UCSF

In preparation for the dramatic morphogenetic events of gastrulation, rapid embryonic cell cycles slow at the mid-blastula transition (MBT). In Drosophila embryos the MBT involves the onset of key properties that distinguish the highly repetitive satellite sequences composing the heterochromatin from the rest of the genome. In early embryos the satellite DNA lacks the characteristic heterochromatic features of late replication and repressive chromatin modifications, like H3K9 methylation and HP1a. My thesis work has explored how the embryo controls the restoration of these conserved features to the heterochromatin. I describe and analyze the results of experiments employing a combination of fly genetics and real time microscopy on developing embryos. At the MBT, down-regulation of Cyclin:Cdk1 activity initiates cell cycle slowing by delaying replication of satellite DNA and extending S phase. I show that Cdk1 activity inhibits the chromatin association of Rif1, a repressor of origin firing. Following Cdk1 down-regulation at the MBT, Rif1 bound selectively to satellite sequences. In the next S phase, Rif1 dissociated from different satellites in a schedule that anticipated their replication. A mutant version of Rif1 lacking potential phosphorylation sites failed to dissociate and dominantly prevented completion of replication. In contrast, loss of Rif1 shortened the post-MBT S phase and rescued embryonic cell cycles disrupted by depletion of Cdc7. Following the Rif1 mediated introduction of late replication, satellite sequences begin acquiring H3K9 methylation and recruit the protein HP1a. By using a novel protein inactivation approach called JabbaTrapping, I show that the K9 methyltransferase Eggless plays a major role in the establishment of this repressive chromatin state. Additionally, live imaging of Eggless suggests that the rapid early cell cycles prevent this enzyme from introducing heterochromatic marks before the MBT. Experimental manipulation of interphase duration showed that cell cycle speed regulates Eggless. By focusing on the initial events in the de novo formation of heterochromatin my work emphasizes how heterochromatin is integrated into the development of the embryo. Through regulation of the two proteins Rif1 and Egg, the embryonic cell cycle program times the appearance of late replication and repressive chromatin modifications, two widely conserved features of heterochromatin.