WWW.JNEUROSCI.ORG
-
The Journal of Neuroscience
QUICK SEARCH:   [advanced]


     
-


HOME  |   SEARCH  |   ARCHIVE  |   SUBSCRIBE  |   CONTACT  |   HELP
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit an eLetter
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (13)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Montesinos, M. L.
Right arrow Articles by Volovitch, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Montesinos, M. L.
Right arrow Articles by Volovitch, M.

 Previous Article  |  Next Article 

The Journal of Neuroscience, May 15, 2001, 21(10):3350-3359

The Neuronal Microtubule-Associated Protein 1B Is under Homeoprotein Transcriptional Control

María Luz Montesinos1, Isabelle Foucher1, Marcus Conradt1, Gaëll Mainguy1, Laurence Robel1, Alain Prochiantz1, and Michel Volovitch1, 2

1 Centre Nationale de la Recherche Scientifique Unité Mixte de Recherche 8542, Ecole Normale Supérieure, 75230 Paris, Cedex 05 France, and 2 University Paris 7, Unité de Formation et de Recherche de Biologie, 75005 Paris, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To identify genes regulated by homeoprotein transcription factors in postnatal neurons, the DNA-binding domain (homeodomain) of Engrailed homeoprotein was internalized into rat cerebellum neurons. The internalized homeodomain (EnHD) acts as a competitive inhibitor of Engrailed and of several homeoproteins (Mainguy et al., 2000). Analysis by differential display revealed that microtubule-associated protein 1B (MAP1B) mRNA is upregulated by EnHD. This upregulation does not require protein synthesis, suggesting a direct effect of the homeodomain on MAP1B transcription. The promoter region of MAP1B was cut into several subdomains, and each subdomain was tested for its ability to bind Engrailed and EnHD and to associate with Engrailed-containing cerebellum nuclear extracts. In addition, the activity, and regulation by Engrailed, of each subdomain and of the entire promoter were evaluated in vivo by electroporation in the chick embryo neural tube. These experiments demonstrate that MAP1B promoter is regulated by Engrailed in vivo. Moreover, they show that one promoter domain that contains all ATTA homeoprotein cognate binding sites common to the rat and human genes is an essential element of this regulation. It is thus proposed that MAP1B, a cytoskeleton protein involved in neuronal growth and regeneration, is under homeoprotein transcriptional regulation.

Key words: neuronal morphogenesis; cytoskeleton; MAP1B; transcriptional targets; homeoproteins; engrailed; in vivo electroporation; differential display


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Homeoproteins are homeogene-coded transcription factors. They are characterized by a highly conserved 60-amino acid DNA-binding domain, the homeodomain (Gehring et al., 1994), and play key roles at all developmental stages. In the mouse, Engrailed homeogene expression starts at the one- and five-somite stages for Engrailed1 (En1) and Engrailed2 (En2), respectively. In the brain, Engrailed transcripts are localized to the mid-hindbrain region, and although En2 is expressed later than En1, both genes show identical transcription patterns at the eight-somite stage (Davidson et al., 1988; Davis and Joyner, 1988; Davis et al., 1988, 1991; McMahon et al., 1992). In the newborn, En1 and En2 (from now on cited collectively as Engrailed) are expressed in large domains of the midbrain and cerebellum, including the substantia nigra, locus coeruleus, and raphe (Davis and Joyner, 1988).

En1 targeting (Wurst et al., 1994) deletes the cerebellum and midbrain, and the mice die at birth. In contrast, En2 mutants are viable and show only a mild phenotype: the cerebellum is reduced in size and shows an abnormal folding pattern (Joyner et al., 1991; Millen et al., 1994). In the nervous system, the En1 phenotype is rescued by the insertion of the En2 coding sequence into the En1 locus (Hanks et al., 1995). This suggests that the diverging phenotypes of both mutants are caused by differences in times and sites of expression and not by differences in the biochemical activities of the proteins.

Although homeoproteins probably regulate the expression of a wide variety of genes, few direct homeoprotein target genes have been identified (Graba et al., 1997; Mannervik, 1999). In vertebrates, identified Engrailed targets are fibroblast growth factor-8 (FGF-8) (Gemel et al., 1999) and Pax-6 (Araki and Nakamura, 1999). RAGS and ELF-1 genes, encoding ligands for Eph-like receptors, have also been proposed as putative Engrailed targets. Indeed, En1 gain of function in the chick embryo rostral tectum provokes an abnormal transcription of the two genes at the site of En1 overexpression (Logan et al., 1996).

Several homeodomains are internalized by cells in culture (for review, see Prochiantz, 2000) and conveyed to their nucleus, where they specifically interfere with transcription (Le Roux et al., 1995). This property has been exploited to antagonize the binding of endogenous homeoproteins to their cognate promoters in physiological conditions. Homeodomain internalization and activity have allowed us to identify BPAG1 as a direct homeoprotein target gene, in a protocol in which EnHD was used as an inducer in a gene trap library of embryonic stem (ES) cells (Mainguy et al., 1999, 2000). BPAG1 is a cytoskeletal protein of the plakin family (Houseweart and Cleveland, 1999) with epidermal and neuronal isoforms.

The present work aimed to identify homeoprotein target genes not in "epiblast-like" ES cells but in postnatal cerebellar neurons. To that end the transcripts expressed by cerebellum cells incubated with or without EnHD were compared by differential display (Liang and Pardee, 1992). We show that the microtubule-associated protein 1B (MAP1B) gene encoding a cytoskeletal protein is a homeoprotein-regulated target.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell cultures. Primary neurons were prepared as follows. Fragments from posterior mesencephalon and cerebellum (rat postnatal day 1) were incubated (5 min, room temperature) in trypsin-EDTA, washed in phosphate buffer plus 33 mM glucose (PBS) and 10% fetal calf serum (FCS), and incubated (10 min, 37°C) with 30 µg/ml DNase I (Sigma, St. Louis, MO). The cells were dissociated mechanically, washed three times with PBS, and plated at a density of 200,000 cells/cm2 on dishes (35 mm diameter) coated with 1.5 µg/ml D,L-polyornithine and 5 µg/ml laminin (Sigma). Culture medium (MSS) consisted of DMEM/F12 (1:1; Life Technologies, Cergy, France) with 33 mM glucose, 2 mM glutamine, 10 mM HEPES, pH 7.4, 9 mM NaHCO3, 5 U/ml penicillin, 5 µg/ml streptomycin. MSS was complemented with 0.1% ovalbumin, 25 µg/ml insulin, 100 µg/ml transferrin, 20 nM progesterone, 60 µM putrescine, and 30 nM selenium. CHP-100 cells (Schlesinger et al., 1976) were grown in RPMI 1640 (Life Technologies, Gaithersburg, MD) plus 15% FCS.

