|
Supervisor:
Prof.
Rick Nicholson
Project Title:
Genetic
and Epigenetic Regulation of the CRH Gene in Pregnancy
Project Summary:
CRH is a stress
responsive hormone produced in the hypothalamus. CRH is also synthesised
in the placenta and decidua. Placental production of CRH is linked to
the determination of gestational length. Decidual expression of CRH
is thought to be important in the regulation of implantation. In placental
cells, CRH expression is stimulated by glucocorticoids while in the
decidua and hypothalamus glucocorticoids inhibit expression.
Sequencing of the CRH gene indicates the presence of two common genetic
haplotypes which mediate altered sensitivity to inflammatory responses.
Abnormal production of CRH is observed in 45% of cases of preterm birth.
Recent advances in the study of epigenetic gene regulation indicate
that methylation of cytosine residues in the promoter and modifications
of histones, particularly the H3 histone by acetylation or by methylation
on the arginine (R) 2 residue or the lysine (4) residue, have profound
effects on the tissue specific expression of genes.
In this project some students will study the different epigenetic regulation
of the Corticotrophin Releasing Hormone (CRH) gene in the placenta,
decidua and umbilical cord (which does not express CRH) to elucidate
the epigenetic mechanisms regulating expression of this key gene, while
other students will test the biological consequences of genetic polymorphisms
of the CRH promoter with particular relevance to preterm birth. These
studies will be performed in cell models of placenta and decidua as
well as by using clinical samples from placenta, decidua and cord tissue.
Students will have the opportunity to use histone deacetylase (HDAC)
inhibitors, overexpression of HDAC proteins, chromatin immunoprecipitation
(ChIP) for methylated and acetylated histones, methylation of DNA and
key transcription factors, and Q-RT-PCR measurement of RNA. Reporter
plasmids containing the common polymorphisms in the CRH promoter will
be constructed and used to determine the effect of the polymorphisms
both on chromatin and on the response to stimuli such as steroid hormones
in transfected cells.
The extraordinary situation where three different human tissues, delivered
at the same time, have three distinctly different patterns of specific
gene expression provides an opportunity to study the epigenetic regulation
of a gene that plays a central role in many biological processes.
|
|
Supervisor:
Prof.
Rick Nicholson
Project Title:
IDENTIFICATION
OF REGULATORY COFACTORS INTERACTING WITH GLUCOCORTICOID RECEPTOR
Project Summary:
CRH is a hypothalamically-derived
hormone that orchestrates a series of neural and endocrine adaptations
known as the "stress response". A challenge to homeostasis
by any stressor initiates the release of CRH from the hypothalamus,
which in turn results in release of adrenocortiotropin hormone (ACTH)
from the pituitary into general circulation. ACTH then acts on the adrenal
cortex resulting in release of glucocorticoids, a steroid hormone, into
the blood. Glucocorticoids act in a negative feedback fashion to terminate
the release of CRH from the hypothalamus. CRH has also been identified
in many tissues outside the hypothalamus; in particular, in the placenta
during human pregnancy. Placental production of CRH has been linked
to the length of gestation in humans and several groups, including ours,
have reported that increased CRH concentrations in maternal blood are
associated with preterm delivery.
Glucocorticoids mediate inhibition of CRH gene expression in the hypothalamus,
whereas these steroid hormones stimulate the expression of CRH in the
placenta. In vitro assays have identified 3 regions of the promoter
that can bind GR but we have shown that these sites are not involved
in the glucocorticoid-mediated stimulation of CRH gene expression in
the placenta. Indeed, we have shown that glucocorticoids stimulate CRH
gene expression in the placenta through the cAMP Regulatory Element
(CRE), presumably through protein-protein interactions with proteins
that bind at the CRE. Other studies have shown one of the regions of
the CRH promoter that can bind GR in vitro is responsible for negative
regulation by glucocorticoids in cAMP stimulated AtT20 cells (a hypothalamic
cell model). Surprisingly, in the absence of this negative regulatory
region the CRH promoter is stimulated by glucocorticoids in AtT20 cells
in a manner similar to that seen in placenta cells.
This Research Project will involve the use of RNAi techniques in addition
to the study of protein-protein interactions between GR and other transcription
factors, and the identification of these factors through several techniques.
|
|
Supervisor: A/Prof.
