Cell fate is determined by programs of gene expression, which are strictly regulated spatio-temporally by a complex network of interacting molecular mechanisms that control the balance between transcription, translation and degradation. In order to elucidate the mechanisms underlying these processes we currently study three proteins: the Drosophila transcription factor Yan, its human orthologue, Tel (ETV6) and a protein that we recently discovered named Mae (Modulator of the Activity of Ets). We study both invertebrate and vertebrate homologues of proteins in order to establish how cellular and biochemical processes have been refined over evolutionary time.
Yan and Tel belong to the Ets family of transcription factors that orchestrate cell growth and differentiation. Genetic analyses of Tel (Wang et al 1997; Wang et al. 1998; Hock et al. 2004) and its Drosophila orthologue Yan (Lai and Rubin, 1992; Rebay and Rubin, 1995; O’Neill et al. 1994; Rogge et al. 1995) have yielded compelling evidence that these proteins are unique Ets repressors that are crucial regulators of progenitor cell differentiation. However, the exact nature of repression by Tel/Yan is incompletely defined. In Drosophila, a protein named Mae (modulator of the activity of Ets) mediates Yan derepression by binding to Yan thereby disrupting Yan self-association and binding to DNA, and sensitizing it to MAPK-dependent down regulation (Baker et al. 2001; Tootle et al. 2003; Qiao et al. 2004). To date, a similar mechanism of regulation of Tel has not been uncovered in vertebrates.
Deciphering the precise mechanisms of action of Yan/Tel is important for understanding the control of stem cell differentiation and tissue patterning. Moreover, perturbation of normal Tel function is an important contributor to the development of cancers, especially leukemias in which at least twenty-two translocations involving Tel have been reported.
Our group takes a multidisciplinary approach encompassing "forward" and "reverse" genetics in Drosophila, functional analyses in human cells and recently zebrafish embryo development, to address three questions:
- What are the mechanisms by which Yan and Tel repress transcription?
- How are Yan and Tel controlled post-translationally? Is Tel activity controlled by a conserved Yan/Mae-like mechanism?
- How do Yan and Tel orchestrate appropriate cell differentiation in vivo
A model is emerging that suggests that monomers of Tel or Yan directly self-associate via their conserved SAM domains and the resulting DNA-bound oligomers (currently of indeterminate length) act as a physical barrier to the transcription-activating apparatus (reviewed in Qiao and Bowie, 2005; Tootle and Rebay, 2005; Vivekanand and Rebay, 2006). In order for cells to differentiate, first they must remove this barrier which (in the case of Drosophila Yan is achieved through phosphorylation and subsequent downregulation of Yan by the Ras/MAPK/Mae pathway). We have uncovered a novel mechanism of control of repression by Tel (and Yan?) that involves PIAS (Protein Inhibitor of Activated STAT) proteins, and are currently determining how Tel/Yan and PIAS collaboratively regulate gene expression.2) Post-translational regulation of Yan and Tel
In order for cells to develop normally the activity of proteins must be carefully controlled, such that they are "switched on" and "off" at the right time. This is particularly important for proteins such as Yan and Tel that function as a barrier to differentiation. We have performed a yeast-2-hybrid screen of proteins that associate with Yan and Tel. By this means we have identified a novel F-box containing protein that associated with both Tel and Yan. F box proteins bind protein substrates and couple them to a conserved ubiquitinating machinery that generally promotes degradation of the substrate. We have established that both Tel and Yan are ubiquitinated and that this process is F-box protein dependent. We are we are now elucidating how this mechanism contributes to Yan/Tel function.
It is now firmly established that multiple covalent modifications of histone tails, many of which occur on lysine residues, play a crucial role in gene expression. More recent work now suggests that modification of non-histone regulatory proteins such as transcription factors will play an equally important role in directly modulating transcription.. We are investigating the role of post-translational modifications in the function of Yan and Tel using a variety of techniques including mass spectrometry and have identified a number of such changes that are indispensable for normal Yan/Tel function.3) How do Yan and Tel orchestrate appropriate cell differentiation in vivo
Our long term goal is to determine the role of Yan/Tel in progenitor cell differentiation and proliferation in vivo. We take an evolutionary view of Yan/Tel function and to that end we exploit the power of forward and reverse genetics in the Drosophila melanogaster model system. As a complementary approach we are now establishing the vertebrate Zebrafish model system in the lab as well as human and mouse cell-based differentiation assays. Since perturbation of normal Tel function is an important contributor to the development of cancers especially leukemias, we are also using primary human leukemic blast cells to investigate how of corruption of the activity of Tel can lead to neoplasia.