CCQ Transgenics

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Lab Head: Dr Graham Kay
grahamK@qimr.edu.au

As well as conducting independent research, the CCQ Transgenic Laboratory provides transgenic and knockout mouse services to members of the Institute. We also can generate knockout and transgenic mice, as well as undertake mouse embryo freezing on a contract research basis to researchers outside the Institute.

X chromosome inactivation
A major focus of our research is studying the Xist gene and its imprinting in X chromosome inactivation. We aim to understand how the Xist gene functions to inactivate one of the two X chromosomes in female mammals to achieve dosage equivalence with males. We are using gene targeting to delete specific portions of the Xist gene leaving it otherwise functional and able to produce a transcript but lacking specific regions of the gene. X inactivation should then be rendered either wholly or partially non-functional. Mutant mouse cells are then studied in vivo and in vitro for their ability to undergo X inactivation.

In collaboration with Prof. Emma Whitelaw (QIMR) we have been investigating genes other than Xist that are involved in X inactivation. A recent ENU mutagenesis screen designed to find genes that establish and/or maintain the genome's epigenetic state identified the MommeD1 mutant as a semidominant suppressor of variegation. When the MommeD1 mutant was bred to homozygousity, it resulted in mid-gestation lethality of female embryos. Homozygous mutant male embryos were unaffected, and when adult were healthy and fertile. This indicated that the gene mutated in MommeD1 may be involved in X inactivation. Subsequent mapping and sequencing identified a nonsense point mutation in the SmcHD1 gene as the mutation in the MommeD1 mice. Analysis of the protein encoded by SmcHD1 reveals a Smc hinge domain and a predicted ATPase domain but no other strong similarity to the Smc proteins, which are essential for faithful chromosome segregation during cellular proliferation. We have shown that in homozygous SmcHD1 mutant female embryos genes on the normally stable inactive X are reactivated at high frequency. This is despite normal localisation of Xist and trimethylation of lysine 27 of histone H3 on the inactive X. We also find that the normally hypermethylated CpG islands on the inactive X are hypomethylated in homozygous SmcHD1 mutant females. These data indicate that SmcHD1 may be involved in the methylation of CpG islands that is associated with the stable maintenance of the inactive X.

Pocket protein (Rb-1, p107 and p130) gene function in cell differentiation, development and cancer
Rb-1 acts by inhibiting cell cycle progression while simultaneously facilitating development. The role of Rb-1 in facilitating cellular differentiation is poorly understood because total knockout of Rb-1 is embryonic lethal due to perturbations in the central nervous system and erythropoiesis. To circumvent this we have developed tissue-specific knockout Rb-1 mice. We have initially directed the knockout to tyrosinase expressing, neural crest derived lineages (melanocytes, RPE, trigeminal nerve, and dorsal root ganglia). Further to this project we developed floxed alleles of the other pocket protein family members, p107 and p130. We are generating animals where multiple combinations of the pocket proteins are tissue specifically deleted to determine which proteins act in different tissues and whether the pocket protein family as a whole is important in the development of specific cancers or whether it is specific individual pocket proteins that are important.

Men1 gene function in cell differentiation, development and cancer In collaboration with Prof. Nick Hayward (QIMR), we have generated both total and tissue-specific knockouts of to the Men1 tumour suppressor gene. Menin, the encoded protein has no homologies to any proteins but interacts with JunD to repress JunD-activated transcription suggesting that menin acts as a tumour suppressor through transcriptional co-repression of unidentified growth-promoting genes. Our Men1 total knockout is embryo lethal, while heterozygously deleted animals develop a range of MEN1 type tumours as they age accurately recapitulating the human MEN1 tumour syndrome. We are now using these total knockout mice in microarray studies to identify genes with expression regulated by the action of menin. We have also developed tissue-specific knockouts to study menin during cellular differentiation and development. Using these animals we have shown that tissue specific knockout of menin also results in tumours. In ongoing studies we will pursue the identification of genes that have their expression regulated by menin to determine the likely consequences of loss or mutation of menin in cancer development. This is anticipated to identify novel pathways to the development of cancer that may identify novel targets for rational drug design and also pinpoint the normal function of menin during development.

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Staff

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Funding

We gratefully acknowledge the support from the following funding agencies:

• The Cancer Council Queensland
• The National Health and Medical Research Council

Student Projects

The Queensland Cancer Fund Transgenic Laboratory has a number of projects available for students.

Key Publications

Fitzpatrick DR, Shirley KM, McDonald LE, Bielefeldt-Ohmann H, Kay GF, and Kelso A. (1998). Distinct methylation of the interferon g (IFN-g) and interleukin (IL-3) genes in newly activated primary CD8+ T lymphocytes: regional IFN-g promoter demethylation and mRNA expression are heritable in CD44high CD8+ T cells. J. Exp. Med. 188:103-117. [pubmed abstract]

McDonald LE, Paterson CA and Kay GF. (1998). Bisulphite genomic sequencing-derived methylation profile of the Xist gene throughout early mouse development. Genomics 54:379-386. [pubmed abstract]

Brown AL and Kay GF (1999). Bex1, a gene with increased expression in parthenogenetic embryos, is a member of a novel gene family on the mouse X chromosome. Human Molecular Genetics. 8:611-619. [pubmed abstract]

Bellomo D, Headrick JP, Silins GU, Paterson CA, Thomas PS, Gartside M, Mould A, Cahill MM, Tonks ID, Grimmond SM, Townson S, Wells C, Little M, Cummings MC, Hayward NK and Kay GF. (2000). Mice lacking the vascular endothelial growth factor-B gene (Vegfb) have smaller hearts, dysfunctional coronary vasculature and impaired recovery from cardiac ischemia. Circ. Res, 86:E29-E35. [pubmed abstract]