Tamas Zakar
Project Title:
POTENTIAL
ROLE OF DECLINING PROSTAGLANDIN METABOLISING ACTIVITY IN PRETERM LABOUR
Project Summary:
Prostaglandin dehydrogenase is the key enzyme in the prostaglandin degradation
pathway. Its principal function is to convert bioactive prostaglandins
to inactive metabolites. In human pregnancy prostaglandin dehydrogenase
restricts the passage of active prostaglandins from the prostaglandin
rich fetal tissues to the uterine muscle. This is critical for the maintenance
of pregnancy, because prostaglandins are potent stimulants of uterine
contractions.
In recent studies, we have shown that post-transcriptional mechanisms
control dehydrogenase levels in uterine tissues at the time of labour
onset. These studies also showed that a premature decline in the activity
may lead to the onset of preterm labour. This project will explore the
role of post-transcriptional control of prostaglandin dehydrogenase
expression in the initiation of premature labour. The first approach
will be to determine whether prostaglandin dehydrogenase mRNA levels
are regulated by the rate of mRNA degradation or by the differential
processing of the primary transcript into functional and non-functional
mRNA splice variants. To achieve this will require the establishment
of a real time RT-PCR procedure that will measure mRNA degradation intermediates
in uterine tissue. Tissue samples obtained following preterm labour
will be analysed to provide information on the method of regulation
in vivo. In the second, in vitro approach, intracellular signalling
pathways will be activated in short-term incubations of the same tissue
with appropriate agonists, and the effect on dehydrogenase mRNA degradation
and transcript processing will be determined. These studies will indicate
if inappropriate changes in regulatory processes lead to a premature
decline in prostaglandin degrading activity and contribute to the causation
of preterm birth.
|
|
Supervisor:
Dr Mark Read
Project Title:
IDENTIFICATION
OF A PROTEIN WITH A CRUCIAL ROLE IN MYOMETRIAL CONTRACTION DURING PARTURITION
Project Summary:
The mechanisms which control the contraction of the uterus
during labour are not well understood. In our studies to identify proteins
in the myometrium with important roles in uterine contraction during
labour we have discovered an important group of proteins which requires
further study. In this project we will examine the role of these proteins
in myometrial muscle contraction, characterise the mechanisms through
which these proteins function and determine their link to labour.
|
|
Supervisor:
Dr Jonathan Hirst
Project Title:
NEUROPROTECTIVE
MECHANISMS IN PREGNANCY: ROLE OF PLACENTAL PRODUCTS IN PROTECTING AGAINST
BRAIN DAMAGE IN THE FETUS AND NEWBORN
Project Summary:
Complications during
pregnancy or at birth may lead to episodes of asphyxia and can result
in neurological impairment in the newborn. The consequent brain injury
may lead to life-long handicaps such as cerebral palsy. The placenta
secretes a number of products that aid in protecting the newborn brain
against asphyxia-induced damage. After birth the adrenal glands take
over from the placenta in providing protection against stressful events
and brain cell death. Preterm neonates often suffer from adrenal insufficiency.
In addition, many preterm neonates are exposed to therapies that suppress
adrenal function and therefore may be particularly vulnerable to brain
damage. This project will examine the expression reductase enzymes that
produce protective steroids, in the placenta from normal and premature
deliveries. The studies will also examine adrenal steroids levels in
umbilical cord blood samples. This data will indicate if adrenal function
is inadequate in these babies and if adrenal production is suppressed
by certain maternal therapies. The overall goal of these studies is
to delineate strategies for maximising neuroprotection in 'at risk'
neonates. The long-term aim is to identify the best therapeutic approaches
for that maintaining steroid-mediated protection following complications
during pregnancy and at birth.
|
|
Supervisor:
Prof.
Rick Nicholson
Project Title:
CHARACTERISATION
OF A MULTIFUNCTIONAL REGULATORY FACTOR
Project Summary:
In our ongoing study
of the regulation of the corticotropin releasing hormone (CRH) gene
we have identified a novel protein capable of binding to the cAMP regulatory
element (CRE), found in the CRH promoter as well as many other gene
promoters. This protein also possesses features suggesting a role in
RNA processing. Therefore, this protein has the potential to function
both as a DNA binding transcription regulatory protein and as a RNA
binding protein involved in RNA splicing. We have named this protein
CREAP, for CRE Associated Protein.
The regulatory
proteins that modulate gene transcription and RNA processing events
have historically been thought to function in one event or the other
but not both. Recent research indicates that substantial cross-talk
occurs between transcriptional and post-transcriptional processes. Indeed,
an increasing number of proteins appear to be multifunctional, participating
in both transcriptional and post-transcriptional events.
