL-3 SP6/T7 (Clontech) was screened by a polymerase chain reaction (PCR) method 6 using primers lor the 5' untranslated region of the mouse 5-HT~c receptor cDNA. One positive clone was isolated and mapped by restriction enzyme analysis. A 1.8 kb Sstl-EcoRl fragment within this clone was gel-isolated, fragmented by sonication and subcloned into M13mpl9 for dideoxy sequence analysis. The same fragment was blunt.ended with E. coli DNA polymerase I large fragment in the presence of all four deoxynucleotide triphosphates and then ligated to the vector pKS + SVOCAT 54 tO create the plasmid KS + SV2CAT. The CAT gene was removed from KS + SV2CAT by digestion with Hpai and EcoRV and replaced with a 23 kb XbaI-EcoR! fragment containing the mouse 5-HTlc cDNA6~ to create the plasmid pKS + 21C. A 2.6 kb Xbal-HindllI cDNA fragment for the rat M t muscarinic acetylcholine receptor 9 was gel-isolated after restriction digestion of the plasmid pGEM7-MI and subcloned downstream from the SV40 promoter in the vector pSV40poly 52 to, create the plasmid pSVpolyM1.
RNA isolation RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction t2. Tissues (either 100 mg total mouse brain or 10 oocytes) were homogenized in 1.0 ml of denaturing solution (4.0 M guanidinium thioojanate, 25 mM sodium citrate, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol). After the addition of 0.1 ml 2.0 M sodium acetate (pH 4.0), 1.0 ml water saturated phenol and 0.2 ml of chloroform-isoamyl alcohol (49:1), the homogenate was vortexed, chilled on ice for 15 min and then centrifuged at 10,000x g for 10 rain at 4°C. The aqueous phase was collected, and the RNA was precipitated by the addition of 1.9 ml of isopropanol and incubated at -20 °C for at least 1 h. The RNA was pelleted and resuspended in 0.3 ml of denaturing solution and reprecipitated with isopropanol. After washing of the pellet with 70% ethanol, the pellet was resuspended in water. Poly(A) + RNA was isolated using an oligo-dT column.
Primer extension The sequence of the 20-mer deoxyoligonucleotide primer 5'-TCCAAGGGCTCAATCATTAA-Y(AU5) is complementary to nucleotides 241-224 of the cDNA sequence in Fig. 1. The primer was phosphorylated using T4 polynucleotide kinase to a specific activity of 1.0× 10s cpm//~g. Whole brain POly(A)+ RNA from 2-week-old mice (80/zg) or total RNA from 10 oocytes was mixed with 1.0x 106 cpm of oligo in 35 /tl of water, incubated at 70°{2 for 10 min and quickly chilled on ice. Primer extension reactions were carried out at 45 °C for 90 min in 50 mM Tris-Ci (pH 8.3), 40 mM KO, 1 mM dithiolthreitol, 6 mM MgCI2, 0.25 mM each of dNTP, 50 /~g/ml actinomycin D, 100 units/mi RNasin, and 400 units of M-MLV H- RT (Superscript, BRL). After extension, the reaction was treated with RNase A (0.2 mg/ml) at 37°C for 30 min, extracted once with phenol/chloroform (1:1), once with chloroform/isoamyl alcohol (49:1) and ethanol precipitated.
Electrophysiology Nuclear injection into Xenopus oocytes was performed as previously described tT'4a. Two to 3 days after injection, individual oocytes were transferred to a recording chamber for voltage-clamp recording. The chamber was continuously superfused with either normal or ligand-containing frog Ringer's solution 6°. The results were recorded both by a microcomputer with the aid of the software pCLAMP (Axon Instruments) and on a Gould 200 dual-channel chart recorder. Data analysis was performed with the same software package.
RESULTS
Isolation and analysis o f the promoter sequence o f the mouse 5.HT!c receptor gene A m o u s e g e n o m i c library in E M B L - 3 SP6/',vT, was s c r e e n e d by a P C R screening method6~ using primers corr,~sponding to the 5' u n t r a n s l a t e d region of m o u s e 5-HT~c r e c e p t o r c D N A 6~ to amplify a 100 bp fragment. O n e positive clone was isolated, a n d restriction m a p ping revealed t h a t it contains a p p r o x i m a t e l y 8 kb of the 5' sequence o f the 5-HT~c r e c e p t o r g e n e (Fig. 1A). T h e P C R f r a g m e n t was located in a 1.8 kb E c o R I - S s t I f r a g m e n t . S e q u e n c e analysis of this f r a g m e n t revealed that it overlaps with the 5' e n d o f the m o u s e c D N A sequence 6~ f r o m b a s e pair 305 a n d the rat c D N A sequence 2s f r o m b a s e pair 177 (Fig. 1B). T h e start site for transcription was m a p p e d by p r i m e r extension with m o u s e brain poly(A) + R N A using a p r i m e r c o r r e s p o n d i n g the 5' e n d o f the m R N A ( A U 5 , Fig. 1B). T h e m a j o r product was 241 bases long (Fig. 2), thus m a p p i n g the transcription initiation site to 566 base pairs u p s t r e a m f r o m the S s t l site (Fig. 1B). This gives the 5 - H T l c r e c e p t o r m R N A a 5' u n t r a n s l a t e d
sequence of 855 nucleotides. Examination of the immediate upstream sequence of the transcription initiation site reveals several notable features (Fig. 1B). The sequences TATGAA at -29, and AAATI'AAT at -21, and CCATG at - 6 6 and CCCCATC at - 8 5 are the closest approximations to TATA and CAAT box consensus sequences with the appropriate spacing reported for many eucaryotic promoters 14,~5,42.There are also a number of sequences in this region that resemble the identified c/s-acting ele. ments or enhancers for transcriptional control of genes. These include a potential AP2 binding site (TFCCC. CATCT) eS, two AP1 binding sites (TGAATCA,TrAA. TCA) 7'3s, as well as consensus sequences for ENKCRE-1 (TACGCCA) 24 and the octamer motif binding factor (GTGCC) s (Fig. 1B). Other notable regions within the 1.8 kb EcoRI-Sstl fragment are two repetitive sequences. One is a purine-pyrimidine repeat at - 1038 consisting of (CA)20 and the other is a TCTG repeat from - 129 (Fig. 1B). Both of these types of repeats are often associated with highly polymorphic regions of the genome and could represent potential polymorphic markers t°. There are also two pairs of direct repeats each of 12 base pairs (A and B, Fig. IB) as well as four pairs of inverted repeats of 9 base pairs (AA~BB) and 8 base pairs (CC, DD).
