Characterisation of potential regulatory elements within the rat preprotachykinin-A promoter

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NeuroscienceLetters 184 (1995) 125-128

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Characterisation of potential regulatory elements within the rat preprotachykinin-A promoter S.C. M e n d e l s o n , J.P. Q u i n n * MRC Brain Metabolism Unit, Royal Edinburgh Hospital, Morningside Park, Edinburgh, EHIO 5HF, UK

Received 14 September1994;revisedversionreceived23 November1994;accepted23 November1994

Abstract

The rat preprotachykinin-A (rPPT-A) gene encodes the precursor of several tachykinin neuropeptides including substance P. Previous studies have demonstrated the presence of multiple DNA sequences important for directing expression of the rPPT-A gene in dorsal root ganglion neurons within a region of the promoter spanning nucleotides -865 and ---47. In order to identify potential cis acting elements, we have carried out DNase I footprinting analysis using a series of constructs containing fragments from this region of the promoter. This study has defined three potential AP-1 complex interactions, two E box binding protein interactions and two dG rich elements, which are potentially bound by complexes related to AP-2 or Spl in this region of the promoter. Keywords: Transcription; Tachykinin; AP-1; bHLH; AP-2; Spl; Substance P

The tachykinins are a family of neuropeptides which display a wide but selective distribution throughout the central and peripheral nervous systems where they are thought to function as neurotransmitters [23]. Rat preprotachykinin-A (rPPT-A) gene expression in vivo is regulated by a wide variety of stimuli including growth factors [14,15], conditioned media [21], inflammation [22], cocaine [12], and factors ]present in innervated target tissues [1]. It is likely that many of the stimuli act at the level of transcription and that the promoter of the rPPT-A gene has to integrate these various stimuli to allow for the correct expression of the gene. It has been shown by deletion analysis of the rPPT promoter that the majority of sequences important for directing expression of the rPPT-A gene in dorsal root ganglia (DRG) neurons lie in a region of the promoter spanning nucleotides -865 to -47 [19]. In order to map DNA binding motifs within this promoter region, this study set out to determi:ae the sites of protein/DNA interactions using the DNase 1 footprinting assay. It is difficult to obtain sufficient quantities of homogeneous nuclear extract from DRG neurons for biochemical analysis, therefore, we performed this initial characterisation of DNA binding motifs within the rPPT-A promoter * Correspondingauthor, Fax: +44 31 537 6110.

using nuclear extract obtained from HeLa cells. HeLa cells have been shown to express a large array of transcription factors many of which are also expressed in DRG neurons, including Octamer binding proteins [28], E box binding proteins [2], AP-1 binding proteins [5], AP-2 [17] and in addition many proteins which are thought to be ubiquitously expressed. Therefore, it is likely that many of the regulatory elements present within the rPPTA promoter will be recognised by proteins or related family members present in both DRG neurons and in HeLa cells. HeLa nuclear extract was prepared as previously described [6]. A series of plasmids were used for the purposes of DNase 1 footprinting analysis. The constructs prPPTflGal 3, prPPT-/~Gal 4, prPPT-flGal 5 and prPPTflGal 6 contain fragments from the rPPT-A promoter spanning nucleotides -865 to +92, -671 to +92, -431 to +92 and -345 to +92, respectively, linked to a lacZ (t3galactosidase) reporter gene and have been described previously [19]. The construct pSM/Q5 contains a fragment spanning nucleotides -160 to +447 which was subcloned into the SmaI site of pUC19. Suitable substrates for DNase I footprinting analysis were generated by digestion of the constructs with SalI in the polylinker sequence in the above plasmids. Resulting linearised plasmids were then dephosphorylated with calf

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S.C. Mendelson, J.P. Quinn I Neuroscience Letters 184 (1995) 125-128

126

LanQ8

DNale 1 ExtZl~'t

A.

123 + + - +

B. + ++

1 + -

2 3 + M+G +

C.

I + -

2 + +

3 M+G

D

123 ++M+G -+

E.