Differential display. Differential display was performed essentially as described by Liang and Pardee (1992) with minor modifications. In brief, 500 ng of total RNA (RNeasy kit; Qiagen, Courtaboeuf, France) from cells treated or not with EnHD (300 ng/105 cells) in the presence of cycloheximide (1 µM) and DNase I (15 µg/ml) were reverse transcribed using Superscript II (Life Technologies), following the supplier's protocol. Before reverse transcription, RNA was systematically treated with RNase-free DNase I (Promega, Charbonnières, France) to eliminate DNA contaminations and repurified (RNeasy kit). Anchored oligo-dT primers used for reverse transcription were 5'-AAG CTT TTT TTT TTT C-3' (HTC primer) or 5'-AAG CTT TTT TTT TTT A-3' (HTA primer). PCR reactions were performed in the presence of 35S-dATP (>1000 Ci/mmol; Amersham Pharmacia Biotech, Orsay, France) with the following cycles: 93°C for 30 sec, 39°C for 1 min, and 72°C for 1 min (3 cycles); 93°C for 30 sec, 43°C for 1 min, and 72°C for 1 min (37 cycles); 72°C for 5 min. PCR products were separated on 6% denaturing sequencing gels that were dried on Whatman 3MM paper under vacuum and subjected to autoradiography. Bands of interest were excised, eluted, reamplified, and cloned in pBluescript (KS) vector (Stratagene, La Jolla, CA). Partial sequencing was performed by standard techniques. Primers leading to PCR amplification of clone 31 were 5'-AAG CTT ATT GGT C-3' and HTC.

RNA isolation and Northern blot analysis. Total RNA (15-20 µg, RNeasy kit) was run in formaldehyde agarose gels and transferred by capillarity to Hybond-N+ membranes (Amersham Pharmacia Biotech), and hybridizations were performed following manufacturer's instructions. Probes were labeled with alpha -32P-dCTP (3000 Ci/mmol; Amersham Pharmacia Biotech) by random priming (Rediprime II system, Amersham Pharmacia Biotech). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe was used as a control for RNA loading. Radioactive signals were quantified on a Fujix BAS 1000 PhosphoImager (Fujifilm Medical Systems, Stamford, CT).

Proteins. Chick En2 homeodomain (amino acids 200-259 of the protein) was produced in Escherichia coli and purified by FPLC on HiTrap heparin-Sepharose columns (Amersham Pharmacia Biotech) (Mainguy et al., 1999). A larger version of EnHD (amino acids 1-9 followed by amino acids 186-289) used in some gel shift assays was produced from expression plasmid pTrc9mEn2Delta SP. This plasmid was obtained by the transfer of myc-tagged En-2 ORF deleted between SmaI and PpuMI sites into a derivative of pTrcHis2A vector (Invitrogen, Groningen, The Netherlands).

Glutathione S-transferase (GST)-En2 and GST proteins were produced in E. coli and purified on Glutathione Sepharose 4B beads (Amersham Pharmacia Biotech), following manufacturer's instructions. GST-En2 fusion (gift from Dr. A. Joliot, Ecole Normale Supérieure, Paris) was prepared by inserting a chick En2 coding sequence into a modified form of pGEX1 (Amersham Pharmacia Biotech).

Nuclear extracts from mouse neonatal (P0) cerebellum and posterior mesencephalon were prepared as in Beckmann et al. (1997). Dissected tissues were homogenized in 5-7 ml of homogenization buffer: 20 mM HEPES, pH 7.9, 100 mM NaCl, 1.5 mM MgCl2, 0.5 mM EDTA, 0.7% Nonidet P-40, 0.5 mM dithiothreitol, 10% (w/v) glycerol, and protease inhibitor mixture Complete 1× (Roche Diagnostics, Meylan, France). After centrifugation (10 min, 2000 × g) and washing with 10 ml of homogenization buffer, the pellets resuspended in 300-500 µl of high-salt buffer were incubated for 30 min at 4°C on a rocker. High-salt buffer composition was 20 mM HEPES, pH 7.9, 0.5 M KCl, 0.5 mM EDTA, 0.5 mM dithiothreitol, 25% (w/v) glycerol, Complete 1× (Roche Diagnostics). Nuclear debris were removed by centrifugation at 14,000 × g for 30 min at 4°C, and supernatants were stored in aliquots at -80°C. Protein concentration was determined by a modified Bradford assay (Bio-Rad, Ivry, France), using bovine serum albumin (BSA) as a standard.

Gel shift assays. DNA fragments were end labeled by filling with Klenow-fragment polymerase and alpha -32P-dCTP. Binding reactions were performed in a final volume of 20 µl (15 mM HEPES, pH 8.0, 0.6 mM dithiothreitol, 6 mM MgCl2, 18% glycerol, 1 µg of salmon sperm DNA, and 10 µg of BSA). Salt concentrations varied with conditions: 100 mM KCl (see Fig. 6A), 80 mM KCl (see Fig. 6B), 135 mM NaCl (see Fig. 5A), or 80 mM KCl plus 40 mM NaCl (see Figs. 5B, 7). After incubation on ice for 30 min, DNA-protein complexes were analyzed on 5% polyacrylamide gels (acrylamide/bisacrylamide, 80:1) in 0.25× TBE buffer (TBE 1× is 100 mM Trizma base, 95 mM boric acid, and 2 mM EDTA) and 2.5% glycerol. For supershift experiments, probes were first incubated with the nuclear extracts for 30 min on ice and then for an additional 30 min with a polyclonal antibody that recognizes both En1 and En2 proteins (Plaza et al., 1997) (a gift from Dr. S. Saule, Institut Curie, Orsay). In that case DNA-protein complexes were analyzed on 4% polyacrylamide gels (acrylamide/bisacrylamide, 60:1) in 0.25× TBE buffer and 2.5% glycerol. Gels were prerun at 4°C for 45 min at 130 V and run at 4°C for 1.5 hr at 240 V, dried, and subjected to autoradiography.

Cell transfection assays and in ovo electroporation. The promoter region of MAP1B (Liu and Fischer, 1996) was amplified by PCR using Vent polymerase (New England Biolabs, Beverly, MA), genomic rat DNA as template, and oligonucleotides pMAP1 (5'-TTA TTG CAG ACC CCC AGT GTG A-3') and pMAP2 (5'-CCT GCC GGC TCT GCT AAA GCC T-3'). The 1.7 kb DNA fragment generated (position -1626 to +60) was cloned in the Klenow-filled HindIII site of pGL2-basic (Promega, Madison, WI) to generate plasmid pMAP-luc, or into the Klenow-filled HindIII site of pbeta gal-basic (Clontech, Palo Alto, CA) to generate pMAP-lacZ.

Plasmid pD-lacZ (see Fig. 8) was obtained by insertion of the Klenow-filled BsaHI-BssHII fragment from pMAP-luc (containing promoter region D) into the Klenow-filled HindIII site of pbeta gal-basic. To generate pE-lacZ (see Fig. 8), plasmid pMAP-luc was cut with HindIII and BssHII, treated with Klenow enzyme, and religated, and the resulting plasmid was cut with BglII and NaeI to obtain promoter fragment E, which was cloned between sites BglII and HindIII (Klenow-filled) of pbeta gal-basic. Plasmid pABCD-lacZ (see Fig. 8) was obtained by insertion of the Klenow-filled HindIII-BssHII fragment from pMAP-luc (containing promoter regions A, B, C, and D) into the Klenow-filled HindIII site of pbeta gal-basic. To obtain pCD-lacZ (see Fig. 8), a fragment containing promoter region C was obtained by MscI-BsaHI digestion of pABCD-lacZ, Klenow-filled, and cloned in the Klenow-filled BglII site of plasmid pD-lacZ.

