Division of Molecular Bioscience
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The Division of Molecular Bioscience has
a broad focus with the general aim of understanding the molecular mechanisms
governing the function of cells and applying this information to the
aberrant molecular events that lead to disease. Areas of research within
the Division relate to many diseases such as diabetes, asthma, allergy,
anaesthetic complications, cancer and viral diseases. Once again the Division was highly successful in obtaining project grants from the National Health & Medical Research Council (NH&MRC), the Australian Research Council (ARC) and other sources. The NHMRC program grant on the Molecular Mechanisms in the Regulation of Allergy and Inflammation commenced this year supporting a continuation of multidisciplinary basic research into the pathogenesis of allergic diseases of the lung and gut. A common link has been identified between the induction of gastrointestinal hypersensitivity and bronchial hyperresponsiveness in asthma with the identification of the cytokine IL-13 as a key therapeutic target. In addition, a novel cytokine binding interface has been identified in the human beta common receptor, an important receptor in eosinophilic inflammation. Professor Young was part of a team that obtained ARC-LIEF funding for mass spectrometry equipment to be located at ANU. Several younger members of our Division were also successful in obtaining funds for their research with Natalia Gousseva awarded an International Union against Cancer Fellowship to travel to France to pursue part of her PhD studies. A large number of PhD students successfully completed their studies in 2003 highlighting the input of the Division in postgraduate education in the University. |
In the last few months of 2003 many of us have relocated our labs and offices to accommodate the workshop in the ground floor. These moves are in preparation for the commencement of Phase 1 of a new building for JCSMR which we hope will add a new dimension to the School. These relocations took place with few dramas thanks to the cooperation of all concerned, especially the workshop guys who put up with all of our indecisions and odd requests.
Dr F Shannon, Head, Division of Molecular Bioscience
Cytokine Gene
Expression Laboratory
Leader: Dr F Shannon
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We now know the sequence of the human genome and of the genomes of many other organisms. This new knowledge is changing the way we approach biological and medical problems. The genes contained within the genome are a code for the proteins that form the working components of the cells that make up the human body. Some genes continuously produce proteins; these proteins are usually required for the day to day workings of all cells. Other genes only produce protein in specific cell types; for example the proteins that are needed for brain function are only produced in cells of the brain. The establishment of the cell-specific pattern of gene expression occurs as the organism develops and inappropriate control leads to developmental defects. An organism can also respond to its environment and it does so very rapidly by altering the pattern of proteins produced from the genome. In humans one of the most highly responsive systems in the body is the immune system which is triggered into action as soon as the body is invaded by a foreign organism. The cells of the immune system respond by rapidly switching on the production of an array of proteins that are required to fight the invasion. Each gene contains a highly tuned molecular switch that allows the production of protein to be switched on and off rapidly in response to the appropriate signals. We are studying the function of these molecular switches in immune cells. The molecular switches of many of the cytokine genes, that are induced in an immune response, are very well defined but there are still a number of important questions that need to be answered if we are to fully understand the activation of genes in an immune response or eventually control the function of these molecular switches. Firstly, we need to know the entire protein composition of these molecular switches. We are applying a functional proteomics approach by purifying the complexes and then identifying the components using mass spectrometry. We are mapping the binding sites of these proteins on cytokine genes in vivo using chromatin immunoprecipitation. We are also examining how the modification of histone proteins, brought about by coactivator complexes, affects chromatin structure across cytokine gene control regions. Secondly, we need to know how endogenous gene expression takes place in the context of packaged DNA in the nucleus. We have developed assays that allow us to measure changes in this packaging (chromatin remodelling) on cytokine genes in primary T cells. We are using these assays to determine the role of specific transcription factors in chromatin remodelling. We are also carrying out structure function analysis of one of the key molecules (c-Rel) involved in cytokine gene transcription in T cells in order to elucidate its mode of action. Thirdly, we are investigating how the signals impinging on the T cell are relayed through intermediate signalling molecules to the molecular switches on the DNA. Finally, we are using a functional genomics approach to determine the overall patterns of gene expression in T cells following activation. This involves expression profiling using Affymetrix microarrays followed by computational analysis of gene control regions to identify common patterns of activity within the molecular switches. |
A functional proteomics analysis of transcription complexes on the GM-CSF gene promoter (Dr Torsten Juelich, Ms Xinxin Chen in collaboration with Dr Adele Holloway)
According to our current model of transcriptional regulation, so-called multi-subunit co-regulator complexes have evolved in the cell to act as an interface between gene specific factors and the general transcriptional apparatus in order to direct transcriptional initiation by the RNA polymerase II machinery. These complexes can either interact directly with the transcriptional machinery (e.g. mediators), or be directed towards chromatin in the vicinity of responsive genes to either modify histones (e.g. histone acetyltransferase complexes) or to remodel the structure of nucleosomes (e.g. ATP-dependent remodelling complexes). Whilst an ever-growing number of co-regulators are being identified, little is known about the nature of such complexes and their role for correct temporal and spatial activation of the cytokine genes such as the GM-CSF gene.
