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The Neuron-Restrictive Silencer Element: a Dual Enhancer͞silencer Crucial for Patterned Expression of a Nicotinic Receptor Gene in the Brain

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The Neuron-Restrictive Silencer Element: a Dual Enhancer͞silencer Crucial for Patterned Expression of a Nicotinic Receptor Gene in the Brain Proc. Natl. Acad. Sci. USA Vol. 94, pp. 5906–5911, May 1997 Neurobiology The neuron-restrictive silencer element: A dual enhancerysilencer crucial for patterned expression of a nicotinic receptor gene in the brain ALAIN BESSIS*, NICOLAS CHAMPTIAUX,LAURENT CHATELIN, AND JEAN-PIERRE CHANGEUX† Neurobiologie Mole´culaire,UA CNRS D1284, De´partementdes Biotechnologies, Institut Pasteur 25y28 rue du Dr Roux, 75724 Paris Cedex 15 Contributed by Jean-Pierre Changeux, March 24, 1997 ABSTRACT The neuron-restrictive silencer element transcriptional repressing activity on promoters that carry the (NRSE) has been identified in several neuronal genes and NRSE sequence (15, 21, 23). confers neuron specificity by silencing transcription in non- In this work, we have analyzed the function of NRSE in the neuronal cells. NRSE is present in the promoter of the promoter of the mouse gene encoding the nAChR b2-subunit neuronal nicotinic acetylcholine receptor b2-subunit gene as well as the importance of its location within the promoter. that determines its neuron-specific expression in the nervous In transgenic mice, we show that upon mutation of NRSE, the system. Using transgenic mice, we show that NRSE may either pattern of expression of the b2-subunit gene promoter dra- silence or enhance transcription depending on the cellular matically changes but remains restricted to the nervous system. context within the nervous system. In vitro in neuronal cells, We confirm that NRSE in vitro can behave either as an NRSE activates transcription of synthetic promoters when enhancer or as a silencer depending both on the primary located downstream in the 5* untranslated region, or at less structure of the promoter and the type of cell line. We further than 50 bp upstream from the TATA box, but switches to a show by antisense experiments that in neurons, REST con- silencer when located further upstream. In contrast, in non- tributes to the activation of transcription via NRSE. A model neuronal cells NRSE always functions as a silencer. Antisense for the regulation of transcription by RESTyNRSE in neurons RNA inhibition shows that the NRSE-binding protein REST is proposed. contributes to the activation of transcription in neuronal cells. MATERIALS AND METHODS The mechanisms that account for transcriptional regulation in neurons are still poorly understood. Yet, increasing numbers Plasmids. NRSE-SVP, an oligonucleotide containing three of transcription factors (1–3) and DNA elements (4, 5) in- NRSE sequences of the b2-subunit gene, was inserted into the volved in neuron-specific transcription are now characterized. NotIyPstI sites of SVP-Luci (described in ref. 4). For NRSE- One of them, the neuron-restrictive silencer element (NRSE; TATA and TATA-Luci, NRSE-SVP and SVP-Luci were re- ref. 6), also called RE (7), behaves as a regulatory sequence of stricted by SalIyNcoI filled with the klenow enzyme. The several neuronal genes (8) by silencing transcription in non- largest restriction fragment was self-ligated. TATAIIB, the neuronal cells (6, 7, 9–14). The silencing activity of NRSE was small PstIyXbaI fragment of 40IIB-MyHC-CAT (ref. 24; primarily studied by transient transfection assays in the pro- kindly provided by Thierry Diagana, Institut Pasteur, Paris) moter of the genes encoding type II sodium channel (NaII; 7) was inserted into SVP-Luci. The NRSE oligonucleotide was and SCG10 protein (6). It was also recognized in the genes then inserted into the NotIyPstI sites to obtain NRSE- encoding synapsin I (9), nicotinic acetylcholine receptor TATAIIB. The plasmids mutated in NRSE were constructed (nAChR) b2-subunit (5), Ng-CAM (11), m4 muscarinic re- using a mutated oligonucleotide (NRSE-Mut). NRSE-spacer- ceptor (12, 13), and choline acetyltransferase (ChAT; 14). On TATA, the 198-bp PstIyBspHI fragment from the rat GluR1 the basis of sequence homologies, Schoenherr et al. (8) recently cDNA (ref. 25; accession number X17184) was inserted into identified about 20 genes carrying a NRSE-like sequence. the PstIyNcoI site of NRSE-SVP. The different lengths of Among these genes, 17 are expressed in the nervous system spacers were obtained by PCR. uTATA-Luci, the BglIySalI and in 10 of them, including the nAChR b2-subunit gene (5), fragment of TATA-Luci, was excised and the plasmid was NRSE is located in the 59 untranslated region (UTR) (ref. 15; self-ligated after action of the T4 DNA polymerase. TATA- see also refs. 16 and 17). Although important functional 272-NRSE, the BglIIyHindIII fragment of NRSE-SVP, was elements have already been found in the 59 UTR or in inserted into the StuIyHindIII sites of uTATA-Luci. uTATA- intragenic positions (4, 18–20), NRSE is, to our knowledge, the 272-Luci, the NRSE sequence, was excised from TATA-272- first element sharing a conserved intragenic position in several NRSE by SacIIyPstI. This plasmid was used to normalize the genes. The functional significance of such an unusual position transcriptional activity of TATA-272-NRSE. TATA-34- remained to be explained. NRSE, the SfiIySacII fragment of TATA-272-NRSE plasmid, A transcription factor of the Gli-Kru¨ppel zinc-finger family was excised. The transcriptional activity of this plasmid was that binds to NRSE has recently been characterized in HeLa normalized to that of uTATA-Luci. The plasmids were se- cells, and named neuron-restrictive silencer factor (NRSF; ref. quenced to rule out mistakes of the Taq DNA polymerase. 