Gene Expression and Epigenomics Group
Current Research Projects:
1. Roles of transcription activators and repressors in
chromatin remodelling at the IL-2 gene promoter.
2. The role of the transcriptional repressor, ZEB, in T cell gene
transcription and beyond.
3. The epigenomic marks that tag inducible genes in T cells.
4. Epigenetics and T cell activation.
5. Gene regulatory networks in T lymphocyte development and activation.
6. A possible epigenetic relationship between IL-2 and IL-21.
7. A role for c-Rel in regulatory T cell development.
1. Roles of transcription activators and repressors
in chromatin remodelling at the IL-2 gene promoter (Seungsoo Lee).
In activated T cells, binding of transcription factors to the interleukin-2
(IL-2) gene promoter region is an important event leading to high level expression
from the gene. This binding results in changes in chromatin structure, allowing
transcriptional initiation and elongation. However, the majority of transcription
factor binding studies with the IL-2 promoter have been performed using in vitro
approaches. In this study, using chromatin immunoprecipitation (ChIP) assays,
we have examined kinetics of transcription factor binding to the endogenous
IL-2 gene promoter in activated T cells. We monitored the kinetics of transcription
factor binding in response to activation of EL-4 T cells at the promoter region.
Initially, we focused on two transcription factor families, NF-?B and AP-1,
that have been postulated to bind to the IL-2 promoter from in vitro studies.
We found that the NF-kB family member, p65/RelA, and the AP-1 family member,
c-jun, bound to the proximal promoter of the IL-2 gene in an activation- and
time-dependent manner. Binding of p65/RelA and c-jun peaked at approximately
2hr following stimulation and gradually decreased, suggesting that binding of
these transcription factors may be involved in the initiation of the IL-2 promoter
chromatin remodelling and transcription initiation. Interestingly, the binding
kinetics of the NF-?B family member, c-Rel were completely different, displaying
a much later increase in binding while the other transcription factors were
decreasing. These data suggest that c-Rel may play a role in maintaining rather
than initiating chromatin remodelling and transcriptional activation of the
IL-2 gene. Binding of these transcription factors to another inducible gene,
GM-CSF, displays similarities as well as contrasts with IL-2. Our findings provide
a more detailed in vivo description of the interaction of transcription factors
with the IL-2 and other gene promoters, and allow us to better examine the functions
of these transcription factors.
2. The role of the transcriptional repressor, ZEB, in
T cell gene transcription and beyond (Jun Wang).
Interleukin-2 is a key cytokine involved in T cell proliferation and differentiation
and may play a role in diseases such as leukaemia, autoimmunity and viral pathogenesis.
Thus, it is important to understand the molecular mechanisms of the induction,
maintenance and repression of IL-2 gene expression in T cells.
ZEB (delta-EF1) is a transcription repressor, originally isolated from T cells,
and shown to repress several T cell genes. The protein contains two Zn finger
domains and a homeobox domain and is expressed during skeletal and muscle development
as well as T cell development. When studying the restriction enzyme accessibility
of the IL-2 promoter chromatin, it was found that the ZEB binding site at -100
upstream from the transcription start site did not become accessible in response
to weak signals (CD3/CD28 which leads to a relatively modest induction of IL-2)
but became fully accessible when T cells were activated with PMA/ionophore/CD28
(which gives a large induction of IL-2 gene transcription). We speculated that
ZEB may maintain a inaccessible chromatin structure across the IL-2 promoter
by targeting corepressor complexes to the site. We have shown that ZEB is expressed
in T cells and mRNA and nuclear protein levels increase following T cell activation.
Over-expression of ZEB can repress IL-2 promoter activity and this repression
is dependent on the ZEB binding site at -100. ZEB cooperates with the co-repressor
CtBP and with HDAC1 to repress IL-2 promoter activity as well as endogenous
IL-2 mRNA expression. Using ChIP assays we have shown that ZEB and HDAC1 bound
to the IL-2 promoter in resting T cells and was transiently removed following
activation. Thus, ZEB may help to recruit a repressor complex to the IL-2 promoter
in resting T cells as well as cells where active repression of the IL-2 gene
is required. We are further investigating the role of ZEB on chromatin remodelling
of the IL-2 gene and the dynamic changes of repressor and activator complexes
across the IL-2 promoter.