The aim of this project is to understand firstly, what co-activator complexes interact with transcription factors known to be important for GM-CSF gene activation, and secondly, in what order those complexes are being recruited to the gene in the nucleus.
To analyse coactivator complexes involved in this process, a functional proteomics approach is being utilized. Different regions of the human GM-CSF promoter are produced by PCR using biotinylated 5' primers and immobilized to streptavidin-conjugated sepharose beads. Bead/oligo conjugates are then incubated with nuclear extracts of either resting or activated Jurkat T cells and samples subjected to 1D or 2D gel electrophoresis, followed by identification of regulator proteins by mass spectrometry. The in vivo binding of proteins identified in these in vitro approaches is being verified using chromatin immunoprecipitation in T cells.
During the course of this work, we have found that certain components of the co-activator complex known as Mediator are in fact up-regulated in response to T cell activation. The Mediator complex is a multi-protein complex that interacts with the basal transcriptional machinery including the polymerase II enzyme and with numerous transcription factors. Up-regulation of such a complex would presumably lead to increased transcriptional efficiency in activated T cells. Further analyses are being carried out to determine the mechanism of up-regulation of these complexes in T cells.
Certain coactivator proteins serve to add modifications to the N-terminal tails of histone proteins. The modification of N-terminal tails of histone proteins is thought to play an important role in chromatin structure. Histone tails can be acetylated, methylated and phosphorylated and the precise nature of the modifications is thought to control nucleosome:nucleosome interactions and thus chromatin accessibility. We are examining the state of histone protein modification across promoter and enhancer regions of the GM-CSF and IL-2 genes in both resting T cells and stimulated T cells to ascertain the role of histone modification in T cell gene transcription. Chromatin immunoprecipitation is being applied to this question generating some novel findings.
The role of transcription factors in chromatin remodelling across cytokine gene promoter (Ms Karen Bunting in collaboration with Dr Steve Gerondakis and Dr Tom Parks)
The structure of chromatin and its remodeling following activation are important aspects of the control of inducible gene transcription. The interleukin-2 (IL-2) gene is an important cytokine that drives the proliferation of T-cells, B-cells and natural killer cells. The expression of this cytokine is induced in a cell specific manner in T-cells following an antigenic stimulus. We have previously shown that one of the critical events leading to increased IL-2 transcription is an alteration in chromatin structure across the 300bp region of the IL-2 gene. Recently, we have shown that IL-2 transcription in CD4+ primary T cells is dependent on c-Rel but not RelA. We found that c-Rel is essential for global changes in chromatin structure across the 300bp IL-2 promoter in response to CD3/CD28 in primary CD4+ T cells but not in response to the pharmacological signals, paralleling the requirement for c-Rel in IL-2 mRNA accumulation. Interestingly, measurement of activation-induced localized changes in chromatin accessibility revealed that accessibility close to the c-Rel binding site is specifically dependent on c-Rel. These results suggest a non-redundant role for c-Rel in generating a correctly remodeled chromatin state across the IL-2 promoter and imply that the strength of the signal determines the requirement for c-Rel.
Precisely how c-Rel carries out these important functions during T cell activation is not well understood. To dissect the role of c-Rel in cytokine gene transcription and chromatin remodelling we are undertaking a structure/function analysis of the c-Rel protein. So far, we have identified a number of mutations in the human c-Rel protein which confer dominant negative activity in a cell line reporter assay. We are, firstly, using these mutants to examine what effects they have on IL-2 and GM-CSF gene transcription and chromatin remodelling in activated T cells. Secondly, we will use a biochemical approach to determine how these single residue substitutions have altered the capacity of these mutant proteins to fold correctly, to form dimers with wild-type c-Rel and other members of the NF-kB family, to bind DNA and/or to interact with transcription coactivator proteins. Using these molecular approaches, we hope to elucidate the biochemical properties of c-Rel and its role in IL-2 and GM-CSF gene transcription.