15) or RE1 silencing transcription factor (REST; ref. 21, see CMV-TSER, the 4100-bp EcoRI fragment of the REST- also ref. 22). In vitro studies revealed that REST exhibits a EXPRESS plasmid (ref. 21; kindly provided by Gail Mandel, University of New York, Stony Brook), was inserted into the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in Abbreviations: nAChR, nicotinic acetylcholine receptor; NRSE, accordance with 18 U.S.C. §1734 solely to indicate this fact. neuron-restrictive silencer element; b-gal, b-galactosidase; 59 UTR, 59 untranslated region; REST, RE1 silencing transcription factor; Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA CNS, central nervous system. 0027-8424y97y945906-6$2.00y0 *e-mail: [email protected]. PNAS is available online at http:yywww.pnas.org. †e-mail: [email protected]. 5906 Downloaded by guest on September 28, 2021 Neurobiology: Bessis et al. Proc. Natl. Acad. Sci. USA 94 (1997) 5907 appropriate sites of pBluescript (Stratagene) and then in the the buffer recommended by the supplier in 2.5 mM MgCl2 HindIIIyXbaI site of pcDNA IAmp (Invitrogen). using the primers 59-GAATCTGAAGA(AyG)CAGTTTGT- Transgenic Mice and Immunohistochemistry. The lucif- GCAT and 59-TTTGAAGTTGCTTCTATCTGCTGT. When erase gene from luciferase plasmids was excised and replaced detecting mRNA from neuroblastomas, we performed a sec- by the nlsLacZ gene. The b2-promoterynlsLacZ fragment was ond PCR amplification using one-tenth of the first amplifica- injected into fertilized oocytes of FVB or B6SJL mice. Staining tion using the following primers: 59-GAAGA(AyG)CAGTT- of tissues was performed as described (5). The integration of TGTGCATCACATC and 59-GTTGCTTCTATCTGCT- the transgene was tested by PCR using DNA extracted from GTTTTGTA. the tail or the yolk sac. Immunohistochemistry was performed Cells and Transfection. Neuroblastomas SK-N-Be and 3T6 on transgenic mice T9 (carrying the mutated promoter), which fibroblasts were grown in DMEM 1 10% fetal calf serum is described in Table 1. Mice were perfused with 4% parafor- (FCS) supplemented with 1% glutamine and 1% streptomycin. maldehyde, the brain was extracted and cut (50-mm sections) PC12 cells were grown in the same medium 1 10% FCS. For using a vibratome (Leitz). The slices were incubated overnight transfection, cells were plated at 1 to 2.105 cells per ml in at 48C in an anti-GFAP serum (Dako), followed by a bioty- 35-mm dishes and transfected with 1 mg of DNA using calcium nilated anti-rabbit antibody solution, and finally revealed by a phosphate the next day. The luciferase activity was measured peroxydase–streptavidin complex. 48 hr later. When plasmid activities were compared, all Gel-Shift Experiments. Nuclear extracts were prepared plasmids were prepared the same day. At least two different from '107 cells as described (5). For binding, 1 nmol of labeled DNA preparations were tested for each plasmid. All transfec- oligonucleotide was mixed with 0.5 mg of protein extract in 10 tions were done in triplicate and repeated at least four times. mM Hepes, pH 8y10% glyceroly0.1 mM EDTAy0.1 M NaCly2 Oligonucleotides. The following oligonucleotides were used: mM DTTy0.1 mg/ml BSAy4 mM MgCl2y4 mM spermidiney1 NRSE, 59-GGCCC(TTCAGCACCACGGACAGCGCT- mM phenylmethylsulfonyl fluoridey1 mg polydIdC in 20 ml. C)3TGCA; NRSEMut, 59-GGCCC(TTCAGCACCACTTAC- The reaction was incubated for 10 min on ice. The DNA– AGCGCTC)3TGCA; and SP1, 59-TCGACTAATCTCCGCC- protein complexes were then analyzed on a 5% polyacrylamide CAGTTC. gel. Reverse Transcription–PCR (RT-PCR). The mRNA (4 mg) RESULTS was hybridized for 5 min at 808C with 50 pmol of poly dT. The synthesis of the cDNA was performed using 400 units of In Vivo Demonstration of the SilenceryEnhancer Function MMLV reverse transcriptase (Gibco) for 45 min at 378Cinthe of NRSE from the nAChR b2-Subunit Gene Promoter. To buffer recommended by the supplier. One-tenth of the reverse understand the role of NRSE in neuronal gene expression in transcription was amplified using Promega’s Taq DNA poly- vivo, transgenic mice were constructed with the 1.2-kbp tran- merase (30 cycles, 948C; 1 min, 558C; 45 sec, 728C; 1 min) in scription control sequences of the b2-subunit gene containing FIG. 1. Point mutation of NRSE in the promoter of the nAChR b2-subunit gene changes the pattern of b-gal expression in transgenic mice.
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