|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
|
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Arx expression (arrowhead; P.C., unpublished).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).
|