1 + -

2 3 + M+G +

Fig. 1. DNase 1 footprinting of the rPPT-A promoter. In (A-E) the data are presented as: lane 1 which contains probe and DNase 1 (1.5 units (A,E); 1.25 units (B,C) and 2.5 units (D)); lane 2 which contains probe (100 ng of singly end labelled DNA fragment), HeLa extract (1 mg) and 30 units of DNase 1; lane 3 shows the G + A Maxam Gilbert sequencing reaction of each DNA probe. Comparison of the products of DNase 1 digestion with Maxam Gilbert sequence reactions of the appropriately labelled fragments allowed the sequence of regions protected from DNase 1 digestion to be determined. For Maxam Gilbert sequencing, singly end-labelled DNA fragments were sequenced using the G + A reaction in the Sigma MaxamGilbert kit according to the manufacturer's instructions (Sigma). Binding reactions containing probe, protein extract, and poly d0-C) (inosine-cytosine) were incubated at room temperature for 20 min, DNase 1 enzyme was then added and incubated at 37°C for 2 min. DNase 1 activity was terminated by the addition of EDTA and SDS and the DNA was separated by gel electrophoresis (6% bis:acrylamide (29:1), 8.3 M urea). The products were separated on 80 x 20 cm gels which necessitated the use of multiple sheets of autoradiographic film, thus leading to the cut and pasting which can be observed for some of the data. (A) The construct pSMI/Q was digested with DNase 1. A footprint was generated (lane 2) which spans nucleotides -67 to --47 relative to the major transcription start site. (B) The construct prPPT-flGal 5 was digested with DNase 1. Three footprints were generated in lane 2 spanning nucleotides -345 to -330, -324 to -308 and -284 to -264. (C) The construct prPPT-flGal 6 was digested with DNase 1. Two footprints were generated in lane 2 spanning nucleotides -198 to -180 and -177 to -155. (D) The construct prPPT-flGal 4 was digested with DNase 1. Two footprints were generated (lane 2) spanning nucleotides -618 to -607 and -575 to -546. (E) The construct prPPT-flGal3 was digested with DNase 1. A footprinted region was generated by HeLa nuclear extract (lane 2) spanning nucleotides -779 to -755. intestinal phosphatase a n d radioactively labelled at both 5' termini by T4 p o l y n u c l e o t i d e kinase. Finally, the constructs prPPT-flGal 3, prPPT-flGal 4, prPPT-flGal 5 and prPPT-flGal 6 were digested with XbaI and pSM/Q5 was digested with HindIII to p r o d u c e two singly end labelled fragments, o n e of almost full length and the other of 1 0 20 bp in length which w o u l d not interfere in the assay. The footprints obtained using these constructs are demonstrated in Figs. 1 A - E , and the data are summarised in Fig. 2. U s i n g construct p r P P T - ~ G a l 5, two sequence elements were identified within the r P P T - A promoter by DNase 1 footprinting analysis, which display homologies to an AP-

1 consensus sequence ( T G A G/C T C A ) [4] (Fig. 1B). The footprint s p a n n i n g nucleotides - 3 2 4 to - 3 0 8 displays a six out of seven base pair match and is termed A P - I ' and the footprint s p a n n i n g nucleotides - 3 4 5 to - 3 3 0 contains a perfect match to an AP-1 consensus sequence and is termed AP-1 (Fig. 2, elements 5 and 6). Additionally, using construct prPPT-flGal 6, an e l e m e n t was identified (Fig. 1C) which is termed A P - 1 / C R E as it displays a six out of seven base pair match to an A P - I consensus sequence and a seven out of eight base pair match to a CREB motif ( T G A C G T C A ) [26] (Fig. 2, e l e m e n t 3). This element s p a n n i n g nucleotides - 1 9 8 to - 1 9 0 has previously been shown to contain e n h a n c e r properties as it will

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s.c. Mendelson, J.P. Quinn / Neuroscience Letters 184 (1995) 125-128

