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First published online 16 September 2008
doi: 10.1242/jcs.028555

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Loss of ATAC-specific acetylation of histone H4 at Lys12 reduces binding of JIL-1 to chromatin and phosphorylation of histone H3 at Ser10

¹ Institute of Biochemistry, Biological Research Center, Temesvári krt. 62, H-6726 Szeged, Hungary
² Chromatin Research Group of HAS, Department of Biochemistry and Molecular Biology, University of Szeged, Közép fasor 52, H-6726 Szeged, Hungary

View larger version (52K): [in this window] [in a new window]   Fig. 1. Decreased level of ATAC-specific H4 acetylation results in reduced phosphorylation of histone H3 at S10. (A) Immunoblots of total protein extracts of wild-type (w1118), dAda2ad189, dAda2bd842, dGcn5E333st and JIL-1z2 late third-instar larvae developed with antibodies specifically recognizing H4K12ac (top), and H4 (bottom). (B) Immunoblots of total protein extracts of wild-type (w1118), dAda2ad189, dGcn5E333st and dAda2bd842 late third-instar larvae developed with antibodies specific for H3S10ph (top), and for H3 (bottom). In A and B, the same membranes shown on the top were reprobed with H3- or H4-specific antibodies. (C) Double labeling with antibodies specific for H3S10ph (top) and for the C-terminal domain of the largest subunit of RNA polymerase 2 (Pol II 7G5; bottom) of polytene squashes from wild-type, dAda2ad189, dAda2bd842, dGcn5E333st larvae. (D) Polytene chromosome squashes of dGcn5-null (dGcn5E333st) third-instar larvae expressing a mutated dGcn5 transgene as indicated, double stained with antibodies specific for H3S10ph (top) and for the C-terminal domain of the largest subunit of RNA polymerase II (Pol II 7G5; bottom).

View larger version (58K): [in this window] [in a new window]   Fig. 2. Ectopic expression of JIL-1 from a transgene in ATAC subunit mutants restores levels of H3S10ph, increases the survival of ATAC mutants and improves male X chromosome structure. (A) Polytene chromosome squashes of wild-type, and JIL-1 transgene expresser dAda2ad189 and dGcn5E333st (JIL-1EGFP/dAda2ad189 and JIL-1EGFP/dGcn5E333st) third-instar larvae double stained with H3S10ph-specific (top) and Pol II-specific (bottom) antibodies. (B) Immunoblot of total protein extracts of (1) wild-type, (2) JIL-1EGFP, (3) dAda2ad189, (4) JIL-1EGFP/dAda2ad189, (5) dGcn5E333st and (6) JIL-1EGFP/dGcn5E333st third-instar larvae developed with H3S10ph- (top) and H3-specific (bottom) antibodies. (C) Lethal phases of dAda2ad189, JIL-1EGFP/dAda2ad189, dGcn5E333st and JIl-1EGFP/dGcn5E333st animals. The percentages of animals perishing in the indicated stages are shown. Numbers of third instar larvae of the particular genotype and sex are given in parentheses. The sex of the animals was determined by observing the presence of testes in L3. (D) The hatching rate of hypomorph dAda2a and dGcn5 mutants in the absence and the presence of a JIL-1EGFP transgene. The genotypes are described in Materials and Methods. Numbers of animals studied were 625 dAda2a hypomorph, 456 Gcn5 hypomorph as controls, 781 dAda2a and 488 dGcn5 carrying JIL-1 transgene. (E) Phase-contrast images of dAda2ad189, JIL-1EGFP/dAda2ad189, dGcn5E333st and JIl-1EGFP/dGcn5sex204/E333st polytene chromosomes of late third-instar male larvae. Arrows indicate the male X chromosomes.

View larger version (51K): [in this window] [in a new window]   Fig. 3. Ectopic spreading of H3K9me2 in dAda2a, dGcn5 and JIL-1 mutants. (A) Polytene chromosome squashes of dAda2ad189-, dGcn5E333st- and JIL-1z2-null homozygous third-instar larvae stained with H3K9me2-specific antibody and DAPI (blue). (B) Polytene nuclei of dAda2ad189-, dGcn5E333st- and JIL-1z2-null homozygous larvae stained with H3K9me2-specific antibody (red) and DAPI (blue). (C) Su(var)3-9 heterozygocy increases the viability of dAda2a- or dGcn5-null animals. The percentages of animals perishing at the indicated developmental stages are shown. The number of animals in each category was over 350. (D) Su(var)3-9 heterozygous background improves the structure of male X chromosomes of dAda2a-null mutants.

View larger version (57K): [in this window] [in a new window]   Fig. 4. The level of JIL-1 expression does not, but JIL-1 binding to chromatin is severely decreased in ATAC mutants. (A) The level of JIL-1 mRNA in ATAC and JIL-1 mutants determined by real-time RT-PCR. (B) Immunoblot of total protein extracts of dAda2ad189- and dAda2bd842-null mutant and wild-type third-instar larvae developed using JIL-1-specific antibody. (C) Immunostaining of salivary glands of ATAC- and JIL-1-null mutants using JIL-1-specific antibody. (D) Immunostaining of polytene chromosomal spreads (top) and `smush' preparations (bottom) of wild-type and dAda2ad189, dGcn5E333st and JIL-1z2 third-instar larvae using JIL-1-specific antibody. Chromosomes of males are shown. The JIL-1 mutant shown is null homozygote.

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