In a collaborative project (Dr Greg Goodall, Hanson Institute, Adelaide; http://www.imvs.sa.gov.au/immunology/research/cytores.htm),
we are investigating the role of ZEB in controlling expression of specific microRNAs
that are critical for mesenchymal to epithelial transition, a process thought
to be a key in cancer metastasis. ZEB appears to be a repressor of specific
microRNA transcription as well as being a target of the microRNAs in a feedback
loop that may play a critical role in maintaining the phenotypic transition.
3. The epigenomic marks that tag inducible genes in
T cells (Chloe Lim).
One of the key regulators of gene transcription is chromatin; the nucleoprotein
complex that packages DNA into a compact higher order structure in the nucleus.
We have studied the changes in chromatin structure that occur across two inducible
cytokine genes in T cells (IL-2 and GM-CSF) in some detail. These genes are
induced with delayed kinetics and are dependent on new protein synthesis for
expression. We find that the promoters of both genes are assembled into nucleosomes
(to different degrees) in the resting state and that histones are lost from
the promoter regions following activation. The GM-CSF gene has constitutive
Brg1 (a subunit of the SWI/SNF complex) association and also has a higher level
of histone acetylation than the surrounding chromatin which may be at least
in part responsible for the constitutive association of Brg1. To expand our
knowledge of the chromatin state of inducible genes in T cell, a group of genes
identified as inducible from microarray data were chosen for further study.
Using chromatin immunoprecipitation (ChIP) and Real-time PCR, we have documented
(i) detailed time courses of gene expression, (ii) histone occupancy across
the promoter and upstream regions of the genes in resting T cells, (iii) the
changes in histone occupancy upon T cell activation, as well as (iv) acetylation
levels for both H3 and H4 in both resting and activated cells. From these data,
we hypothesise that the basal chromatin state can be clearly correlated with
the kinetics of response of the genes. Consequently, we hope to elucidate the
general mechanism of transcriptional regulation of genes expressed by activated
T cells in the context of chromatin.
4. Epigenetics and T cell activation (Jun Fan).
Epigenetic mechanisms, which involve DNA and histone modifications, can result
in the heritable silencing of genes without a change in their coding sequence.
Epigenetic mechanisms play an important role in many human diseases. Disruption
of the balance of epigenetic networks can cause several major pathologies, including
cancer.
We are interested in the epigenetic changes (DNA methylation and histone modifications)
that occur in inducible genes during T cell differentiation or T cell activation.
How epigenetic marks are initiated, maintained, and what functions of the cell
and organism they affect, is not only key to a fundamental biological understanding
of our genome, but also pivotal for many applications in medicine and biotechnology.
Recent studies in our lab showed that the promoter-enhancer region of the IL-2
gene demethylates in T lymphocytes following cell activation, while the gene
expression of the IL-2 is increased. Our current research is focusing on understanding:
(1) the mechanism of the epigenetic changes, (2) the correlation between demethylating
activity and gene expression and (3) the correlation between DNA methylation
and histone modifications. Exciting new developments in sequencing technology
have made it easier to examine methylation changes on a global level. We plan
to utilise "whole genome" sequencing to examine changes to epigenetic
modifications in T cells during development and activation.
5. Gene regulatory networks in T lymphocyte development
and activation (Kristine Hardy).
T lymphocytes play an important role in the adaptive immune response to foreign
pathogens and potentially to cancer cells. Naive T lymphocytes in the periphery
become activated when they encounter foreign peptide antigens presented to them
by other cells, with the appropriate co-stimulatory signals. If lymphocytes
recognised self-peptides a dangerous auto-immune response results, so removal
of developing lymphocytes that can recognise self-antigens is undertaken in
the thymus (negative selection). Defects in this process can lead to diseases
such as diabetes. Much is still to be learned about the transcription factors
that regulate both positive and negative selection in the thymus. Large scale
gene expression studies show many transcription factors changing in expression
during the selection process and it is clear that their functional interaction
is important in determining gene expression patterns and thus functional outcomes.