Expression plasmids coding for CMV promoter-driven myc-tagged chick En2 (pCL9mEn2) (Mainguy et al., 2000), En2Delta HD1 (pCL9mEn2Delta H1; derived from pTL1mEn2Delta H1) (Joliot et al., 1998), and mouse Hoxa5 (pCL9A5m; derived from pSP9A5m) (Chatelin et al., 1996) were transfected by electroporation. For cell electroporation, a Bio-Rad Gene Pulser II apparatus and 4-mm-gap cuvettes were used (260 V and 1050 µF in 350 µl of culture medium). In Figure 4A, 0.5 × 106 cells were transfected with 2 µg of reporter plasmid and the indicated amounts of expression plasmid, plus empty parental vector (pCL9m) to keep the total amount of DNA constant. In Figure 4B, 106 cells were transfected with 2 µg of reporter and 6 µg of expressing plasmid, in duplicate. Frequency and nuclear localization of transfected homeoproteins were verified by immunocytochemistry with anti-myc 9E10 antibody (Evan et al., 1985). Luciferase activity was measured 24 hr after transfection (Le Roux et al., 1995) in a Lumat luminometer (Berthold, Bald Wilbald, Germany).

Electroporation of chick embryos at stage HH9-11 (Hamburger and Hamilton, 1951) was performed as described (Muramatsu et al., 1997), using a BTX Electrosquareporator T820 apparatus (four pulses of 25 V and 50 msec) (Genetronics, San Diego, CA). The appropriate reporter plasmid and control or En2 expression plasmids were injected into the neural tube with a micropipette. After 24 hr of incubation at 37°C, embryos were fixed and stained for beta -galactosidase activity (Hogan et al., 1994).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of MAP1B cDNA by differential display

To find genes controlled by Engrailed we looked for modifications in expression profiles of postnatal rat cerebellum neurons on treatment by EnHD. Postnatal day 1 cerebellums were dissociated, plated for 4 hr, and incubated for another 12 hr with or without EnHD (in duplicate), in the presence of cycloheximide to prevent protein synthesis and regulation of indirect targets (Mainguy et al., 1999). Cycloheximide was not toxic because its removal allowed all neurons to resume differentiation (data not shown). In these conditions EnHD was internalized by the cells and could be visualized in the nucleus by immunocytochemistry as reported earlier (Mainguy et al., 1999). Expression profiles were then analyzed by differential display (Liang and Pardee, 1992).

Total RNA samples were reverse transcribed in duplicate with one of two different anchored oligo-dT primers (HTA or HTC primers). The subsets of mRNAs defined by each of these primers were amplified by PCR using 12 different 13-mers with arbitrary sequences in combination with the corresponding oligo-dT primer. Samples derived from EnHD-treated and control cells showed a small number of differentially expressed bands (Fig. 1A, asterisks). One cDNA, more abundant in treated than in control cells (Fig. 1A), was reamplified by PCR, cloned, and partially sequenced (Fig. 2B). Clone 31 contains a 1.1 kb sequence present in the 3' untranslated region (nucleotide positions 1838-2961; GenBank accession number: AF115776) of MAP1B (Meixner et al., 1999) (Fig. 2). Differential expression of MAP1B was confirmed by Northern blot analysis using RNA isolated from rat cerebellum neurons, incubated or not with EnHD in the presence of cycloheximide. A representative Northern blot showing that EnHD increases the expression level of MAP1B by twofold is presented in Figure 1B.



View larger version (29K):
[in this window]
[in a new window]
  		Figure 1.   Identification of clone 31 (MAP1B gene) by differential display. A, Duplicate RT and PCR reactions were performed and run in adjacent gel lanes. The positions of some differentially expressed bands are indicated with asterisks. The band corresponding to clone 31 is also indicated. B, Northern blot of total RNA from control or EnHD-treated cells probed with clone 31 or GAPDH cDNA fragments.



View larger version (49K):
[in this window]
[in a new window]
  		Figure 2.   Localization of clone 31 in the MAP1B genomic locus. A, Schematic representation of the rat MAP1B locus [modified from Kutschera et al. (1998) and Meixner et al. (1999)]. The most frequent MAP1B transcripts (90% of total MAP1B mRNA) are encoded by exons 1-7 (solid lines indicate splicing). Alternative MAP1B transcripts are encoded by exon 3U/3-7 (splicing indicated by a dashed line) and by exon 3A/3-7. Clone 31 localization is shown. B, Sequence alignments of clone 31 5' (top) and 3' (bottom) regions with mouse MAP1B 3' untranslated region (UTR) and rat expressed-sequence tags (EST) AW530133 and AI008511. Residues that differ from clone 31 are shaded in black.

Regulation of MAP1B promoter activity by homeoproteins

The promoter region of the rat MAP1B gene (Liu and Fischer, 1996, 1997) contains two independent TATA boxes and several regulatory motifs, including two cAMP-responsive elements, an Sp1 site, a "neuronal motif," and a TCC repeat motif (Fig. 3A). The MAP1B promoter region (nucleotide positions -1626 to +60) was cloned in front of a luciferase reporter gene (pGL2bas promoter-less vector). This construct (pMAP-luc) was tested by transient transfection of a human neuroepithelial cell line (CHP-100) (Mainguy et al., 1999). Figure 4A illustrates that transfecting increasing amounts of a plasmid expressing En2 leads to a dose-dependent activation of the MAP1B promoter.



View larger version (35K):
[in this window]
[in a new window]
  		Figure 3.   MAP1B promoter region. A, Promoter of the rat MAP1B gene: the two TATA boxes, 5' capping sites, and putative regulatory motifs described previously (Liu and Fischer, 1996, 1997) are shown. The positions of ATTA or TAAT sites are indicated by filled circles and numbered 1-16. Asterisks indicate ATTA sites conserved in the putative human MAP1B promoter (GenBank accession number: AC021318). Positions of fragments A, B, C, D, and E used in gel-shift assays and restriction sites used for their isolation are also indicated. B, Sequence comparison of rat and human MAP1B promoters showing one of the two conserved regions. A BLAST search using the rat MAP1B promoter sequence (GenBank accession number: U55276) was made against the unfinished human genome. Conserved ATTA/TAAT sites are boxed; the nonconserved TAAT site is indicated by a solid line. Residues that differ from rat sequence are shaded in black.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 4.   Regulation of MAP1B promoter activity in a neuroepithelial cell line. A, Cotransfection of CHP-100 cells with the MAP1B-luciferase reporter plasmid pMAP-luc and increasing amounts of En2-expressing plasmid (DNA was kept constant by adding the empty plasmid). B, Cotransfection of CHP-100 cells with pMAP-luc and empty vector (control) or with a plasmid expressing full-length En2 protein (En2), En2 deleted in the homeodomain (En2Delta HD1), or full-length Hoxa5 protein (Hoxa5). Luciferase activity of cell extracts after 24 hr is indicated in relative light units (RLU).