Coordinated kinase signalling pathways involved in interleukin-2 gene regulation (Ms Anna Moore, Ms Donna Woltring in collaboration with Dr Sudha Rao)
Signals derived from the T cell receptor and costimulatory receptors are
mediated by two major kinase signalling pathways; the PI3-Kinase/Akt pathway
and the Protein Kinase C (PKC) pathway predominantly through the PKC theta
(PKC) isoform. Activation of these signalling pathways has been linked to
the downstream activation of the NFkB/Rel
family of transcription factors. Recent evidence pinpoints the NF-B family
member, c-Rel, as critical for IL-2 gene transcription following T cell
activation. We hypothesise a direct signalling link from PKC to c-Rel to
control cytokine gene transcription.
Using a specific PKC inhibitor, Rottlerin, we have shown that both c-Rel
mRNA synthesis as well as c-Rel translocation to the nucleus require PKC
in primary CD4+ T cells. In contrast, c-Rel mRNA synthesis is not dependent
on PKC in a mouse T cell line, EL-4, implying a signalling defect in these
cells. Inhibition of PKC also leads to reduced chromatin remodelling of
the IL-2 proximal promoter after stimulation in both the EL-4 T cell line
and primary CD4+ T cells implying that chromatin remodelling is at least
partially dependent on PKC signalling. Interestingly, in CD4+ T cells the
PKC message is down-regulated following stimulation and this effect is enhanced
by Rottlerin treatment. This data supports the essential role of PKC and
c-Rel in IL-2 gene activation and suggests PKC signals upstream of c-Rel
leading to IL-2 gene activation. Mice where the PKC theta gene has been
deleted have been used to further confirm the link between PKC theta and
c-Rel.
On the other hand, the PI3-kinase/Akt pathway does not appear to play a role in chromatin remodelling but is critical for the overall rate of transcription.
Deciphering the role of c-Rel in T cells by a genome-wide analysis approach (Dr Nancy Wilkinson, Dr Joanne Attema, Ms Donna Woltring in collaboration with Dr Sudha Rao, Dr Steve Gerondakis, Dr Gareth Denyer and Dr Charmaine Simeonovic)
Until very recently, we have only been able to study the production of protein from one gene at a time. As a result of the human genome project, technology has been developed that allows us to study tens of thousands of genes at one time. This technology is called microarray technology and its use to study the expression of genes is referred to as gene expression profiling. The use of this technology allows us to visualize the response of the cell as a whole and not just the response of individual genes. The advantages of such a view are that we can now visualize regulatory networks within the cell and compare the function of these networks in disease states. Thus, we can identify pathways within the cell that are malfunctioning in disease opening up the possibility of targeting these pathways for disease treatment.
We are using microarray technology and gene expression profiling to gain insights into the function of T cells when they are activated by an immune signal. With the aid of computational analyses, we have discovered groups of genes that are co-expressed and have identified common molecular switches.
By using animal models where key control proteins are deleted, we can visualize the global impact of these key proteins on cell function. Interestingly, we have found that c-Rel appears to function as both an activator and repressor of transcription in T-cells. We have identified groups of genes that are coregulated by c-Rel and further analyses of the promoters of these genes have shown that a combination of c-Rel and members of the Ets transcription factor family may form a common control module in T cells.
We have also analysed expression profiles from T cells lacking a closely related protein, RelA and can identify genes that are dependent on one or other of these proteins as well as genes where either protein will suffice to drive transcription. It has recently been shown that c-Rel is required for immune rejection in animal models of cardiac and pancreatic islet transplants. We are using the data obtained above as well as expression profiling on transplanted tissue to decipher the molecular role of c-Rel in transplant rejection.
Cytokine
Molecular Biology and Signalling Group
Leader: Professor I Young
Interleukin-5 (IL-5) is a hormone-like protein which plays
a key role in the regulation of immune responses to helminth parasites
and to allergens. This cytokine regulates inducible white blood cell
formation in response to infection by helminth parasites or to allergen
exposure and is principally responsible for controlling the eosinophilic
inflammation characteristic of
asthma and allergy. This inflammatory response is primarily orchestrated
by the inducible production of IL-5 by T lymphocytes which results in
enhanced production of eosinophils in the bone marrow. IL-5 then cooperates
with other cytokines like eotaxin which promote the migration of the eosinophils
to the affected site. Our group has had a long term interest in defining
the biological role, regulation and signalling mechanism for IL-5. It
is expected that these fundamental studies will provide opportunities
for the development of novel therapeutic approaches for the treatment
of allergic disease and also information relevant to understanding some
leukemias.
