Epigenetic telomere protection by Drosophila DNA damage response pathways.

Analysis of terminal deletion chromosomes indicates that a sequence-independent mechanism regulates protection of Drosophila telomeres. Mutations in Drosophila DNA damage response genes such as atm/tefu, mre11, or rad50 disrupt telomere protection and localization of the telomere-associated proteins HP1 and HOAP, suggesting that recognition of chromosome ends contributes to telomere protection. However, the partial telomere protection phenotype of these mutations limits the ability to test if they act in the epigenetic telomere protection mechanism. We examined the roles of the Drosophila atm and atr-atrip DNA damage response pathways and the nbs homolog in DNA damage responses and telomere protection. As in other organisms, the atm and atr-atrip pathways act in parallel to promote telomere protection. Cells lacking both pathways exhibit severe defects in telomere protection and fail to localize the protection protein HOAP to telomeres. Drosophila nbs is required for both atm- and atr-dependent DNA damage responses and acts in these pathways during DNA repair. The telomere fusion phenotype of nbs is consistent with defects in each of these activities. Cells defective in both the atm and atr pathways were used to examine if DNA damage response pathways regulate telomere protection without affecting telomere specific sequences. In these cells, chromosome fusion sites retain telomere-specific sequences, demonstrating that loss of these sequences is not responsible for loss of protection. Furthermore, terminally deleted chromosomes also fuse in these cells, directly implicating DNA damage response pathways in the epigenetic protection of telomeres. We propose that recognition of chromosome ends and recruitment of HP1 and HOAP by DNA damage response proteins is essential for the epigenetic protection of Drosophila telomeres. Given the conserved roles of DNA damage response proteins in telomere function, related mechanisms may act at the telomeres of other organisms.

Drosophila atm/telomere fusion is required for telomeric localization of HP1 and telomere position effect.

Terminal deletions of Drosophila chromosomes can be stably protected from end-to-end fusion despite the absence of all telomere-associated sequences. The sequence-independent protection of these telomeres suggests that recognition of chromosome ends might contribute to the epigenetic protection of telomeres. In mammals, Ataxia Telangiectasia Mutated (ATM) is activated by DNA damage and acts through an unknown, telomerase-independent mechanism to regulate telomere length and protection. We demonstrate that the Drosophila homolog of ATM is encoded by the telomere fusion (tefu) gene. In the absence of ATM, telomere fusions occur even though telomere-specific Het-A sequences are still present. High levels of spontaneous apoptosis are observed in ATM-deficient tissues, indicating that telomere dysfunction induces apoptosis in Drosophila. Suppression of this apoptosis by p53 mutations suggests that loss of ATM activates apoptosis through a DNA damage-response mechanism. Loss of ATM reduces the levels of heterochromatin protein 1 (HP1) at telomeres and suppresses telomere position effect. We propose that recognition of chromosome ends by ATM prevents telomere fusion and apoptosis by recruiting chromatin-modifying complexes to telomeres.

Drosophila melanogaster MNK/Chk2 and p53 regulate multiple DNA repair and apoptotic pathways following DNA damage.

We have used genetic and microarray analysis to determine how ionizing radiation (IR) induces p53-dependent transcription and apoptosis in Drosophila melanogaster. IR induces MNK/Chk2-dependent phosphorylation of p53 without changing p53 protein levels, indicating that p53 activity can be regulated without an Mdm2-like activity. In a genome-wide analysis of IR-induced transcription in wild-type and mutant embryos, all IR-induced increases in transcript levels required both p53 and the Drosophila Chk2 homolog MNK. Proapoptotic targets of p53 include hid, reaper, sickle, and the tumor necrosis factor family member EIGER: Overexpression of Eiger is sufficient to induce apoptosis, but mutations in Eiger do not block IR-induced apoptosis. Animals heterozygous for deletions that span the reaper, sickle, and hid genes exhibited reduced IR-dependent apoptosis, indicating that this gene complex is haploinsufficient for induction of apoptosis. Among the genes in this region, hid plays a central, dosage-sensitive role in IR-induced apoptosis. p53 and MNK/Chk2 also regulate DNA repair genes, including two components of the nonhomologous end-joining repair pathway, Ku70 and Ku80. Our results indicate that MNK/Chk2-dependent modification of Drosophila p53 activates a global transcriptional response to DNA damage that induces error-prone DNA repair as well as intrinsic and extrinsic apoptosis pathways.

Structure and expression patterns of Drosophila TULP and TUSP, members of the tubby-like gene family.

Tubby is a mouse gene that may provide a model for adult-onset obesity in humans. It is a member of a four gene family in mammals that collectively encode the Tubby-like proteins (TULPs), putative transcription factors which share similar 260 amino acid 'tubby domains' at their C-termini. The mammalian genome also encodes distant relatives of TULPs, which have been called TUSPs (tubby domain superfamily proteins). We have characterized the transcription unit of the single Drosophila TULP homolog, analyzed the expression pattern of the Drosophila TULP and TUSP genes, and determined the evolutionary relationships between the Drosophila proteins and members of the tubby domain superfamily in other organisms. Interestingly, like its mammalian homologs, Drosophila TULP is principally expressed in the embryonic central and peripheral nervous systems. This suggests that mammalian and Drosophila TULPs may possess some conserved functional properties in the nervous system. The Drosophila TUSP gene is also expressed in the central nervous system and olfactory organ but in few other peripheral sensory organs.

The Drosophila Hox gene deformed sculpts head morphology via direct regulation of the apoptosis activator reaper.

Hox proteins control morphological diversity along the anterior-posterior body axis of animals, but the cellular processes they directly regulate are poorly understood. We show that during early Drosophila development, the Hox protein Deformed (Dfd) maintains the boundary between the maxillary and mandibular head lobes by activating localized apoptosis. Dfd accomplishes this by directly activating the cell death promoting gene reaper (rpr). One other Hox gene, Abdominal-B (Abd-B), also regulates segment boundaries through the regional activation of apoptosis. Thus, one mechanism used by Drosophila Hox genes to modulate segmental morphology is to regulate programmed cell death, which literally sculpts segments into distinct shapes. This and other emerging evidence suggests that Hox proteins may often regulate the maintenance of segment boundaries.

Hox protein mutation and macroevolution of the insect body plan.

A fascinating question in biology is how molecular changes in developmental pathways lead to macroevolutionary changes in morphology. Mutations in homeotic (Hox) genes have long been suggested as potential causes of morphological evolution, and there is abundant evidence that some changes in Hox expression patterns correlate with transitions in animal axial pattern. A major morphological transition in metazoans occurred about 400 million years ago, when six-legged insects diverged from crustacean-like arthropod ancestors with multiple limbs. In Drosophila melanogaster and other insects, the Ultrabithorax (Ubx) and abdominal A (AbdA, also abd-A) Hox proteins are expressed largely in the abdominal segments, where they can suppress thoracic leg development during embryogenesis. In a branchiopod crustacean, Ubx/AbdA proteins are expressed in both thorax and abdomen, including the limb primordia, but do not repress limbs. Previous studies led us to propose that gain and loss of transcriptional activation and repression functions in Hox proteins was a plausible mechanism to diversify morphology during animal evolution. Here we show that naturally selected alteration of the Ubx protein is linked to the evolutionary transition to hexapod limb pattern.