Outlook

Certainly, one recurrent theme for Trap230 and the Mediator kinase subcomplex is its involvement in the control of differentiation. Intriguingly, as seems to be the case for HDAC1 (Stadler et al., 2005), which is generally thought to be a repressor and has been proposed as an opponent of the Mediator complex (Kwon and Lee, 2001), although they share some phenotypes, the Mediator kinase subcomplex appears to be involved in activation as well as in its canonical repression of genes, and has been implicated in both maintaining an undifferentiated cell state and promoting cell differentiation, depending on the context. Its cdk and cyclin components link it to the cell cycle, making this role more feasible. Apparently, phosphorylation of Trap240 and possibly also Trap230 through a Ras/cAMP pathway seems to be recurrently involved in this function. Moreover, the proposed chromatin remodelling activity in the subunit may come to aid there, and complex differential regulation of the Mediator kinase submodule through other Mediator components, co- and transcription factors as well as directly through secondary modifications like phosphorylation or possibly sumoylation explain the fact that Trap230 and Trap240 have been identified in Drosophila as members of both the Trithorax and Polycomb group, maintaining either an active or repressed transcriptional state. Indeed, Trap230/Trap240 are involved in regulating the activity or expression of a number of homeobox transcription factors in various organisms.

Differentiation is inhibited - respective an undifferentiated state maintained - in presence of the kinase complex in case of yeast cells, where it represses entry into G0/stationary phase. This is also the case for much of the Sox9 functions, which through Wnt signalling have been implicated in maintaining undifferentiated cell states and require Trap230 to be relayed. On the other hand, in C. elegans vulval development, Drosophila eye development and Dictyostelium spore formation, Trap230 appears to promote differentiation.

Moreover, another (or possibly the same) connection exists between Trap230/the Mediator kinase subcomplex and regulation of cell affinity in yeast, mammalian cells, Drosophila and zebrafish. Whether this is an indirect effect, eg. through modulating Wnt/Sox9 signalling and thereby the composition of the ECM, the actin

DISCUSSION 86

cytoskeleton bound to β-catenin or the planar cell polarity pathway, or a more direct function, remains to be seen.

Clearly, the emerging picture of Mediator is that of an all-integrative platform for transcriptional regulation as well as other DNA-related activities, at which signalling pathways and transcription factor activities converge and cross-talk. It is a complex and versatile machinery with manifold regulatory features. Different proteins may bind to different subunits, modify them, eg. by phosphorylation, sterically inhibit each other or be brought in contact with one another. The chromatin structure is altered to bring enhancer- and promoter-bound proteins in contact with one another, nucleosomes are removed and acetylated, individual Mediator components may modify each other and regulate each other, submodules may be recruited depending on the context – and ultimately, structure and state of the entire complex are thus modified to intricately regulate the activity of pol II for transcribing the gene close to which Mediator is bound.

Such a complex system is probably more than the sum of its components, yet, understanding the function of individual subunits will promote the understanding of the whole. Therefore, it is essential to analyse a component like Trap230 and study its function. Again, analysing individual instances of Trap230 activity will help to get an idea, like its interaction with Sox9 or a possible interaction with Tbx5 and resulting effects. Nevertheless, it will be most important to understand the general idea behind it and find the rules that this piece in the whole framework obeys to. One may think of Trap230 as a protein which randomly interacts with some transcription factor or other as a competence factor for its activity, yet I believe the emerging picture to be more specific. A number of possibilities present themselves for now, which may not encompass the whole scope yet and can also include the truth in a combination of them. Trap230 may be specific for relaying transcription factor activity downstream of certain signalling pathways and not others – so far, the Wnt, Ras, Notch, Hedgehog and JNK pathways have been proposed. It may be specific for certain tissues, although its ubiquitous expression suggests otherwise. Its specificity may lie in a role for regulating cell cycle and proliferation, as has been implicated at several points also for other components of the Mediator kinase subunit.

Alternatively, it may serve to regulate cell-cell or cell-matrix affinity as a general

DISCUSSION 87

rule, as some data suggest. Or, it may be specific for a set of transcription factors with common structural or other features, like those which need to synergise with others for their function, as Sox and T-box proteins do. Moreover, unknown functions of Trap230 may remain to be found, like enzymatic activities or a role in the cytoplasm. These possible functions may also differ from one species or phylum to the other – or be conserved. It remains to be seen.

Another important point are the possible implications of Trap230 and Mediator defects for human diseases. They have been linked to neurodegenerative and neuropsychiatric diseases, as well as to congenital defects of many organs and cancer.

In summary, both a detailed analysis of specific Trap230-interactions and a broader analysis, eg. by microarray studies, should help to provide us with a clearer picture of its function within the Mediator complex. The same holds true for its other components, and comparative analysis of the clockwork components will be important to understand how the Mediator clock ticks.

4 M ATERIALS AND M ^ETHODS

MATERIALS AND METHODS 89