Interplay between Two Epigenetic Marks

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Interplay between Two Epigenetic Marks Lianna M. Johnson, Xiaofeng Cao, Steven E. Jacobsen Current Biology Volume 12, Issue 16, Pages 1360-1367 (August 2002) DOI: 10.1016/S0960-9822(02)00976-4

Figure 1 Analysis of the Centromeric 180-bp Repeats (A) A schematic diagram of 180-bp centromeric repeats. The arrow above each repeat indicates a single HpaII/MspI restriction site. The arrows below the repeats indicate primers that bind to the repetitive DNA generating a PCR ladder. (B) DNA from each of the indicated lines was digested with either HpaII or MspI and was probed with the 180-bp centromeric repeat probe. (C) ChIP analysis of mutant lines was performed with dimethyl-lysine 9 histone H3 (H3-K9me) antibodies. Primers specific for either ACTIN (lower panel) or 180-bp repeats were used. PCR reactions (180 bp) were stopped after 23 cycles, while ACTIN was allowed to go to 36 cycles. “No AB” refers to the no antibody control. Current Biology 2002 12, 1360-1367DOI: (10.1016/S0960-9822(02)00976-4)

Figure 2 Analysis of the Ta3 Retrotransposon (A) A schematic diagram of Ta3. The arrows indicate HpaII/MspI restriction sites, with the sizes of fragments shown in bp. The Ta3 probe spanned the 2194-, 1742-, and 746-bp fragments. Two regions were amplified for ChIP analysis, the left LTR (region 1) and the middle portion (region 2). (B) A Southern blot comparing DNA methylation at Ta3 for each of the mutant lines. (C) Results of ChIP assays using either dimethylated H3-K9 antibodies or acetylated H3 (H3-Ac) antibodies, and results of RT-PCR from the same mutant lines. ChIP PCR reactions were stopped after 34 cycles. Current Biology 2002 12, 1360-1367DOI: (10.1016/S0960-9822(02)00976-4)

Figure 3 Analysis of the Ta2 Retrotransposon (A) Results of ChIP assays using dimethylated H3-K9 antibodies and amplifying Ta2 and ACTIN in a multiplex reaction. (B) Results of RT-PCR from the same mutant lines. Current Biology 2002 12, 1360-1367DOI: (10.1016/S0960-9822(02)00976-4)

Figure 4 Analysis of the SUPERMAN Gene (A) DNA from the 5′ portion of the SUP gene was amplified from ChIP preparations. This region contains an intron and the start of the coding region (M: initiating methionine). (B) ChIP using dimethylated H3-K9 antibodies. SUP and ACTIN were amplified in a multiplex PCR reaction. Current Biology 2002 12, 1360-1367DOI: (10.1016/S0960-9822(02)00976-4)

Figure 5 Model for the Relationship between Histone H3-K9 Methylation and DNA Methylation DDM1 is required for both KYP-dependent H3-K9 methylation and MET1-dependent DNA methylation. CpNpG methylation is controlled by CMT3 through H3-K9 methylation. CpNpG methylation is also reduced in met1 mutant plants (dashed line), but this seems to be an indirect effect, as MET1 does not appear to methylate CpNpG sites [41]. Derepression of transcriptional silencing due to loss of DNA methylation causes a decrease in H3-K9 methylation levels, possibly through nucleosome exchange. Current Biology 2002 12, 1360-1367DOI: (10.1016/S0960-9822(02)00976-4)