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doi: 10.1242/10.1242/dev.00414

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Joint regulation of the MAP1B promoter by HNF3ß/Foxa2 and Engrailed is the result of a highly conserved mechanism for direct interaction of homeoproteins and Fox transcription factors

¹ CNRS UMR 8542, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris Cedex 05, France
² Université Paris 7, UFR de Biologie, 2 place Jussieu, 75005 Paris, France

View larger version (55K): [in a new window]   Fig. 2. Binding of Foxa2 to fragment C of MAP1B promoter. (A) Purified GST (lanes 2-4; 20, 40 or 80 ng) or GST-Foxa2 fusion proteins (lanes 5-7; 20, 40 or 80 ng; lane 9: 20 ng) were incubated with radioactive MAP1B promoter fragment C (lanes 1-7) or fragments C plus E (lanes 8-9). Migration of the free probe is indicated on the left. GST-Foxa2 binds to fragment C, but not E, in a dose-dependent manner; GST alone shows no binding activity. (B) Binding of 1 µg nuclear extracts from P0 mice cerebellum to fragments C, D, E and C + E. Migration of free probes is indicated on the left. Both fragments C and D are retarded by the cerebellar nuclear extracts. The complex bound to fragment C contains Foxa2, as demonstrated by the supershift induced by the addition of the anti-Foxa2 antibody (4C7 Ab). No supershift is observed when the 4C7 antibody is used in the absence of cerebellum nuclear extracts. No supershift is observed when another — unrelated — monoclonal antibody (anti-myc, 9E10) is used instead of the 4C7 antibody (CTRL Ab, right panel).

View larger version (42K): [in a new window]   Fig. 4. Foxa2 inhibits En2SP binding to the HF1 and HF2 sites of MAP1B promoter. (A) Binding of En2SP and Foxa2 to the HF1 site. The HF1 probe sequence, highlighting the ATTA (square box) and Foxa2 (oval box) sites, is represented above the EMSA panels. Purified En2SP or GST-Foxa2 fusion proteins were incubated independently (lanes 2-7), or in combination (lanes 8-9). Lane 1: free HF1 probe; lanes 2-3: 25 and 50 ng of En2SP, respectively; lanes 4-5 and 6-7 show two different autoradiographic exposures of the same gel area (4-5: long exposure; 6-7: short exposure). Gels were loaded with 40 ng (lanes 4 and 6) or 120 ng (lanes 5 and 7) of GST-Foxa2. An interaction of Foxa2 with HF1 is visible in lane 5 (double arrowhead). At low concentration (lane 2: 25 ng) En2SP gives one retarded band, while at a higher one (lane 3: 50 ng) it generates two retarded bands (arrowheads). Co-incubation of GST-Foxa2 with En2SP leads to an inhibition of En2SP binding (compare lane 3 to lanes 8 and 9). (B) Binding test for En2SP and Foxa2 to the HF2 site. The probe sequence, highlighting the ATTA (square box) and Foxa2 (oval box) sites, is represented above the EMSA panel. Purified En2SP or GST-Foxa2 fusion protein were incubated independently (lane 2: 50, lane 3, 100 ng of En2SP; lane 4: 120, lane 5, 200 ng of GST-Foxa2) or together (lane 6-9) with the HF2 probe. En2SP was preincubated with HF2 for 20 minutes, before adding Foxa2. En2SP binds HF2 (arrowhead), GST-Foxa2 does not bind by itself (lanes 4,5) but dissociates En2SP from HF2 (compare lane 2 with lanes 6-7, and lane 3 with lanes 8-9).

View larger version (57K): [in a new window]   Fig. 1. Conserved Foxa2 consensus binding sites in the MAP1B promoter. Schematic representation of the MAP1B promoter highlighting potential Foxa2 binding sites (asterisks) on previously characterized A, B, C, D and E fragments (modified from Montesinos et al., 2001). Fragment C rat (top) and human (bottom) sequences have been aligned. Four ATTA (square boxes) and five Foxa2 consensus binding sites (oval boxes, core underlined) are conserved between rodent and human. Note that, in two regions, the putative homeoprotein and Foxa2 sites overlap (HF1 and HF2). Residues that differ between rat and human sequences are shaded in black.

