The Hilton laboratory is interested in understanding the molecular regulation of cell proliferation and differentiation, with particular emphasis on the haematopoietic or blood cell forming system. We currently have two major programs, the first focussed on cytokines, their cell surface receptors and signal transduction pathways; the second focussed on uncovering pathways regulating hemopoiesis using a combination of forward and reverse mouse genetics, genomics, proteomics and bioinformatics. In addition to our basic research, the laboratory is committed to developing clinical links and to applying our research through collaboration with the biopharmaceutical industry.

Read below for further information on the Hilton laboratory and see the Prospective Students web page for information about projects available in the Division of Molecular Medicine.

Multi-functional cytokines and haemopoietin receptors
Cytokines are secreted proteins that allow cells to communicate with one another. Cytokines act by binding to multi-subunit receptors expressed on the surface of responsive cells and activating the Janus Kinase/Signal Transduction and Activator of Transcription (JAK/STAT) pathway. While some cytokines, such as G-CSF and TPO affect a single lineage of blood cells; others, like leukaemia inhibitory factor (LIF) have diverse effects. For example, while we purified LIF on the basis of its ability to induce the macrophage differentiation of blood cells, we also found that it could inhibit the differentiation of embryonic stem (ES) cells, leading to its sale as ESGRO and use in laboratories throughout the world. We have also identified a number of cytokine receptors including the interleukin-11 receptor and a shared component of the interleukin-4 and interleukin-13 receptors. In collaboration with Zenyth Therapeutics we produced a panel of antagonistic human antibodies to the human IL-13 receptor, which are currently being developed as a treatment for asthma in conjunction with Merck. Using a similar strategy we are also collaborating with Prof. Ian Wicks to determine whether inhibitors of G-CSF action have a place in the treatment of inflammatory diseases such as arthritis. This project is being commercialised by Zenyth Therapeutics and Murigen Therapeutics.

Limiting cytokine action: Suppressors Of Cytokine Signalling (SOCS) Proteins
Many cytokines are a double-edged sword – having beneficial effects in the body’s fight against infection, yet playing a role in the onset and development of inflammatory disease. This implied to us that the body must have mechanisms by which it keeps the effects of cytokines under strict control. Using an expression cloning approach we discovered a family of SH2 domain containing proteins, which we named the Suppressors Of Cytokine Signalling or SOCS proteins, that have proven to be the major negative regulators of cytokine action. Studies in collaboration with the Division of Cancer and Haematology and many other laboratories have established that SOCS proteins are expressed rapidly following stimulation of the JAK/STAT pathway by cytokines. Once produced, SOCS proteins terminate JAK/STAT signalling by binding to signalling complexes. Inhibition of signal transduction occurs both through interaction of the SH2 domain of SOCS proteins with key phosphotyrosine residues in activated signalling components and the recruitment of ubiquitin ligase machinery via a conserved 40 amino acid motif that we named the SOCS Box. This leads to polyubiquitylation of signalling proteins and their degradation by the proteasome.

In order to understand the physiological role of the SOCS proteins we have generated or obtained null alleles of all eight family members. Six of these have been created in collaboration with Dr Warren Alexander in the Division of Cancer and Haematology, one was obtained from Prof. James Ihle and one was obtained using a TILLING approach. Analysis of these mice has yielded important and surprising insights into the specificity of SOCS proteins. SOCS1 for example plays a crucial role in limiting the production and action of IFNγ, SOCS2 controls body size through regulation of growth hormone action and SOCS3 acts as a feedback inhibitor of the LIF/IL6 group of cytokines and of G-CSF. In contrast, the roles of SOCS4, SOCS5, SOCS6 and SOCS7 have been more difficult to define. Our current studies are focussed on understanding the redundancy in this system, by generating and analysing mice that lack two or more functional SOCS genes.

The SOCS Box; A link between generic and substrate-specific components of E3 ubiquitin ligases

In addition to the canonical SOCS proteins that contain a central SH2 domain, we found several large and poorly characterized protein families that contain a C-terminal SOCS box and a variety of other protein-protein interaction domains including SPRY domains, WD40 repeats and ankyrin repeats. We hypothesize that each of these SOCS box containing proteins targets a specific protein for polyubiquitylation and degradation and are taking a variety of proteomic and genetic approaches to identify these substrates.

Genetic Dissection Of Blood Cell Production
Genetics has been successfully employed in lower organisms including yeast, nematodes, insects and fish to unravel the molecular regulation of complex processes such as development, cell division and cell death. Two factors have combined to make large-scale mouse genetics feasible and accessible to even small laboratories; (1) the demonstration in the early 1980s that ENU is a powerful germ cell mutagen in the mouse and (2) the completion early this century of the mouse genome project.

