Mammalian systems may employ mechanisms similar to Drosophila heterochromatin to regulate the stability of repeated DNAs. Human Alu repeats and heterochromatin on human chromosome 1 (band 1q12) both contain ‘fragile’ sites associated with chromosomal rearrangements found in malignant cancers [60,61]. Vulnerability of these DNA elements is also highly correlated with their chromatin composition [62,63]. A more detailed understanding of how H3K9 methylation helps stabilize Drosophila heterochromatin would help direct efforts to elucidate how repeated DNAs are maintained in mammals. Materials and Methods Fly stocks All fly stocks were raised at 22uC. We received the grp06034, rodEY04576, mit(1)155, and p5311-1B-1 flies from the Bloomington stock center. The lokP6 flies are from Michael Brodsky, dcr2L811fsx from Richard Carthew, mit(1)15S2M from Michael Goldberg, smc1exc461 from Scott Hawley, mei-W684572 from Kim McKim, Su(var)3-9 alleles 6 and 17 from Gunter Reuter, mei-4129D from Tin Tin Su, and lokP6, grpfs1 [64] flies from Michael Brodsky and Kent Golic. Fly crosses were performed using standard genetic techniques. Su(var)3- 9null flies used in all experiments were transheterozygotes of alleles 6 and 17 produced from (6/17) mothers, so they lacked both maternal and zygotic Su(var)3-9 protein. dcr-2 mutant flies were also produced from homozygous mutant mothers. rodEY04576 Su(var)3- 9null flies, p5311-1B-1 Su(var)3-9null flies, and smc1exc46, Su(var)3-917 flies were made by meiotic recombination and scored by PCR reactions, using template DNA from single flies and primers that distinguish wild type from mutated DNA sequences. Antibodies Rabbit antibodies that recognize cH2Av (1:250 dilution) were purchased from Rockland (Gilbertsville, PA). The rabbit anti- Rad51 antibody (1:100 dilution after direct labeling) was provided by Jim Kadonaga, and was directly labeled as previously described [65]. The mouse anti-C(3)G antibody (1:500 dilution) was provided by Scott Hawley [66], and the rabbit anti-PCNA antibody (1:100 dilution) was provided by Daryl Henderson [67]. Rabbit anti-PH3 (1:1000 dilution) was purchased from Upstate (Charlottesville, VA). The anti-CycA mouse monoclonal antibody (1:20 dilution) was purchased from the Developmental Studies Hybridoma Bank (Iowa City, IA). Alexa dye-conjugated secondary antibodies were purchased from Invitrogen (Carlsbad, CA) and used at 1:500 dilution. Rhodamine-conjugated anti-DIG antibody was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA) and used at a 1:100 dilution. TUNEL assay The TUNEL assay was performed in whole-mount tissues that were fixed with 4% paraformaldehyde in PBS and 0.2% of TritonX-100 (PBST), then washed in PBST and permeabilized overnight with PBST. Tissues were incubated with TUNEL buffer (16 TUNEL buffer from Roche, 2.5 mM CoCl2, 0.2% TritonX- 100) for 10 min, then in TUNEL buffer, dNTPs (final concentra- tions of 10 uM of dATP, dCTP, and dGTP, 3.3 uM dTTP, and 6.6 uM DIG-dUTP) and TdT enzyme (20 U/ml final concentra- tion; purchased from Roche Diagnostics (Mannheim, Germany) for 3 hours at 37uC. To analyze the percentage of cells in apoptosis, brain and imaginal disc tissues were squashed onto slides into single cell layer using standard techniques. The slides were washed extensively with PBST, incubated with TUNEL buffer, dNTPs and TdT enzyme for 2 hours at 37uC. After the TUNEL assay, DIG signals were detected via standard IF Requirement for Heterochromatin Stability procedures using rhodamine-labeled anti-DIG antibody from Jackson ImmunoResearch (West Grove, Pennsylvania). Developmental stage analysis % fertilization. Flies were allowed to lay eggs for 4 hours at 25uC on soft agar plates containing yeast paste. Eggs were incubated for 6 hours at 25uC, then fixed using standard methods [68]. Nuclei in the fixed eggs were visualized by DAPI staining. The percentages of fertilized eggs were calculated by the formula: ((total number of eggs – the number of eggs containing one or two nuclei)/total number of eggs)6100%. % hatched eggs. Flies were allowed to lay eggs overnight at 25uC on soft agar plates containing yeast paste, and the numbers of eggs laid were counted. The eggs were allowed to incubate at 25uC for 30 hours, and the numbers of unhatched eggs were counted (which includes unfertilized eggs). The percentages of hatched eggs were calculated by the formula: ((number of eggs laid – number of unhatched eggs)/number of eggs laid)6100%. % eclosion. Flies were allowed to lay eggs overnight in a bottle containing fly food at 25uC. The bottles were incubated at 25uC for 2 weeks. The eclosion percentages were calculated by the formula: (number of hatched pupa cases/total number of pupae)6100%. Lifespan analysis More than 120 flies from each genotype, one day after eclosion, were separated into female and male populations, and passed onto new vials every other day and incubated at 25uC. Each vial contained approximately 20 flies. Dead flies were counted every other day. When all flies died, the total number of flies was summed from the numbers of dead flies. The viability percentages were calculated by dividing the number of flies alive at specific time periods by the total number of flies. IF, FISH, and IF-FISH of whole-mount tissues and squashed tissues Whole mount IF was performed as previously described [34,69]. Germaria were dissected within 24 hours of mating, fixed with 4% paraformaldehye in PBS, 0.3% Triton-X-100, and washed for 1 hour in PBS, 0.3% Triton-X-100. Germaria were permeabilized in PBS, 0.3% Triton-X-100 for 3 nights, blocked in PBS with 5% milk and 0.3% Triton-X-100. Fixed germaria were incubated in primary and secondary antibody solutions for .4 hours and washed for .1 hour. FISH was performed as previously described [5] using 100 ng of each probe. In combined IF-FISH experiments, tissues were fixed after IF then FISH analysis was performed. FISH probes targeting Drosophila satellite DNAs were made by 39-end labeling of oligonucleotides (sequences are homologous to Drosophila satellite DNAs) with aminoallyl-dUTP (Sigma, St. Louis, MO) using the TdT enzyme, followed by conjugation to Alexa ester dyes (Invitrogen). Microscopy, volumetric, and colocalization analysis All images were captured using an Applied Precision Deltavi- sion microscope (Issaquah, Washington) and deconvolved by the SoftWorx software (also from Applied Precision), using the conservative algorithm with 5 to 8 iterations. The SoftWorx- deconvolved images were converted to TIFF files and then into image stacks for volumetric analysis with the Metamorph 7.0 software (Molecular Devices; Downingtown, PA). The Metamorph 7.0 volumetric analysis application identified individual foci within the image stacks, and the foci were then manually counted. For foci localization and colocalization studies, optical sections of deconvolved images were enhanced for contrast and counted PLoS Genetics | www.plosgenetics.org 11 March 2009 | Volume 5 | Issue 3 | e1000435