Signal transduction pathways that regulate Dictyostelium gene expression

Research

The quantum evolutionary leap, from unicellularity to multiucellularity, occurred many times and in many different ways in the eukaryotes. Dictyostelium discoideum is a free-living soil amoeba that undergoes a remarkable, facultative transition to multicellularity when subjected to starvation. The initially separate cells aggregate together in response to pulses of cyclic AMP (cAMP) that they synthesise and secrete as a chemoattractant. They first form themselves into a migratory slug that displays most of the defining features of a multi-cellular organism. During their subsequent development, to form a fruiting body, the cells differentiate as either spore cells or stalk cells (Fig 1).

Dictyostelium is much valued as a model eukaryotic cell, to study processes such as chemotaxis and phagocytosis, but we wish to understand how its remarkable developmental cycle is effected. The answers are in part known but there remain very large gaps in our understanding. cAMP and another small organic molecules, the polyketide DIF, play key roles. cAMP induces the differentiation of prespore cells and they secrete DIF. DIF then induces a proportion of the uncommitted cells to differentiate as prestalk cells. We are tracing this signaling pathway upwards, from a DIF-responsive promoter that directs expression of EcmA, an extracellular matrix protein. We have identified a Myb transcription factor, MybE, and a bZip transcription factor, DimB, that co-operatively mediate prestalk gene induction by DIF (Fig 2). When cells are exposed to DIF DimB is rapidly serine-threonine phosphorylated at a site near its C terminus and it moves to the nucleus and binds to the ecmA promoter.

Two other major components in the signalling pathways for stalk cell differentiation are STAT (Signal Transducer and Activator of Transcription) proteins. They are conserved in structure and function with the STAT proteins that mediate cytokine signalling in animal cells. Cytokines control many vital animal cell functions and the study of STATs in a diverged but genetically amenable organism such as Dictyostelium is providing insights into mammalian STAT signalling pathways (Fig 3). One of the two STAT proteins, STATa, is activated, i.e. it is tyrosine phosphorylated and moves to the nucleus, in response to cAMP signalling. STATa directs the differentiation of the tip-organiser cells; a sub-set of the prestalk cells that control slug integrity and behaviour. The second, STATc, is activated by a non-paradigmatic pathway wherein DIF acts to cause repression of a STATc specific protein tyrosine phosphatase: PTP3. This repression is bought about by DIF induced serine phosphorylation of PTP3 at two sites.

Thus the STATc, and DimB DIF signaling pathways have both been traced to the regulated phosphorylation of specific sites within pathway intermediates and the cognate kinases are being purified. Our overriding goal now is to understand how all of the different signal transduction pathways that regulate these transcription factors are structured and integrated to direct cellular differentiation.

Fig 1 An electron micrograph of a late stage in the development of a Dictyostelium culminant (fruiting body). They are typically about 1mm high and contain 100,000 cells: 20,000 dead, highly vacuolated stalk cells supporting 80,000 spore cells. Work from this laboratory showed that the prestalk and stalk cells are heterogeneous, with different cell types located in different parts of the developing structures. This is indicated by false colouring, in red, of a sub-set of the prestalk cells that are just entering the entrance to the stalk (image with the kind courtesy of Drs. Larry Blanton and Mark Grimson).

Fig 2 Cell type specific expression of a lacZ reporter construct containing a Myb and a bZIP transcription factor binding site, both contained within just 22 nucleotides of promoter DNA. The 22-mer, from within the promoter of the DIF-inducible ecmA gene, was multimerised four times and inserted upstream of a heterologous cap site and TATA box. This was in turn linked to a lacZ reporter. Stable transformants were generated using the construct and the cells were developed to various stages then stained for ß-galactosidase activity.

Fig 3 The crystal structure of Dictyostelium STATa, determined in collaboration with Dr. Christoph Mueller and his group at the EMBL and compared with the structure of metazoan STAT3. STATs dimerise via mutual interactions of their single SH2 domains (SH2 in the figure) and single tyrosine phosphorylation sites (pTyr in the figure). The target DNA for STAT3 is shown in grey and the interacting amino acids are asterisked. The STATa structure proved highly informative because dimeric transcription factors usually only dimerise in the presence of their target DNA. STATa crystallised without target DNA and this showed for the first time that unliganded STATs exist as a highly extended, semi-linear structure.

