Heterochromatin-induced gene silencing in Drosophila
Final Report Abstract
The project was based on molecular cloning of the Su(var)3-9 gene, a key factor of heterochromatic gene silencing. SU(VAR)3-9 is the major heterochromatin specific H3K9 methyltransferase. We further discovered functional interdependence in heterochromatin association of SU(VAR)3-9 and HP1. The HP1 protein binds H3K9me2/me3 and interacts with the SU(VAR)3-9 HMTase to ensure its heterochromatin association. In the project we performed thorough enzymatic and functional analysis of SU(VAR)3-9 using a large collection of more than 40 mutations. Clonal analysis in Su(var)3-9 null mutants indicated early embryonic establishment of heterochromatic gene silencing and stable maintenance throughout development. We furthermore demonstrated a role of E(Z) dependent H3K27 methylation in heterochromatic gene silencing. By molecular analysis of new Su(var) genes an essential role of H4K20me3 in heterochromatic gene silencing was found. The studies revealed a molecular pathway of heterochromatic gene silencing which is conserved between Drosophila and mammals. H3K9 methylation by SU(VAR)3-9 controls through interaction with HP1 consecutive H4K20 trimethylation in pericentric heterochromatin which is catalyzed by the SUV4-20 HMTase. Molecular characterization of Su(var)3-1 mutations finally identified a role of the JIL-1 kinase in controlling the balance between eu- and heterochromatic chromatin states at the breakpoint of PEV rearrangements. Spreading of heterochromatin into euchromatic regions requires an intact C-terminus of JIL-1, which is probably involved in control of H3S10 de-phosphorylation. With molecular cloning of Su(var)3-3 the Drosophila homologue of the mammalian LSD1 demethylase was identified and an essential role of this demethylase in establishment of heterochromatin demonstrated using the Drosophila model. Developmental analysis revealed early embryonic establishment of heterochromatin by the SU(VAR)3-3/RPD3/SU(VAR)3-9/HP1 protein complex which probably also contains the SU(VAR)3-7 and SU(VAR)2-1 proteins as shown by more recent studies. SUV4-20 and H4K20me3 are recruited subsequently through interaction with HP1 and might play an important role as maintenance function. It is surely interesting to resolve in consecutive work how the other about 80 molecularly unknown Su(var) genes are involved in these processes. Based on previous genetic analysis of Su(var) genes and the new genetic tools generated by our work in the DrosDel EU project an efficient methodology for molecular isolation of the whole Su(var) gene complement in Drosophila could be established including mainly duplication/deficiency and SNP mapping.