Functional analysis of the promoter region in Xenopus oocytes
Cell type-specific promoters have been shown to have some activity when injected into Xenopus
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Fig. 1. A: restriction map of A MG4A3. The 8.0 kb Xhol-$stl fragment contains the 5' sequence for the 5-HT~c receptor gene. The 1,8 kb EcoRl--Sstl fragment indicated with cross-hatching was the region analyzed further in this study. B: nucleotide sequence of the 1.8 kb EcoRI-Sstl fragment. The sequence of the 5-HTlc promoter is shown, together with 584 base pairs upstream from the cap site (?) determined by primer extension in this study. The nucleotides are numbered with the cap site as + 1. The putative TATA and CAAT boxes are indicated by the symbol e. Primer binding sites (PCR primers 1 and 2, AUS) as well as the CA- and TCTG repeats are underlined. The transcription start site in oocytes is indicated by an asterisk. The sequence was searched for putative transcription factor binding sites. Sequences showing homology to the consensus sequences associated with the binding of a number of transcription factors are indicated by the symbol v. Direct repeats A and B as well as inverted repeats AA, BB, CC and DD are indicated by arrows.
Fig. 2. Primer extension analysis of mouse brain poly(A) + RNA. Samples were analyzed on a 6% sequencing gel along with a sequencing ladder using a known DNA template as the size marker (lanes A, C, G and T). Lane 1, primer extension using 80 ,¢g of poly(A) + isolated from 2-week-old mouse brain. The length (in bases) of the major extension product is indicated.
resulting plasmid, pKS+21C, was injected into the nucleus of Xenopus oocytes. Another plasmid containing the muscarinic M n receptor cDNA ~ downstream from the SV40 promotor (pSVpolyM1, Fig. 3) was co-injected as an internal control. Two days post injection, oocytes were assayed for serotonin- and acetylcholineinduced currents. Representative traces of membrane current are shown in Fig. 4. Oocytes injected with 0.25 ng DNA did not respond to serotonin. This amount of DNA was shown to be below the threshold for oocyte expression s°. Accuracy of nuclear injection was demonstrated by the oocyte's response to acetylcholine (Fig. 4A). Oocytes injected with 2.5 ng DNA produced both serotonin- and acetylcholine-induced currents (Fig. 4B). The size of the depolarizing currents ranged from 500 to 2000 nA in response to 1.0 ~M serotonin. The presence of serotonin-induced depolarizing membrane currents suggests that the 5-HT~c receptor was functionally expressed under the promoter activity of the 5' flanking sequence of its gene. Cell-type specific genes, when injected alone into oocytes, often produce multiple RNA species with 5' termini located both upstream and downstream of the original cap site 5°. Using the RNA isolated from oocytes injected with 2.5 ng of pKS+21C DNA, primer
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Fig. 3. Schematic diagrams of the plasmids pKS+21C and pSVpolyM1. The 1.8 kb fragment of the 5-HTlc receptor promoter was cloned in front of the mouse 5-HTic receptor cDNA. The rat M 1 muscarinic acetylcholine receptor was cloned downstream of the SV40 promoter.
region reveals that 5-HT~c mRNA has a fairly lon~ 5'-noncoding region of 855 bases. Only one-fourth of the 5'-noncoding sequences from vertebrate mRNAs scored by Kozak were more than 100 bases in length3~. 5'-noncoding sequences of most vertebrate mRNAs are from 20-100 nucleotides in length. One group of messages with longer 5'-noncoding sequences are the mRNAs derived from proto-oncogenes 32'33. The association of the 5-HT~c receptor with proto-oncogenes is not new. It has been demonstrated that ectopic expression of the 5-HT~c receptor in NIH-3T3 cells is both
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extension experiments showed a single transcription initiation site for the reporter mRNA (Fig. 5). This site is 36 base pairs downstream from the transcription initiation site mapped with mouse brain poly(A) + RNA (Fig. 2). The altered transcription initiation in oocytes suggests that brain nuclear proteins may be required for transcription initiation at the original position. DISCUSSION
We report here the isolation and characterization of the 5' sequence of the mouse 5-HT~c receptor gene. Mapping of the transcription initiation site within this
Fig. 5. Primer extension analysis of reporter mRNA isolated from
Xenopus oocytes. Samples were analyzed on a. 6% sequencing gel along with a sequencing ladder (lanes A, C, G and T). The length of the major extension product is indicated.
