1. Purification of chromatin modifiers and structural chromatin complexes (people: HP1-Jochen Abel, Andreas Thomae, Irene Vetter. HMTs-Jörn Boeke, Simone Vollmer)
We have isolated three histone methyltransferase complexes that are involved in the establishment of stable histone methylation marks. Two belonging to the system involved in the regulation of homeobox gene and one that plays a crucial role in heterochromatin formation. The latter one contains the previously identified methyltransferase SU(VAR)3-9 in a complex with the deacetylase Rpd3. Our findings that the spreading of heterochromatin requires the concerted action of a deacetylase as well as a methyltransferase provided a first model of how chromatin spreading may occur at a molecular level. To further characterise the enzymatic properties of dSU(VAR)3-9, we expressed and purified the enzyme in bacteria and analysed the requirements of various protein domains for full catalytic capacity. It turned out that the N-terminus of SU(VAR)3-9 mediates dimerisation of the enzyme, which significantly increases its ability to methylate histones in vitro. This N-terminal region is also interacting with a variety of other heterochromatic molecules such as HP-1 and dSU(VAR)3-7 making it a likely candidate for a regulatory domain.
2. Mass spectrometry of histone modifications (people: Ana Villar-Garea, Tilman Schlunk)
In order to quantitatively study global changes in histone modifications, we have developed methods to use mass spectrometry (MS) to fully describe the posttranslational modification pattern of bulk histones isolated from native sources such as Drosophila embryos of different developmental stages. The use of MS also proved to be extremely helpful when analysing enzymatic activities in vitro and determine their specificity. In order to study the effect of distinct chomatin structures on embryonic development and cellular differentiation, we identified several small molecules that inhibit histone methyltransferases in vitro and in vivo. One of the identified inhibitors specifically interferes with SU(VAR)3-9 activity and reduces the level of lysine 9 methylation in vivo.
3. Chromatomics (people: Ignasi Forne, Julian Hupf, Viola Sansoni)
We will continue to use mass spectrometry to study histone modifications. However, we will extend the analyses to study modification patterns within specific chromatin domains such as the centromere or the telomere. These methods will allow us to study specific histone modification patterns in an unbiased and sensitive manner. Furthermore the use of stable isotope labelling with amino acids in cell culture (SILAC) methods will help us to dissect the dynamic aspect of the establishment and maintenance of histone modifications in living cells by pulse labelling of the newly synthesized histones and following their fate before and chromatin assembly. In order to achieve the highest possible resolution to distinguish for example between histone acetylation and trimethylation or to get a better identification of isolated peptides we will need a new high resolution mass spectrometer with MSn capabilities such as a LTQ Orbitrap Hybrid mass spectrometer (Thermo Electron).
4. Small Molecules that target epigenetic modifiers (people: Ana Villar-Garea, Simone Vollmer)
We will use the inhibitor that we isolated to study the kinetics of heterochromatin formation by analysing the binding of heterochromatin specific proteins and heterochromatin specific histone modification patterns following treatment with a histone methyltransferase inhibitor. We will look for further regulatory molecules that target epigenetic regulators in collaboration with a pharmaceutical company, Chroma Therapeutics in Cambridge. One of the central goals of this collaboration is the generation of small molecules that will enable researchers to generate specific stem cells in the test tube with a much higher efficiency making stem cell therapies more likely to be successful.