The En2-mediated activation of MAP1B promoter was not observed when an En2Delta HD1-expressing plasmid was used (Fig. 4B). En2Delta HD1 (Joliot et al., 1998) is an En2 mutant lacking amino acids 36-46 in the homeodomain; it is still addressed to the nucleus but does not bind DNA (data not shown). To test whether other homeoproteins were also active, pMAP-luc and Hoxa5 coding plasmid were cotransfected in CHP-100 cells. Figure 4B shows that Hoxa5 also regulates MAP1B promoter activity ex vivo.

Identification of Engrailed-binding sites within the MAP1B promoter region

The MAP1B promoter contains several putative homeoprotein binding sites (ATTA or TAAT sites) (Fig. 3). Five DNA fragments (from A to E) covering the entire MAP1B promoter region were generated (Fig. 3A), and each fragment was used in gel-shift experiments. Fragments A and E (417 and 138 bp, respectively) contain one ATTA site (overlapping the TATA-2 box in the case of fragment E); fragments B (421 bp) and C (547 bp) contain 10 and 7 ATTA sites, respectively; fragment D (398 bp) contains no ATTA sequence. Purified EnHD (Fig. 5A) and GST-En2 protein (Fig. 5B) retarded probes A, B, C, and D (the latter to a lesser extent) but failed to retard probe E even when a sevenfold excess of GST-En2 protein was used in the binding reaction (data not shown).



View larger version (54K):
[in this window]
[in a new window]
  		Figure 5.   EnHD and full-length En2 bind to the MAP1B promoter. A, Purified EnHD (-, no protein; +, 50 ng of EnHD) was incubated with 1 ng of the indicated MAP1B promoter fragment (from A to E). B, Two different amounts (625 ng or 1.25 µg) of full-length En2 (GST-En 2) protein or 5 µg of GST protein were incubated with 0.4 ng of the indicated MAP1B promoter fragment (from A to E). Designation of MAP1B probes refers to promoter regions indicated in Figure 3A. Note that the five probes are of different sizes and thus migrate with different velocities.

Gel-shift assays were then performed with nuclear extracts from P0 mouse cerebella. Probes A, B, C, and D (although to a lesser degree), but not E, were retarded (Fig. 6A), indicating the existence, in the nuclear extract, of one or more factors that specifically recognize the DNA fragments. That Engrailed is one of these factors was confirmed by a supershift experiment using a polyclonal anti-Engrailed antibody (Fig. 6B). Finally, to test whether Engrailed forms an active complex with factors in the nuclear extract, a supershift experiment was achieved by adding GST-En2. Figure 7 illustrates that GST-En2 (Fig. 7A), but not GST alone (Fig. 7B), interacts with one or several factors. Taken together, these results demonstrate that Engrailed is present in the extracts and recognizes different fragments of the MAP1B promoter in an appropriate molecular context.



View larger version (65K):
[in this window]
[in a new window]
  		Figure 6.   Cerebellum nuclear extracts bind the MAP1B promoter, and the binding complex contains Engrailed. A, Binding of increasing amounts (0, 0.5, 1, or 2 µg) of nuclear extract from P0 mice cerebellum to fragments A, B, C, D, and E (0.4 ng). B, Supershift assay of fragments A, B, C, D, E, and C+E (0.5 ng each) using an anti-Engrailed antibody (2.5 µl of a dilution 1:10) and 1 µg of nuclear extract. Designation of MAP1B probes refers to promoter regions indicated in Figure 3A. Note that the five probes are of different sizes and thus migrate with different velocities.



View larger version (68K):
[in this window]
[in a new window]
  		Figure 7.   Enhanced retardation of MAP1B promoter by GST-En2 and cerebellum nuclear extract. A, Band-shift assays with MAP1B probes A to E (0.4 ng) with (+) or without (-) 2 µg of cerebellum nuclear extract and/or 1.25 µg of GST-En2. B, Band-shift assays with fragments A to E (0.4 ng) with (+) or without (-) 2 µg of cerebellum nuclear extract and/or 10 µg of GST. Designation of MAP1B probes refers to promoter regions indicated in Figure 3A. Note that the five probes are of different sizes and thus migrate with different velocities.

Repression of MAP1B promoter activity by Engrailed in the neural tube of chick embryos

The results described above demonstrate that Engrailed binds different fragments in the MAP1B promoter and regulates the expression of this promoter in transfected neuroepithelial cells. To better sustain the idea that MAP1B is regulated by Engrailed and to identify which domains in the promoter are involved, several lacZ-reporter constructions (Fig. 8) were expressed in the neural tube of chick embryos at stage HH9-11 (Hamburger and Hamilton, 1951), and their expression was analyzed 1 d later.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 8.   MAP1B reporter plasmids for in ovo electroporations. The indicated regions of the MAP1B promoter were fused to a lacZ-reporter gene. ATTA sites are indicated with filled circles. Letters refer to regions indicated in Figure 3A.

Full-length MAP1B promoter is expressed in vivo and repressed by Engrailed (Fig. 9A). On the basis of cell transfection assays, Liu and Fischer (1996) have proposed that the two MAP1B TATA boxes, in association with their adjacent regulatory cis-elements, function independently and confer neuron-specific expression. To test whether each TATA box is repressed by Engrailed in the developing chick neural tube, pD-lacZ and pE-lacZ were electroporated with or without Engrailed. Plasmid pD-lacZ contains TATA box1 (TATA-1) and associated upstream motifs (Fig. 3A), and plasmid pE-lacZ contains TATA box2 (TATA-2) and the upstream Sp1 motif (Fig. 3A). As illustrated in Figure 9, only pD-lacZ is active (Fig. 9B), whereas pE-lacZ shows no basal neural activity (Fig. 9C) and neither construct expression is regulated by Engrailed. Because some activity was observed in adjacent non-neural tissue in several embryos, it is likely that TATA-2 can drive lacZ expression but lacks the motifs necessary for neural expression.



View larger version (45K):
[in this window]
[in a new window]
  		Figure 9.   Activity of MAP1B promoter fragments in the developing chick neural tube. Chick embryos (HH9-11) were co-electroporated with empty (control) or En2-expressing (En2) plasmids, together with the following lacZ-reporter plasmids: A, pMAP-lacZ; B, pD-lacZ; C, pE-lacZ; D, pABCD-lacZ; E, pCD-lacZ. Embryos were stained for beta -galactosidase activity 24 hr after electroporation. Histograms show the percentage of electroporated embryos for each condition showing strong (+++/++), weak (+), or no (-) beta -galactosidase activity. The number of embryos for each condition is indicated on the top of the corresponding bar. Examples of beta -galactosidase expression in each experimental condition are shown on the right side of the Figure.

