Fig 2: Bqt4-rich domains ensure faithful inheritance of heterochromatin.(A) Schematic of system designed to move replication forks to the NE. Coexpression of Pola-GFP and Man1-GBP recruits Pola-associated (replicating) chromatin to Man1-rich subdomains of the NE. Images below schematic show remobilization of Pol a-GFP to colocalize with Man1-GBP-mCherry encircling the nucleus. (B) In cells lacking Man1-GBP, Pola-GFP shows diffuse localization within the nuclear interior. (C) ChIP-seq experiments were performed as described in text and Methods, using H3K9Me2 antibody (ab1220, Abcam). (D) Ribbon line-plot of enrichment of H3K9Me over the ade6+ locus in three independent WT isolates. The solid blue line indicates median; width of the ribbon indicates range of enrichment levels among the three isolates. (E–F) Ribbon line-plot of enrichment of H3K9Me2 at centromere of Chr III. The central core region is indicated by the yellow box. The sharp peaks flanking the core region (F) align to small repetitive regions (~25 bp) present in multiple regions across the genome; it is therefore not possible to determine their source.

Fig 3: Western blot analysis to show specificity of antibodies used for ChIP assays in the study.Crude extract (A) and isolated nuclei (B) were probed with antibodies against histone 3, H3K9me2 (Abcam Cat# ab1220), H3K27me3 (Millipore Cat# 07-449) and H3K4me3 (Millipore Cat# 04-745), respectively.DOI: http://dx.doi.org/10.7554/eLife.06671.013

Fig 4: ChIP-PCR assays for selected flowering genes and heterochromatic loci confirm ChIP-qPCR.(A) ChIP-PCR analysis of various histone 3 modifications in flowering genes in different genetic backgrounds. (B) ChIP-PCR analysis of various histone 3 modifications in TEs in different genetic backgrounds. ChIP assays were conducted on 9-day-old seedlings using antibodies specific for H3K9me2 (Abcam Cat# ab1220), H3K27me3 (Millipore Cat# 07-449), and H3K4me3 (Millipore Cat# 04-745). The PCRs were done with 22 cycles for the input samples and with 30 cycles after ChIP.DOI: http://dx.doi.org/10.7554/eLife.06671.014

Fig 5: ChIP-qPCR analyses of H3K4me3, H3K9me2, and H3K27me3 in TrAP-regulated loci in vivo.(A) TrAP-activated transposons in heterochromatic regions contained reduced H3K9me2 and H3K27me3 but did not show consistent variation in H3K4me3 marks. (B) TrAP-upregulated flowering genes showed consistently reduced H3K9me2 and H3K27me3 marks compared to wild-type Col-0. (C) TrAP-downregulated genes displayed variable changes of H3K9me2 and H3K27me3 marks and no obvious changes of H3K4me3 mark. (D) Tubulin (TUB8) was used as internal control for all the ChIP experiments; the percentage enrichment vs input is shown. ChIP assays were conducted on 11-day-old seedlings using antibodies specific for H3K9me2 (Abcam, Cat# ab1220), H3K27me3 (Millipore, Cat# 07-449), and H3K4me3 (Millipore, Cat# 04-745). Enrichment of H3K4me3 and H3K27me3 in each locus is normalized to that of TUB8; H3K9me2 enrichment is plotted as percentage of input. The standard deviation (SD) was calculated from at least three biological repeats.DOI: http://dx.doi.org/10.7554/eLife.06671.012