Kent G. Golic

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

ABSTRACT 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.

Loss of Hsp70 in drosophila is pleiotropic, with effects on thermotolerance, recovery from heat shock and neurodegeneration

The heat-shock response is a programmed change in gene expression carried out by cells in response to environmental stress, such as heat. This response is universal and is characterized by the synthesis of a small group of conserved protein chaperones. In Drosophila melanogaster the Hsp70 chaperone dominates the profile of protein synthesis during the heat-shock response. We recently generated precise deletion alleles of the Hsp70 genes of D. melanogaster and have used those alleles to characterize the phenotypes of Hsp70-deficient flies. Flies with Hsp70 deletions have reduced thermotolerance. We find that Hsp70 is essential to survive a severe heat shock, but is not required to survive a milder heat shock, indicating that a significant degree of thermotolerance remains in the absence of Hsp70. However, flies without Hsp70 have a lengthened heat-shock response and an extended developmental delay after a non-lethal heat shock, indicating Hsp70 has an important role in recovery from stress, even at lower temperatures. Lack of Hsp70 also confers enhanced sensitivity to a temperature-sensitive lethal mutation and to the neurodegenerative effects produced by expression of a human polyglutamine disease protein.

Safeguarding gene drive experiments in the laboratory

Multiple stringent confinement strategies should be used whenever possible Gene drive systems promote the spread of genetic elements through populations by assuring they are inherited more often than Mendelian segregation would predict (see the figure). Natural examples of gene drive from Drosophila include sex-ratio meiotic drive, segregation distortion, and replicative transposition. Synthetic drive systems based on selective embryonic lethality or homing endonucleases have been described previously in Drosophila melanogaster (1–3), but they are difficult to build or are limited to transgenic populations. In contrast, RNAguided gene drives based on the CRISPR/Cas9 nuclease can, in principle, be constructed by any laboratory capable of making transgenic organisms (4). They have tremendous potential to address global problems in health, agriculture, and conservation, but their capacity to alter wild populations outside the laboratory demands caution (4–7). Just as researchers working with self-propagating pathogens must ensure that these agents do not escape to the outside world, scientists working in the laboratory with gene drive constructs are responsible for keeping them confined (4, 6, 7).

Telomere Loss Provokes Multiple Pathways to Apoptosis and Produces Genomic Instability in Drosophila melanogaster

Telomere loss was produced during development of Drosophila melanogaster by breakage of an induced dicentric chromosome. The most prominent outcome of this event is cell death through Chk2 and Chk1 controlled p53-dependent apoptotic pathways. A third p53-independent apoptotic pathway is additionally utilized when telomere loss is accompanied by the generation of significant aneuploidy. In spite of these three lines of defense against the proliferation of cells with damaged genomes a small fraction of cells that have lost a telomere escape apoptosis and divide repeatedly. Evasion of apoptosis is accompanied by the accumulation of karyotypic abnormalites that often typify cancer cells, including end-to-end chromosome fusions, anaphase bridges, aneuploidy, and polyploidy. There was clear evidence of bridge–breakage–fusion cycles, and surprisingly, chromosome segments without centromeres could persist and accumulate to high-copy number. Cells manifesting these signs of genomic instability were much more frequent when the apoptotic mechanisms were crippled. We conclude that loss of a single telomere is sufficient to generate at least two phenotypes of early cancer cells: genomic instability that involves multiple chromosomes and aneuploidy. This aneuploidy may facilitate the continued escape of such cells from the normal checkpoint mechanisms.

Imprinted control of gene activity in Drosophila

Genetic imprinting is defined as a reversible, differential marking of genes or chromosomes that is determined by the sex of the parent from whom the genetic material is inherited [1]. Imprinting was first observed in insects where, in some species, most notably among the coccoids (scale insects and allies), the differential marking of paternally and maternally transmitted chromosome sets leads to inactivation or elimination of paternal chromosomes [2]. Imprinting is also widespread in plants and mammals [3,4], in which paternally and maternally inherited alleles may be differentially expressed. Despite imprinting having been discovered in insects, clear examples of parental imprinting are scarce in the model insect species Drosophila melanogaster. We describe a case of imprint-mediated control of gene expression in Drosophila. The imprinted gene - the white+ eye-color gene - is expressed at a low level when transmitted by males, and at a high level when transmitted by females. Thus, in common with coccoids, Drosophila is capable of generating an imprint, and can respond to that imprint by silencing the paternal allele.

