Projects
Research projects
Hematopoietic transcription factor complexes
The hematopoietic stem cell (HSC) is responsible for the formation of all of the blood cell types. HSC differentiation involves coordinated and changing transcription, often by functionally conserved genes. For example, in mammals such a set of transcription factors (including Gata2, Tal1, Lmo2, Ldb1, Eto2, Gata1 and Runx1/Aml-1) is required for the differentiation of HSCs.
The laboratory has shown that each of these factors forms a number of different protein complexes that carry out different functions. For example the Gata1 TF forms a number of different complexes which can activate or repress transcription.
We performed single-step purification of transcription factor (TF) complexes in erythroid cells combined with mass spectrometry to characterize the composition of TF complexes. This approach led us to successfully identify new binding partners of essential hematopoietic TFs. These newly identified binding partners have essential roles in hematopoiesis as revealed by morpholino-mediated knock-down experiments in zebrafish embryo. We combine these data with genome-wide identification of TF binding sites using ChIP-Seq technology (chromatin immunoprecipitation coupled to high throughput sequencing) in order to map the different complexes to different gene loci. Taken altogether, these data lead us to build a comprehensive map of TF interactions and gene activation/repression during the course of hematopoietic cells differentiation.
Globin gene switching
The β globin genes are arranged in the same order in the β globin locus as their order of expression during development. The expression of the different genes at different stages is known as globin gene switching. The locus is characterized by a number of 3 dimensional interactions between different regulatory regions and these interactions change during development. β thalassemia and sickle cell anaemia which together are the most widespread genetic disease in the world, are usually characterized by mutations in or deletions of the adult β gobin gene, leaving the remainder of the locus unaltered. One of the primary aims of the laboratory is to understand the switch from the human foetal γ genes to the adult β gene with the aim to reverse this switch in patients. A number of different strategies are exploited to achieve this aim. In addition to the characterisation of transcription factor complexes (see above) a number of in vivo and functional approaches are also in progress. The first is crosslinking all of the protein complexes to the β globin promoter in vivo when the gene is silenced followed by the purification of the promoter and the characterisation of the factors by mass spectrometry. Subsequent functional studies will determine which of the factors are important for the silencing of the genes using mouse and zebrafish.
The second approach is purely based on function and involves a screen for β globin gene activation in reporter cells using shRNA libraries or heavy chain only antibody libraries. We are also collaborating with a number of laboratories to determine why patients react differently to hydroxyurea treatment using expression studies.
Finally we are interested in the three dimensional structure of complex gene loci in vivo and in particular that of the β globin locus as a model system. To that end we use 3C, 4C and ChIP methods in combination with confocal microscopy, 4pi microscopy and spatially modulated illumination (SMI) microscopy.
