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. 2012 Feb;121(1):79-90.
doi: 10.1007/s00412-011-0344-7. Epub 2011 Nov 10.

The role of MOF in the ionizing radiation response is conserved in Drosophila melanogaster

Manika P Bhadra  1 , Nobuo Horikoshi, Sreerangam N C V L Pushpavallipvalli, Arpita Sarkar, Indira Bag, Anita Krishnan, John C Lucchesi, Rakesh Kumar, Qin Yang, Raj K Pandita, Mayank Singh, Utpal Bhadra, Joel C Eissenberg, Tej K Pandita
Affiliations

The role of MOF in the ionizing radiation response is conserved in Drosophila melanogaster

Manika P Bhadra et al. Chromosoma. 2012 Feb.

Abstract

In Drosophila, males absent on the first (MOF) acetylates histone H4 at lysine 16 (H4K16ac). This acetylation mark is highly enriched on the male X chromosome and is required for dosage compensation in Drosophila but not utilized for such in mammals. Recently, we and others reported that mammalian MOF, through H4K16ac, has a critical role at multiple stages in the DNA damage response (DDR) and double-strand break repair pathways. The goal of this study was to test whether mof is similarly required for the response to ionizing radiation (IR) in Drosophila. We report that Drosophila mof mutations in males and females, as well as mof knockdown in SL-2 cells, reduce post-irradiation survival. MOF depletion in SL-2 cells also results in an elevated frequency of metaphases with chromosomal aberrations, suggesting that MOF is involved in DDR. Mutation in Drosophila mof also results in a defective mitotic checkpoint, enhanced apoptosis, and a defective p53 response post-irradiation. In addition, IR exposure enhanced H4K16ac levels in Drosophila as it also does in mammals. These results are the first to demonstrate a requirement for MOF in the whole animal IR response and suggest that the role of MOF in the response to IR is conserved between Drosophila and mammals.

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Conflict of interest statement

Conflict of interest statement None.

Figures

Fig. 1
Fig. 1
MOF depletion results in decreased cell numbers post-irradiation. a Western blot analysis of MOF levels in SL-2 cells 72 h after transfection with MOF–RNAi or control GFP-RNAi. b SL-2 cells with and without depletion of MOF were irradiated with 20 or 40 Gy and the cell numbers determined at the indicated time points, means of three independent experiments. MOF-depleted cells exposed to IR had significantly reduced cell number than control irradiated cells. (Differences from control are statistically significant according to Student’s t test, *P<0.05)
Fig. 2
Fig. 2
Comparison of wild-type and mof mutant male Drosophila survival post-irradiation. Male third instar larvae, irradiated as embryos, were scored as mof4 or mof+(FM7) at the third instar larval stage. Mean survival from three to four experiments is presented for each dose point
Fig. 3
Fig. 3
Frequency of metaphases with chromosome aberrations at different time points of post irradiation. SL-2 cells, with or without MOF knockdown, were irradiated with gamma rays and the metaphases were analyzed. a 12 h after exposure to 3 Gy, b 5 h after irradiation with 2.5 Gy, and c 2 h after irradiation with 2 Gy. For each experiment, 200 metaphases were analyzed for chromosome aberrations and each experiment was repeated at least three times. Mean of three independent experiments is presented
Fig. 4
Fig. 4
Excessive spontaneous apoptosis is associated with mof mutation. Apoptotic cells in wild-type and mof1 mutant male larval eye imaginal discs 4 h after irradiation with 25 Gy were detected by staining with acridine orange, which stains solely the apoptotic cells
Fig. 5
Fig. 5
DNA damage checkpoint response in wing imaginal discs of wild-type and Drosophila mof mutant. Larval wing discs from wild-type and mof1 mutant male were immunostained with anti-phospho histone H3 antibody before irradiation and 1 h after irradiation with 25 Gy of gamma rays. The mitotic cells in the discs were stained brightly in the confocal images
Fig. 6
Fig. 6
Determination of Chk1, p53, and mei-41 protein levels in larvae extracts of wild-type and mof1 mutant Drosophila male larvae with and without irradiation. Larvae were irradiated with 25 Gy of gamma rays and the proteins extracted within 1 h post-irradiation. a Western blot and b histogram representing the relative amount of proteins estimated from triplicate blots
Fig. 7
Fig. 7
Male X-chromosome staining for A MOF or B H4K16ac at different time points post-irradiation in SL-2 cells. C Cells showing the status of male X chromosomes detected by MOF staining (MOF color is green and DNA is blue): a daughter cells showing similar intensity of MOF staining of X chromosomes at late anaphase; b one daughter cell showing MOF staining whereas the other lacks MOF staining at telophase; and c two daughter cells sharing three MOF signals at telophase
Fig. 8
Fig. 8
MOF and H4K16ac immunostained wild-type male Drosophila polytene salivary gland X-chromosomes. Drosophila were irradiated with 25 Gy and the salivary gland chromosomes, fixed and immunostained
Fig. 9
Fig. 9
Increased cellular H4K16ac levels in SL-2 cells following irradiation. Cells were irradiated with either 25 or 50 Gy and the H4K16ac levels measured at different time points post-irradiation by Western blot analysis. Relative H4K16ac levels were normalized relative to Histone H4 levels. Differences from control (unirradiated) cells are statistically significant according to Student’s t test, *P<0.05; **P<0.001)
Fig. 10
Fig. 10
Effect of IR on MOF and H4K16ac levels in Drosophila larvae. a Wild-type and mof1 mutant third instar larvae were irradiated (25 Gy), and the histones extracted within 1 h of irradiation. Quantitative Western blot analysis was carried out with H4K16ac antibody. The same blots were re-probed with antibody to total histone H4 as a gel loading control. b Histogram representing the relative amount of H4K16ac/total histone H4 (H4K16ac/H4) as estimated from triplicate blot analysis
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