Fig. 5. Arx and Pax4 mutually inhibit the transcription of one another through a direct interaction. (A) A sequence analysis of a 50 kb region centered on the Arx locus was performed, comparing Mus musculus with Homo sapiens (H/M), Rattus norvegicus (M/R) or Danio rerio (M/D). Despite an overall high similarity, comparison with the zebrafish sequence reveals the presence of only a few highly homologous zones, in the homeobox region and also in a domain located 11 to 14 kb downstream of the Arx gene (highlighted in red in the alignment). No obvious similarity was found upstream of the Arx gene. The mouse Arx locus is represented underneath the result of the alignment (exons are shown as black rectangles), as are the three typical -helices of the homeodomain. The NheI-EcoRV 9.7 kb region encompassing the 3' conserved region was further used for the generation of transgenic animals using the ß-galactosidase reporter gene lacZ. (B) ß-Gal staining of such animals demonstrates a recapitulation of the Arx expression pattern at E10.5 (see Collombat et al., 2003). The pattern of labelling in the pancreas corresponds perfectly to endogenous Arx expression (arrowhead; P.C., unpublished). (C) A search for Pax4-binding sites (P4BS) within the 9.7 kb region demonstrated that a labelled 37 bp DNA fragment, located 8.5 kb downstream of the NheI site (red rectangle in A), can efficiently interact with Pax4, as evidenced by EMSA analysis (lane 3). A competition assay with increasing concentrations of unlabelled P4BS further demonstrates the specificity of the interaction (lanes 4-6). Lane 1 shows the Pax4-binding-site consensus used as control (C). (D) The specificity of the interaction Pax4-P4BS was validated by ChIP analysis. No binding was detected in COS-cells (lanes 2-4), whereas, in ß-cells (lanes 5-7), PCR and sequencing reactions confirmed that Pax4 binds to P4BS (lane 7; P, P4BS-specific primers; C, control primers). (E) A reporter assay study indicates that Pax4 interacts with P4BS and acts as a transcriptional inhibitor. (F) Representation of the Pax4 locus, as well as the encoded homeodomain and the SmaI-NcoI 0.9 kb Pax4 enhancer region. (G) This 0.9 kb region was subdivided into five overlapping domains that were tested for recognition by the Arx protein. The 400 to 600 bp region (ArBS) can interact with Arx (blue rectangle in F,G). By means of EMSA and competition assay, Arx was found to preferentially interact with ArBS (lanes 2 and 3-5, respectively). (H) Using ChIP assay on isolated pancreas, an Arx-ArBS interaction is observed in E14 pancreas (lanes 5-7) but not in hind limb (lanes 2-4). (I) A reporter assay analysis suggests that Arx can bind to ArBS and acts as a transcription inhibitor. P, minimal promoter; Luc, luciferase gene. Results are presented as mean of luciferase activity ±s.e.m. Statistically significant differences of reporter activity, comparing cells transfected with the indicated reporters alone or with a Pax4-encoding vector (n=3), were estimated using Student's t-test (^***P<0.001).