Biology Reference
In-Depth Information
A
Receptor interacting
domain (ID)
Repressor interacting domains
(RDs)
1988
2349 2498
N-CoR
myc-ID monomer
myc
NLS
dnN-CoRs
myc NLS
myc-ID dimer
B
C
5
6
Tg
Wt
-
T3
+
T
3
Onset of
metamorphosis
30
*
*
54
1500
20
*
52
1000
10
500
50
0
0
48
FLAG-TR
a/
RXR
+ + + + +
-
+ + +
-
myc-ID monomer
-
-
+
-
46
myc-ID dimer
-
-
-
-
-
+
0 5 10 15 20 25 30
Days of heat shock
Figure 10.3 Transgenic expression of a dominant negative corepressor leads to prema-
ture metamorphosis. (A) Schematic representation of dnN-CoRs. X. laevis N-CoR interacts
with unliganded TR via the receptor ID near the C-terminus. The repressor interacting
domains (RDs) located in the N-terminal part are required for the recruitment of other
co-repressors such as TBL1/TBLR1 and HDAC3. One dnN-CoRs, myc-ID monomer, com-
prises the ID (amino acids 1988
2349) fused to an N-terminal peptide containing a
myc tag and a NLS. The other dnN-CoR, myc-ID dimer, consists of two IDs in the same
directions connectedby a linker sequence. (B) Of the two dnN-CoRs, myc-IDdimer relieves
reporter gene repression by unliganded TR more efficiently than myc-ID monomer. The
mRNAs for FLAG-tagged TR and RXRwith or without themRNA formyc-IDdimer ormono-
mer were injected into X. laevis oocyte cytoplasm. The TRE-containing firefly luciferase
reporter vector together with the control Renilla luciferase plasmid was then injected into
the nucleus. After overnight incubation with or without T3, the oocytes were lysed and
assayed for luciferase activities. The ratio of firefly luciferase activity to Renilla luciferase
activity was determined as a measure of the reporter gene transcription level. The result
was expressed as a percentage of the basal transcription level that was obtained from the
oocytes without TR/RXR mRNA injection. Note that both dnN-CoRs relieved the
unliganded TR-induced repression, but the derepression was more potent with the
myc-ID dimer (left graph,
*
p
<
0.05). Neither of dnN-CoRs affected the liganded-TR
induced reporter gene activation (right graph). (C) Transgenic (Tg) tadpoles expressing
the dnN-CoR myc-ID dimer under the control of a heat shock-inducible promoter initiate
metamorphosis earlier than wild type (Wt) siblings. Wild type and transgenic sibling ani-
mals at stage 46 were treated daily with heat shock every day to induce dnN-CoR expres-
sion and their developmental stages were determined every 5 days for 30 days. Note that
at the end of the 30-day experiments, Tg animals had initiated metamorphosis (passed
stage 55) whileWt ones were one stage behind (a delay of about 7 days).
*
p
-
values
0.05
<
(Tg vs. Wt). See for
Sato, Buchholz, Paul, and Shi (2007)
more details.
