Person:

Joyce, Eric F.

The pairing of homologous chromosomes is a fundamental feature of the meiotic cell. In addition, a number of species exhibit homolog pairing in nonmeiotic, somatic cells as well, with evidence for its impact on both gene regulation and double-strand break (DSB) repair. An extreme example of somatic pairing can be observed in Drosophila melanogaster, where homologous chromosomes remain aligned throughout most of development. However, our understanding of the mechanism of somatic homolog pairing remains unclear, as only a few genes have been implicated in this process. In this study, we introduce a novel high-throughput fluorescent in situ hybridization (FISH) technology that enabled us to conduct a genome-wide RNAi screen for factors involved in the robust somatic pairing observed in Drosophila. We identified both candidate “pairing promoting genes” and candidate “anti-pairing genes,” providing evidence that pairing is a dynamic process that can be both enhanced and antagonized. Many of the genes found to be important for promoting pairing are highly enriched for functions associated with mitotic cell division, suggesting a genetic framework for a long-standing link between chromosome dynamics during mitosis and nuclear organization during interphase. In contrast, several of the candidate anti-pairing genes have known interphase functions associated with S-phase progression, DNA replication, and chromatin compaction, including several components of the condensin II complex. In combination with a variety of secondary assays, these results provide insights into the mechanism and dynamics of somatic pairing.

Drosophila PR-Set7 or SET8 is a histone methyltransferase that specifically monomethylates histone H4 lysine 20 (H4K20). L(3)MBT has been identified as a reader of methylated H4K20. It contains several conserved domains including three MBT repeats binding mono- and dimethylated H4K20 peptides. We find that the depletion of PR-Set7 blocks de novo H4K20me1 resulting in the immediate activation of the DNA damage checkpoint, an increase in the size of interphase nuclei, and drastic reduction of cell viability. L(3)mbt on the other hand stabilizes the monomethyl mark, as L(3)mbt-depleted S2 cells show a reduction of more than 60% of bulk monomethylated H4K20 (H4K20me1) while viability is barely affected. Ploidy and basic chromatin structure show only small changes in PR-Set7-depleted cells, but higher order interphase chromatin organization is significantly affected presumably resulting in the activation of the DNA damage checkpoint. In the absence of any other known functions of PR-Set7, the setting of the de novo monomethyl mark appears essential for cell viability in the presence or absence of the DNA damage checkpoint, but once newly assembled chromatin is established the monomethyl mark, protected by L(3)mbt, is dispensable.