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Humpty dumpty defines a new gene family required for S phase

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  • Biology
  • Genetics
  • Biology


The full complement of proteins required for the proper regulation of genome duplication are yet to be described. Developmental amplification of eggshell (chorion) genes during Drosophila oogenesis is a genetic model system for the cell cycle regulation of DNA replication. Here I use this system to molecularly identify and characterize the gene corresponding to the thin eggshell mutant fs(3)272-9, which I have named humpty dumpty (hd). ^ Analysis of Hd protein sequence indicates that it is conserved from plants to humans, but the function of this gene family is unknown. In Drosophila , expression of Hd protein peaks during late G1 and S phase, and it responds to the E2F1/Dp transcription factor. Published microarrays showed that expression of the human hd ortholog also peaks at G1/S, indicating that both its sequence and cell cycle expression are conserved. ^ I show that hd is essential for DNA amplification in the Drosophila ovary, and that it also is required for genomic DNA replication during development. Developmental mosaic analysis of hd mutants, and RNAi in Kc cells, reveal that cells depleted of Hd fail to proliferate. These cells have incomplete genomic replication, suffer DNA damage, and accumulate at G2/M likely due to activation of an incomplete replication checkpoint. Confocal analysis indicates that most Hd protein is found in numerous nuclear foci that do not co-localize with chromatin. Some of these foci span the nuclear envelope, while others are in the cytoplasm, suggesting that it may be transported in and out of the nucleus. ^ Purification of proteins that physically interact with Hd identified RNA splicing and translation factors, among others. Immunofluorescent microscopy indicates that some Hd nuclear bodies do indeed contain RNA splicing proteins, and genetic experiments indicate that hd thin eggshell alleles are synthetically lethal with a few splicing and replication mutants. ^ The data suggest that hd defines a new gene family likely required for S phase in all multi-cellular eukaryotes. Its cellular location and association with proteins involved in RNA metabolism hint that a further investigation into the function of hd may unveil a previously unknown mechanism for the control of genome duplication. ^

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