We will expand
scientific knowledge in this emerging area by characterising the functions
of this protein. The CREAP protein has the ability to link transcriptional
and post-transcriptional regulatory events. Alternative splicing is
a potential feature of the majority of mammalian genes, and an understanding
of the mechanisms involved is essential to eukaryotic biology.
In this project
we will perform structure function studies of CREAP to understand how
it functions as a DNA binding transcription factor. Simultaneously we
will perform studies to understand how CREAP functions as an RNA binding
factor involved in RNA splicing.
|
|
Supervisor: A/Prof.
Tamas Zakar
Project Title:
ROLE
OF CHANGES IN PROGESTERONE RECEPTOR EXPRESSION IN THE TRIGGERING OF
LABOUR
Project Summary:
The maintenance
of pregnancy requires adequate levels of the steroid hormone progesterone
in the maternal circulation. In most mammalian species, progesterone
levels fall in late gestation leading to the onset labour. In a few
other mammals including all primates and the guinea pig, labour occurs
in the presence of high progesterone levels, and the mechanism that
triggers labour is unknown. Recent studies suggest that changes in the
ratio of progesterone receptor isoforms determine the responsiveness
of uterus to progesterone. These changes in expression may result in
"functional" progesterone withdrawal and lead to labour onset.
This project will test this possibility using pregnant guinea pigs as
the experimental model. Gestational tissues will be obtained at advancing
stages of late pregnancy, and progesterone receptor subtype mRNA and
protein levels will be measured by real time RT-PCR and Western blotting,
respectively. The cellular and sub-cellular localization of progesterone
receptor isoforms will be determined by immunohistochemistry. We expectedly
to detect changes in receptor subtype expression that would lead to
decreased progesterone responsiveness with approaching labour. These
studies will determine if drugs target specific progesterone receptor
isoforms may be used to suppress premature labour.
|
|
Supervisors:
Dr Mark Read, Dr
Giavanna Angeli
Project Title:
MEDIATED
SIGNALLING MECHANISMS IN FETAL MEMBRANES
Project Summary:
Premature
birth occurs in 5-10% of births and is associated with 70-80% of neonatal
mortality. Arguably a greater problem exists among the survivors of
preterm births who are at high risk for intellectual handicap. Approximately
50% of cases of cerebral palsy are related to preterm birth. Unfortunately
the rate of preterm birth has not changed significantly in the last
30 years and trends indicate that it may be increasing. Our inability
to diagnose and treat patients with pre-term labour is largely due to
our ignorance of the fundamental mechanisms that control the process
of normal human labour.
Corticotropin-releasing
hormone (CRH) is a 41 amino acid polypeptide that exerts a wide spectrum
of effects in the CNS and in peripheral tissues. During pregnancy, the
placenta and other intrauterine tissues produce and secrete immunoreactive
CRH. CRH is secreted into the mother's circulation in large amounts
during the third trimester of human pregnancy and may play an important
role in the onset of labour. CRH exerts a number of functions within
the intrauterine environment including stimulation of prostaglandin
production in the fetal membranes and maintenance of the placental blood
flow.
Placentally derived
CRH plays a major role in the mechanisms controlling human pregnancy
and parturition. It has been suggested that there is a CRH placental
clock that is active from the early stages of pregnancy and determines
the length of gestation and the timing of parturition. Generally CRH
receptors are coupled to cAMP, such as occurs in the hypothalamus and
myometrium. Fetal and placental membranes express high levels of CRH
receptors, specifically CRH-R1?, along with other splice variants CRH-R1c
and CRH-R1d. However, CRH receptors in human placental and fetal membranes
do not couple to cAMP and appear to be preferentially linked to mechanisms
leading to an increase in intracellular inositol phosphate levels. These
mechanisms are poorly understood and their importance in pregnancy and
labour needs to be determined. This project involves an examination
of CRH mediated signal transduction pathway in JEG-3 choriocarcinoma
cells, a cell line derived from human fetal membranes. Specifically
the cell signalling pathways activated by CRH via CRH-1a receptors will
be determined. The methodologies employed in this study will include
recombinant gene technology, cellular biochemistry and pharmacological
approaches.
|
| |
| NOTE |
| Additional projects may
be available with these and other supervisors. Potential students are encouraged
to view the web-pages of individual researchers on the Senior
Research Staff page and arrange to speak directly with them if you are
interested in their work. |
