![]() Error bars represent SEM of three biological replicates. Right: Quantification of H3 K36me 3 signal after normalizing to total H3 signal. Extract from a set2∆ strain was used to confirm specificity of the H3 K36me 3 antibody. Library plasmids were transformed into KY812 for plasmid shuffling. ( A) Left: western blot analysis of H3 K36me 3 levels in H3 and H4 mutant strains. All heatmaps and metagene plots were generated using deepTools2 ( Ramírez et al., 2014 Ramírez et al., 2016) using 25 bp bins. Arrows above gene names indicate directionality of transcription. IGV-visualized BAM-file snapshots depict presence or absence of strand-specific read-pairs between genes. Regions were chosen to be void of neighboring ncRNA loci. Data represent log-scaled, spike-in normalized 4tU-seq read density over the plus (+) and minus (-) strands. ( C) Browser tracks visualized in IGV ( Thorvaldsdóttir et al., 2013) depicting aberrant transcription 5’ and 3’ to divergent (top), convergent (middle), and tandem (bottom) gene pairs in the indicated strains. ( B) Metagene plots show averaged intensity over regions displayed in the difference heatmaps shown in panel A. Heatmap rows represent 1186 divergent, 1981 convergent, and 2976 tandem protein-coding genes showing 500 bp upstream of the +1 nucleosome ( Brogaard et al., 2012) and 500 bp downstream of the CPS ( Ozsolak et al., 2010). ( A) Heatmaps showing log2-fold change in spike-in normalized 4tU-seq read counts in H3 R52A or H3 T45A mutants relative to WT at divergent, convergent, and tandem genes. All heatmaps and metagene plots were generated with deepTools2 ( Ramírez et al., 2014 Ramírez et al., 2016) using 25 bp bins. Antisense heatmaps are the reverse of the sense heatmaps plotting (from left to right on the x-axis) the region from 500 bp upstream of the CPS to 500 bp downstream of the +1 nucleosome (dotted line) on the non-coding strand. ( C) Expression of regions antisense to the 6205 protein-coding genes shown in Figure 3A in the H3 T45A and H3 R52A mutants compared to WT. Heatmaps plot the region 500 bp upstream of the +1 nucleosome to 500 bp downstream of the CPS (dotted line) and are sorted by gene length. ( B) Heatmaps sorted by gene length and showing spike-in normalized FLAG-Rpb3 ChIP-seq read counts (gray scale) and log2-fold change between the H3 R52A mutant and WT at 6205 protein-coding genes. Box and whisker plots were generated in R Studio ( RStudio Team, 2016) and p-values were calculated using the Wilcoxon-Ranked sum test (***p<0.001). ( A) Box plots comparing log2-transformed spike-in normalized RNA-seq read counts in a window 150 bp downstream of the CPS. Structure from PDB ID 1ID3 ( Luger et al., 1997). H2A, H2B, H3, and H4 are colored in pink, yellow, lilac, and green, respectively. Due to its buried location, H3 Q93 is not marked. ( E) X-ray crystal structure of the nucleosome denoting histone residues (highlighted in red) identified in the termination reporter screen and through northern analysis. The northern blots are representative of three independent experiments. ![]() Arrows correspond to those shown in the locus diagrams in panel C. ( D, F) Northern blot analysis to assess transcription readthrough of SNR genes in ( D) H3 and H4 mutants (plasmids shuffled into KY812) and ( F) H2A mutants (plasmids shuffled into KY943). For SNR13, the probe overlaps the downstream gene, TRS31, and detects a readthrough transcript of SNR13 (black), as well as the full-length TRS31 transcript (green). The intergenic SNR48 probe detects a single readthrough transcript. The intergenic SNR47 probe detects two read-through transcripts, as indicated by the long black and short blue arrows. The black bar over each locus denotes the probe position. ( C) Diagrams of three snoRNA loci analyzed for termination readthrough by northern analysis. For each strain, a 10-fold dilution series (starting at 1 × 10 8 cells/mL) was plated to SC-Trp as a growth control and to SC-His-Trp + 0.5 mM 3-aminotriazole (3-AT), a competitive inhibitor of the HIS3 gene product, to identify mutants expressing the HIS3 gene. Library plasmids ( TRP1-marked, CEN/ARS) ( Nakanishi et al., 2008) expressing the histone gene mutations were introduced by plasmid shuffling into strains expressing the SNR47 termination reporter (KY3220 left) or the reporter control (KY3219 right). ( B) Yeast dilution assays to monitor growth of strains expressing the indicated H3 and H4 derivatives as the only source of H3 or H4. Black arrows denote the transcripts produced from each reporter in wild type (WT) and termination mutant backgrounds. ![]() ( A) Yeast strains contain either an SNR47 transcription termination reporter (top, KY3220) or a control transcription cassette lacking the SNR47 terminator (bottom, KY3219) integrated at the LEU2 locus. ![]()
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