We are using computational approaches to identify over- and under- represented
transcription factor binding motifs in co-regulated genes sets from our own
and other microarray data sets. We have shown that gene sets that are co-regulated
in positive or negative selection are enriched for genes with certain transcription
factor binding motifs. We have tested our model on a separate group of genes
to those represented on the original microarrays and predicted their expression
during selection. These studies reveal that we can predict many of the genes
either up-regulated only in positive selection or down-regulated only in negative
selection. We have also examined the representation of the motifs in other types
of T cells and this gives us further information on which groups of genes are
co-regulated.
The non-obese diabetes (NOD) mouse has a defect in negative selection. There
appears to be a global signalling defect in the thymocytes and the defect is
due to the combined effect of several genetic loci. In collaboration with the
Immunogenomics
Group we are breeding congenic mice that will allow us to separate the effects
of the loci on chromosome 2, 1, 7 and 15. We hypothesise that some of the loci
will give rise to the global defect, while others will have more localised effects
and will be especially interesting to us if they are due to mis-regulation of
a transcription factor. Having developed a putative network, we can test sentinel
genes (genes whose expression is indicative of the activity of a particular
transcription factor) for mis-regulation of that transcription factor and determine
which defects in the gene regulatory network result from various disease causing
gene mutations. It also allows us to see which transcription factors play a
key role in regulating T cell selection and activation.
6. A possible epigenetic relationship between IL-2
and IL-21 (Guobing Chen).
Interleukin 21 (IL-21) is a novel cytokine which was first identified in 2000.
It is homologous to IL-2 and IL-15, and its receptor is formed from a complex
of the IL-21 receptor (IL-21R), which is most closely related to the IL-2 receptor
? chain, and the common gamma chain (?c), which is also a component of the receptors
of IL-2, IL-4, IL-7, IL-9 and IL-15. It has been shown that IL-21 has a broad
range of influences on immune cells, including T cells, B cells, nature killer
(NK) cells and dendritic cells (DCs). It has also been found to have a remarkable
pharmacological effect on some cancers. The phase I and II clinical studies
with recombinant human IL-21 (rhIL-21) have obtained approval from the U.S.
Food and Drug Administration (FDA), mainly focusing on metastatic melanoma and
renal cell carcinoma.
IL-21 is only expressed in activated CD4+ T cells through TCR recognition or
the calcium ionophore signal pathway. It cannot be detected in resting CD4+
cells, CD8+ T cells, CD19+ B cells, CD14+ monocytes and CD56+ NK cells. According
to all available genomic databases, the IL-21 and IL-2 gene loci are on the
same chromosome, approximately the same distance (100kb) apart and with no other
genes located between them in all species. This shows a remarkable degree of
conservation in chromosomal arrangement and begs the questions as to the function
of the genomic arrangement. Given that IL-21 is only expressed in activated
CD4+ T cells with delayed kinetics compared to IL-2, a hypothesis can be made
that IL-21 expression is based on IL-2 expression and the consequent opening
of the compact chromosomal structure between the IL-21 and IL-2 loci. The confirmation
of the hypothesis will build on the study of kinetics of IL-21 and IL-2 gene
expression, the order of the chromatin remodelling events on both genes, and
the investigation of the long-distance control of both genes.
7. A role for c-Rel in regulatory T cell development
(Lina Ma).
During thymopoiesis, a unique program of gene expression promotes the
development of CD4 regulatory T (Treg) cells. Treg cells suppress lymphocyte
effector activity and play an important role in protecting the body from autoimmune
diseases. Whilst the forkhead transcription factor Foxp3, is essential for maintaining
the gene expression necessary for Treg cell function, other transcription factors
are emerging as important determinants of Treg cell development. We are currently
using mice that lack the NF-?B family member c-Rel, to look at the role of c-rel
in Treg development and function.
