The Unit on Molecular Morphogenesis is exploring molecular mechanisms
in amphibian metamorphosis. The control of metamorphosis by thyroid hormone
(TH) offers a unique paradigm in which to study genes that are important
for postembryonic organ development. During metamorphosis, different organs
undergo vastly different changes. Some, like the tail, undergo complete
resorption while others, such as the limb, are developed de novo.
Most larval organs persist through metamorphosis, but, for the frog, they
are dramatically remodeled. For example, tadpole intestine in Xenopus
laevis is a simple tubular structure consisting primarily of a single
layer of primary epithelial cells. During metamorphosis, specific cell
death and selective cell proliferation and differentiation transform the
intestine into a multiply folded adult epithelium with elaborate connective
tissue and muscles. The wealth of knowledge from past research and the
ability to manipulate amphibian metamorphosis both in vivo and
in vitro in organ culture offer an excellent opportunity to study
the developmental function of thyroid hormone receptors (TRs) and the
underlying mechanisms in vivo and to identify and functionally characterize
genes that are critical for postembryonic organ development in vertebrates.
Function and Mechanism of Gene Regulation by TR during Frog Development
Amano, Jones, Rouse, Sachs,a Shi in
collaboration with Wadeb
We have proposed a dual TR function model that is based on our earlier
studies of the oocyte and developing embryos and tadpoles. According to
the model, the heterodimers between TR and RXR (9-cis retinoic
acid receptor) activate gene expression during metamorphosis when TH is
present, while, in premetamorphic tadpoles, they repress gene expression
to prevent metamorphosis, thus ensuring a tadpole growth period. Our studies
have since provided strong support for and mechanistic insights into such
a model. First, chromatin immunoprecipitation (ChIP) assay shows for the
first time that TR/RXR are bound to thyroid hormone response elements
(TREs) in the target genes in premetamorphic tadpoles. In support of a
role of histone acetylation in gene regulation by TR/RXR, we have demonstrated
that blocking the deacetylase function with the drug trichostatin A (TSA)
can activate TH response genes in the intestine of premetamorphic tadpoles,
just like TH. Furthermore, treatment of premetamorphic tadpole with TH
leads to increased histone acetylation at the TRE regions as the target
genes are being activated. In addition, TH treatment causes a reduction
in the association of the histone deacetylase Rpd3 with TH response genes.
Together, these studies suggest a role for the deacetylase complex in
gene repression by unliganded TR in premetamorphic tadpoles (Sachs et al., 2000, 2001).
Interestingly, our studies with TSA also showed that, during metamorphosis,
histone deacetylases are required at a step or steps downstream of gene
activation by TH-bound TR. Thus, histone deacetylases plays a role at
two distinct steps during frog development (Sachs et al., 2001).
To investigate how histone deacetylases participate in development, we
have begun to characterize frog histone deacetylase complexes. To isolate
TR complexes that contain the TR-interaction corepressor N-CoR, we made
use of the fact that the frog egg contains all necessary components for
gene regulation. We were able to purify three N-CoR complexes (Jones et
al., 2001), two of which possess histone deacetylase activity, and
one of which contains Sin3, Rpd3, and RbAp48. They are the first such
complexes to be purified, even though interactions among Sin3, Rpd3, and
RbAp48 have been known for some time. The second complex contains a Sin3-independent
histone deacetylase while the third lacks histone deacetylase activity.
Our complexes differ from similar mammalian complexes reported by a few
other laboratories, demonstrating the complexity of gene repression mediated
by N-CoR. Immunoprecipitation studies show that N-CoR binds to unliganded
TR expressed in the frog oocyte, confirming that N-CoR complexes are involved
in repression by unliganded TR. The failure to bring precipitate Sin-3
by anti-TR antibody suggests that the first complex is unlikely to be
involved in gene repression by unliganded TR. Our current studies aim
at identifying the components of the complexes and investigating which
complex is involved in gene repression by TR during frog development.
We are also employing the recently developed transgenesis technology in
Xenopus to analyze TR function directly during metamorphosis. Preliminary
data showed that overexpressing a dominant negative TR in the tadpole
intestine had no effect on tadpole development but interfered with adult
epithelial morphogenesis, consistent with the role of TR in mediating
the effects of TH during tissue transformation.
Roles of Matrix Metalloproteinases during TH-Induced Tissue Remodeling
Amano, Damjanovski,c Shi in collaboration
with Ishizuya-Okad
In a second area of research, we have been focusing on TH-response genes
encoding matrix metalloproteinases (MMPs), which are extracellular enzymes
capable of digesting various extracellular matric (ECM) components. Our
earlier studies led us to propose that the MMP stomelysin-3 (ST3) is directly
or indirectly involved in ECM remodeling, which in turn influences cell
behavior. By using intestinal organ cultures, we have now shown that ST3
expression is associated with ECM remodeling and cell death in TH-induced
intestinal remodeling in vitro. Studies with primary cell cultures of
the intestine have shown that ECM inhibits TH-induced larval epithelial
cell death in vitro. These observations prompted us to investigate whether
ST3 is required for epithelial transformation in organ cultures. By using
a function-blocking antibody against the catalytic domain of ST3, we have
demonstrated that blocking ST3 function inhibits TH-induced apoptosis
of larval intestinal epithelial cells and the invasion of the proliferating
adult epithelial cells into the connective tissue. These effects are accompanied
by an inhibition in the remodeling of the basal lamina or basement membrane,
which is the ECM that separates the connective tissue from the epithelium.
