Gene Expression and Epigenomics Group
Current Research Projects:
1. Mechanism of action of the NF-kB
transcription factor, c-Rel.
2. Gene regulatory networks in T lymphocyte development and activation.
3. The role of the transcriptional repressor, ZEB, in T cell gene
transcription and beyond.
4. The epigenomic marks that tag inducible genes in T cells.
5. A possible epigenetic relationship between IL-2 and IL-21.
6. A role for c-Rel in regulatory T cell development.
7. Network and Transcription factor analysis of disease-associated
gene groups.
8. Regulation of interferon-responsive genes in T cells by c-Rel.
1. Mechanism of action of the NF-kB
transcription factor, c-Rel (Karen Bunting).
It is well established that the NF-kB family of transcription
factors plays a major role in the control of inducible gene expression. c-Rel,
a member of the NF-kB family, appears to play a specific
role in the activated transcription and chromatin remodelling of genes involved
in T lymphocyte immune responses and in T cell survival and growth. c-Rel is
a proto-oncogene and has been associated with specific cancers. It is also implicated
in immune-related diseases and transplant rejection. Despite this, the molecular
mechanisms by which c-Rel controls these events at individual gene loci and
in the coordinate regulation of multiple genes during T cell activation remain
poorly defined. We have investigated the role of c-Rel in T cell gene transcription
using both gain- and loss-of-function experiments in a T cell line and in CD4+
T cells. Using a combination of reporter assays, expression profiling analysis
(microarrays) and measurement of endogenous gene transcription to investigate
the effects of c-Rel over-expression, we have shown that c-Rel is a limiting
factor for the inducible responses of a subset of c-Rel-regulated genes in T
cells. Moreover, we have used mutational analysis to show that the specific
activity of c-Rel on several of these genes, including GM-CSF, IL-2 and ICAM-1,
is dependent on the presence of conserved residues within the c-Rel Rel Homology
Domain. Computational analysis of the cis-regulatory features of c-Rel-dependent
genes identified in genome-wide analyses of activated c-Rel-/- and c-Rel-over-expressing
T cells has uncovered a transcriptional module which is common to the proximal
promoters of a number of these genes and has been used to predict novel NF-kB/c-Rel
targets. We are currently investigating the effects of over-expression of the
closely-related family member, RelA, to determine whether the c-Rel-responsive
genes identified in this study are selectively controlled by c-Rel. We are also
performing further structure:function studies using functional rescue experiments
in c-Rel-/- T cells to determine structure requirements for activity on a number
of endogenous genes.
2. 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.
3. 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.
4. 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.
Furthermore, there are studies showing that nuclear localization of chromosomes
may influence gene expression. Thus, we are keen to examine co-localization
and nuclear location of these sets of genes in T cells and determine if the
chromatin state, histone occupancy and histone modification correlate with the
nuclear location of these genes. We plan to use (i) fluorescence in situ hybridization
(FISH) together with immunostaining to examine colocalization of genes and also
the location of Pol II in the nucleus, (ii) chromosome conformation capture
(3C) assays to examine frequency of interaction between gene loci, and (iii)
antibodies to heterochromatin protein 1 (HP1), a protein localized to heterochromatin
marks, to examine location of genes relative to heterochromatin marks. Consequently,
we hope to elucidate the general mechanism of transcriptional regulation of
genes expressed by activated T cells in the context of chromatin.
5. 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
b chain, and the common gamma chain (gc),
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.
6. A role for c-Rel in regulatory T cell development
(Stephanie Palmer).
In preparation
7. Network and transcription factor analysis of disease-associated
gene groups (Jaime Pena).
The phenotype that a particular mutation causes is typically related to the
biological process in which the gene is involved and its interactions with other
molecules of the system. The understanding of multigenic diseases is a multifaceted
process and cannot be explained solely by the study of the parts. As more genomes
are sequenced and more literature becomes available, the complexity of the pathways
increase and the understanding of multigenic diseases becomes more challenging;
using bioinformatics it is possible to study a set of genes instead of focusing
on one simple gene.
Rheumatoid arthritis (RA) is the most common systemic autoimmune disease, affecting
1% of the adult population worldwide and has a heritability estimated at 60%.
The aetiology of RA is still incompletely understood and like most complex human
diseases, it appears that at least some of its heterogeneity reflects the action
of different genetic factors, interactions between genes, or between genes and
environment.
Recent publications describe different methods to identify and study sets of
genes based on computational methods. This global viewpoint can help us to obtain
a better understanding of disease mechanisms in a generalized manner. We are
combining different bioinformatics approaches to study a set of Rheumatoid Arthritis
Associated Genes (RAAG): Gene categorization to study gene association patterns,
Network Analysis to identify relevant associations and functional links between
genes involved in RA; and Transcription Factor Analysis to trace gene co-regulation
and look for new genes associated to RA that share the same level of co-regulation.
8. Regulation of interferon-responsive genes in T cells (Anny
Kwok).
