Rotary Bone Marrow Research
Analysis of a novel family of genes crucial for prevention human skin diseases and wound healing
Supervisors: Dr Tomasz Wilanowskiand Professor Stephen Jane
Location: Rotary Bone Marrow Research Laboratories, Royal Melbourne Hospital
Contact: Dr Tomasz Wilanowski T: 9342 8125 Email: wilanowski@wehi.edu.au Professor Stephen Jane T: 9342 8641 Email: jane@wehi.edu.au
Our research team studies a highly conserved family of mammalian genes, the Grainyhead family. Knockout studies in mice have shown that these genes play essential roles in a range of developmental events including neural tube closure and embryonic wound repair (Nature Medicine 9: 1513-1519, 2003; Science 308: 411-413, 2005). The Grainyhead genes encode transcription factors which act through target genes to mediate their effects. The project offered here will involve identification of Grhl target genes relevant to neural tube closure and wound healing. This is likely to have impact on the treatment of children with spina bifida, and in the development of new approaches to wound repair.
Skills - Molecular biology, cell culture, knockout models, bioinfomatics, and more.
Studying the role of self-renewal in causing T cell leukaemia
Supervisor names: Dr Matthew McCormack, Dr David Curtis
Location: Bone Marrow Research Laboratories, Royal Melbourne Hospital
Contact details: Dr Matthew McCormack T: 9342-8948 Email: mccormack@wehi.edu.au or Dr David Curtis T: 9342 8444 Email: dcurtis@wehi.edu.au
Project description: Whilst transcription factor Lmo2 is a principle oncogene in human T cell leukaemia, its cellular effects on T cell development remain poorly defined. We have recently shown that Lmo2 causes long-term persistence of early T cells via the process of self-renewal. This process involves early T cells replicating themselves whilst retaining the ability to mature, and normally never occurs during T cell development. This project will establish in vitro model systems for studying self-renewal caused by Lmo2 and test whether inhibition of various signalling pathways can interfere with this process. This will identify potential therapeutic approaches for preventing leukaemia development.
Identifying target genes of Lmo2 in haemopoietic (blood) stem cells
Supervisors: Dr Matthew McCormack, Dr David Curtis
Location: Bone Marrow Research Laboratories, Royal Melbourne Hospital
Contact details: Dr Matthew McCormack T: 9342-8948 Email: mccormack@wehi.edu.au or Dr David Curtis T: 9342 8444 Email: dcurtis@wehi.edu.au
Project description: The transcription factor Lmo2 has crucial roles in haemopoietic (blood) stem cell development and T cell leukaemia. With regard to blood development, the critical targets of this transcription factor remain poorly defined. Using microarray analysis of T cells overexpressing Lmo2 we have shown that Lmo2 induces expression of several genes that are normally only expressed in haemopoietic stem cells (HSCs). This implies that Lmo2 normally regulates these genes in HSCs. This project will use molecular and cellular techniques to demonstrate the regulation of key HSC genes by Lmo2.
bHLH factors and Haemopoietic Stem Cell cycling
Supervisor: Dr David Curtis
Location: Bone Marrow Research Laboratories, Royal Melbourne Hospital
Contact details: Dr David Curtis T: 9342-8444 Email: dcurtis@wehi.edu.au
Haemopoietic stem cells (HSC) are primitive cells that maintain the blood system throughout life. To achieve this there must be tight control of HSC cycling such that most HSCs are in a “quiescent” or non-dividing state. Restriction of HSC cycling is achieved by factors that govern the entry of HSCs into the cell cycle. One such regulator is p21, also known as cyclin-dependent kinase inhibitor 1A (CDKN1A). p21 serves as a negative regulator of HSC cycling by regulating the activity of cyclin-dependent kinase proteins. However, the factors that regulate p21 activity are largely unknown. We have data suggesting that the bHLH factors, Scl and Lyl1 are important repressors of p21. HSCs in mice lacking Scl or Lyl1 show a 3-fold reduction in HSC cycling, and this correlates with increased p21 expression. HSCs lacking both Scl and Lyl1 cannot grow and we hypothesize that this is due to high levels of p21. The aims and experiments of this project may include
- Determine cell cycle status and p21 levels of Scl/Lyl1 deficient HSCs using RT-PCR and in vivo labelling with BrdU.
- Determine the response of Scl/Lyl1-deficent mice to cell cycle-dependent cyctotoxics.
- Generate in vitro assays for deletion of Scl to assess early changes in gene expression.
- Use an in vitro promoter assay to determine if Scl and Lyl1 repress p21.
- Perform ChIP assay using a ‘stem cell’ line to determine if Scl and Lyl1 can bind to the p21 promoter and if so characterise other protein partners.
- Perform knockdown experiments of p21 using lenti-viral RNAi and genetic approaches to determine if this can rescue the growth defects of Scl/Lyl1-null HSCs.
Analysis of a novel family of genes crucial for prevention of human skin diseases
Supervisor names: Dr Charbel Darido, Professor Stephen jane
Location: Bone Marrow Research Laboratories, Royal Melbourne Hospital
Contact details: Dr Charbel Darido T: 9342 8125 Email: darido@wehi.edu.au Professor Stephen Jane T: 9342 8641 Email: jane@wehi.edu.au
Our research team discovered and studies a highly conserved family of mammalian genes, the Grainyhead family. Knockout studies in mice have shown that these genes play essential roles in a range of developmental events involving the skin, including wound repair, and barrier formation (Nature Medicine 9: 1513-1519, 2003; Science 308: 411-413, 2005). They may also be important in the context of skin cancer. Grainyhead genes encode transcription factors which act through target genes to mediate their effects. The project offered here will involve identification of Grhl target genes relevant to skin diseases. This is likely to have impact on the treatment of a range of human diseases including skin barrier defects wound healing and and cancer.