activate reporter gene expression from a heterologous minimal promoter in both HeLa cells and DRG primary culture cells [ 18]. Two footprints were generated which contain an E-box consensus sequence (CANNTG), recognised by dimeric protein complexes composed of members of the basic helix-loop-helix and b~sic helix-loop-helix leucine zipper (bHLH and bHLH-Zip) families of proteins [20]. One of these footprints, termed E-box 1 obtained with the pSM/Q5 construct, spans nucleotides -67 to --47 (Fig. IA and Fig. 2, element 1) and contains the sequence CACGTG. The second footprint, termed E-box 2 obtained with construct prPPT-flGal 6, spans nucleotides -177 to -155 (Fig. 1C and Fig. 2, element 2)'and contains the sequence CAGATG. Two footprints were also generated within the rPPT-A promoter which are rich in dG residues. One of the footprints, obtained with construct prPPT-flGal 5, spanning nucleotides -284 to -264 (Fig. 1B) which we term PRE1 (purine-rich element 1) is rich in both dG and dA resides (Fig. 2, element 4). The other element (PRE2) obtained with construct prPPT-flGal 4 is rich in dG residues and spans nucleotides -576 to -546 (Fig. 1D and Fig. 2, element 7). Both of these footprints contain regions of sequence displaying homologies to an AP-2 consensus sequence (GN4GGG) [27] and the consensus sequence for a number of Zn 2÷ finger binding proteins including Spl (G/T G/A GGCG G/T G/A G/A C/T) [9]. A second footprint obtained with prPPT-flGal 4 was observed in this promoter region spanni~Lg nucleotides -618 to -607 (Fig. 1 and Fig. 2, element 8). Finally, DNase 1 footprinting analysis using prPPTflGal 3 identified a pote:atial regulatory element covering a region of the rPPT-A promoter which is adjacent to an element previously demonstrated to bind specifically to single-stranded DNA binding proteins [25]. A footprint was generated by HeLa nuclear extract which spans nucleotides -779 to -755 (Fig. 1E and Fig. 2, element 9).

A summary of the footprinting data is present in Fig. 2 which gives the DNA sequence of the region of the rPPTA promoter analysed (i.e. -865 to --47) (sequenced by Carter and Krause [3]) and the positions of the protein/ DNA interaction sites identified within this region of the promoter. These identified DNA elements protected from DNase I digestion are likely to be involved in both tissue-specific and stimulus-dependent gene regulation. For example, proteins encoded by the fos and jun gene families which recognise the AP-1 consensus elements and E-box binding proteins have been shown to exhibit tissue-specific and stimulus-dependent gene expression [4,11,13,24]. rPPT-A mRNA expression has been best characterised in DRG, where a characterised regulator of expression in adult rat primary cultures is NGF [15]. Two types of cis acting elements identified in this study, E box and AP-1/ CRE elements, are candidates for mediating this response. DRG neurons have been shown to express high levels of the E-box binding protein neuron-specific homologue of stem cell leukaemia factor (NSCL), which is expressed in the developing nervous system where it is thought to play a role in neuronal development [2]. Additionally, it has been shown that the expression of the bHLH protein mammalian achaete-scute homologous-1 (MASH-l) is upregulated by NGF in PC12 cells [13]. Members of the AP-1 family of transcription factors and the CREB protein are also modulated or induced in response to NGF [10,16,24]. Identification of purine-rich motifs, which may serve as binding sites for AP-2, Spl or other zinc fingercontaining proteins, indicate a potential role for these elements in the induction of rPPT-A mRNA in models of inflammation, mRNA encoding AP-2 has been detected in DRG neurons [17] and it has been shown that the levels of both AP-2 and rPPT-A mRNA increase in DRG neurons in response to adjuvant mediated inflammation in vivo [7,8]. Therefore, it is possible that AP-2 binds to one

-865

C T G C A G A G C T C C A A A G G T A A G C A T C C A G C C T'FFCTAGTCC C C C A A C A A G G C T A A A G G G G A G A G A G G C A C A A T T A T C C T C T T C C C A C C C C T

-775 -685

T C T G C C T T C A ~ G G T G T G C C T G G G A A G A A G C T G T A G G G G A A C A A A A G A T G C CTTAGAATC43 C T G A ~ T A AGTTCTACAT GAGAAAGGAG 9 G T T T A A A T T C CTCT'FI~CCC T A A A T G T A A A A C A A A C C T G C C T T C A T C C T C T G A A G C ~ A GACCGGAAAC ACTTTTGCAG TGCTAGAGAA

-595

ATGAGAATAT TCTGACTGAT TTGG~A

-505

8 GG~TTGG ~TGTGT TCCAGCCCTA GATATAACAC CTCATAAACC TTAAGACACA 7 T A A A G T A G A A A T G A A A G G A A A A C C C C G C T T G C T T C A T C C C T C T G A A G T G C TTGCTC4~TGT C T T A G T A T T A T T C A C A A G G T T'FI~CTGCTC

