The neuronal microtubule-associated protein 1B is under homeoprotein transcriptional control

Abstract

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.

Fig. 1.

Identification of clone 31 (MAP1Bgene) 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 withasterisks. 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.

Fig. 2.

Localization of clone 31 in theMAP1B genomic locus. A, Schematic representation of the rat MAP1B locus [modified fromKutschera et al. (1998) and Meixner et al. (1999)]. The most frequentMAP1B transcripts (90% of total MAP1BmRNA) 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.

Fig. 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 numbered1-16. Asterisks indicate ATTA sites conserved in the putative human MAP1Bpromoter (GenBank accession number: AC021318). Positions of fragmentsA, 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 ratMAP1B 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.

Fig. 4.

Regulation of MAP1B promoter activity in a neuroepithelial cell line. A, Cotransfection of CHP-100 cells with theMAP1B-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 (En2ΔHD1), or full-length Hoxa5 protein (Hoxa5). Luciferase activity of cell extracts after 24 hr is indicated in relative light units (RLU).

Fig. 5.

EnHD and full-length En2 bind to theMAP1B promoter. A, Purified EnHD (−, no protein; +, 50 ng of EnHD) was incubated with 1 ng of the indicatedMAP1B promoter fragment (from A toE). 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 Ato 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.

Fig. 6.

Cerebellum nuclear extracts bind theMAP1B 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 fragmentsA, 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 MAP1Bprobes refers to promoter regions indicated in Figure3A. Note that the five probes are of different sizes and thus migrate with different velocities.

Fig. 7.

Enhanced retardation of MAP1Bpromoter by GST-En2 and cerebellum nuclear extract. A, Band-shift assays with MAP1B probes A toE (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.

Fig. 8.

MAP1B reporter plasmids for in ovoelectroporations. The indicated regions of the MAP1Bpromoter were fused to a lacZ-reporter gene. ATTA sites are indicated with filled circles.Letters refer to regions indicated in Figure3A.

Fig. 9.

Activity of MAP1Bpromoter 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 β-galactosidase activity 24 hr after electroporation. Histograms show the percentage of electroporated embryos for each condition showing strong (+++/++), weak (+), or no (−) β-galactosidase activity. The numberof embryos for each condition is indicated on the top of the corresponding bar. Examples of β-galactosidase expression in each experimental condition are shown on the right side of the Figure.