Fragment D contains no ATTA sequence and is not repressed by Engrailed. We thus tested whether fragment ABC, which contains all ATTA sequences, confers regulation. Figure 9D illustrates the expression of pABCD-lacZ in the neural tube and its repression by En2. Little difference was observed between pABCD-lacZ and the full-length promoter (Fig. 9, compare A and D). Aligning human and rat sequences highlights two homologous MAP1B promoter regions and, in particular, shows that only ATTA sites at positions 9, 10, 12, and 13 of the rat promoter (Fig. 3B) are conserved between rat and human. Because fragment C contains all of these conserved sites (Fig. 3), pCD-lacZ was electroporated in the chick neural tube with or without Engrailed. Figure 9E confirms that motifs in fragment C mediate the regulation of MAP1B promoter by Engrailed.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study demonstrates that MAP1B is a homeoprotein target and, most probably, an Engrailed target. This conclusion is based on the following series of experiments. First, EnHD internalization in primary cerebellum neurons provokes an accumulation of MAP1B mRNA in the presence of cycloheximide. Second, cerebellum nuclear extracts retard several fragments of the MAP1B promoter, and Engrailed is present in the retarded complex. Finally, the promoter is active and regulated by Engrailed in vivo.

Technology

A differential display approach was associated with the internalization of EnHD by cerebellum primary neurons. The molecular basis of homeodomain internalization has been well established, and it is now clear that specific properties of the third helix are involved (Prochiantz, 1996; Derossi et al., 1998). The absence of chiral receptors and the translocation across artificial lipid layers (Thoren et al., 2000) explain why homeodomains gain direct access to the cytoplasm and thereafter, through the nuclear pores, to the nucleus.

Internalized homeodomains compete with endogenous homeoproteins for cognate binding sites in the genome. This was shown by the use of the homeodomain of Antennapedia (AntpHD) and by its mutated version where glutamine in position 50 has been replaced by an alanine (AntpHD-50A). Indeed, AntpHD-50A is internalized and conveyed to the nucleus but does not bind DNA with high affinity (Le Roux et al., 1993) and, contrary to AntpHD, does not regulate gene expression (Le Roux et al., 1995; Mainguy et al., 1999).

Advantageously, homeodomains internalized by all cells in culture only bind accessible sites in an unmodified chromatin context. Moreover, cycloheximide can be added to identify direct targets. Another interesting aspect of this approach is its relative absence of specificity: wild-type homeodomains with a glutamine in position 50 will recognize a large number of sequences accessible to various homeoproteins of the Q50 family. This is why homeodomain internalization will reveal targets shared by several homeoproteins. Target induction by homeodomains is thus a first step in the identification process as illustrated by this study and by previous work in which BPAG1 was identified as a homeoprotein target (Mainguy et al., 2000).

In vitro promoter analysis

The MAP1B promoter region (Liu and Fischer, 1996) shows 16 putative homeoprotein binding sites. Gel-shift experiments demonstrating Engrailed binding to four promoter fragments (fragments A, B, C, and D) support a direct regulation of MAP1B expression by Engrailed. In fact, the use of a protein synthesis inhibitor (cycloheximide) in the differential display protocols prevents cascade effects and favors the isolation of targets directly regulated by EnHD.

The ATTA motif present in fragment E (which is not retarded) overlaps the second TATA box of the MAP1B promoter (Liu and Fischer, 1996) and probably does not represent a physiological homeoprotein-binding site. In contrast, fragment D does not contain any ATTA site but forms a complex with EnHD or En2. However, a comparison of bound versus free probe in gel-shift experiments (same amounts of probe and protein for all fragments) suggests that the relative affinity of Engrailed for the probes is C>= B>A>D (Fig. 5B) and that the affinity between En2 and D is low.

Gel-shift experiments gave similar results when purified EnHD, GST-En2, or cerebellum nuclear extracts were used. Engrailed is abundant in the cerebellum at P0 and in the nuclear extracts (data not shown). As demonstrated by the supershift experiments, Engrailed is part of the retarded complexes formed between the probes and proteins in the nuclear extract. Although in these retarded complexes Engrailed might not bind directly to the promoter, the fact that purified En2 binds directly and that nuclear extracts and purified En2 show the same order of relative affinities for the five fragments is very much in favor of a direct binding.

Enhanced retardation of MAP1B promoter probes when En2 is added to cerebellum nuclear extracts (Fig. 7) suggests the presence of cofactors. Groucho/TLE and Exd/Pbx are recognized cofactors of Engrailed in invertebrates and vertebrates. Groucho/TLE proteins do not bind DNA but are co-repressors of several transcription factors, including Hairy-related, Runt domain, and Engrailed proteins (for review, see Fisher and Caudy, 1998; Chen and Courey, 2000). In contrast, Pbx homeoproteins have the ability to modulate the binding activity of Hox and Engrailed through cooperative DNA binding (Chang et al., 1995; van Dijk et al., 1995). In rat embryos, Pbx1 and Engrailed show overlapping expression patterns (Roberts et al., 1995), and Pbx1 is detected by Western blot in cerebellum nuclear extracts (data not shown). However, the possibility that added purified En2 binds Pbx1 or other cofactors present in the extract has not been investigated.

Ex vivo and in ovo promoter activity

Transfections in the neuroepithelial cell line CHP-100 provided some interesting information. First, Engrailed regulates MAP1B promoter activity, and this regulation is lost when 11 amino acids are deleted in the homeodomain. It was indeed verified that the mutated protein is synthesized and accumulates in the nucleus. Second, Hoxa5, another member of the Q50 homeoprotein family, also regulates MAP1B promoter expression. This supports the idea that different Q50 homeoproteins may regulate the same target genes (Biggin and McGinnis, 1997), including MAP1B. This is not a surprise because homeoproteins are region-specific transcription factors, whereas MAP1B is expressed in all neurons and not specifically in the mid-hindbrain.

En2 represses the MAP1B promoter after electroporation in the chick neural tube, a gain of function protocol that permits the study of gene activity in vivo (in ovo, rather), thus in a physiological context. This repression is in agreement with the differential display results because internalized EnHD antagonizes Engrailed activity (Mainguy et al., 2000) and leads to an upregulation of the MAP1B transcript in cultured midbrain and cerebellum neurons. The in ovo experiments show that overexpressed Engrailed completely downregulates MAP1B promoter activity. This does not mean that Engrailed-expressing neurons do not express MAP1B but that Engrailed acts in conjunction with other transcription factors to regulate the appropriate levels of the protein. In fact, it must be kept in mind that the activity of a transcription factor is both dose and context dependent. For example, in Drosophila, Engrailed either activates or represses polyhomeotic, and the two opposite effects depend both on Engrailed concentration (high concentrations are inhibitory) and on the presence of Exd as a cofactor (necessary for activation) (Serrano and Maschat, 1998). Another example is given in this study because Engrailed is a repressor of MAP1B promoter activity in neuronal cells and an activator in CHP-100 cells.

Expression in the chick embryo of the different reporter constructs demonstrates that only TATA box1 (and not TATA box2, in fragment E) is active in the nervous system. These experiments also show that fragment D is not regulated by Engrailed in vivo and suggest that the lower in vitro affinity of Engrailed for fragment D (in comparison with the other fragments) is physiologically relevant. This has allowed us to use the TATA box of fragment D to test C domain activity by electroporating pCD-lacZ and to show that this domain, which contains all ATTA sequences conserved between rat and human, permits regulation by Engrailed. pMAP-lacZ, pABCD-lacZ, and pCD-lacZ constructions are active in the mid-hindbrain region (Fig. 9) where Engrailed is normally expressed, and repression follows Engrailed electroporation. As already discussed in the case of polyhomeotic, this illustrates that MAP1B regulation by Engrailed is probably dose dependent and that the presence of physiological amounts of Engrailed is compatible with MAP1B expression.

