Proc. Natl. Acad. Sci. USA Vol. 95, pp. 3233–3238, March 1998 Neurobiology The neural restrictive silencer element can act as both a repressor and enhancer of L1 cell adhesion molecule gene expression during postnatal development PEKKA KALLUNKI*, GERALD M. EDELMAN, AND FREDERICK S. JONES† Department of Neurobiology, The Scripps Research Institute and The Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037 Contributed by Gerald M. Edelman, December 23, 1997 ABSTRACT The cell adhesion molecule L1 mediates ax- genic mice, a native L1lacZ gene construct containing an 18-kb onal guidance during neural development and mutations in its segment of the L1 gene (spanning the region from the pro- gene result in severe neurological defects. In previous studies, moter to the fourth exon) produced a tissue-specific pattern of we identified the promoter for the L1 gene and showed that a expression that was neurally restricted. However, a similar neural restrictive silencer element (NRSE) was critical for construct lacking the NRSE called L1lacZDN, although gen- preventing ectopic expression of L1 during early embryonic erating a neural pattern that was similar to the native L1 development. In the present study, we have investigated the transgene, also showed extensive ectopic expression in several role of the NRSE in the regulation of L1 expression during nonneural tissues, primarily in mesenchymal derivatives of the postnatal development. In gel mobility shift experiments, the neural crest (16). NRSE formed DNA–protein complexes with nuclear extracts The NRSE has been shown to silence several genes whose prepared from the brains of postnatal mice. To examine the expression is restricted to the nervous system (17). A tran- influence of the NRSE on postnatal patterns of L1 expression scription factor that binds to the NRSE called the neural in vivo, we compared the expression of two lacZ transgene restrictive silencer factor (NRSF), also known as the RE-1 constructs, one containing the native L1 gene regulatory silencing transcription factor (REST), (18, 19) has been char- sequences (L1lacZ) and another (L1lacZDN) lacking the acterized. RESTyNRSF is a member of the Gli–Kruppel NRSE. Newborn mice carrying the L1lacZDN showed en- family of zinc-finger proteins. During early neural develop- hanced b-galactosidase expression relative to L1lacZ in the ment, RESTyNRSF is expressed ubiquitously in nonneural brain and ectopic expression in nonneural tissues. In contrast cells and in neuronal precursors but is not expressed in to L1lacZ mice, however, L1lacZDN mice showed an unex- postmitotic neurons (18, 19). These observations suggest that pected loss, during postnatal development and in the adult, of RESTyNRSF silences neurally expressed genes containing the b-galactosidase expression in several neural structures, in- NRSE in nonneural cells and in neural progenitors up to the cluding the neural retina, cerebellum, cortex, striatum, and time of neural differentiation. In postmitotic neurons, repres- hippocampus. These data support the conclusion that the sion of NRSE-containing genes would be released in the NRSE not only plays a role in the silencing of L1 expression absence of RESTyNRSF, allowing local activators of tran- in nonneural tissues during early development but also can scription to induce expression of these genes. function as a silencer and an enhancer of L1 expression in the Although the L1 cell adhesion molecule is first expressed nervous system of postnatal and adult animals. during the differentiation of postmitotic neurons from neuro- epithelial precursors (20), the most abundant expression of L1 Cell adhesion molecules (CAMs) play fundamental roles in the mRNA and protein occurs during the period of postnatal development of the nervous system. During embryogenesis, development (21). At this time, there is an extensive outgrowth CAMs participate in axonal guidance, fasciculation, and syn- of neurons and formation of synaptic connections. In the apse formation (1, 2). L1 is an integral membrane CAM present study, we examine the expression of the native and containing six Ig domains and five fibronectin type III repeats NRSE-mutated L1 transgenes during postnatal development (3). Other proteins with this overall domain structure include of the mouse nervous system. We show that the NRSE is a Ng-CAM, Nr-CAM, neurofascin, chL1, and neuroglian (4–8). silencer of L1 gene expression in nonneural tissues but behaves L1 mediates homophilic neuron–neuron adhesion and is ex- as both a silencer and enhancer of L1 expression in different pressed predominantly by postmitotic neurons and by periph- cells of the nervous system during postnatal development. eral glia (9–12). Recent studies have revealed that mutations