Biondi C, Gartside M, Tonks I, Paterson C, Hayward NK and Kay GF. (2002). Targeting and conditional inactivation of the murine Men1 locus using the Cre recombinase: loxP system. Genesis 32:150-151. [pubmed abstract]

Wanstall JC, Gambino A, Jeffery TK, Cahill MM, Bellomo D, Hayward NK and Kay GF. (2002). Vascular endothelial growth factor-B-deficient mice show impaired development of hypoxic pulmonary hypertension. Cardiovasc Res. 55:361-368. [pubmed abstract]

Maxwell M, Bjorkman J, Nguyen T, Sharp P, Finnie J, Paterson C, Tonks I, Paton BC, Kay GF and Crane DI. (2003). Pex13 inactivation in the mouse disrupts peroxisome biogenesis and leads to a Zellweger syndrome phenotype. Mol Cell Biol. 23:5947-5957. [pubmed abstract]

Tonks I, Nurcombe V, Paterson C, Zournazi A, Prather C, Mould A and Kay GF. (2003). Tyrosinase-Cre mice for tissue-specific gene ablation in neural crest and neuroepithelial-derived tissues. Genesis 37:131-138.[pubmed abstract]

Mould AW, Tonks ID, Cahill MM, Pettit AR, Thomas R, Hayward NK and Kay GF. (2003). Vegfb gene knockout mice display reduced pathology and synovial angiogenesis in both antigen-induced and collagen-induced models of arthritis. Arthritis and Rheumatism 48:2660-2669.[pubmed abstract]

Hughes, C.M., Rozenblatt-Rosen, O., Milne, T.A., Copeland, T.D., Levine, S.S., Lee, J.C., Hayes, D.N., Shanmugam, K.S., Bhattacharjee, A., Biondi, C.A., Kay, G.F., Hayward, N.K., Hess, J.L. and Meyerson, M. (2004). Menin associates with a trithorax family histone methyltransferase complex and with the hoxc8 locus. Mol Cell 13:587-597.[pubmed abstract]

Biondi, C.A., Gartside, M.G., Waring, P., Loffler, K.A., Stark, M.S., Magnuson, M.A., Kay, G.F. and Hayward, N.K. (2004). Conditional inactivation of the MEN1 gene leads to pancreatic and pituitary tumorigenesis but does not affect normal development of these tissues. Mol Cell Biol 24:3125-3131. [pubmed abstract]

Tonks, I., Hacker, E., Irwin, E., Muller, K., Keith, P., Mould, A., Zournazi, A., Pavey, S., Hayward, N., Walker, G. and Kay, G.F. (2005) Melanocytes in conditional Rb-/- mice are normal in vivo but exhibit proliferation and pigmentation defects in vitro. Pigment Cell Res 18:252-264. [pubmed abstract]

Mould, A., Greco, S., Cahill, M., Tonks, I., Bellomo, D., Patterson, C., Zournazi, A., Nash, A., Scotney, P., Hayward, N.K. and Kay, G.F. (2005). Transgenic over-expression of VEGF-B isoforms by endothelial cells potentiates postnatal vessel growth in vivo and in vitro. Circ Res. 97:e60-70.[pubmed abstract]

Tonks, I.D., Hayward, N.K. and Kay, G.F. (2006) Pocket protein function in melanocyte homeostasis and neoplasia. Pigment Cell Res 19:260-283.[pubmed abstract]

Findlay, J. K., Gear, M. L., Illingworth, P. J., Junk, S. M., Kay, G., Mackerras, A. H., Pope, A., Rothenfluh, H. S., and Wilton, L. (2007). Human embryo: a biological definition. Human Reproduction 22:905-911.[pubmed abstract]

Loffler, K. A., Biondi, C. A., Gartside, M., Waring, P., Stark, M., Serewko-Auret, M. M., Muller, H. K., Hayward, N. K., and Kay, G.F. (2007). Broad tumor spectrum in a mouse model of multiple endocrine neoplasia type 1. Int J Cancer 120:259-267. [pubmed abstract]

Loffler, K. A., Biondi, C. A., Gartside, M. G., Serewko-Auret, M. M., Duncan, R., Tonks, I. D., Mould, A. W., Waring, P., Muller, H. K., Kay, G.F., and Hayward, N. K. (2007). Lack of augmentation of tumor spectrum or severity in dual heterozygous Men1 and Rb1 knockout mice. Oncogene 26:4009-4017.[pubmed abstract]

Mould, A. W., Duncan, R., Serewko-Auret, M., Loffler, K. A., Biondi, C., Gartside, M., Kay, G. F., and Hayward, N. K. (2007). Global expression profiling of murine MEN1-associated tumors reveals a regulatory role for menin in transcription, cell cycle and chromatin remodelling. International Journal of Cancer 121:776-783.[pubmed abstract]

Mould, A.W., Scotney, P., Greco, S.A., Hayward, N.K., Nash, A. and Kay. G.F. (2008) Prophylactic, but not therapeutic activity of a monoclonal antibody that neutralizes the binding of VEGF-B to VEGFR-1 in a murine collagen-induced arthritis model. Rheumatology 47:263-266. [pubmed abstract]

Blewitt, M.E., Gendrel, A-V., Pang, Z., Sparrow, D.B., Whitelaw, N., Craig, J.M., Apedaile, A., Hilton, D.J., Dunwoodie, S.L., Brockdorff, N., Kay, G.F. and Whitelaw, E. (2008) SmcHD1, containing a structural-maintenance-of chromosomes hinge domain, has a critical role in X inactivation. Nature Genetics 40:663-669. [pubmed abstract]

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