Collaborative studies with the Allergy and Inflammation Research Group
and the Gene Targeting Laboratory on the role of IL-5 in allergic lung
disease have continued. The present emphasis is to define the roles played
by IL-5 and eotaxin in regulating eosinophilic inflammation and airways
hyperresponsiveness using mice deficient in one or both cytokines. The
results indicate that eosinophils play a key role in the orchestration
of allergic inflammation and airways dysfunction. These and other studies
carried out by the Cytokines and Inflammation Group, have formed the basis
of our NH&MRC Program Grant on the Molecular Mechanisms in the Regulation
of Allergy and Inflammation which commenced in 2003.
The receptor system for IL-5 is shared with two related cytokines, IL-3
and GM-CSF, which are also involved in the regulation of blood cell formation
and inflammation. One of the major problems in cell signalling is to understand
how these cytokines regulate blood cell growth and function by binding
and activating their cell-surface receptors. In a collaborative project
with Paul Carr and David Ollis (Research School of Chemistry) using X-ray
crystallography we have determined the structure of the complete extracellular
domain of the beta common receptor which is the major signalling entity
of the IL-5 receptor and is central to the signalling of all three cytokines.
The receptor was expressed in insect cells and its crystallization and
derivatization involved extensive use of site-directed mutagenesis to
improve crystal quality and to solve the phase problem. The novel dimer
configuration of the receptor gives new insights into receptor activation.
James Murphy, Tanya Soboleva and Jinglong Chen used site-directed mutagenesis
to define the residues of the beta common and beta IL-3 receptor which
are involved in forming the activated receptor complex and to investigate
the role of N-glycosylation in receptor function. Madga Sakowska, Mark
Young, Sally Ford, Janine Inggs and Jane Olsen have prepared the activated
complex of the closely related beta-IL-3 receptor and carried out crystallization
trials. Further structural studies should give a better understanding
of the process of receptor activation and provide opportunities to develop
drugs capable of controlling this important receptor system. Such drugs
could be useful in treating asthma, allergy or cancer.
The group has also participated in another project in the area of functional genomics with Hugh Campbell (Research School of Biological Sciences) and the Gene Targeting Laboratory. In this work, the functions of the mammalian homologues of two interesting genes, flightless and small optic lobes, with functions in development and behaviour in the fruit fly Drosophila are being investigated by gene targeting in mice. Investigations to date have shown a function for mammalian flightless in early embryonic development, remarkably analogous to its role in Drosophila. This cross-organism approach uses information gained from studies with the simpler fruit fly to better understand development and brain function in mammals.
Gene Targeting
Leader: Dr K Matthaei
Creating mice with a pre-determined genetic makeup
A major aim of modern biology is to understand how normal gene activities
give rise to the structure and behaviour of complex organisms. In particular
it is important to study the function of genes and their derangement’s involved
in human diseases. In most cases however, it is impossible to achieve these
studies directly in the human. It is easier therefore, to carry out such
studies in a more manipulable system such as the mouse. However, natural
mutations occur in a serendipitous manner, i.e. by chance. To find
a mutation that mimics a particular human disease is therefore difficult.
However, given a knowledge of the nucleotide sequence of a gene, it is now
possible to make changes to the corresponding endogenous gene of an embryonal
stem cell and to produce a mouse that is homozygous for the desired mutation.
This procedure is called gene targeting.
Gene targeting involves firstly the use of recombinant DNA technology to
modify a cloned gene (usually to stop the function of the gene). At the
same time a cultured cell line of embryonic stem (ES) cells is generated
by culturing cells from an early mouse embryo (a blastocyst). The ES cells
are totipotent and can be used to regenerate live normal animals (i.e.
it is possible to select a single ES cell and produce a whole mouse from
that cell, see below). Whilst in tissue culture the modified gene is introduced
into the ES cells and the normal gene is replaced by the mutated (functionally
inactive) gene. The modified ES cells are then micro-injected into another
mouse embryo and the ES cells become integrated. These blastocysts are
re-implanted into pseudo-pregnant mice and give rise to live chimæric offspring
that consist of the modified injected cells as well as the normal cells.
Since the injected cells can also contribute to the testis of these mice,
the breeding of a chimæra with a normal mouse gives rise to an animal carrying
the genes of the modified stem cell (including the mutated gene). Interbreeding
of the heterozygous (F1) siblings finally yields transgenic animals homozygous
for the desired mutation (usually a deletion or a "gene knockout"
mouse). In this way co-isogenic animals can be generated, i.e. animals
which are identical to the original mouse strain except that the function
of a single gene has been deleted thereby allowing the study of the loss
of this gene in vivo.