within the human L1 gene result in several congenital disor- MATERIALS AND METHODS ders including X chromosome-linked hydrocephalus, mental retardation, aphasia, shuffling gait, and adducted thumbs, and Nuclear extracts from P3 and P16 mouse brains were prepared agenesis of the corpus callosum (13–15). as described (22). To generate a probe for gel mobility shift In an effort to identify factors that control neural patterns analyses, a pair of complementary oligonucleotides containing the NRSE from the L1 gene (59-ggccgcTCCAGCACCACG- of L1 gene expression, we have characterized (16) the pro- 9 moter of the mouse L1 gene and found that a single neural GACAGCAGAgc-3 ) was annealed to form duplex DNA. Additional nucleotides (shown in lowercase type) were in- restrictive silencer element (NRSE) composed of no more 9 than 21 nucleotides repressed the expression of the L1 gene in cluded to create NotI5 protruding ends. Five picomoles of nonneural cells but had little or no effect on L1 promoter NRSE probe were labeled by using the Klenow fragment of 32 y activity in neuroblastoma cells. In experiments using trans- Escherichia coli DNA polymerase and [ P]dCTP (3,000 Ci y The publication costs of this article were defrayed in part by page charge Abbreviations: NRSE, neural restrictive silencer element; REST NRSF, neural restrictive silencer factoryRE-1 silencing transcription payment. This article must therefore be hereby marked ‘‘advertisement’’ in factor; P, day of postnatal development; CAM, cell adhesion molecule. accordance with 18 U.S.C. §1734 solely to indicate this fact. *Present address: Department of Neurobiology, H. Lundbeck AyS, © 1998 by The National Academy of Sciences 0027-8424y98y953233-6$2.00y0 Ottiliavej 9, Copenhagen-Valby, DK2500 Denmark. PNAS is available online at http:yywww.pnas.org. †To whom reprint requests should be addressed. 3233 Downloaded by guest on October 1, 2021 3234 Neurobiology: Kallunki et al. Proc. Natl. Acad. Sci. USA 95 (1998) mmol; 1 Ci 5 37 GBq; DuPontyNEN). The probe was separated by electrophoresis on a 10% polyacrylamide gel, eluted, and resuspended in water at 20,000 cpmyml. Binding reactions and electrophoresis of DNA–protein complexes were performed as described (23). The construction of L1lacZ and L1lacZDN transgenes have been described (16) and the constructs are diagrammed in Fig. 1. L1 transgenes were excised from plasmids by digestion with restriction enzymes XmaI and SnaBI. Transgenic mice were established by standard oocyte microinjection techniques (24). Genomic DNA isolated from tails of progeny was screened for the presence of either transgene by PCR using the TissueAmp kit (Qiagen, Chatsworth, CA). Animals positive for the trans- genes were mated to establish individual lines. Males from these transgenic lines were then mated with C57BLy6 females and postnatal day 1 (P1), P5, P10, and adult mice were sacrificed and analyzed for the presence of either transgene. At least six offspring from two transgenic lines carrying a single copy of either the L1lacZ or L1lacZDN transgenes were analyzed to ensure that the lacZ expression patterns observed were consistent and stable at different sites of transgene integration. Sagittal and transverse sections were taken and examined for expression of b-galactosidase. Embryos were fixed in 0.2% FIG. 2. DNA–protein complexes formed between brain nuclear glutaraldehydey1% formaldehyde in PBS, transferred through extracts and the NRSE from the L1 gene. Nuclear extracts were prepared from the brains of four different mice, two staged at P3 an ascending gradient of sucrose to 24% sucroseyPBS, frozen m (animals 7.5 and 7.7) and two staged at P16 (animals 25–4 and 25–5). in Tissue-Tek (Miles), and sectioned (20 m) on a cryomic- Binding reactions with the 32P-labeled NRSE probe were performed rotome. Sections were attached to poly-(L-lysine)-coated slides in the absence (lane 1) or presence (lanes 2–9) of nuclear extract. In and stained for b-galactosidase in PBS containing 3 mM binding reactions labeled C, a 200-fold molar excess of unlabeled K3Fe(CN)6,3mMK4Fe(CN)6, and 5-bromo-4-chloro-3- competitor NRSE probe was included. indolyl b-D-galactoside (1 mgyml), mounted with 50% glyc- erol, and photographed with bright-field optics. To adequately To evaluate the influence of the NRSE on L1 expression in evaluate differences in the intensity of lacZ expression pro- vivo, the patterns of L1lacZ and L1lacZDN transgenes were duced by L1lacZ and L1lacZDN constructs, reaction times for compared at different stages during postnatal development of the histochemical staining procedure were kept constant for the mouse. At P1, the L1lacZ transgene showed widespread each set of sections that were compared. expression of b-galactosidase