198 necessary and sufficient to produce transformed foci which, when introduced into mice, lead to the generation of tumors 27. Thus, neurotransmitters and neurotransmitter receptors have the potential to act as regulators of neural cell growth ts. The frequent presence of long leader sequences in critical regulatory genes suggests that such sequences may constitute an important part of gene regulation in vertebrates 33 and could play an important role in the regulation of translation of the mouse 5-HT~c message. Comparison of the DNA sequence upstream from the transcriptional initiation site of the 5-HT~c gene with consensus binding sequences for transcription factors revealed the presence of several potential promoter and enhancer elements. The AAATTAAT at -21 may serve as a potential TATA box in that it contains a T r A A element found to provide the principal TATA box function for the rat brain creatine kinase gene t9 and that it is AT-rich. A second potential TATA box at - 2 9 (TATGAA) has been found in one of the aeu-globulin genes57. Either one of these sequences could serve as a TATA promoter element. The presence of another classical promoter element, the CAAT box, is less obvious. The sequences at - 6 6 (CCATG) and at - 85 (CCCCATC) bear some resemblence to the consensus CAAT box 14,~5,but the homologies are low. In light of this, it is of interest to note that a number of G protein-coupled receptor genes have also been shown to lack a CAAT box 3°,4L45. Several consensus enhancer sequences were also present in this 1.8 kb region. One such sequence, GTGCC at -69, has been shown to bind the octamer motif binding factor in vitro ~. Another sequence (TACGCCA) overlapping with the transcription start site, characterizes the ENKCRE-1 element of the proenkephalin gene 24. This sequence comprises one of the cAMP- and phorboi ester-inducible enhancers of the neurotransmitter proenkephalin gene and ~..ppears to bind a distinct factor ENKTF.1 ~3. There are also two potential binding sites (at - 1 8 and -27) for the enhancer protein AP-1 with the consensus sequence T(T,G)ANTCA7,35. Studies suggest that this particular transcription factor may be modulated by pharmacological agents known to stimulate protein kinase C. Another sequence, T r c c CCATCT found at -87, is a potential AP-2 binding site. This transcription factor binding site forms a cell type-specific enhancer element whose activity is increased in response to treatment of cells with phorbol esters and agents that elevate cAMP 25. The presence of a number of potential cAMP- and phorbol ester-responsive elements suggests a possible role for these second messengers in the regulation of 5-HT~¢ receptor transcription. In addition, the density
of overlapping sequences predicted to bind transcription factors emphasizes the potential importance of the region immediately upstream from the transcription start site. Functional studies will be required to determine if these sequence elements do represent transcription factor binding domains. Promoter sequences for several other neurotransmitter receptors have been isolated and analysed. These include the human and hamster/32-adrenergic receptor genes 3°, the human D~A-dopamine receptor gene 41 and the human 5-HTtA receptor gene 45. They share many characteristics of the promoters for housekeeping genes, including a G + C-rich content, multiple potential binding sites for the transcription factor SP1 and, in some cases, a lack of well-defined consensus sequences for TATA or CAAT boxes. The promoter for the mouse 5-HT~c receptor gene does not appear to conform to these neurotransmitter receptor promoters in that it does not contain G + C-rich regions or potential SP1 binding sites. However, it does share characteristics with human D~A-dopamine receptor gene as both have potential AP1 and AP2 binding sites. Recent years have seen rapid progress in the characterization of cis-acting regulatory elements and the isolation of trans-active protein factors that bind to them. Typically, this has been accomplished by transient expression gene transfer into cultured mammalian cells. Nuclear injection in Xenopus oocytes offers certain advantages over cultured cells for characterizing tissue-specific regulatory elements and transcription factors 23,5°. One advantage is that the copy number of the introduced gene in oocytes can be predetermined in microinjection whereas the copy number of a transfected gene in cultured cells is quite variable. This is important because of the accumulating evidence that gene expression is often regulated by a fine balance between gene dosage and the concentration of modulating factors in the cell 3'26'34'55'56.Another advantage of the oocyte system is that it is possible to manipulate the nuclear environment of the oocyte by coinjection with nuclear extracts from different cell types 23'44'50. This allows for analysis of both activators and repressors in the same system as well as the ability to study the role of nuclear proteins in differentiated cells from various tissue sources and brain regions, without the necessity for establishing a cell culture from the tissue of interest, The oocyte expression system has been used to study a number of genes including the chicken skeletal a actin gene ~, the chicken 5-aminolevulinate synthase gene 36, the herpes simplex virus thymidine kinase gene 39, the rabbit /3 globin promoter s°, the endogenous viteUogenin promoter 3s and the trans-activation
199
of the mouse IL-2 gene 44. We have shown in this study that the 5-HT~c receptor promoter, when injected into Xenopus oocytes, has characteristics similar to that observed for other cell type-specific genes. First, nanogram quantities of DNA are required to activate the 5-HTmc promoter in oocytes as has been shown for other tissue-specific genes44'5°(Fig. 4), whereas viral promoters such as the SV40 promoter require only picogram quantities 39'4s. This activity of the 5-HT~c promoter is probably due to the binding of general transcription factors in the oocyte to basic promoter elements present in the 5-HT~c receptor gene. A second characteristic of tissue-specific genes expressed in oocytes is the presence of multiple reporter RNA species with transcription initiation sites both upstream and downstream from the original cap site 5°. Initiation of transcription from the 5-HT~c promoter in oocytes is from a cryptic site 36 base pairs downstream from the cap site mapped with mouse brain mRNA. Thus it appears that, although the 5-HTlc promoter is functional in oocytes, certain nuclear proteins in the mouse brain may be required for authentic initiation of transcription. The nuclear injection assay in Xenopus oocytes will be useful to further analyze the c/s-acting elements in the promotor region of the 5-HT~c receptor gene and to identify both positive and negative trans-acting factors that regulate tissue-specific expression of the receptor. Acknowledgements. We thank Dr. Alexander Stacey for SVpoly vector, Dr. Tom Bonner for M I receptor cDNA, Drs. Maureen Harrington and Lucy Carr for helpful discussions, and Weiyin Li for technical assistance. This work was supported in part by a grant from the NIH (NS 28190) and a grant from Eli Lilly & Company to L.Y.L.J.B. and L.S.B. were supported by an NIH training grant (T32HD07373) and J.L. was supported by a John B. Hickam Memorial postdoctoral fellowship from the American Heart Association, Indiana Affiliate, Inc.
