Journal:

Article Title: DNA Hypermethylation in Drosophila melanogaster Causes Irregular Chromosome Condensation and Dysregulation of Epigenetic Histone Modifications

doi: 10.1128/MCB.23.7.2577-2586.2003

Figure Lengend Snippet: DNA hypermethylation causes changes in epigenetic histone modifications. (A) Immunodetection of acetylated histone H4 (acH4) on chromosome preparations from larval neuroblasts. There was no detectable difference between flies expressing full-length or deleted Dnmt3a protein. (B) Similarly, H3-K9 acetylation (acH3) was found to be very low on mitotic chromosomes and did not change upon DNA methylation. (C) Immunodetection of K9-methylated histone H3 (mH3). Chromosomes from Dnmt3a-overexpressing flies showed a strongly increased level of H3 methylation compared to controls. The majority of K9-methylated H3 staining was located in pericentromeric heterochromatin. (D) Phosphorylation of H3-S10 (pH3) was strongly reduced on chromosomes from Dnmt3a-overexpressing flies. Only the telomere-proximal regions of the chromosome arms remained stained. Control chromosomes showed widespread and homogeneous phosphorylation of H3. Bars, 2 μm.

Article Snippet: The following antibodies were used for immunofluorescence: rat antibromodeoxyuridine (α-BrdU; Harlan Sera Laboratories, 1:2); rabbit anti-5-methylcytosine (Megabase Research Products, 1:100); rabbit anti-H3 (Cell Signaling, 1:200); rabbit anti-Cid ( 25 ) (1:200); rabbit anti-acetylated H4 (Upstate Biotechnology, 1:200); rabbit anti-acetylated H3-K9 (Cell Signaling, 1:200); rabbit anti-4x-methylated H3-K9 ( 51 ) (1:2,000); and mouse anti-phosphorylated H3-S10 (Cell Signaling, 1:50).

Techniques: Immunodetection, Expressing, DNA Methylation Assay, Methylation, Staining

Journal:

Article Title: DNA Hypermethylation in Drosophila melanogaster Causes Irregular Chromosome Condensation and Dysregulation of Epigenetic Histone Modifications

doi: 10.1128/MCB.23.7.2577-2586.2003

Figure Lengend Snippet: Rescue of DNA hypermethylation-induced phenotype by Su(var)3-9 mutations in H3-K9 methyltransferase. (A) Survival of flies was calculated by determining their stage of developmental arrest during pupal development. Flies overexpressing Dnmt3a in a wild-type Su(var)3-9 background are shown as a grey line, and flies overexpressing Dnmt3a in a heterozygous Su(var)3-9 null mutant background are shown as a black line. The mutant allele as such showed no detectable effect on fly development (dashed black line). For each experiment, we analyzed several independent crosses. (B) Rescued adult flies were viable and fertile and had a normal appearance. (C) Western analysis indicated larger amounts of methylated K9-H3 both in third-instar larvae overexpressing Dnmt3a in a wild-type background and in a heterozygous Su(var)3-9 mutant background. See text for details. (D to F) Rescue of the polytene chromosome phenotype. Bars, 10 μm. (D) No polytene chromosomes could be prepared from flies overexpressing Dnmt3a in a wild-type background. (E) The presence of a heterozygous mutant Su(var)3-9 allele resulted in the appearance of polytene chromosomes that were similar to wild-type chromosomes. (F) Overexpression of the catalytically inactive Dnmt3a protein combined with a heterozygous Su(var)3-9 mutation had no effect on polytene chromosome morphology. (G) Quantification of genomic DNA methylation levels in rescued (Dnmt3a; Sv39/+) and nonrescued (Dnmt3a; +/+) pupae. The reduction of H3-K9 methylation in rescued flies resulted in a significant decrease of the genomic DNA methylation level (P < 0.001, as determined by a standard t test).

Article Snippet: The following antibodies were used for immunofluorescence: rat antibromodeoxyuridine (α-BrdU; Harlan Sera Laboratories, 1:2); rabbit anti-5-methylcytosine (Megabase Research Products, 1:100); rabbit anti-H3 (Cell Signaling, 1:200); rabbit anti-Cid ( 25 ) (1:200); rabbit anti-acetylated H4 (Upstate Biotechnology, 1:200); rabbit anti-acetylated H3-K9 (Cell Signaling, 1:200); rabbit anti-4x-methylated H3-K9 ( 51 ) (1:2,000); and mouse anti-phosphorylated H3-S10 (Cell Signaling, 1:50).

Techniques: Mutagenesis, Western Blot, Methylation, Over Expression, DNA Methylation Assay