Unlike many large national mouse mutagenesis centres, which have broad programs examining many different phenotypes, our approach has been to focus almost exclusively on the haematopoietic system. We have also borrowed a number of tricks, (e.g. modifier and sensitised screens) developed by geneticists using lower organisms such as yeast. Rather than beginning with wild type mice and identifying mutants with an aberrant phenotype, modifier screens commence with a mouse with a pre-existing phenotype and aim to identify mutations that make this phenotype more severe (enhancers) or less severe (suppressors). We are one of the first laboratories to employ this strategy in vertebrates and are particularly excited about this approach because, not only may it shed light on the in vivo regulation of complex biological such haematopoiesis, but also because of its potential to pin-point new targets for the treatment of human disease. Just as most ENU-induced mutations cause loss of function, most small molecule therapies also reduce the function of proteins to which they bind. Accordingly, screens for genes that lead to amelioration of disease when mutated should provide genome-wide access to novel in vivo validated targets for pharmaceutical discovery.

In collaboration with the laboratories of Dr Warren Alexander in the Division of Cancer and Haematology and Dr Benjamin Kile in this Division, our focus has been on dissecting platelet formation; a process that includes elements common to other blood cell lineages, including self-renewal of haematopoietic stem cells and lineage commitment, as well as unique events like polyploidization via endoreduplication, pro-platelet formation and platelet shedding. Many of our studies have commenced with a mouse model of human inherited thrombocytopenia, in which the gene encoding the thrombopoietin receptor, Mpl, has been mutated by homologous recombination in ES cells. Mpl-/- mice have approximately 10% of the normal number of platelets and a corresponding decrease in megakaryocytes, megacaryocyte progenitors and stem cells. Despite their stem cell deficit, Mpl-/- mice have normal numbers of other peripheral blood cells, implying the presence of a compensation mechanism within the haematopoietic system. We have used Mpl-/- in an ENU mutagensis screen to identify suppressors of thrombocytopenia and to find enhancers of the stem cell phenotype that manifest as a multi-lineage defect in the peripheral blood. In addition, we have carried out a conventional ENU mutagenesis screen using Mpl+/+ wild type mice and identified pedigrees with thrombocytopenia and thrombocytosis. To date we have identified more than 30 mutant pedigrees with defects in stem cells and/or the platelet lineage. The causative mutation in 12 of these has been identified and in an additional 10 cases genes in the candidate interval are currently being sequenced. These pedigrees are being intensively studied within our laboratory at both the biological and molecular level.

Defining Pathways
Through our genetic screens we have identified suites of mutations that have similar effects – for example, Plt3, Plt4, Plt6, Plt8, Plt10, Plt11 and Plt15 all elevate the platelet count of Mpl-/- mice. Plt3 and Plt4 mice harbour independent mutations in the gene encoding the transcription factor c-Myb, while Plt6 mice contain a mutation in the gene for the histone acetyl transferase and c-Myb binding partner, p300. Consistent with the function of Myb and p300 in the same complex, Plt3, Plt4 and Plt6 mice show stem cell defects involving an increased commitment to the megakaryocyte lineage at the expense of red cell and B cell production. Plt8 and Plt10 in contrast contain a mutation in a gene that is a component of the polycomb repressive complex 2 (PRC2) which acts by methylating lysine 27 of Histone 3. Many interesting questions arise when analysing these mutants; (1) Do each of the mutations exert their effect using the same biological mechanism – i.e. does mutation of Suz(12) or the mutations in the Plt11 and Plt15 mice, also bias differentiation down the megakaryocyte lineage? (2) Do c-Myb, p300 and Suz(12) act in the same molecular pathway or separate pathways? (3) As has occurred successfully in lower organisms, can we use epistasis analyses in which the phenotype of double mutants are compared to single mutants to infer pathway relationships. (4) ENU-induced mutants provide finger-holds in biologically important pathways; can we combine genetics, genomics, proteomics and bioinformatic to flesh out other components of these pathways?

Stem cells shown at the left of the diagram are the ultimate source of all blood cells. The key property of stem cells, which distinguishes them from progenitors, is their capacity for self-renewal and their ability to produce all lineages of blood. Some more primitive progenitor cells retain the ability to form several lineages of cells. These are known as multipotent progenitors (CMP=common myeloid progenitor, MEP=megakaryocyte-erythroid progenitor, GMP=granulocyte-macrophage progenitor, CLP=common lymphoid progenitor) and they give rise to progenitors with restricted potential known as colony-forming cells or CFCs.

1. Starr R, Willson TA, Viney EM, Murray LJ, Rayner JR, Jenkins BJ, Gonda TJ, Alexander WS, Metcalf D, Nicola NA, Hilton DJ.