-415

AAGTTATTTG GCTGTCCTCA AAGCGCAATA TTCCCTGATG CCTCTTGAGA GAAAAGTTCC CTAAGTCCGA AGCATGAGTC ACTTCGCTCA

-145

6 GTTTTGATGA GTAATCTCAG GTGTCACTGA ACCTTGTTCG GAAGAAGAGG GGAG~C GTCAGAT'FI~ C A G A C G G A A G A A A A C A G G T C ~5 4 TCTCTC,G A T T G G A T G G C G A G A C C T C G A C T T C C C T A A A A T T G C G T C A T T T C G A A C C C A A T T T G G T C C A G A T G T T A T G G A C T C C G A C G G G T T 3 2 A C C G T C T C G G AA.ACTCTATC A C G C A A G C A A AAGGCGAC4243 G G C G G C T A A T T A A A T A T T G A G C A G A A A G T C G C G T G G G G A G A G T G T C A C G T

-55

Q~C~CAG

-325 -235

1 GCTCAT

Fig. 2. The sequenceof the PPr-A promoteris shown from-865 to ---40.The sequencesprotectedfrom DNase 1 digestionare underlinedand numbered 1-9.

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s.c. Mendelson, J.P. Quinn I Neuroscience Letters 184 (1995) 125-128

or a c o m b i n a t i o n o f the purine-rich motifs identified within the r P P T - A p r o m o t e r to m e d i a t e the transcriptional response to inflammation. T h e functional significance of these putative transcription factor binding sites will be investigated by microinjection of reporter g e n e constructs driven by the r P P T - A p r o m o t e r containing mutations of these elements, into D P G neurons in culture. W e w o u l d like to thank N o r m a Brearley for the careful preparation o f the manuscript. S M is a recipient of a M R C student fellowship. [1] Barakat-Walter, I., Affolter, H.U. and Droz, B., Expression of substance P and preprotachykinin mRNA by primary sensory neurons in culture: regulation by factors present in peripheral and central target tissues, Mol. Brain Res., 10 (1991) 107-114. [2] Begley, C.G., Lipkowitz, S., G6bel, V., Mahon, K.A., Bertness, V., Green, A.R., Gough, N.M. and Kirsch, I.R., Molecular characterization of NSCL, a gene encoding a helix-loop-helix protein expressed in the developing nervous system, Proc. Natl. Acad. Sci. USA, 89 (1992) 38-42. [3] Carter, M.S. and Krause, J.E., Structure, expression, and some regulatory mechanisms of the rat preprotachykinin gene encoding substance P, neurokinin A, neuropeptide K, and neuropeptide y, J. Neurosci., 10 (1990) 2203-2214. [4] Curran, T. and Franza, Jr., B.R., Fos and Jun: the AP-I connection, Cell, 55 (1988) 395-397. [5] De Felipe, C. and Hunt, S.P., The differential control of c-jun expression in regenerating sensory neurons and their associated glial cells, J. Neurosci., 14 (1994) 2911-2923. [6] Dignam, J.D., Martin, P., Shastry, B.S. and Roeder, R.G., Eukaryotic gene transcription with purified components Methods Enzymol., 101 (1983) 582-598. [7] Donaldson, L.F., Harmar, A.J., McQueen, D.S. and Seckl, J.R., Increased expression of preprotachykinin, calcitonin gene-related peptide, but not vasoactive intestinal peptide messenger RNA in dorsal root ganglia during the development of adjuvant monoarthritis in the rat, Mol. Brain Res., 16 (1992) 143-149. [8] Donaldson, L.F., McQueen, D.S. and Seckl, J.R., Expression of mRNA encoding the transcription factor AP-2 increases in dorsal root ganglion neurons in response to adjuvant injection in the rat in vivo, Proc. Phys. Soc., C165 (1993) 15. [9] Dynan, W.S. and Tjian, R., The promoter specific transcription factor Spl binds to upstream sequences in the SV40 early promoter, Cell, 35 (1983) 79-87. [10] Ginty, D.D., Bonni, A. and Greenberg, M.E., Nerve growth factor activates a ras-dependent protein kinase that stimulates c-fos transcription via phosphorylation of CREB, Cell, 77 (1994) 713-725. [11] Guillemot, F. and Joyner, A.L., Dynamic expression of the murine Achaete-Scute homologue Mash-1 in the developing nervous system, Mech. Dev., 42 (1993) 171-185. [12] Hurd, Y.L., Brown, E.E., Finlay, J.M., Fibiger, H.C. and Gerfen, C.R., Cocaine self-a,4,ninistration differentially alters mRNA expression of striatal peptides, Mol. Brain Res., 13 (1992) 165-170.