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reaction mediated by a single primer: cloning of genomic sequences adjacent to a serotonin receptor protein coding region, Nuci. Acids Res., 19 (1991) 7155-7160. 46 Pazos, A., Hoyer, D. and Palacios, J.M., The binding of serotonergic ligands to the porcine choroid plexus: characterization of a new type of serotonin recognition site, Fur. J. Pharmacoi., 106 (1984) 539-546. 47 Peroutka, S.J., 5-Hydroxytryptamine receptor subtypes, Annu. Rev. Neurosci., 11 (1988)45-60. 48 Pfaff, S.L., Tamkun, M.M. and Taylor, W.L., pOEV: a Xenopus oocyte protein expression vector, Anal. Biochem., 188 (1990) 192-199. 49 Roth, B.L., Hamblin, M. and Ciaranello, R.D., Regulation of 5-HT e and 5-HTIc serotonin receptor levels, Neuropsychopharmacology, 3 (1990) 427-433. 50 Rungger, D., Muster, L., Boeck, R. and Nichols, A., Tissuespecific trans-activation of the rabbit/]-globin promoter in Xenopus oocytes, Differentiation, 44 (1990) 8-17. 51 Saltzman, A.G., Morse, B., Whitman, M.M., Ivanshchenko, Y., Jaye, M. and Felder, S., Cloning of the human serotonin 5-HT2 and 5-HTIC receptor subtypes, Biochem. Biophys. Res. Commun., 181 (1991) 1469-1478. 52 Stacy, A. and Schnieke, A., SVpoly: a versatile mammalian expression vector, Nucl. Acids Res., 18 (1990) 2829. 53 Steinbeisser, H., AIonso, A., Epperlein, H.H. and Trendelenburg, M.F., Expression of mouse histone HI ° promoter sequences following microinjection into Xenopus oocytes and developing embryos, Int. J. Dev. Biol., 33 (1989) 361-368. 54 Tsonis, P.A., Manes, T., Millan, J.L. and Goetinck, P.F., CAT constructs with convenient sites for cloning and generating deletions, Nucl. Acids Res., 15 (1988) 7745. 55 Umek, R.M., Friedman, A.D. and McKnight, S.L., CCAAT-enhancer binding protein: a component of a differentiation switch, Science, 251 (1991) 288-292. 56 Wientraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, M., Benezra, R., Blackwell, T.K., Turner, D., Rupp, R., Hollenberg, S., Zhuang, Y. and Lassar, A., The myoD gene family: Nodal point during specification of the muscle cell lineage, Science, 251 (1991) 761-766. 57 Winderickx, J, Van Dijck, P., Dirckx, L., Volckaert, G,, Rombauts, W., Heyns, W. and Verhoven, G,, Comparison of the 5' upstream putative regulatory sequences of three members of the a2u-globulin gene family, Fur. J. Biochem., 165 (1987) 521-529. 58 Yagaloff, K.A. and Hartig, P.R., t'~l.Lysergic acid diethylamide binds to a novel serotonergic site on rat choroid plexus epithelial cells,/. Neurosci,, 5 (1985) 3178-3183. 59 Yagaloff, K.A., Lozano, G., VanDyke, T., Levine, AJ. and Hartig, P.R., Serotonin 5-HTIc receptors are expressed at high density on choroid plexus tumors from transgenic mice, Brain Res., 385 (1986) 389-394. 60 Yoshii, K., Yu, L., Mayne, K.M., Davidson, N. and Lester, H.A., Equilibrium properties of mouse-Torpedo acetylcholine receptor hybrids expressed in Xenopus oocytes, J. Gen. Physiol., 90 (1987) 553-573. 61 Yu, L., Nguyen, H., Le, H., Bloem, L.J., Kozak, C.A., Hoffman, B.J., Snutch, T.P., Lester, H.A., Davidson, N. and Liibbert, H., The mouse 5-HTtc receptor contains eight hydrophobic domains and is X-linked, Mol. Brain Res., 11 (1991) 143-149. 62 Zelent, A., Mendelsohn, C., Kastner, P., Krust, A., Gamier, J.-M., Ruffenach, F., Leroy, P. and Chambon, P., Differentially expressed isoforms of the mouse retinoic acid receptor are generated by usage of two promoters and alternative splicing, EMBO J., 10 (1991) 71-81.