View larger version (15K): [in a new window]   Fig. 3. Ex vivo regulation of the MAP1B promoter by Foxa2 and Engrailed. (A) Primary cultures of mouse mes-metencephalic neurons were transfected with the MAP1B promoter fused to a luciferase reporter (pMAP-luc), or with a modified version of this promoter, in which a 43 bp fragment containing both the HF1 and HF2 sites was deleted (pMAPHF1+2-luc). The expression of the deleted promoter is about threefold that of the full length promoter, indicating that the 43 bp fragment including HF1 and HF2 has regulatory functions. The data are the results of pooling three independent experiments. (B) Expression levels of En1, En2 and Foxa2 mRNAs in E13 mes-metencephalic neurons or N2A cells, as determined by real-time RT-PCR. PCR was performed on cDNA derived from 2 ng of RNA (see Materials and Methods). Results are expressed as a percentage of GAPDH mRNA expression level (100% being the level of GAPDH mRNA expression in mes-metencephalic neurons). Significant amounts of En1, En2 and Foxa2 mRNAs were detected in mes-metencephalic cultures. Foxa2 mRNA low abundance is probably due to the fact that Foxa2 is expressed only by cells located in the ventral mes-metencephalon (the engrailed gene being, by contrast, expressed by most cells of the mes-metencephalon). In N2A cells, En2 mRNA level is extremely low and En1 and Foxa2 mRNAs are not detected (nd). (C) N2A cells were co-transfected with various concentrations of Foxa2- or En2-expressing plasmids, alone or in combination, together with the MAP1B-luciferase reporter plasmid (pMAP-luc). Control cells were transfected with empty pCL9m plasmid. Transfection of high concentration of En2-expressing plasmid activates the promoter. Transfection of low concentration of Foxa2-expressing plasmid has no effect by itself but partially antagonizes the En2-dependent activation of the MAP1B promoter. In a similar way, activation of MAP1B promoter by high concentration of Foxa2 plasmid is partially antagonized by a low dose of En2 plasmid. The data are the results of pooling three independent experiments.

View larger version (57K): [in a new window]   Fig. 5. Foxa2 associates with Engrailed through its winged-helix/Forkhead box domain. (A) 35S-labeled Foxa2 was incubated with bacterially expressed GST or GST-En2 immobilized on glutathione beads, then analysed by SDS-PAGE. Foxa2 binds to GST-En2 but not to GST. (B) Foxa2 constructs used to identify its domain(s) interacting with Engrailed. CRI to CRIV domains of Foxa2 are indicated. Numbers refer to amino acids positions. (C) Each construct was produced and labeled in vitro, and used in a pull-down experiment with GST-En2 immobilized on glutathione beads. Bound proteins were visualized after SDS-PAGE and autoradiography. All constructs containing the CRI domain of Foxa2 bind to Engrailed, and this domain (148-257) is sufficient to mediate this binding. Positions of molecular mass markers (in kDa) are indicated on the left.

View larger version (46K): [in a new window]   Fig. 6. Identification of the domains of Engrailed interacting with Foxa2. (A) Engrailed truncated constructs produced as GST fusions to identify Foxa2-binding domains within the En2 sequence. The homeodomain (EH4), as well as other previously characterized domains of Engrailed [EH1-5 (Duboule, 1994)] are indicated. Numbers refer to amino acids positions. (B) 35S-labeled Foxa2 was incubated with immobilized GST-fusion proteins. Bound radioactive protein was visualized, following SDS-PAGE, using autoradiography. Left panel illustrates that the homeodomain and the 146-199 domain fully retain Foxa2 (compare with input). Right panel demonstrates that, compared with the full 146-199 domain, the two sub-domains 146-167 and 168-199 bind Foxa2 poorly.

View larger version (53K): [in a new window]   Fig. 7. Binding of Foxa2 to Hoxa5, Lim1 and Gsc homeoproteins through its Forkhead box domain. (A) 35S-labeled homeoproteins Hoxa5, Gsc, Lim1, and Engrailed (En2), as well as 35S-labeled Krox20 were incubated with immobilized GST or GST-Foxa2. All four homeoproteins bind Foxa2 but Krox20 does not. Positions of molecular mass markers (in kDa) are indicated on the left. (B) Mapping of the domain of Foxa2 involved in its interaction with Hoxa5, Gsc and Lim1. 35S-labeled Forkhead box domain (148-257) and its flanking N-terminal (1-148) and C-terminal (257-458) domains were challenged for binding to GST-Hoxa5, GST-Gsc and GST-Lim1. In all three cases, only the Forkhead box domain of Foxa2 binds to the homeoprotein.

View larger version (21K): [in a new window]   Fig. 8. Homeoprotein domains interacting with Foxa2, as revealed in pull-down assays. Summary of the protein domains within the four homeoproteins studied (other than Engrailed) that bind Foxa2 [Hoxa5, Gsc, Lim1 (this work) and Otx2 (Nakano et al., 2000)]. Radioactive fragments of the homeoproteins were tested for binding to Foxa2 (+ indicates positive binding; — indicates absence of binding). In all cases, the homeodomain (HD) binds Foxa2. Moreover, three homeoproteins out of four have, like Engrailed, one additional interacting domain, located either in their N-terminal domain (Hoxa5, Gsc) or in their C-terminal (Otx2) sequence. HP, Hexapeptide; GEH/Eh1, Gsc-Engrailed homology/Eh1 domain; LIM, LIM domains; Tail, tail domain of Otx2.