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Li, Wang, et al., 2000; Shi et al., 2012; Yoon et al., 2003; Zhang et al., 2002;
Zhang & Lazar, 2000
). Consistently, TBLR1 is also recruited by unliganded
TR to endogenous target genes in premetamorphic tadpoles and released
during metamorphosis, accompanied by local increase in histone acetylation
(
Sachs et al., 2002; Tomita et al., 2004
), suggesting that such corepressor
complexes and histone deacetylation are important for the repression of
TH target genes and premetamorphic animal development.
To investigate the role of corepressors in premetamorphic tadpoles,
we generated two dominant negative forms of the
X. laevis
N-CoR
(dnN-CoRs) that contained only the receptor interacting domain (ID)
near the C-terminus of N-CoR (
Fig. 10.3
A). The first dnN-CoR, myc-
ID monomer, comprised just the ID fused to an N-terminal peptide con-
taining the myc tag and a nuclear localizing sequence (NLS). The second
dnN-CoR, myc-ID dimer, is similar to myc-ID monomer except with
two copies of the ID in the same directions connected by a linker sequence.
In the reconstituted frog oocyte transcription system where one can study
transcription of a reporter gene in the context of chromatin, both dnN-
CoRs were able to compete against wild type N-CoR for binding to
unliganded TR (
Sato et al., 2007
). More importantly, they reduced the
repression of the TH target promoter by TR in the absence of TH with
the myc-ID dimer more effective, while having no effect on the activation
by TR in the presence of TH (
Fig. 10.3
B) (
Sato et al., 2007
).
To investigate the role of corepressor complex in TR function during
development, we carried out transgenesis to express the myc-ID under the
control of a heat shock-inducible promoter. In the absence of heat
shock, the dnN-CoR was not expressed and the transgenic tadpoles and
frogs were apparently normal. As this dominant N-CoR binds only to
unliganded TR, it is expected to affect TR function only during the devel-
opmental stages 45-55 when TR expression is high while TH levels are
low (
Fig. 10.1
), a window period of about 30 days (
Nieuwkoop &
Faber, 1956
). Thus, we subjected transgenic and their wild type siblings
to daily heat shock treatment, starting at stage 46. Heat shock treatment
led to overexpression of the dnN-CoR and upregulation of most of the
TH-response genes analyzed (
Sato et al., 2007
). More importantly, trans-
genic animals developed faster, reaching stage 55, the onset of metamor-
phosis, by as much as 7 days earlier than the wild type siblings within the
30-day experiment (
Sato et al., 2007
)(
Fig. 10.3
C). These observations
demonstrated that the recruitment of HDAC-containing corepressor
complexes is indeed important for gene repression by unliganded TR in