Gene targeting therefore allows the ability to study the function of a cloned gene in the context of the whole mammal by creating mutant mice defective in specific genes. This is particularly important since, with gene targeting, mouse models can be created for studying human genetic diseases and also provides a powerful approach to the development of somatic gene therapy. Moreover, it is possible to also add genes using similar processes resulting in “transgenic” mice.
Our laboratory has generated a number of different "knockout" and “transgenic” mouse mutants using C57BL/6 or BALB/c ES cells that are at different stages of investigation. These include mouse models of asthma, nerve regeneration, xenograft rejection, parasite-host relationships, hypertension, drug detoxification and cancer.
Biomolecular
Structure Laboratory
Leader: Dr MG Casarotto
One of the fascinating properties of biological molecules is their remarkable
ability to trigger a range of biological responses by adopting distinct
three-dimensional structures. The range of structural diversity forms the
basis of a wide range of scientific disciplines ranging from molecular recognition
and drug design through to protein folding and design. The Biomolecular
Structure Laboratory at the JCSMR seeks to carry out research that explores
how the structural properties of biological molecules can impact on biological
processes involved in nature and biotechnology. This laboratory is dedicated
to investigating how bioactivity is governed by molecular shape and recognition.
Although the main focus of our research is from a structural perspective,
an integrated approach involving complementary techniques such as molecular
biology, kinetics and molecular modeling are routinely employed in the laboratory.
Research projects currently under investigation relate to a wide range of
diseases and applications including cancer, malaria, heart
disease, muscular dystrophy and virus related illnesses such
as AIDS and Ross River fever.
A number of projects are currently the focus of our research efforts; these include (1) the structure, specificity and mechanism of enzyme systems and includes dihydrofolate reductase, glutathione-S-transferases and chitinases (chitin degrading enzymes) (2) structural and functional studies of muscle related proteins (3) the role of ion channels in virus associated proteins.
(1) The mechanism by which enzyme systems function is central to the development of effective therapies associated with these systems. The enzyme dihydrofolate reductase is the target for an extensive range of diseases such as cancer, malaria, and bacterial infections and we have used NMR structural data, molecular biology and enzymology to determine how this enzyme functions. Enzymes, such as glutathione S-transferases (GSTs), are involved in the metabolism of chemical toxins and mutagens as well as of therapeutic agents. A detailed understanding of their specificity and mechanism is crucial if one is to be able to predict the metabolism of foreign compounds. Efforts are underway to structurally characterise the different classes of GSTs and investigate the way that these enzymes recognize a wide range of compounds.
Chitinases are sugar degrading enzymes that specifically target chitin. Both chitin and chitinases are widespread in nature, occurring in a range of organisms and are consequently, of major biotechnological interest. We are actively involved in the structural study of a chitinase with the view of investigating its binding and enzyme properties. This project is supported by the Hellenic Republic Ministry for Development.
(1) For skeletal and heart muscle to function properly careful regulation of calcium levels must occur. In muscle two proteins, the dihydropyridine and ryanodine receptors play key roles in triggering the release of calcium. We are using high resolution NMR spectroscopy to investigate how these proteins function by firstly determining the structure of various regions of these proteins and then using this structural information to determine how they interact. As a result of this work we have designed a series of peptides and peptido-mimetic analogues which have the ability to regulate calcium levels in both skeletal and cardiac muscle. Such peptide therapies may hold the key to designing new drugs which may be beneficial in the treatment of a range of muscle related diseases such as heart failure, malignant hypothermia and muscular dystrophy. This project is supported by the National Heart Foundation, the NH&MRC and the ARC.
(2) Many membrane proteins are essential components for the survival of viruses and we are targeting several proteins which form ion channels. The aim is to design "blockers" of the ion channels based on a structural knowledge of these ion channels. In one case, we have identified a drug which slows the replication of the AIDs virus. Work is currently underway to chemically and structurally optimise the effectiveness of this drug. This approach will give rise to a new generation of drugs to treat diseases such as HIV AIDS, hepatitis C and Ross River Fever. This project is supported by the ARC and Biotron Ltd.
Facilities
The laboratory is well equipped with access to state-of-the-art facilities
including two high field Nuclear Magnetic Resonance spectrometers (Varian,
Inova 500 & 600) and a network of Silicon Graphics workstations running
the latest structure-related software. A new 800 MHz NMR spectrometer will
also be operational in 2004. Other facilities include stopped-flow instrumentation
capable of performing CD, fluorescence and UV kinetic analysis which is housed
in a well appointed molecular biology laboratory.