194
BRESM 70556
Analysis of the promoter sequence and the transcription initiation site of the mouse 5-HT c serotonin receptor gene L a u r a J. B l o e m *, Y a n Chen, J i a n Liu, L e i g h a n S. Bye a n d Lei Y u Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202 (USA) ~'Accepted 15 September 1992)
Key words: Xenopus oocyte; Nuclear injection; Neurotransmitter receptor
The serotonin lc (5-HTIc) receptor is found in many brain regions, but is particularly enriched on the epithelial cells of the choroid plexus. A major challenge in neurobiology is to delineate the molecular processes that regulate the specific pattern of neuronal gene expression in the brain. As an initial step towards identi~ing cis-acting DNA sequences that control the expression of the 5-HTtc receptor, we have isolated the promoter sequence of its gene. Sequence analysis of a 1.8 kb fragment indicated that the 3' end of this fragment overlaps with the 5' untranslated region of the 5-HTtc receptor mRNA, and primer extension using mouse brain poly(A)+ RNA mapped the transcription initiation site within this fragment. There are a number of sequence elements upstream from the transcription initiation site that are homologous to regulatory elements found in other eucaryotic genes. To determine the promoter activity, a plasmid was constructed that contains this fragment as promoter region and the eDNA for the 5-HTtc receptor as the reporter. When injected into the nucleus of Xenopus oocytes, this construct resulted in functional expression of the reporter gene. Primer extension using the RNA extracted from the injected oocytes indicated a single transcription initiation site of the reporter mRNA. These results suggest that the 5-HTIc receptor was functionally expressed under the promoter activity of the 1.8 kb 5' sequence of its gene. This system will be useful for further analysis of the cis-acting elements in the promotor region of the 5-HTtc receptor gene and the trans.acting factors that regulate tissue-specific expression of the receptor.
INTRODUCTION
The diverse physiological effects of the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) are mediated by specific membrane receptors that have been classified into pharmacologically distinct subtypes~'2t,47. The 5-HTtc receptor is one of the major serotonin receptor subtypes in the brain. It is present at high density on the epithelial cells of the choroid plexus 22'46'5s'59. The eDNA for the receptor has been cloned in mouse 37,6~, rat 28 and human 5~. Using the cDNA as probe, in situ hybridization studies have shown that the 5-HT~c receptor is expressed in a variety of cortical and sub. :tica'. neurons in addition to the choroid plexus ~- 40,43 A major goal of neurobiology is to define in molecular terms the processes that regulate the regional ex-
pression pattern of neurotransmitter receptors. Regulation of receptor gene expression can be at the transcriptional level, at the translational level, and by posttranslational modifications 49. Many eucaryotic genes have been studied that are expressed only in certain cells and tissues and are regulated at the level of transcription 2't6'~9'26'56'62. Studies have suggested that c/s-acting sequences consisting of both positive and negative upstream regulatory sequences as well as enhancer elements are responsible for tissue-specific expression of a gene. Presumably, tissue-specific transcription factors interact with cis-acting DNA sequences to control the level of RNA synthesis in different cell typos 29'42. Little is known about the molecular processes that regulate the regional expression pattern of the 5-HTmc receptor. We report here the isolation of the 5' se-
Correspondence: L. Yu, Department of Medical and Molecular Genetics, Indiana University School of Medicine, 975 West Walnut Street, Indianapolis, IN 46202, USA. Fax: (1) (317) 274-2387 or (317) 274-1069. * Present address: Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
195 q u e n c e o f the m o u s e 5-HT~c r e c e p t o r gene a n d the analysis o f its p r o m o t e r activity. This work has b e e n p r e s e n t e d in abstract form 4'5. MATERIALS A N D METHODS
Library screening and recombinant DNA A mouse genomic library in EMBL-3 SP6/T7 (Clontech) was screened by a polymerase chain reaction (PCR) method 6 using primers lor the 5' untranslated region of the mouse 5-HT~c receptor cDNA. One positive clone was isolated and mapped by restriction enzyme analysis. A 1.8 kb Sstl-EcoRl fragment within this clone was gel-isolated, fragmented by sonication and subcloned into M13mpl9 for dideoxy sequence analysis. The same fragment was blunt.ended with E. coli DNA polymerase I large fragment in the presence of all four deoxynucleotide triphosphates and then ligated to the vector pKS + SVOCAT 54 tO create the plasmid KS + SV2CAT. The CAT gene was removed from KS + SV2CAT by digestion with Hpai and EcoRV and replaced with a 23 kb XbaI-EcoR! fragment containing the mouse 5-HTlc cDNA6~ to create the plasmid pKS + 21C. A 2.6 kb Xbal-HindllI cDNA fragment for the rat M t muscarinic acetylcholine receptor 9 was gel-isolated after restriction digestion of the plasmid pGEM7-MI and subcloned downstream from the SV40 promoter in the vector pSV40poly 52 to, create the plasmid pSVpolyM1.
