Characterisation of Kame, a novel JmjC domain-containing protein in Dictyostelium discoideum

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Microbiology, School of Life Sciences, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

Jumonji C domain-containing proteins were first identified with a role in the modulation of histone methylation marks. This family of proteins is broken up into 7 distinct subgroups based on domain architecture and their ability to antagonise specific histone methylation marks. However one subgroup remains with no distinct role. These proteins belong to the JmjC-domain-only subgroup and as their name suggests, they contain only the highly conserved JmjC domain. Little is known about this particular subgroup; but they are widely distributed amongst all known organisms where the JmjC domain family has been identified. Dictyostelium discoideum has been used as a model to gain insight into processes such as cellular differentiation, signal transduction and phagocytosis (Annesley and Fisher, 2009). It was one of the first organisms shown to contain a member of the JmjC-domain-only subgroup (then identified as the orphan group) (Clissold and Ponting, 2001). This led to the first part of my study which included the identification of the JmjC domain-containing proteins in D. discoideum. Using the JmjC domain from the first identified protein, CbfA, as the initial query sequence I found a further 12 D. discoideum proteins containing this domain and my proposal for their nomenclature was accepted by the curators at dictyBase. The sequences of these proteins were then studied bioinformatically to identify their key features including putative localisation, possible subgroup distinctions and tertiary structure. Phylogenetic relationships to other members of the Dictyostelia and eukaryotes were also analysed. This identified an Dictyostelia-specific subgroup of proteins within the JmjCdomain-only subgroup containing JcdA Kame and JcdB. The second part of this project was to characterise one of the D. discoideum JmjC domain-containing proteins to understand the role these proteins play in cells. Kame was chosen due to its unique domain architecture and it being a Dictyostelid-specific protein. Phenotypic analysis was conducted and identified a role for Kame in growth and development. The unique domain architecture allowed further experiments aimed at understanding the role of the JmjC domain and the function, if any, of the N-terminal region of Kame. Together with the bioinformatics the resulting functional studies show that Kame has 2 clear domains - an. N-terminal domain and the JmjC domain. The N-terminus was found to have a possible regulatory effect on the Kame protein, in that expression of the N-terminus alone mimics the phenotypic changes observed in strains in which Kame expression was reduced by antisense inhibition (Chapter 5). It is possible that the N-terminal domain controls access to the catalytic cleft of the β barrel of the JmjC domain by functioning as a “lid”, which in the right conditions binds to a target protein and opens up the barrel making the active site accessible to the substrate. Expressing the JmjC domain alone caused the same phenotypic changes as overexpressing the full protein. This suggests that in the absence of the N-terminus, the JmjC domain is active or accessible constantly to the substrate and therefore can carry out its function. When both domains are expressed in trans they were able, to different degrees, to restore the phenotypic aberrations seen when either domain was expressed alone. This suggests that even when the two domains of the protein are separated they can interact to restore function as a whole protein. This also clearly demonstrates that both parts of the Kame protein are essential to correct functioning of the protein. Although there are still many questions remaining to be answered about JmjC domain-containing proteins in D. discoideum, this study has opened up a line of research that may shed light on the roles of this family as a whole, not only in Dictyostelium but also in other organisms. It also suggests that JmjC domain-containing proteins will have roles outside of the currently known histone demethylation.