A family of cytokine-inducible inhibitors of signalling.

Nature. 1997 Jun 26;387(6636):917-21

PMID: 9202125 [PubMed - indexed for MEDLINE]

2. Alexander WS, Starr R, Fenner JE, Scott CL, Handman E, Sprigg NS, Corbin JE, Cornish AL, Darwiche R, Owczarek CM, Kay TW, Nicola NA, Hertzog PJ, Metcalf D, Hilton DJ.

SOCS1 is a critical inhibitor of interferon gamma signaling and prevents the potentially fatal neonatal actions of this cytokine.

Cell. 1999 Sep 3;98(5):597-608

PMID: 10490099 [PubMed - indexed for MEDLINE]

3. Croker BA, Metcalf D, Robb L, Wei W, Mifsud S, DiRago L, Cluse LA, Sutherland KD, Hartley L, Williams E, Zhang JG, Hilton DJ, Nicola NA, Alexander WS, Roberts AW.

SOCS3 is a critical physiological negative regulator of G-CSF signaling and emergency granulopoiesis.

Immunity. 2004 Feb;20(2):153-65

PMID: 14975238 [PubMed - indexed for MEDLINE]

4. Carpinelli MR, Hilton DJ, Metcalf D, Antonchuk JL, Hyland CD, Mifsud SL, Di Rago L, Hilton AA, Willson TA, Roberts AW, Ramsay RG, Nicola NA, Alexander WS.

Suppressor screen in Mpl-/- mice: c-Myb mutation causes supraphysiological production of platelets in the absence of thrombopoietin signaling.

Proc Natl Acad Sci U S A. 2004 Apr 27;101(17):6553-8

PMID: 15071178 [PubMed - indexed for MEDLINE]

5. Majewski IJ, Blewitt ME, de Graaf CA, McManus EJ, Bahlo M, Hilton AA, Hyland CD, Smyth GK, Corbin JE, Metcalf D, Alexander WS, Hilton DJ.

Polycomb repressive complex 2 (PRC2) restricts hematopoietic stem cell activity.

PLoS Biol . 2008 Apr 15;6(4):e93

PMID: 18416604 [PubMed - in process]

Faculty Member:	Doug Hilton, BSc Mon BSc(Hons) Melb FAA PhD Melb
Scientific Coordinator/Alliance Manager:	Fiona McGrath, BAppSc(Hons) RMIT
Administrative Assistant:	Maria Markovic, BA RMIT DipEd LaT
Senior Postdoctoral Fellow:	Rachel Burt, BSc PhD Melb
Senior Postdoctoral Fellow:	Chris Greenhalgh, BAgSc(Hons) PhD Melb
	(Jointly with Cancer & Haematology Division)
Senior Postdoctoral Fellow:	Toby Sargeant, BComp(Hons) BSc MComp Mon PhD Melb
Senior Postdoctoral Fellow:	Tracy Willson, BSc(Hons) PhD Mon
Senior Postdoctoral Fellow:	Jian-Guo Zhang, BSc Xinjiang PhD Melb
	(Jointly with Cancer & Haematology Division)
Postdoctoral Fellow:	Ann Cornish, BSc(Hons) PhD Melb
	(Jointly with Autoimmunity & Transplantation Division)
Postdoctoral Fellow:	Marnie Blewitt, BSc(Hons) PhD Syd
Postdoctoral Fellow:	Doug Hacking, BCh BM Oxon BMedSci Dundee PhD Weatherall
Postdoctoral Fellow:	Ian Majewski, BSc(Hons) UWA PhD Melb
Postdoctoral Fellow:	James Murphy, BSc(Hons) Canterbury PhD ANU
Postdoctoral Fellow:	Aaron Robinson, BBiotech Flinders PhD Adelaide
Postdoctoral Fellow:	Gillian Tannahill, BSc(Hons) PhD Queen's
Computational Scientist:	Jarny Choi, BSc(Hons) PhD Melb
Computational Scientist:	James Wettenhall, BEng(Hons) BSc(Hons) Melb
Research Assistant:	Tracey Baldwin, BAppSc RMIT
Research Assistant:	Miha Pakusch, BSc(Hons) Melb
Postgraduate Student:	Carolyn de Graaf, BInfTech Swinburne BBiomedSc BSc(Hons) Melb
Postgraduate Student:	Kylie Greig, BSc(Hons) Melb
Postgraduate Student:	Sarah Kinkel, BA BSc(Hons) Melb
Postgraduate Student:	Huei Leong, BBiomedSc(Hons) Melb
Postgraduate Student:	Lisa Mielke, BSc Melb
Undergraduate Student:	Leila Varghese, BSc Melb