In Drosophila, Engrailed, and Futsch (a MAP1B-like gene) (Hummel et al., 2000; Roos et al., 2000) are ectopically expressed in Tramtrack mutants (Xiong and Montell, 1993; Giesen et al., 1997). Because Tramtrack (a zinc-finger transcription factor) binds the Engrailed promoter (Read and Manley, 1992), an appealing possibility is that a direct genetic interaction between Engrailed and MAP1B has been conserved in several species, including human, rat, chick, and possibly Drosophila.

Physiological significance

MAP1B, a major component of the neuronal cytoskeleton (Bloom et al., 1984, 1985), is the earliest MAP expressed during brain development (Riederer et al., 1986), reaching highest expression levels 2-3 d after birth. In the adult, MAP1B is downregulated (Safaei and Fischer, 1989; Garner et al., 1990), except in regions that retain high levels of axonal growth and synaptic plasticity, for example, the olfactory bulb, the hippocampus, and Purkinje cells in the cerebellum (Sato-Yoshitake et al., 1989; Schoenfeld et al., 1989). Although the precise function of MAP1B remains unclear, the phenotype of MAP1B knock-out mice suggests a role in neuronal differentiation (Edelmann et al., 1996; Takei et al., 1997; Gonzalez-Billault et al., 2000). This view is confirmed by antisense experiments showing that MAP1B downregulation in PC12 cells or cultured cerebellum macro-neurons reduces neurite outgrowth (Brugg et al., 1993; DiTella et al., 1996).

In conclusion, this report adds MAP1B to the list of established cytoskeleton genes that interact with one or several homeogenes. This list contains beta 3-tubulin (Serrano et al., 1997), centrosomin (Li and Kaufman, 1996), MAP2 (Ding et al., 1997), calponin (Morgan et al., 1999), NF68 (Biagioni et al., 2000), and BPAG1 (Mainguy et al., 1999, 2000). The fact that homeoprotein transcription factors regulate the expression of several components of the cytoskeleton, and probably of adhesion molecules (Edelman and Jones, 1993), provides a molecular basis for their well established functions in tissue and cell morphogenesis (Joliot et al., 1991; Bloch-Gallego et al., 1993; Le Roux et al., 1993).


    FOOTNOTES

Received Dec. 21, 2000; revised Feb. 27, 2001; accepted Feb. 28, 2001.

This work was supported by the European Community Biotechnology program (BIOT980227) and by the Association Française de lutte contre les Myopathies. M.L.M. is an European Molecular Biology Organization and Fondation pour la Recherche Medicale fellow. We thank E. Ipendey and S. Dupas for excellent technical assistance and Dr. S. Saule for providing us with polyclonal anti-Engrailed antibody. We also thank Drs. C. Goridis and M. Wassef for their critical reading of this manuscript, as well as Drs. F. Nothias, B. Lesaffre, and A. Trembleau for helpful discussions.

Correspondence should be addressed to Dr. Alain Prochiantz, Unité Mixte de Recherche 8542, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris, Cedex 05 France. E-mail: prochian{at}wotan.ens.fr.

Dr. Conradt's present address: MEMOREC Stoffel GmbH, Stoeckheimer Weg 1, 50829 Cologne, Germany.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

