Functional characterization of dASCIZ in gene expression and development in Drosophila melanogaster
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Abstract
A protein may perform various functions inside a cell through multiple signaling networks and help in fine-tuning cellular activities required to maintain complex physiology. A large number of proteins have been described that act as both stress response proteins as well as transcription factors in gene expression related to development. These functions may or may not be interlinked depending on the protein under consideration. For example, Sp1 was initially described to act as a transcription factor regulating the expression of a large number of essential genes. Later, it was also implicated in DNA damage response and in recent studies, has been shown to be a substrate for ATM-kinase-mediated phosphorylation with kinetics comparable to H2AX. Deletion of Sp1 sensitizes the cell to DNA damage induced by ionizing radiation. Like Sp1, Atmin (or ASCIZ) is another such protein with roles in both stress response and development. Atmin defines an NBS1 independent arm of ATM-kinase signaling under conditions of non-IR stress like hypotonic stress or oxidative stress. Studies from the knockout mouse work shows Atmin plays a crucial role in vivo as a regulator of organ development during embryonic growth that is independent of its function in stress response. However, analysis of its role in post-embryonic development has not been possible as murine mutants studied so far, are embryonic lethals. In this study, we have characterized an EP- insertion line EP(3)3709 as dASCIZ mutant and used it to investigate the roles of the protein during post-embryonic stages of 3rd larval instar and metamorphosis (equivalent of adult development in sexually mature humans). EP(3)3709 homozygotes survived till the pupal phase but did not eclose as adult flies. Mutant pupae displayed impaired metamorphosis with majority of the population showing little or no morphogenesis of adult head, wings, legs and abdomen. Expression of key ecdysone inducible genes including Broad Complex was significantly reduced in the mutant pupae compared to WT controls. Disruption of dASCIZ also affects DNA synthesis and cell division in mutant brains resulting in small size than the WT counterparts. The mutant larvae displayed a more transparent appearance than the WT controls suggesting a possible reduction in fat tissue. The increased transparency of the mutant larvae coupled with a complete failure to eclose suggest possible fat body malfunction and energy deficits. The lethality associated with the EP(3)3709 homozygotes could be rescued by ectopically expressing the transgenic reporter protein dASCIZ-HA-mCitrine by the Act5C-Gal4 driver. This observation first established, EP(3)3709 is a true mutant allele of dASCIZ. Second, it indicated, in absence of suitable antibodies capable of detecting endogenous dASCIZ, the behavior of the transgenic reporter protein dASCIZ-HA-mCitrine could be studied to predict the possible roles of the endogenous protein. Exploiting the presence of tags on the transgenic reporter protein (HA or Hemagglutinin in our case), the chromatin association property of dASCIZ-HA-mCitrine was analyzed by polytene chromosomes squash using anti-HA antibody. dASCIZ-HA-mCitrine localized braodly to the interbands regions of polytene chromosomes with an extensive overlap of signal with active RNApolII at multiple sites. In addition, dASCIZ-HA-mCitrine also showed a characteristic staining pattern in localization on polytene chromosomes before and after heat shock.
Analysis of roles of dASCIZ in metamorphosis and in the preceding stage of 3rd larval instar will give us a more holistic overview of its range of functions in a fruitfly model. Also our studies may help predict the involvement of its human counterpart in adult development.
On a broader scale, an understanding of the pathways involved in the two seemingly independent branches of Atmin function, DNA damage response and development, using different mutants alleles in different model organisms will provide a more detailed picture about the regulation of proteins with dual functions in gene expression and the DNA damage response.