Allergy
and Inflammation Research Group
Leader: Dr P Foster
The aim of this research is to understand the cellular and
molecular networks that underlie the development of disease processes associated
with asthma and allergic disorders. We also focus on understanding how respiratory
viral and bacterial infections regulate the immune system and their role
in protecting or predisposing to allergic disorders and chronic respiratory
infections.
The worldwide incidence, morbidity, and mortality of allergic asthma and
allergies are increasing at a dramatic rate. Deaths from asthma have now
reached over 180,000 worldwide annually. In the USA alone 15 million people
are thought to suffer from asthma and this disorder is now the most common
cause of childhood absence from school. Allergic disease of the gastrointestinal
tract in response to food allergens is also of growing concern in developed
countries. One of the major precipitating factors of asthma, particularly
in children, is viral infections of the respiratory tract.
A predominant clinical feature of allergic disease is a persistent inflammation
at the site of disease. In asthma, inflammation is localized to the airway
wall. Currently, it is thought that inflammatory cells (white blood cells)
induce asthma by releasing substances that damage the lining of the airways
and induce constriction (narrowing of the airways), mucus production and
remodeling of tissue. The inflammatory response in the asthmatic lung and
in other allergic diseases is a very complex mixture of cells and molecules
and it is not clear which factors play the major role in inducing disease.
Allergic asthma is recognized as a chronic inflammatory disease of the airways
that is characterized by reversible airways obstruction in association with
aberrant CD4+ T helper 2 (Th2 cell) lymphocyte responses to common
environmental stimuli. Indeed, the hallmark features of allergic asthma,
elevated serum immunoglobulin E (IgE), mucus hypersecretion, eosinophilia
and enhanced bronchial reactivity (airways hyperreactivity [AHR]) to non-specific
spasmogenic stimuli have all been linked to the effector functions of Th2
cytokines (e.g. interleukin- (IL)-4, 5, 9, 10 and 13). Collectively, it
is these pathogenic processes that are thought to promote airways obstruction
in asthma, which predisposes to wheezing, shortness of breath and
life-threatening limitations in airflow. Th2 mediated immune processes have
also linked to the pathogenesis of allergic diseases of the gastrointestinal
tract. Viral infections of the lung play a key role in exacerbating asthma
and eosinophils and T -lymphocytes are also thought to underpin aspects
of viral induced asthmatic episodes.
Research in our Group focuses on two major areas:
1. Identifying the key cells and molecules which underpin the pathogenesis
of allergic disease of the lung, skin and gastrointestinal tracts and those
that underpin the pathogenesis of respiratory viral infections.
2. Developing strategies that will direct the immune response away from
the harmful Th2 inflammatory response to that which is protective or non-responsive.
The long-term goal of this research is to identify novel therapeutic approaches
for the treatment of asthma and allergy and viral-induced disease of the
lung.
Our experimental approach is integrative, employing state-of-the-art molecular
genetic techniques in association with model systems to identify the role
of inflammatory cells and molecules in the events that underpin disease.
Projects within the group focus on the role of individual Th2 cytokines
in disease pathogenesis and characterization of the downstream signaling
pathways employed by these molecules to induce disease.
Ubiquitin
Laboratory
Leader: Dr R Baker
The Ubiquitin Laboratory investigates enzymes that control the small protein ubiquitin, which becomes attached to other proteins, and can either signal their rapid destruction (proteolysis; if multiple ubiquitins are attached), or control their trafficking around the cell (single ubiquitin). We are interested in the consequences of abnormal proteolysis or trafficking due to defects in the ubiquitin system.
Most of our research focuses on a family of enzymes called ubiquitin-specific proteases (USPs), that have the ability to cleave ubiquitin from such marked proteins, and thus slow or prevent their destruction, and/or control their location in the cell. In this context, some USPs can be considered "proof-reading" components of the system, safeguarding a target from inappropriate ubiquitination, and imposing a further level of regulation on the pathway. Other USPs function to recycle ubiquitin after protein degradation at the proteasome, the large complex that degrades ubiquitinated proteins.