The results support the argument that ST3 is directly or indirectly involved
in ECM remodeling, which in turn influences cell behavior (Ishizuya-Oka
et al., 2000).
To investigate directly the roles of MMPs in developing animals, we are
employing the transgenic approach to express wild-type and mutant MMPs
in Xenopus embryos and tadpoles. In our initial study, we overexpressed
Xenopus MMPs stromelysin-3 (ST3) and collagenases-4 (Col4) under the control
of a ubiquitous promoter and observed that embryos with overexpressed
ST3 or Col4, but not the control green fluorescent protein (GFP), died
in a dose-dependent manner during late embryogenesis. The specificity
of this embryonic lethal phenotype was confirmed by the failure of a catalytically
inactive mutant of ST3 to affect development. Finally, overexpression
of a MMP of the mammalian membrane type also led to late embryonic lethality
in Xenopus embryos, suggesting that membrane type-MMPs have functions
in vivo for ECM remodeling, in addition to being activators of other proMMPs.
These data, together with the developmental expression of several MMPs
during Xenopus development, suggest that MMPs play important roles during
mid- to late embryogenesis and that proper regulation of MMP genes is
critical for tissue morphogenesis and organogenesis (Damjanovski et al.,
2001). The data also suggest that alteration in MMP expression during
metamorphosis may alter TH-dependent tissue remodeling process. We have
therefore begun to investigate MMP function during metamorphosis by using
tissue-specific promoters to drive the expression of MMPs during metamorphosis.
Mechanism of Transcriptional Regulation of HIV LTR by TR
Hsia, Shi
In a separate study supported by an NIH Intramural AIDS Targeted Antiviral
Program (IATAP) grant, we have investigated the roles of TR/RXR in the
regulation of HIV LTR, which controls the transcription of the AIDS virus.
Using the frog oocyte as an in vivo system that allows assembly of the
LTR into chromatin through a replication-coupled chromatin assembly process,
thus mimicking somatic cells, we have demonstrated that unliganded TR/RXR
heterodimers repress LTR in the context of chromatin, whereas the addition
of T3 relieves the repression and further activates the promoter (Hsia
et al., 2001). To investigate how TR regulates HIV LTR, we have determined
the binding of TR to the putative TREs in the LTR in vivo by using ChIP
assay. The results demonstrated for the first time that both TR, most
likely as a homodimer, and TR/RXR heterodimers are bound constitutively
to the putative TREs in the LTR in chromatin and that transcriptional
repression by unliganded TR leads to a decrease in histone acetylation
levels at the LTR. Furthermore, we have found that the activation of the
promoter by TH causes chromatin disruption. The results suggest that chromatin
remodeling plays an important role in LTR regulation by TH in vivo.
|
PUBLICATIONS
- Damjanovski
S, Amano T, Li Q, Pei D, Shi Y-B. Overexpression of matrix metalloproteinases
leads to lethality in transgenic Xenopus laevis: implications
for tissue-dependent functions of matrix metalloproteinases during late
embryonic development. Dev Dyn 2001a;221:37-47.
- Damjanovski S, Sachs LM, Shi Y-B. Function of thyroid hormone receptors
during amphibian development. In: Methods of molecular medicine: thyroid
hormone receptors. Totowa, NJ: Humana Press, Inc., 2001b, in press.
- Hsia
VSC, Wang H, Shi Y-B. Involvement of chromatin and histone acetylation
in the regulation of HIV-LTR by thyroid hormone receptor. Cell Res 2001;11:8-16.
- Ishizuya-Oka
A, Li Q, Amano T, Damjanovski S, Ueda S, Shi Y-B. Requirement for
matrix metalloproteinase stromelysin-3 in cell migration and apoptosis
during tissue remodeling. J Cell Biol 2000;150:1177-1188.
- Jones
PL, Sachs LM, Rouse N, Wade PA, Shi Y-B. Multiple N-CoR complexes
contain distinct histone deacetylases. J Biol Chem 2001;276:8807-8811.
- Sachs
LM, Amano T, Rouse N, Shi Y-B. Involvement of histone deacetylase
at two distinct steps in gene regulation during intestinal development
in Xenopus laevis. Dev Dyn 2001;222:280-291.
- Sachs
LM, Shi Y-B. Targeted chromatin binding and histone acetylation
in vivo by thyroid hormone receptor during amphibian development.
Proc Natl Acad Sci USA 2000;97:13138-13143.
aL.M. Sachs, former member; present
address: CNRS, Paris, France.
bP. Wade, Emory University, Atlanta,
GA
cS. Damjanovski, former member; present
address, University of Western Ontario, Ontario, Canada.
dA. Ishizuya-Oka, Dokkyo University School
of Medicine, Tochigi, Japan
|