Skills - A wide range of skills will be taught including molecular biology, cell culture, knockout models, and bioinfomatics. This is an ideal project for a student who wishes to pursue higher studies in the future. Two positions are available.
Modulating apoptosis in myelodysplasia
Supervisors: Dr Chris Slape and Dr David Curtis
Location: Bone Marrow Research Laboratories, Royal Melbourne Hospital
Contacts: Dr Chris Slape T: 9342-8948 Email: slape@wehi.edu.au or Dr David Curtis T: 9342 8444 Email: dcurtis@wehi.edu.au
Project description: Myelodysplasia is the most common hematologic malignancy with limited effective therapies. The major clinical problems of MDS are low blood counts requiring blood transfusions and antibiotics for recurrent infections. Studies of human MDS suggest that aberrant cell death (apoptosis) is central to the cause of low blood counts. We have generated transgenic mouse models of human MDS that have low blood counts. The aims of this project are to determine the mechanism(s) of apoptosis in transgenic mice and translate these findings to cases of human MDS.
Techniques: This project uses genetically modified mouse strains, retroviral expression systems as well as human samples. Techniques learnt will include mouse handling/breeding/drug administration, tissue harvesting, cell culture, DNA and RNA isolation, gene expression analyses including quantitative RT-PCR and Western blotting, flow cytometry and cell sorting.
Significance: This work will form the pre-clinical data for subsequent human clinical trials in MDS.
Drug therapies targeting the cancer stem cell
Supervisors: Dr David Curtis and Dr Matthew McCormack
Location: Bone Marrow Research Laboratories, Royal Melbourne Hospital
Contacts: Dr Matthew McCormack T: 9342 8948 Email: mccormack@wehi.edu.au or Dr David Curtis T: 9342-8444 Email: dcurtis@wehi.edu.au
Project description: We have identified the cancer stem cell in a transgenic mouse model of acute lymphoblastic leukemia. The frequency of this abnormal stem cell can be measured using transplant assays. The aim of this project is to determine the effect of new therapuetic drugs on the cancer stem cell.
Techniques: T-cells from transgenic mice will be transplanted into wild-type mice, and then mice will be treated with various agents including the mTOR inhibtior Rapamycin, the HDAC inhibtior vorinostat and the differentiating agent arsenic trioxide. The effects of these drugs will be determined by flow cytometry and gene expression studies.
Signficiance: These studies will provide the rationale for testing of these drugs in human cases of acute lymphoblastic leukemia.
bHLH factors and Haemopoietic and Leukemic Stem Cell cycling
Supervisors: Dr David Curtis and Dr Matthew McCormack
Location: Bone Marrow Research Laboratories, Royal Melbourne Hospital
Contacts: Dr Matthew McCormack T: 9342 8948 Email: mccormack@wehi.edu.au or Dr David Curtis T: 9342-8444 Email: dcurtis@wehi.edu.au
Project descripiton: Haemopoietic stem cells (HSC) are primitive cells that maintain the blood system throughout life. To achieve this there must be tight control of HSC cycling such that most HSCs are in a “quiescent” or non-dividing state. Restriction of HSC cycling is achieved by factors that govern the entry of HSCs into the cell cycle. One such regulator is p21, also known as cyclin-dependent kinase inhibitor 1A (CDKN1A). p21 serves as a negative regulator of HSC cycling by regulating the activity of cyclin-dependent kinase proteins. However, the factors that regulate p21 activity are largely unknown. We have data suggesting that the bHLH factors, Scl and Lyl1 are important repressors of p21. HSCs in mice lacking both Scl and Lyl1 cannot grow and have p21 levels 10-fold higher than normal. We hypothesize that bHLH factors regulate the quiescence of HSCs by controlling the levels of p21. This project will study the cell cycle characteristics of HSCs lacking Scl and Lyl1. Furthermore, you will determine if reducing levels of p21 by either siRNA or using p21 knockout mice can rescue the growth defects seen in Scl/Lyl1-deficient HSCs.
Techniques: Cell cycle analyses including BrdU-labelling and Hoechst pyronin staining, siRNA and/or analysis of p21 knockout mice, DNA and gene expression techniques, cell culture, flow cytometry.
Significance: This work will provide a better understanding of regulators of stem cell growth. These findings may be important for improved uses of blood stem cells.
Targeting the inflammatory response of myocardial infarction to improve heart function
Supervisors: Dr David Curtis and Professor Alex Bobik
Location: Bone Marrow Research Laboratories, Royal Melbourne Hospital and Cell Biology Laboratory, Baker Heart Research Institute
Contacts: T: 9342-8444 Email: dcurtis@wehi.edu.au
Project description: We have shown that mice lacking the G-CSF receptor have impaired heart function after myocardial infarction (MI) (Kanellakis et al. Circ. Res 2006). In contrast, mice lacking the GM-CSF receptor have improved heart function after MI. We postulate that these findings are due to differences in macrophage subsets that accumulate within the infarct within the first 48 hours. This project will examine the effects of an inhibitor of GM-CSF on heart function in a mouse model of MI.
Techniques: Analyses of myocaridal tissue using flow cytometry and gene expression methods. Assistnace with cardiac surgery including invasive cardiac catheter monitoring. Cell culture. Western blotting.
Significance: These studies will provide data essential for design of potential human trials using the inhibtior of GM-CSF.