[13] Johnson, J.E., Birren, S.J. and Anderson, D.J., Two rat homologues of Drosophila achaete-scute specifically expressed in neuronal precursors, Nature, 346 (1990) 858-861. [14] Kessler, J.A. and Black, I.B., Nerve growth factor stimulates development of substance P in the embryonic spinal cord, Brain Res., 208 (1981) 135-145. [15] Lindsay, R.M. and Harmar, A.J., Nerve growth factor regulates expression of neuropeptide genes in adult sensory neurons, Nature, 337 (1989) 362-264. [16] Milbrandt, J., Nerve growth factor rapidly induces c-fos mRNA in PC12 rat pheochromocytoma cells, Proc. Natl. Acad. Sci. USA, 83 (1986) 4789-4793. [17] Mitchell, P.J., Timmons, P.M., Hebert, J.M., Rigby, P.W.J. and Tjian, R., Transcription factor AP-2 is expressed in neuronal crest cell lineages during mouse embryogenesis, Genes Dev., 5 (1991) 105-119. [18] Morrison, C.F., McAllister, J., Dobson, S.P., Mulderry, P.K. and Quinn, J.P., An activator element within the preprotachykinin-A promoter, Mol. Cell. Neurosci., 5 (1994a) 165-175. [19] Mulderry, P.K., Chapman, K.E., Lyons, V. and Harmar, A.J., 5'Flanking sequences from the rat preprotachykinin gene direct high-level expression of a reporter gene in adult rat sensory neurons transfeeted in culture by microinjection, Mol. Cell. Neurosci., 4 (1993) 164-172. [20] Murre, C., McCaw, P.S., Vaessin, H., Candy, M., Jan, LY., Jan, Y.N., Cabrera, C.V., Buskin, J.N., Hauschka, S.D., Lassar, A.B., Weintraub, H. and Baltimore, D., Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence, Cell, 58 (1989) 537-544. [21] Nawa, H. and Sah, D.W.Y., Different biological activities in conditioned media control the expression of a variety of neuropeptides in cultured sympathetic neurons, Neuron, 4 (1990) 279-287. [22] Noguchi, K. and Ruda, M.A., Gene regulation in an ascending noeiceptive pathway: inflammation-induced increase in preprotachykinin mRNA in rat lamina I spinal projection neurons, J. Neurosci., 12 (1992) 2563-2572. [23] Otsuka, M.S., Konishi, M., Yanagisawa, A., Tsunoo, A. and Akagi, H., Role of substance P as a sensory transmitter in the spinal cord and sympathetic ganglia, CIBA Foundation Symposium 91: Substance P in the Nervous System, Pitman, London, 1982. [24] Quinn, J.P., Variation in the composition of the AP-1 complex in PCI2 cells following induction by NGF and TPA, Mol. Cell. Neurosci., 2 (1991) 253-258. [25] Quinn, J.P. and McAllister, J., The preprotachykinin A promoter interacts with a sequence specific single stranded DNA binding protein, Nucleic Acids Res., 21 (1993) 1637-1641. [26] Roesler, W.J., Vandenbark, G.R. and Hanson, R.W., Cyclic AMP and the induction of eukaryotic gene transcription, J. Biol. Chem., 263 (1988) 9063-9066. [27] Williams, T. and Tjian, R,, Analysis of the DNA-binding and activation properties of the human transcription factor AP-2, Genes Dev., 5 (1991) 670-682. [28] Wood, J.N., Lillycrop, K.A., Dent, C.L., Ninkina, N,N., Beech, M.M., Willoughby, J.J., Winter, J. and Latchman, D.S., Regulation of expression of the neuronal POU protein Oct-2 by nerve growth factor, J. Biol. Chem., 267 (1992) 17787-17791.