Epigenetic control of pluripotency in Drosophila primordial and ovarian germ line stem cells
Entwicklungsbiologie
Zusammenfassung der Projektergebnisse
In our project we studied epigenetic control of lineage commitment underlying development of primordial germ line stem cell (PGCs) and of adult ovarian stem cells (GSCs). Chromatin of cleavage nuclei is transcriptionally inert, shows abundant histone acetylation (H3K9ac, H4K8ac and H4K14ac) and low levels of histone methylation. Chromatin differentiation in blastoderm nuclei is initiated at cycle 9-10 in early embryogenesis by global deacetylation and polar differentiation of eu- and heterochromatin. In PGCs histone deacetylation and H3K4 di- and tri-methylation is significantly delayed and initiated at the end of germ cell migration. Like somatic nuclei PGCs nuclei show polar chromatin differentiation but pericentromeric heterochromatin forms no chromocenter. Chromatin indexing in PGCs suggests that transcriptional quiescence is controlled in addition at the chromatin level. Most abundant histone modifications in PGCs are H3K4me1, H3K9ac, H3K36me2, H3K27me1, H3K27me2 and H3K27me3. No staining in PGCs is found for H3K4me2 and H3K4me3, two histone modification marks associated with active genes. The histone demethylase LSD1 has a protective function against H3K4me4 in PGCs. LSD1 and the methyltransferases E(Z) and SETDB1 are required for maintenance of stemness. RNAi down regulation of E(z), SetDB1 and Su(var)2-5 (HP1) impairs GSC development. Ecdysone signaling controls chromatin differentiation in blastoderm and PGC nuclei. For genetic dissection of chromatin regulation in PGCs we established a novel monitoring system for silencing processes in female germ line cells. The system is based on a collection of transgene inserts for the female germ line specific fs(1)K10 gene. In these studies the functional role of the basic chromatin proteins HP1, SU(VAR)3-9, SetDB1, LSD1, LID, SUV4-20, E(Z), PC, JIL1, SU(VAR)2-1 and PIWI in germ line chromatin regulation was revolved. Several of these factors induce intergenerational effects. A second set of genetic tools isolated represent chromosomal rearrangements juxtaposing the fs(1)K10 gene to pericentromeric heterochromatin. The rearrangements revealed a completely different epigenetic control of pericentric heterochromatin in PGCs. With SU(VAR)2-1 we identified a novel regulator of genome wide histone deacetylation. Su(var)2-1 mutations strongly impair heterochromatic gene silencing and global gene expression. Our data suggest that SU(VAR)2-1 controls recruitment of histone deacetylases. Its function is essential for stem cell and female germ line development. The Su(var)2-1 gene is highly conserved in insects and crustaceans but it still remains to be resolved whether functional orthologs in higher organisms are found.