Highly Efficient Sex Chromosome Interchanges Produced By I-CreI Expression in Drosophila

The homing endonuclease I-CreI recognizes a site in the gene encoding the 23S rRNA of Chlamydomonas reinhardtii. A very similar sequence is present in the 28S rRNA genes that are located on the X and Y chromosomes of Drosophila melanogaster. In this work we show that I-CreI expression in Drosophila is capable of causing induced DNA damage and eliciting cell cycle arrest. Expression also caused recombination between the X and Y chromosomes in the heterochromatic regions where the rDNA is located, presumably as a result of a high frequency of double-strand breaks in these regions. Approximately 20% of the offspring of males expressing I-CreI showed exceptional inheritance of X- and Y-linked markers, consistent with chromosome exchange at rDNA loci. Cytogenetic analysis confirmed the structures of many of these products. Exchange between the X and Y chromosomes can be induced in males and females to produce derivative-altered Y chromosomes, attached-XY, and attached-X chromosomes. This method has advantages over the traditional use of X rays for generating X-Y interchanges because it is very frequent and it generates predictable products.

Evolution of Gene Sequence in Response to Chromosomal Location

Evolutionary forces acting on the repetitive DNA of heterochromatin are not constrained by the same considerations that apply to protein-coding genes. Consequently, such sequences are subject to rapid evolutionary change. By examining the Troponin C gene family of Drosophila melanogaster, which has euchromatic and heterochromatic members, we find that protein-coding genes also evolve in response to their chromosomal location. The heterochromatic members of the family show a reduced CG content and increased variation in DNA sequence. We show that the CG reduction applies broadly to the protein-coding sequences of genes located at the heterochromatin:euchromatin interface, with a very strong correlation between CG content and the distance from centric heterochromatin. We also observe a similar trend in the transition from telomeric heterochromatin to euchromatin. We propose that the methylation of DNA is one of the forces driving this sequence evolution.

BIOSAFETY. Safeguarding gene drive experiments in the laboratory.

Multiple stringent confinement strategies should be used whenever possible Gene drive systems promote the spread of genetic elements through populations by assuring they are inherited more often than Mendelian segregation would predict (see the figure). Natural examples of gene drive from Drosophila include sex-ratio meiotic drive, segregation distortion, and replicative transposition. Synthetic drive systems based on selective embryonic lethality or homing endonucleases have been described previously in Drosophila melanogaster (1–3), but they are difficult to build or are limited to transgenic populations. In contrast, RNAguided gene drives based on the CRISPR/Cas9 nuclease can, in principle, be constructed by any laboratory capable of making transgenic organisms (4). They have tremendous potential to address global problems in health, agriculture, and conservation, but their capacity to alter wild populations outside the laboratory demands caution (4–7). Just as researchers working with self-propagating pathogens must ensure that these agents do not escape to the outside world, scientists working in the laboratory with gene drive constructs are responsible for keeping them confined (4, 6, 7).

RNA-Guided Nucleases: A New Era for Engineering the Genomes of Model and Nonmodel Organisms

In 1946, H. J. Muller was awarded the Nobel Prize in Medicine for his discovery with Drosophila melanogaster , some 20 years earlier, that exposure to X-rays caused mutation ([Muller 1927][1]). This led to the identification of a large number of Drosophila mutants and chromosome rearrangements, many

The NADPH Metabolic Network Regulates Human αB-crystallin Cardiomyopathy and Reductive Stress in Drosophila melanogaster

Dominant mutations in the alpha-B crystallin (CryAB) gene are responsible for a number of inherited human disorders, including cardiomyopathy, skeletal muscle myopathy, and cataracts. The cellular mechanisms of disease pathology for these disorders are not well understood. Among recent advances is that the disease state can be linked to a disturbance in the oxidation/reduction environment of the cell. In a mouse model, cardiomyopathy caused by the dominant CryABR120G missense mutation was suppressed by mutation of the gene that encodes glucose 6-phosphate dehydrogenase (G6PD), one of the cells primary sources of reducing equivalents in the form of NADPH. Here, we report the development of a Drosophila model for cellular dysfunction caused by this CryAB mutation. With this model, we confirmed the link between G6PD and mutant CryAB pathology by finding that reduction of G6PD expression suppressed the phenotype while overexpression enhanced it. Moreover, we find that expression of mutant CryAB in the Drosophila heart impaired cardiac function and increased heart tube dimensions, similar to the effects produced in mice and humans, and that reduction of G6PD ameliorated these effects. Finally, to determine whether CryAB pathology responds generally to NADPH levels we tested mutants or RNAi-mediated knockdowns of phosphogluconate dehydrogenase (PGD), isocitrate dehydrogenase (IDH), and malic enzyme (MEN), the other major enzymatic sources of NADPH, and we found that all are capable of suppressing CryABR120G pathology, confirming the link between NADP/H metabolism and CryAB.