Publications

1. Eichinger, et al. (2005) The genome of the social amoeba Dictyostelium discoideum. Nature, 435, 43-57. * JGW is one of the six joint senior authors.
2. Sugden, C.J., Roper, J.R. and Williams, J.G. (2005) Engineered gene over-expression as a method of drug target identification. Biochem and Biophys. Res. Comm. 334, 555-560.
3. Hudson, J.R., Hsu, D., Guo, K., Zhukovskaya, N.V., Liu, P., Williams, J.G., Pears, C.J. and Lakin, N.D. (2005). DNA-PKcs dependent signaling of DNA damage in Dictyostelium discoideum. Curr Biol., 15, 1880-1885.
4. Zhukovskaya, N.V., Fukuzawa, M., Yamada, Y., Araki, T. and Williams, J.G. (2006) Dictyostelium bZIP transcription factor DimB regulates prestalk-specific gene expression. Development, 133, 439-448.
5. Fukuzawa, M., Zhukovskaya, N.V., Yamada, Y., Araki, T. and Williams, J.G. (2006) Regulation of Dictyostelium prestalk-specific gene expression by a SHAQKY family MYB transcription factor. Development. 133, 1715-1724.
6. Stremcki, L., Bloomfield, G., Araki, T., Dalton, E., Skelton, J., Schilde, C., Harwood, A., Williams, J.G., Ivens, A. and Pears, C.J. (2007) Proteomic and micro-array analyses of the Dictyostelium Zak1-GSK-3 signalling pathway reveals a role in early development. Eukaryotic Cell, 6. 245-52.
7. Tsujioka, M.*, Zhukovskaya, N.*, Yamada, Y., Fukuzawa,M., Ross, S. and Williams, J. G. (2007) Dictyostelium Myb transcription factors function at culmination as activators of ancillary stalk differentiation. Eukaryotic Cell, 6, 568-70. *equal co-authors.
8. Araki, T., Langenick, J., Gamper, M., Firtel, R.A., Williams, J.G. (2008) Evidence that DIF-1 and hyper-osmotic stress activate a Dictyostelium STAT by inhibiting a specific protein tyrosine phosphatase. Development. 135:1347-53.
9. Langenick, J., Araki, T., Yamada, Y., Williams, J.G. (2008) A Dictyostelium homologue of the metazoan Cbl proteins regulates STAT signalling. J Cell Sci. 200: 3524-30.
10. Yamada, Y., Wang, H.Y., Fukuzawa, M., Barton, G.J., Williams, J.G. (2008) A new family of transcription factors. Development. 135:3093-101.
11. Gaudet, P., Williams, J.G., Fey, P., Chisholm, R.L. (2008) An anatomy ontology to represent biological knowledge in Dictyostelium discoideum. BMC Genomics. 9:130.
12. Wang, H. Y. and Williams, J. G. (2009). Identification of a target for CudA, the transcription factor which directs formation of the Dictyostelium tip organiser. Int J Dev Biol 54, 161-5.
13. Araki, T., van Egmond, W. N., van Haastert, P. J. and Williams, J. G. (2010). Dual regulation of a Dictyostelium STAT by cGMP and Ca2+ signalling. J Cell Sci 123, 837-41.
14. Yamada, Y., Kay, R. R., Bloomfield, G., Ross, S., Ivens, A. and Williams, J. G. (2010). A new Dictyostelium prestalk cell sub-type. Dev Biol 339, 390-7.
15. Sugden, C., Ross, S., Bloomfield, G., Ivens, A., Skelton, J., Mueller-Taubenberger, A. and Williams, J. G. (2010) Two novel Src homology 2 domain proteins interact to regulate Dictyostelium gene expression during growth and early development. J Biol Chem 285, 22927-35.
16. Williams, J.G. (2010) Dictyostelium finds new roles to model.  Genetics 185, 717-26.
17. Wang, H.Y. and William, J.G. (2010) Synergy between two transcription factors directs gene expression in Dyctyostelium tip-organiser cells.  Int J Dev Biol 54, 1301-7.
18. Nunez-Corcuera, B., Birch, J. and William, J. G. (2011) A SET/MYND chromatin re-modelling protein regulates Dictyostelium prespore patterning.  Int J Dev Biol 55, 205-8.
19. Sugden, C., Ross, S., Annesley, S.J., Cole, C., Bloomfield, G., Ivens, A., Skelton, J., Fisher, P.R., Barton, G. and Williams, J. G. (2011) A Dictyostelium SH2adaptor protein required for correct DIF-1 signaling and pattern formation.  Dev Biol 353, 290-301.
20. Yamada, Y., Nunez-Corcuera, B. and William,s J.G. (2011) DIF-1 regulates Dictyostelium basal discdifferentiation by inducing the nuclear accumulation of a bZIP transcription factor.  Dev Biol 354, 77-86.