RNA isolation RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction t2. Tissues (either 100 mg total mouse brain or 10 oocytes) were homogenized in 1.0 ml of denaturing solution (4.0 M guanidinium thioojanate, 25 mM sodium citrate, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol). After the addition of 0.1 ml 2.0 M sodium acetate (pH 4.0), 1.0 ml water saturated phenol and 0.2 ml of chloroform-isoamyl alcohol (49:1), the homogenate was vortexed, chilled on ice for 15 min and then centrifuged at 10,000x g for 10 rain at 4°C. The aqueous phase was collected, and the RNA was precipitated by the addition of 1.9 ml of isopropanol and incubated at -20 °C for at least 1 h. The RNA was pelleted and resuspended in 0.3 ml of denaturing solution and reprecipitated with isopropanol. After washing of the pellet with 70% ethanol, the pellet was resuspended in water. Poly(A) + RNA was isolated using an oligo-dT column.
Primer extension The sequence of the 20-mer deoxyoligonucleotide primer 5'-TCCAAGGGCTCAATCATTAA-Y(AU5) is complementary to nucleotides 241-224 of the cDNA sequence in Fig. 1. The primer was phosphorylated using T4 polynucleotide kinase to a specific activity of 1.0× 10s cpm//~g. Whole brain POly(A)+ RNA from 2-week-old mice (80/zg) or total RNA from 10 oocytes was mixed with 1.0x 106 cpm of oligo in 35 /tl of water, incubated at 70°{2 for 10 min and quickly chilled on ice. Primer extension reactions were carried out at 45 °C for 90 min in 50 mM Tris-Ci (pH 8.3), 40 mM KO, 1 mM dithiolthreitol, 6 mM MgCI2, 0.25 mM each of dNTP, 50 /~g/ml actinomycin D, 100 units/mi RNasin, and 400 units of M-MLV H- RT (Superscript, BRL). After extension, the reaction was treated with RNase A (0.2 mg/ml) at 37°C for 30 min, extracted once with phenol/chloroform (1:1), once with chloroform/isoamyl alcohol (49:1) and ethanol precipitated.
Electrophysiology Nuclear injection into Xenopus oocytes was performed as previously described tT'4a. Two to 3 days after injection, individual oocytes were transferred to a recording chamber for voltage-clamp recording. The chamber was continuously superfused with either normal or ligand-containing frog Ringer's solution 6°. The results were recorded both by a microcomputer with the aid of the software pCLAMP (Axon Instruments) and on a Gould 200 dual-channel chart recorder. Data analysis was performed with the same software package.
RESULTS
Isolation and analysis o f the promoter sequence o f the mouse 5.HT!c receptor gene A m o u s e g e n o m i c library in E M B L - 3 SP6/',vT, was s c r e e n e d by a P C R screening method6~ using primers corr,~sponding to the 5' u n t r a n s l a t e d region of m o u s e 5-HT~c r e c e p t o r c D N A 6~ to amplify a 100 bp fragment. O n e positive clone was isolated, a n d restriction m a p ping revealed t h a t it contains a p p r o x i m a t e l y 8 kb of the 5' sequence o f the 5-HT~c r e c e p t o r g e n e (Fig. 1A). T h e P C R f r a g m e n t was located in a 1.8 kb E c o R I - S s t I f r a g m e n t . S e q u e n c e analysis of this f r a g m e n t revealed that it overlaps with the 5' e n d o f the m o u s e c D N A sequence 6~ f r o m b a s e pair 305 a n d the rat c D N A sequence 2s f r o m b a s e pair 177 (Fig. 1B). T h e start site for transcription was m a p p e d by p r i m e r extension with m o u s e brain poly(A) + R N A using a p r i m e r c o r r e s p o n d i n g the 5' e n d o f the m R N A ( A U 5 , Fig. 1B). T h e m a j o r product was 241 bases long (Fig. 2), thus m a p p i n g the transcription initiation site to 566 base pairs u p s t r e a m f r o m the S s t l site (Fig. 1B). This gives the 5 - H T l c r e c e p t o r m R N A a 5' u n t r a n s l a t e d
sequence of 855 nucleotides. Examination of the immediate upstream sequence of the transcription initiation site reveals several notable features (Fig. 1B). The sequences TATGAA at -29, and AAATI'AAT at -21, and CCATG at - 6 6 and CCCCATC at - 8 5 are the closest approximations to TATA and CAAT box consensus sequences with the appropriate spacing reported for many eucaryotic promoters 14,~5,42.There are also a number of sequences in this region that resemble the identified c/s-acting ele. ments or enhancers for transcriptional control of genes. These include a potential AP2 binding site (TFCCC. CATCT) eS, two AP1 binding sites (TGAATCA,TrAA. TCA) 7'3s, as well as consensus sequences for ENKCRE-1 (TACGCCA) 24 and the octamer motif binding factor (GTGCC) s (Fig. 1B). Other notable regions within the 1.8 kb EcoRI-Sstl fragment are two repetitive sequences. One is a purine-pyrimidine repeat at - 1038 consisting of (CA)20 and the other is a TCTG repeat from - 129 (Fig. 1B). Both of these types of repeats are often associated with highly polymorphic regions of the genome and could represent potential polymorphic markers t°. There are also two pairs of direct repeats each of 12 base pairs (A and B, Fig. IB) as well as four pairs of inverted repeats of 9 base pairs (AA~BB) and 8 base pairs (CC, DD).
