SETDB1 Is Involved in Postembryonic DNA Methylation and Gene Silencing in Drosophila
Figure 5
DSETDB1-mediated tri-methylation of H3-K9 propagates spreading of DNA methylation and silencing of Rb.
(A) Digital images of ethidium bromide-stained agarose gels showing reaction products for the PDE and Exon-I of Rb indicated in (A) in DNA pools obtained by ChIP. Chromatin was isolated from S2 cells transiently expressing GFP (control) and S2 cells co-expressing GFP and dSETDB1, dSETDB1(H775L), or dSETDB1(R436C). Chromatin was immunoprecipitated with antibodies and agarose beads described in Figure 2C. PCR assays detected the PDE, PPE and Exon-I in immunoprecipitated DNA pools. (A,C) Input represents the amount of target DNA present in 1% of the chromatin used for ChIP. (B) Digital images of ethidium bromide-stained agarose gels detecting the target DNA sequences for dSETDB1 in Antp, CG2136 and Rt1b{}779 (see Figure S9) in DNA pools obtained by ChIP. Chromatin was isolated from S2 cells transiently expressing GFP and dSETDB1(R436C). Chromatin was immunoprecipitated with antibodies and agarose beads described in Figure 2C. (C) Schematic representation of the Rb locus. Boxes mark the position of exons I (Exon-I), II, and VIII. The positions of the promoter distal enhancer element (PDE), promoter proximal enhancer element (PPE), and Exon-I fragments detected in ChIP assays are indicated. (D) Digital images of ethidium bromide stained agarose gels showing the reaction products of methylation-sensitive restriction analyses of genomic DNA isolated from cells described in (B). Genomic DNA was isolated, incubated with bovine serum albumin (BSA) (mock), the methylation sensitive restriction endonuclease HpaII, or the methylation-insensitive restriction enzyme MspI. PCR assays monitored the presence of the PDE, Exon-I, and the promoter region of Peepsqueak (Psq) in treated DNA pools.