  • Araki I, Nakamura H (1999) Engrailed defines the position of dorsal di-mesencephalic boundary by repressing diencephalic fate. Development 126:5127-5135[Abstract].
  • Beckmann AM, Matsumoto I, Wilce PA (1997) AP-1 and Egr DNA-binding activities are increased in rat brain during ethanol withdrawal. J Neurochem 69:306-314[Web of Science][Medline].
  • Biagioni S, Ciuffini L, De Jaco A, Vignoli AL, Augusti-Tocco G (2000) Activation of neurospecific gene expression by antennapedia homeobox peptide. Int J Dev Neurosci 18:93-99[Medline].
  • Biggin MD, McGinnis W (1997) Regulation of segmentation and segmental identity by Drosophila homeoproteins: the role of DNA binding in functional activity and specificity. Development 124:4425-4433[Abstract].
  • Bloch-Gallego E, Le Roux I, Joliot AH, Volovitch M, Henderson CE, Prochiantz A (1993) Antennapedia homeobox peptide enhances growth and branching of embryonic chicken motoneurons in vitro. J Cell Biol 120:485-492[Abstract/Free Full Text].
  • Bloom G, Schoenfeld T, Vallee R (1984) Widespread distribution of the major polypeptide component of MAP 1 (microtubule-associated protein 1) in the nervous system. J Cell Biol 98:320-330[Abstract/Free Full Text].
  • Bloom G, Luca F, Vallee R (1985) Microtubule-associated protein 1B: identification of a major component of the neuronal cytoskeleton. Proc Natl Acad Sci USA 82:5404-5408[Abstract/Free Full Text].
  • Brugg B, Reddy D, Matus A (1993) Attenuation of microtubule-associated protein 1B expression by antisense oligodeoxynucleotides inhibits initiation of neurite outgrowth. Neuroscience 52:489-496[Web of Science][Medline].
  • Chang CP, Shen WF, Rozenfeld S, Lawrence HJ, Largman C, Cleary ML (1995) Pbx proteins display hexapeptide-dependent cooperative DNA binding with a subset of Hox proteins. Genes Dev 9:663-674[Abstract/Free Full Text].
  • Chatelin L, Volovitch M, Joliot AH, Perez F, Prochiantz A (1996) Transcription factor hoxa-5 is taken up by cells in culture and conveyed to their nuclei. Mech Dev 55:111-117[Web of Science][Medline].
  • Chen G, Courey AJ (2000) Groucho/TLE family proteins and transcriptional repression. Gene 249:1-16[Web of Science][Medline].
  • Davidson D, Graham E, Sime C, Hill R (1988) A gene with sequence similarity to Drosophila engrailed is expressed during the development of the neural tube and vertebrae in the mouse. Development 104:305-316[Abstract].
  • Davis CA, Joyner AL (1988) Expression patterns of the homeo box-containing genes En-1 and En-2 and the proto-oncogene int-1 diverge during mouse development. Genes Dev 2:1736-1744[Abstract/Free Full Text].
  • Davis CA, Noble-Topham SE, Rossant J, Joyner AL (1988) Expression of the homeo box-containing gene En-2 delineates a specific region of the developing mouse brain. Genes Dev 2:361-371[Abstract/Free Full Text].
  • Davis CA, Holmyard DP, Millen KJ, Joyner AL (1991) Examining pattern formation in mouse, chicken and frog embryos with an En-specific antiserum. Development 111:287-298[Abstract].
  • Derossi D, Chassaing G, Prochiantz A (1998) Trojan peptides: the penetratin system for intracellular delivery. Trends Cell Biol 8:84-87[Web of Science][Medline].
  • Ding M, Robel L, James AJ, Eisenstat DD, Leckman JF, Rubenstein JL, Vaccarino FM (1997) Dlx-2 homeobox gene controls neuronal differentiation in primary cultures of developing basal ganglia. J Mol Neurosci 8:93-113[Web of Science][Medline].
  • DiTella MC, Feiguin F, Carri N, Kosik KS, Caceres A (1996) MAP-1B/TAU functional redundancy during laminin-enhanced axonal growth. J Cell Sci 109:467-477[Abstract].
  • Edelman GM, Jones FS (1993) Outside and downstream of the homeobox. J Biol Chem 268:20683-20686[Free Full Text].
  • Edelmann W, Zervas M, Costello P, Roback L, Fischer I, Hammarback JA, Cowan N, Davies P, Wainer B, Kucherlapati R (1996) Neuronal abnormalities in microtubule-associated protein 1B mutant mice. Proc Natl Acad Sci USA 93:1270-1275[Abstract/Free Full Text].
  • Evan G, Lewis G, Ramsay G, Bishop J (1985) Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol 5:3610-3616[Abstract/Free Full Text].
  • Fisher AL, Caudy M (1998) Groucho proteins: transcriptional corepressors for specific subsets of DNA-binding transcription factors in vertebrates and invertebrates. Genes Dev 12:1931-1940[Free Full Text].
  • Garner CC, Garner A, Huber G, Kozak C, Matus A (1990) Molecular cloning of microtubule-associated protein 1 (MAP1A) and microtubule-associated protein 5 (MAP1B): identification of distinct genes and their differential expression in developing brain. J Neurochem 55:146-154[Web of Science][Medline].
  • Gehring WJ, Qian YQ, Billeter M, Furukubo-Tokunaga K, Schier AF, Resendez-Perez D, Affolter M, Otting G, Wuthrich K (1994) Homeodomain-DNA recognition. Cell 78:211-223[Web of Science][Medline].
  • Gemel J, Jacobsen C, MacArthur CA (1999) Fibroblast growth factor-8 expression is regulated by intronic engrailed and Pbx1-binding sites. J Biol Chem 274:6020-6026[Abstract/Free Full Text].
  • Giesen K, Hummel T, Stollewerk A, Harrison S, Travers A, Klambt C (1997) Glial development in the Drosophila CNS requires concomitant activation of glial and repression of neuronal differentiation genes. Development 124:2307-2316[Abstract].
  • Gonzalez-Billault C, Demandt E, Wandosell F, Torres M, Bonaldo P, Stoykova A, Chowdhury K, Gruss P, Avila J, Sanchez MP (2000) Perinatal lethality of microtubule-associated protein 1B-deficient mice expressing alternative isoforms of the protein at low levels. Mol Cell Neurosci 16:408-421[Web of Science][Medline].
  • Graba Y, Aragnol D, Pradel J (1997) Drosophila Hox complex downstream targets and the function of homeotic genes. BioEssays 19:379-388[Web of Science][Medline].
  • Hamburger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo. J Morphol 88:49-92[Web of Science].
  • Hanks M, Wurst W, Anson-Cartwright L, Auerbach AB, Joyner AL (1995) Rescue of the En-1 mutant phenotype by replacement of En-1 with En-2. Science 269:679-682[Abstract/Free Full Text].
  • Hogan B, Beddington R, Costantini F, Lacy E (1994) In: Manipulating the mouse embryo. A laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  • Houseweart MK, Cleveland DW (1999) Cytoskeletal linkers: new MAPs for old destinations. Curr Biol 9:R864-866[Web of Science][Medline].
  • Hummel T, Krukkert K, Roos J, Davis G, Klambt C (2000) Drosophila Futsch/22C10 is a MAP1B-like protein required for dendritic and axonal development. Neuron 26:357-370[Web of Science][Medline].
  • Joliot A, Pernelle C, Deagostini-Bazin H, Prochiantz A (1991) Antennapedia homeobox peptide regulates neural morphogenesis. Proc Natl Acad Sci USA 88:1864-1868[Abstract/Free Full Text].
  • Joliot A, Maizel A, Rosenberg D, Trembleau A, Dupas S, Volovitch M, Prochiantz A (1998) Identification of a signal sequence necessary for the unconventional secretion of Engrailed homeoprotein. Curr Biol 8:856-863[Web of Science][Medline].
  • Joyner AL, Herrup K, Auerbach BA, Davis CA, Rossant J (1991) Subtle cerebellar phenotype in mice homozygous for a targeted deletion of the En-2 homeobox. Science 251:1239-1243[Abstract/Free Full Text].
  • Kutschera W, Zauner W, Wiche G, Propst F (1998) The mouse and rat MAP1B genes: genomic organization and alternative transcription. Genomics 49:430-436[Medline].
  • Le Roux I, Joliot AH, Bloch-Gallego E, Prochiantz A, Volovitch M (1993) Neurotrophic activity of the Antennapedia homeodomain depends on its specific DNA-binding properties. Proc Natl Acad Sci USA 90:9120-9124[Abstract/Free Full Text].
  • Le Roux I, Duharcourt S, Volovitch M, Prochiantz A, Ronchi E (1995) Promoter-specific regulation of gene expression by an exogenously added homedomain that promotes neurite growth. FEBS Lett 368:311-314[Medline].
  • Li K, Kaufman TC (1996) The homeotic target gene centrosomin encodes an essential centrosomal component. Cell 85:585-596[Web of Science][Medline].
  • Liang P, Pardee AB (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967-971[Abstract/Free Full Text].
  • Liu D, Fischer I (1996) Two alternative promoters direct neuron-specific expression of the rat microtubule-associated protein 1B gene. J Neurosci 16:5026-5036[Abstract/Free Full Text].
  • Liu D, Fischer I (1997) Structural analysis of the proximal region of the microtubule-associated protein 1B promoter. J Neurochem 69:910-919[Web of Science][Medline].
  • Logan C, Wizenmann A, Drescher U, Monschau B, Bonhoeffer F, Lumsden A (1996) Rostral optic tectum acquires caudal characteristics following ectopic engrailed expression. Curr Biol 6:1006-1014[Web of Science][Medline].
  • Mainguy G, Erno H, Montesinos ML, Lesaffre B, Wurst W, Volovitch M, Prochiantz A (1999) Regulation of epidermal bullous pemphigoid antigen 1 (BPAG1) synthesis by homeoprotein transcription factors. J Invest Dermatol 113:643-650[Medline].
  • Mainguy G, Montesinos ML, Lesaffre B, Zevnik B, Karasawa M, Kothary R, Wurst W, Prochiantz A, Volovitch M (2000) An induction gene trap for identifying a homeoprotein-regulated locus. Nat Biotechnol 18:746-749[Web of Science][Medline].
  • Mannervik M (1999) Target genes of homeodomain proteins. BioEssays 21:267-270[Web of Science][Medline].
  • McMahon AP, Joyner AL, Bradley A, McMahon JA (1992) The midbrain-hindbrain phenotype of Wnt-1-/Wnt-1- mice results from stepwise deletion of engrailed-expressing cells by 9.5 days postcoitum. Cell 69:581-595[Web of Science][Medline].
  • Meixner A, Wiche G, Propst F (1999) Analysis of the mouse MAP1B gene identifies a highly conserved 4.3 kb 3' untranslated region and provides evidence against the proposed structure of DBI-1 cDNA. Biochim Biophys Acta 1445:345-350[Medline].
  • Millen KJ, Wurst W, Herrup K, Joyner AL (1994) Abnormal embryonic cerebellar development and patterning of postnatal foliation in two mouse Engrailed-2 mutants. Development 120:695-706[Abstract].
  • Morgan R, Hooiveld MH, Pannese M, Dati G, Broders F, Delarue M, Thiery JP, Boncinelli E, Durston AJ (1999) Calponin modulates the exclusion of Otx-expressing cells from convergence extension movements. Nat Cell Biol 1:404-408[Web of Science][Medline].
  • Muramatsu T, Mizutani Y, Ohmori Y, Okumura J (1997) Comparison of three nonviral transfection methods for foreign gene expression in early chicken embryos in ovo. Biochem Biophys Res Commun 230:376-380[Web of Science][Medline].
  • Plaza S, Langlois MC, Turque N, LeCornet S, Bailly M, Begue A, Quatannens B, Dozier C, Saule S (1997) The homeobox-containing Engrailed (En-1) product down-regulates the expression of Pax-6 through a DNA binding-independent mechanism. Cell Growth Differ 8:1115-1125[Abstract].
  • Prochiantz A (1996) Getting hydrophilic compounds into cells: lessons from homeopeptides. Curr Opin Neurobiol 6:629-634[Web of Science][Medline].
  • Prochiantz A (2000) Messenger proteins: homeoproteins, TAT and others. Curr Opin Cell Biol 12:400-406[Web of Science][Medline].
  • Read D, Manley JL (1992) Alternatively spliced transcripts of the Drosophila tramtrack gene encode zinc finger proteins with distinct DNA binding specificities. EMBO J 11:1035-1044[Web of Science][Medline].
  • Riederer B, Cohen R, Matus A (1986) MAP5: a novel brain microtubule-associated protein under strong developmental regulation. J Neurocytol 15:763-775[Web of Science][Medline].
  • Roberts VJ, van Dijk MA, Murre C (1995) Localization of Pbx1 transcripts in developing rat embryos. Mech Dev 51:193-198[Web of Science][Medline].
  • Roos J, Hummel T, Ng N, Klambt C, Davis GW (2000) Drosophila Futsch regulates synaptic microtubule organization and is necessary for synaptic growth. Neuron 26:371-382[Web of Science][Medline].
  • Safaei R, Fischer I (1989) Cloning of a cDNA encoding MAP1B in rat brain: regulation of mRNA levels during development. J Neurochem 52:1871-1879[Web of Science][Medline].
  • Sato-Yoshitake R, Shiomura Y, Miyasaka H, Hirokawa N (1989) Microtubule-associated protein 1B: molecular structure, localization, and phosphorylation-dependent expression in developing neurons. Neuron 3:229-238[Web of Science][Medline].
  • Schlesinger HR, Gerson JM, Moorhead PS, Maguire H, Hummeler K (1976) Establishment and characterization of human neuroblastoma cell lines. Cancer Res 36:3094-3100[Abstract/Free Full Text].
  • Schoenfeld TA, McKerracher L, Obar R, Vallee RB (1989) MAP 1A and MAP 1B are structurally related microtubule associated proteins with distinct developmental patterns in the CNS. J Neurosci 9:1712-1730[Abstract].
  • Serrano N, Maschat F (1998) Molecular mechanism of polyhomeotic activation by Engrailed. EMBO J 17:3704-3713[Web of Science][Medline].
  • Serrano N, Brock HW, Maschat F (1997) beta3-tubulin is directly repressed by the engrailed protein in Drosophila. Development 124:2527-2536[Abstract].
  • Takei Y, Kondo S, Harada A, Inomata S, Noda T, Hirokawa N (1997) Delayed development of nervous system in mice homozygous for disrupted microtubule-associated protein 1B (MAP1B) gene. J Cell Biol 137:1615-1626[Abstract/Free Full Text].
  • Thoren PE, Persson D, Karlsson M, Norden B (2000) The antennapedia peptide penetratin translocates across lipid bilayers: the first direct observation. FEBS Lett 482:265-268[Web of Science][Medline].
  • van Dijk MA, Peltenburg LT, Murre C (1995) Hox gene products modulate the DNA binding activity of Pbx1 and Pbx2. Mech Dev 52:99-108[Web of Science][Medline].
  • Wurst W, Auerbach AB, Joyner AL (1994) Multiple developmental defects in Engrailed-1 mutant mice: an early mid-hindbrain deletion and patterning defects in forelimbs and sternum. Development 120:2065-2075[Abstract].
  • Xiong WC, Montell C (1993) tramtrack is a transcriptional repressor required for cell fate determination in the Drosophila eye. Genes Dev 7:1085-1096[Abstract/Free Full Text].