Functional analysis of the promoter region in Xenopus oocytes
Cell type-specific promoters have been shown to have some activity when injected into Xenopus
196
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Fig. 1. A: restriction map of A MG4A3. The 8.0 kb Xhol-$stl fragment contains the 5' sequence for the 5-HT~c receptor gene. The 1,8 kb EcoRl--Sstl fragment indicated with cross-hatching was the region analyzed further in this study. B: nucleotide sequence of the 1.8 kb EcoRI-Sstl fragment. The sequence of the 5-HTlc promoter is shown, together with 584 base pairs upstream from the cap site (?) determined by primer extension in this study. The nucleotides are numbered with the cap site as + 1. The putative TATA and CAAT boxes are indicated by the symbol e. Primer binding sites (PCR primers 1 and 2, AUS) as well as the CA- and TCTG repeats are underlined. The transcription start site in oocytes is indicated by an asterisk. The sequence was searched for putative transcription factor binding sites. Sequences showing homology to the consensus sequences associated with the binding of a number of transcription factors are indicated by the symbol v. Direct repeats A and B as well as inverted repeats AA, BB, CC and DD are indicated by arrows.
Fig. 2. Primer extension analysis of mouse brain poly(A) + RNA. Samples were analyzed on a 6% sequencing gel along with a sequencing ladder using a known DNA template as the size marker (lanes A, C, G and T). Lane 1, primer extension using 80 ,¢g of poly(A) + isolated from 2-week-old mouse brain. The length (in bases) of the major extension product is indicated.
resulting plasmid, pKS+21C, was injected into the nucleus of Xenopus oocytes. Another plasmid containing the muscarinic M n receptor cDNA ~ downstream from the SV40 promotor (pSVpolyM1, Fig. 3) was co-injected as an internal control. Two days post injection, oocytes were assayed for serotonin- and acetylcholineinduced currents. Representative traces of membrane current are shown in Fig. 4. Oocytes injected with 0.25 ng DNA did not respond to serotonin. This amount of DNA was shown to be below the threshold for oocyte expression s°. Accuracy of nuclear injection was demonstrated by the oocyte's response to acetylcholine (Fig. 4A). Oocytes injected with 2.5 ng DNA produced both serotonin- and acetylcholine-induced currents (Fig. 4B). The size of the depolarizing currents ranged from 500 to 2000 nA in response to 1.0 ~M serotonin. The presence of serotonin-induced depolarizing membrane currents suggests that the 5-HT~c receptor was functionally expressed under the promoter activity of the 5' flanking sequence of its gene. Cell-type specific genes, when injected alone into oocytes, often produce multiple RNA species with 5' termini located both upstream and downstream of the original cap site 5°. Using the RNA isolated from oocytes injected with 2.5 ng of pKS+21C DNA, primer
197
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Fig. 3. Schematic diagrams of the plasmids pKS+21C and pSVpolyM1. The 1.8 kb fragment of the 5-HTlc receptor promoter was cloned in front of the mouse 5-HTic receptor cDNA. The rat M 1 muscarinic acetylcholine receptor was cloned downstream of the SV40 promoter.
region reveals that 5-HT~c mRNA has a fairly lon~ 5'-noncoding region of 855 bases. Only one-fourth of the 5'-noncoding sequences from vertebrate mRNAs scored by Kozak were more than 100 bases in length3~. 5'-noncoding sequences of most vertebrate mRNAs are from 20-100 nucleotides in length. One group of messages with longer 5'-noncoding sequences are the mRNAs derived from proto-oncogenes 32'33. The association of the 5-HT~c receptor with proto-oncogenes is not new. It has been demonstrated that ectopic expression of the 5-HT~c receptor in NIH-3T3 cells is both
1
ACGT
extension experiments showed a single transcription initiation site for the reporter mRNA (Fig. 5). This site is 36 base pairs downstream from the transcription initiation site mapped with mouse brain poly(A) + RNA (Fig. 2). The altered transcription initiation in oocytes suggests that brain nuclear proteins may be required for transcription initiation at the original position. DISCUSSION
We report here the isolation and characterization of the 5' sequence of the mouse 5-HT~c receptor gene. Mapping of the transcription initiation site within this
Fig. 5. Primer extension analysis of reporter mRNA isolated from
Xenopus oocytes. Samples were analyzed on a. 6% sequencing gel along with a sequencing ladder (lanes A, C, G and T). The length of the major extension product is indicated.