We use mice as a model organism to study human disease. One USP (USP4) can cause cancer when overproduced, and it interacts with the Retinoblastoma tumour-suppressor protein and other related tumour suppressors. We have found that this USP shuttles in and out of the nucleus in normal cells, but in cancer cells it becomes trapped in the nucleus. Interfering with tumour suppressor protein levels can also change its location in the cell. Our current efforts are aimed at determining how the nuclear localisation of this USP is linked to cancer, and how it traffics in and out of the nucleus.
We have also shown that another USP, USP2, interacts with components of the Ras signalling pathway, a pathway involved in regulating cell growth and often mutated in cancer. USP2 may regulate this signalling pathway by controlling the ubiquitination and localisation of signalling molecules and receptors.
We also study USP enzymes using yeast as a model system, both because it is very amenable to genetic and biochemical studies, and also because the ubiquitin system is so highly conserved between yeast and humans that insights we gain in yeast can be applied to our studies on mouse and human enzymes. We have found that Ubp6, one of a family of 16 USPs in yeast, is a previously unknown integral part of the proteasome, and its major job appears to be in removing ubiquitin chains from proteins as they are destroyed, thus allowing proteasome clearing and ubiquitin recycling. Interestingly, recent work from another lab has shown that defects in the mouse version of this enzyme, Usp14, result in neurodegenerative disease, suggesting that a lack of free ubiquitin is at fault. We will obtain these mutant mice to test this hypothesis.
Our results show that USPs can play important roles in regulating the ubiquitin pathway, and through this, processes such as cell growth and cell development. Abnormal USP function may lead to cancer and disease.
Chromatin and Transcriptional
Regulation Laboratory
Leader: Dr D Tremethick
Chromatin and transcriptional regulation during development
The fate of the eukaryotic cell at all stages of its life cycle is dependent upon the accurate readout of genes encoded by DNA. For example, the development of a single cell into multicellular organism requires precise temporal and spatial regulation of gene transcription. Consequently, certain diseases and developmental disorders are often associated with, and likely to be caused by, aberrant gene expression. It has become increasingly clear over the last decade that eukaryotic gene regulation at the level of transcription is strictly connected to the structural organization of the genome. The basis for this structural organization is the nucleosome. Our overall aim is to understand how chromatin structure contributes to the regulation of transcription during development.
Remarkably, a typical eukaryotic cell contains approximately two meters of DNA, which can be squeezed into a nucleus of about 20 µm in diameter. This packaging of DNA is achieved by a hierarchical scheme of folding and compaction into a protein-DNA ensemble called chromatin. At the first level of organization, approximately two superhelical turns of DNA are wrapped around a protein complex consisting of eight histone molecules. This complete unit, the nucleosome, forms the basic building block of chromatin and is further reorganised into a regular array to form a chromatin fibre. How this fibre subsequently folds into higher order structures is not yet understood. This protein-induced folding of DNA into a complex three-dimensional structure has profound implications with respect to understanding how gene expression is regulated.
As a result of this compaction of the eukaryotic genome, the conformation and accessibility of DNA is dramatically altered. The compaction of a gene into chromatin clearly impedes the transcription process. The cell has therefore devised mechanisms, which reversibly de-compact or remodel chromatin to allow transcription factor binding by altering the stability of protein-DNA interactions in underlying nucleosomes. The multi-subunit structure of the nucleosome (consisting of four histone-dimer subunits) is ideally suited for performing these opposing roles; nucleosomes are both stable enough to compact DNA while at the same time labile enough to allow access of DNA to transcription factors. This lability can be enhanced by targeted modification of the histone proteins or by changing the biochemical composition of the nucleosome. The hypothesis that we are examining is that the stability of protein-DNA interactions in underlying nucleosomes can be altered by the replacement of major histone types with specific histone variants.
There are two important stages with regard to the accurate transcriptional regulation of a gene. (1) The establishment of gene activity, which for most genes occurs during early mammalian development, and (2) the subsequent maintenance of this gene activity throughout many rounds of cell division during the life of the organism. Major global transitions in chromosome and chromatin structure occur early in development when cell lineage and tissue-specific transcriptional patterns of gene expression are established. Very little is known about these structural changes and the mechanisms by which these changes differentially regulate gene transcription. However, it is clear that chromatin plays a fundamental causal role in determining patterns of gene activity.
An important way to control chromatin function is to alter the biochemical make-up of the nucleosome by replacing an individual histone with a histone variant. Interestingly, most histone variants belong to the histone H2A family implying that H2A plays a unique role in the nucleosome. Modulating nucleosomal and higher-order chromatin structure through variation in H2A will have an impact on all nuclear functions.