Copyright © 2001 Society for Neuroscience  0270-6474/01/21103350-10$05.00/0


This article has been cited by other articles:


Home page
 Mol. Biol. CellHome page
L. Zhao, L. Ku, Y. Chen, M. Xia, P. LoPresti, and Y. Feng
QKI Binds MAP1B mRNA and Enhances MAP1B Expression during Oligodendrocyte Development
Mol. Biol. Cell, October 1, 2006; 17(10): 4179 - 4186.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
C. Southwood, C. He, J. Garbern, J. Kamholz, E. Arroyo, and A. Gow
CNS Myelin Paranodes Require Nkx6-2 Homeoprotein Transcriptional Activity for Normal Structure
J. Neurosci., December 15, 2004; 24(50): 11215 - 11225.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
R. Lu, H. Wang, Z. Liang, L. Ku, W. T. O'Donnell, W. Li, S. T. Warren, and Y. Feng
The fragile X protein controls microtubule-associated protein 1B translation and microtubule stability in brain neuron development
PNAS, October 19, 2004; 101(42): 15201 - 15206.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
I. Foucher, M. L. Montesinos, M. Volovitch, A. Prochiantz, and A. Trembleau
Joint regulation of the MAP1B promoter by HNF3{beta}/Foxa2 and Engrailed is the result of a highly conserved mechanism for direct interaction of homeoproteins and Fox transcription factors
Development, May 1, 2003; 130(9): 1867 - 1876.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Nakayama, H. Murakami, N. Maeyama, N. Yamashiro, A. Sakakibara, N. Mori, and M. Takahashi
Role for RFX Transcription Factors in Non-neuronal Cell-specific Inactivation of the Microtubule-associated Protein MAP1A Promoter
J. Biol. Chem., January 3, 2003; 278(1): 233 - 240.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
I. Foucher, M. Volovitch, M. Frain, J. J. Kim, J.-C. Souberbielle, L. Gan, T. G. Unterman, A. Prochiantz, and A. Trembleau
Hoxa5 overexpression correlates with IGFBP1 upregulation and postnatal dwarfism: evidence for an interaction between Hoxa5 and Forkhead box transcription factors
Development, September 1, 2002; 129(17): 4065 - 4074.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit an eLetter
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (13)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Montesinos, M. L.
Right arrow Articles by Volovitch, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Montesinos, M. L.
Right arrow Articles by Volovitch, M.

-

Home  |   Search  |   Archive  |   Subscribe  |   Contact  |   Help
-
Copyright 2010 by Society for Neuroscience ONLINE ISSN: 1529-2401
-