198 necessary and sufficient to produce transformed foci which, when introduced into mice, lead to the generation of tumors 27. Thus, neurotransmitters and neurotransmitter receptors have the potential to act as regulators of neural cell growth ts. The frequent presence of long leader sequences in critical regulatory genes suggests that such sequences may constitute an important part of gene regulation in vertebrates 33 and could play an important role in the regulation of translation of the mouse 5-HT~c message. Comparison of the DNA sequence upstream from the transcriptional initiation site of the 5-HT~c gene with consensus binding sequences for transcription factors revealed the presence of several potential promoter and enhancer elements. The AAATTAAT at -21 may serve as a potential TATA box in that it contains a T r A A element found to provide the principal TATA box function for the rat brain creatine kinase gene t9 and that it is AT-rich. A second potential TATA box at - 2 9 (TATGAA) has been found in one of the aeu-globulin genes57. Either one of these sequences could serve as a TATA promoter element. The presence of another classical promoter element, the CAAT box, is less obvious. The sequences at - 6 6 (CCATG) and at - 85 (CCCCATC) bear some resemblence to the consensus CAAT box 14,~5,but the homologies are low. In light of this, it is of interest to note that a number of G protein-coupled receptor genes have also been shown to lack a CAAT box 3°,4L45. Several consensus enhancer sequences were also present in this 1.8 kb region. One such sequence, GTGCC at -69, has been shown to bind the octamer motif binding factor in vitro ~. Another sequence (TACGCCA) overlapping with the transcription start site, characterizes the ENKCRE-1 element of the proenkephalin gene 24. This sequence comprises one of the cAMP- and phorboi ester-inducible enhancers of the neurotransmitter proenkephalin gene and ~..ppears to bind a distinct factor ENKTF.1 ~3. There are also two potential binding sites (at - 1 8 and -27) for the enhancer protein AP-1 with the consensus sequence T(T,G)ANTCA7,35. Studies suggest that this particular transcription factor may be modulated by pharmacological agents known to stimulate protein kinase C. Another sequence, T r c c CCATCT found at -87, is a potential AP-2 binding site. This transcription factor binding site forms a cell type-specific enhancer element whose activity is increased in response to treatment of cells with phorbol esters and agents that elevate cAMP 25. The presence of a number of potential cAMP- and phorbol ester-responsive elements suggests a possible role for these second messengers in the regulation of 5-HT~¢ receptor transcription. In addition, the density
of overlapping sequences predicted to bind transcription factors emphasizes the potential importance of the region immediately upstream from the transcription start site. Functional studies will be required to determine if these sequence elements do represent transcription factor binding domains. Promoter sequences for several other neurotransmitter receptors have been isolated and analysed. These include the human and hamster/32-adrenergic receptor genes 3°, the human D~A-dopamine receptor gene 41 and the human 5-HTtA receptor gene 45. They share many characteristics of the promoters for housekeeping genes, including a G + C-rich content, multiple potential binding sites for the transcription factor SP1 and, in some cases, a lack of well-defined consensus sequences for TATA or CAAT boxes. The promoter for the mouse 5-HT~c receptor gene does not appear to conform to these neurotransmitter receptor promoters in that it does not contain G + C-rich regions or potential SP1 binding sites. However, it does share characteristics with human D~A-dopamine receptor gene as both have potential AP1 and AP2 binding sites. Recent years have seen rapid progress in the characterization of cis-acting regulatory elements and the isolation of trans-active protein factors that bind to them. Typically, this has been accomplished by transient expression gene transfer into cultured mammalian cells. Nuclear injection in Xenopus oocytes offers certain advantages over cultured cells for characterizing tissue-specific regulatory elements and transcription factors 23,5°. One advantage is that the copy number of the introduced gene in oocytes can be predetermined in microinjection whereas the copy number of a transfected gene in cultured cells is quite variable. This is important because of the accumulating evidence that gene expression is often regulated by a fine balance between gene dosage and the concentration of modulating factors in the cell 3'26'34'55'56.Another advantage of the oocyte system is that it is possible to manipulate the nuclear environment of the oocyte by coinjection with nuclear extracts from different cell types 23'44'50. This allows for analysis of both activators and repressors in the same system as well as the ability to study the role of nuclear proteins in differentiated cells from various tissue sources and brain regions, without the necessity for establishing a cell culture from the tissue of interest, The oocyte expression system has been used to study a number of genes including the chicken skeletal a actin gene ~, the chicken 5-aminolevulinate synthase gene 36, the herpes simplex virus thymidine kinase gene 39, the rabbit /3 globin promoter s°, the endogenous viteUogenin promoter 3s and the trans-activation
199
of the mouse IL-2 gene 44. We have shown in this study that the 5-HT~c receptor promoter, when injected into Xenopus oocytes, has characteristics similar to that observed for other cell type-specific genes. First, nanogram quantities of DNA are required to activate the 5-HTmc promoter in oocytes as has been shown for other tissue-specific genes44'5°(Fig. 4), whereas viral promoters such as the SV40 promoter require only picogram quantities 39'4s. This activity of the 5-HT~c promoter is probably due to the binding of general transcription factors in the oocyte to basic promoter elements present in the 5-HT~c receptor gene. A second characteristic of tissue-specific genes expressed in oocytes is the presence of multiple reporter RNA species with transcription initiation sites both upstream and downstream from the original cap site 5°. Initiation of transcription from the 5-HT~c promoter in oocytes is from a cryptic site 36 base pairs downstream from the cap site mapped with mouse brain mRNA. Thus it appears that, although the 5-HTlc promoter is functional in oocytes, certain nuclear proteins in the mouse brain may be required for authentic initiation of transcription. The nuclear injection assay in Xenopus oocytes will be useful to further analyze the c/s-acting elements in the promotor region of the 5-HT~c receptor gene and to identify both positive and negative trans-acting factors that regulate tissue-specific expression of the receptor. Acknowledgements. We thank Dr. Alexander Stacey for SVpoly vector, Dr. Tom Bonner for M I receptor cDNA, Drs. Maureen Harrington and Lucy Carr for helpful discussions, and Weiyin Li for technical assistance. This work was supported in part by a grant from the NIH (NS 28190) and a grant from Eli Lilly & Company to L.Y.L.J.B. and L.S.B. were supported by an NIH training grant (T32HD07373) and J.L. was supported by a John B. Hickam Memorial postdoctoral fellowship from the American Heart Association, Indiana Affiliate, Inc.
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