Our work focuses on a variant of histone H2A referred to
as H2A.Z. The presence and high level of conservation from yeast to man
(across species, the amino acid sequence of H2A.Z is more conserved than
the amino acid sequence of major H2A) shows that H2A.Z plays an important
and specific role in chromosome function. This function is essential since
in Drosophila and Tetrahymena null mutants die. We also found that the
H2A.Z gene is essential for mouse survival with the defect occurring early
in development around the time of implantation. However, despite being
essential, nothing is known about the specific functional and structural
consequences of having H2A.Z incorporated into chromatin.
To begin to understand why H2A.Z is essential for survival, we adopted
an in vivo approach. To search for the unique feature(s) of H2A.Z required
for its function, we performed amino acid swap experiments in which residues
unique to Drosophila H2A.Z were replaced with equivalently positioned
histone H2A residues. Mutated H2A.Z genes encoding modified versions of
this histone were transformed into Drosophila and tested for their ability
to rescue null mutant lethality. Most interestingly, we discovered that
the unique and essential feature of H2A.Z lies outside the histone fold
in the carboxy-terminal domain. This C-terminal region maps to a short
alpha-helix in H2A that is partly buried deep inside the nucleosome and
partly exposed on the surface of the nucleosome. A region immediately
adjacent to this short alpha-helix, located at the surface of the nucleosome,
was also found to be important for adult Drosophila survival. Together,
this region forms part of a docking domain, a domain involved in stabilising
the interaction between the H2A/H2B dimer with the H3/H4 tetramer and
in contributing to surface features of the nucleosome.
Recently, we solved the crystal structure of a nucleosome
containing H2A.Z (in collaboration with Karolin Luger from Colorado State
University). The overall structure is similar to the previously reported
structure containing major H2A. In part, consistent with our prediction,
distinct localised changes in the docking domain result in a subtle destabilisation
between the dimer and the tetramer. However, there is also a stabilisation
between the two H2A.Z molecules at the back of the nucleosome. Interestingly,
salt dissociation experiments and a FRET analysis revealed that H2A.Z
actually increases the overall stability of the nucleosome. Potentially
even more significant, the amino changes in the docking domain of H2A.Z
results in an altered nucleosomal surface that includes a metal ion, a
more extensive acidic patch, and a larger hole in the centre of the nucleosome.
Our current favoured hypothesis is that these surface changes may create
a highly specific interaction interface for other nuclear proteins like
chromatin remodelling factors and/or modulate nucleosome-nucleosome interactions.
Most recently we tested the prediction that incorporation of H2A.Z into
chromatin alters intra- and/or inter- nucleosomal interactions. This was
determined by examining whether this histone variant alters the folding
pathway from an extended nucleosomal array to highly compacted heterochromatin.
In summary, we carried out the first biochemical analysis of a homogenous
preparation of H2A.Z-containing nucleosomal arrays. H2A.Z uniquely affects
chromatin condensation; intra-nucleosomal interactions are accentuated
while fibre-fibre interactions and the subsequent formation of condensed
heterochromatin is inhibited. These data suggest that a major function
of H2A.Z is to generate a conformational intermediate in the chromatin-folding
pathway poised to be assembled into a specialised functional chromosomal
domain.
The prediction, from these structural studies, that H2A.Z
functions to establish a specialised higher-order chromatin domain was
recently tested. Using early mouse embryos at a time when H2AZ null mice
die, confocal immunofluorescence experiments demonstrated that H2A.Z was
located at constitutive heterochromatin that surrounds the centromere.
To investigate the role of H2AZ at this specialised domain. We developed
a new inducible RNAi strategy. We show that in the absence of H2A.Z, the
genome becomes highly unstable and that this instability is the result
of defects in the chromosome segregation process. A principal cause of
these defects is a disruption to the structure of constitutive heterochromatin.
We conclude that H2A.Z is essential for the accurate transmission of chromosomes
by ensuring proper centromere function.
Most interestingly, it appears that H2A.Z may have more than one role by also having a specific function during early vertebrate development. Endogenous H2A.Z function was disrupted during Xenopus laevis development using double-stranded H2A.Z RNA interference and a dominant-negative H2A.Z mutant protein. Both of these strategies resulted in abnormal development following gastrulation. Notably, changing the stabilizing histidine residues located on the surface of a H2A.Z nucleosome specifically interfered with gastrulation movements and impaired notochord formation. Consistent with this finding, endogenous H2A.Z expression is enriched in the notochord. Taken together, these results argue that key determinants located on the surface of an H2A.Z nucleosome also play an important specific role during embryonic patterning.