Numerous Serine/Threonine Kinases Affect Blood Cell Homeostasis in Drosophila melanogaster.

Blood cells in Drosophila serve primarily innate immune responses. Various stressors influence blood cell homeostasis regarding both numbers and the proportion of blood cell types. The principle molecular mechanisms governing hematopoiesis are conserved amongst species and involve major signaling pathways like Notch, Toll, JNK, JAK/Stat or RTK. Albeit signaling pathways generally rely on the activity of protein kinases, their specific contribution to hematopoiesis remains understudied. Here, we assess the role of Serine/Threonine kinases with the potential to phosphorylate the transcription factor Su(H) in crystal cell homeostasis. Su(H) is central to Notch signal transduction, and its inhibition by phosphorylation impedes crystal cell formation. Overall, nearly twenty percent of all Drosophila Serine/Threonine kinases were studied in two assays, global and hemocyte-specific overexpression and downregulation, respectively. Unexpectedly, the majority of kinases influenced crystal cell numbers, albeit only a few were related to hematopoiesis so far. Four kinases appeared essential for crystal cell formation, whereas most kinases restrained crystal cell development. This group comprises all kinase classes, indicative of the complex regulatory network underlying blood cell homeostasis. The rather indiscriminative response we observed opens the possibility that blood cells measure their overall phospho-status as a proxy for stress-signals, and activate an adaptive immune response accordingly.

Phospho-Site Mutations in Transcription Factor Suppressor of Hairless Impact Notch Signaling Activity During Hematopoiesis in Drosophila.

The highly conserved Notch signaling pathway controls a multitude of developmental processes including hematopoiesis. Here, we provide evidence for a novel mechanism of tissue-specific Notch regulation involving phosphorylation of CSL transcription factors within the DNA-binding domain. Earlier we found that a phospho-mimetic mutation of the Drosophila CSL ortholog Suppressor of Hairless [Su(H)] at Ser269 impedes DNA-binding. By genome-engineering, we now introduced phospho-specific Su(H) mutants at the endogenous Su(H) locus, encoding either a phospho-deficient [Su(H) S269A ] or a phospho-mimetic [Su(H) S269D ] isoform. Su(H) S269D mutants were defective of Notch activity in all analyzed tissues, consistent with impaired DNA-binding. In contrast, the phospho-deficient Su(H) S269A mutant did not generally augment Notch activity, but rather specifically in several aspects of blood cell development. Unexpectedly, this process was independent of the corepressor Hairless acting otherwise as a general Notch antagonist in Drosophila. This finding is in agreement with a novel mode of Notch regulation by posttranslational modification of Su(H) in the context of hematopoiesis. Importantly, our studies of the mammalian CSL ortholog (RBPJ/CBF1) emphasize a potential conservation of this regulatory mechanism: phospho-mimetic RBPJ S221D was dysfunctional in both the fly as well as two human cell culture models, whereas phospho-deficient RBPJ S221A rather gained activity during fly hematopoiesis. Thus, dynamic phosphorylation of CSL-proteins within the DNA-binding domain provides a novel means to fine-tune Notch signal transduction in a context-dependent manner.

Drosophila Xrcc2 regulates DNA double-strand repair in somatic cells.

Genomic integrity is challenged by endo- and exogenous assaults that are combated by highly conserved DNA repair mechanisms. Repair of DNA double-strand breaks (DSBs) is of particular importance, as DSBs inflict chromosome breaks that are the basis of genomic instability. High fidelity recombination repair of DSBs relies on the Rad51 recombinase, aided by several Rad51 paralogs. Despite their significant contribution to DSB repair, the individual roles for Rad51 paralogs are incompletely understood. Drosophila serves as a metazoan model for DNA damage repair at the organismal level. Yet, only two out of four Rad51 paralogs have been studied so far and both are restricted to meiotic recombination repair. Using CRISPR/Cas9 technology, we have generated the first X-ray repair cross complementing 2 (xrcc2) null mutant in Drosophila. Like any other Drosophila Rad51 homologue, loss of xrcc2 does not affect fly development. We found that Drosophila xrcc2 - despite a specific expression in ovaries - is not essential for meiotic DSB repair, but supports the process. In contrast, xrcc2 is required for mitotic DNA damage repair: the mutants are highly sensitive towards various genotoxic stressors, including ionizing radiation, which significantly increase mortality. Moreover, loss of xrcc2 provokes chromosome aberrations in mitotic larval neuroblasts under unstressed conditions and enduring chromosomal breaks as well as persistent repair foci after irradiation exposure. Together these results demonstrate that xrcc2 plays a crucial role in combating genotoxic insult by controlling DSB repair in somatic cells of Drosophila.