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The Set2/Rpd3S pathway suppresses cryptic transcription without regard to gene length or transcription frequency.

Lickwar CR, Rao B, Shabalin AA, Nobel AB, Strahl BD, Lieb JD - PLoS ONE (2009)

Bottom Line: It was previously reported that this "cryptic" transcription occurs preferentially in long genes, and in genes that are infrequently transcribed.Our conclusions differ with those reported previously because we obtained a higher-resolution dataset, we accounted for the fact that gene length and transcriptional frequency are not independent variables, and we accounted for several ascertainment biases that make cryptic transcription easier to detect in long, infrequently transcribed genes.These new results and conclusions have implications for many commonly used genomic analysis approaches, and for the evolution of high-fidelity RNA polymerase II transcriptional initiation in eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

ABSTRACT
In cells lacking the histone methyltransferase Set2, initiation of RNA polymerase II transcription occurs inappropriately within the protein-coding regions of genes, rather than being restricted to the proximal promoter. It was previously reported that this "cryptic" transcription occurs preferentially in long genes, and in genes that are infrequently transcribed. Here, we mapped the transcripts produced in an S. cerevisiae strain lacking Set2, and applied rigorous statistical methods to identify sites of cryptic transcription at high resolution. We find that suppression of cryptic transcription occurs independent of gene length or transcriptional frequency. Our conclusions differ with those reported previously because we obtained a higher-resolution dataset, we accounted for the fact that gene length and transcriptional frequency are not independent variables, and we accounted for several ascertainment biases that make cryptic transcription easier to detect in long, infrequently transcribed genes. These new results and conclusions have implications for many commonly used genomic analysis approaches, and for the evolution of high-fidelity RNA polymerase II transcriptional initiation in eukaryotes.

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Detection of cryptic initiation sites in S. cerevisiae strains lacking Set2.(A) YGL142C exhibits cryptic initiation in the set2Δ strain (red, supF 29.7), but not wild-type (blue). Plotted along the length of YGL142C are z scores of wild type (Wt) RNA raw probe intensity values (blue); set2Δ RNA/Wt RNA (red) and set2Δ/Wt ratios from Li et al. 2007 (green). The transition probe detected by our algorithm is marked by a dashed red line and red arrow. The solid lines represent the average z score for Wt (blue) and set2Δ/Wt values (red) before and after the cryptic initiation event. This cryptic initiation event was confirmed by Northern blotting (not shown). (B) Genes identified as containing cryptic initiation sites according to our liberal (supF 3.0, 1193 genes) or conservative (supF 9.0, 429 genes) criteria were compared to the genes identified as containing a cryptic initiation event from Li et al. 2007 (1033 genes, orange). Shown at far right is the average intersection of 429 genes chosen at random with the Li et al set.
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pone-0004886-g001: Detection of cryptic initiation sites in S. cerevisiae strains lacking Set2.(A) YGL142C exhibits cryptic initiation in the set2Δ strain (red, supF 29.7), but not wild-type (blue). Plotted along the length of YGL142C are z scores of wild type (Wt) RNA raw probe intensity values (blue); set2Δ RNA/Wt RNA (red) and set2Δ/Wt ratios from Li et al. 2007 (green). The transition probe detected by our algorithm is marked by a dashed red line and red arrow. The solid lines represent the average z score for Wt (blue) and set2Δ/Wt values (red) before and after the cryptic initiation event. This cryptic initiation event was confirmed by Northern blotting (not shown). (B) Genes identified as containing cryptic initiation sites according to our liberal (supF 3.0, 1193 genes) or conservative (supF 9.0, 429 genes) criteria were compared to the genes identified as containing a cryptic initiation event from Li et al. 2007 (1033 genes, orange). Shown at far right is the average intersection of 429 genes chosen at random with the Li et al set.

Mentions: We selected two significance thresholds which led to family-wise error rates of roughly 10−10 and 10−26, and identified 1193 and 429 genes, respectively, with at least one cryptic initiation event (Table S1). Individual genes identified by the algorithm as containing cryptic initiation events typically exhibit internal transitions that are clear by visual inspection (Figure 1A). Furthermore, genes characterized previously as containing cryptic transcripts in set2Δ strains, including FLO8 (supF = 3.62) STE11 (supF = 9.02) and PCA1 (supF = 70.9) were identified by our algorithm. Using the more stringent cutoff, 59% of the genes we identified were identified previously as containing a cryptic initiation event [12]. We find this concordance striking, especially because the earlier study made use of a different microarray platform, a different RNA labeling method, and a different method of identifying cryptic initiation sites (Figure 1B). Indeed, examination of individual loci reveals that the raw data from this study and the previous study are consistent with each other (Figure 1A). The concordance of our raw data with the raw data from the lower-resolution study, along with the utility of our data in the identification of genes previously characterized as containing cryptic transcripts, provides support for our experimental design, analysis methods, and threshold selections.


The Set2/Rpd3S pathway suppresses cryptic transcription without regard to gene length or transcription frequency.

Lickwar CR, Rao B, Shabalin AA, Nobel AB, Strahl BD, Lieb JD - PLoS ONE (2009)

Detection of cryptic initiation sites in S. cerevisiae strains lacking Set2.(A) YGL142C exhibits cryptic initiation in the set2Δ strain (red, supF 29.7), but not wild-type (blue). Plotted along the length of YGL142C are z scores of wild type (Wt) RNA raw probe intensity values (blue); set2Δ RNA/Wt RNA (red) and set2Δ/Wt ratios from Li et al. 2007 (green). The transition probe detected by our algorithm is marked by a dashed red line and red arrow. The solid lines represent the average z score for Wt (blue) and set2Δ/Wt values (red) before and after the cryptic initiation event. This cryptic initiation event was confirmed by Northern blotting (not shown). (B) Genes identified as containing cryptic initiation sites according to our liberal (supF 3.0, 1193 genes) or conservative (supF 9.0, 429 genes) criteria were compared to the genes identified as containing a cryptic initiation event from Li et al. 2007 (1033 genes, orange). Shown at far right is the average intersection of 429 genes chosen at random with the Li et al set.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654109&req=5

pone-0004886-g001: Detection of cryptic initiation sites in S. cerevisiae strains lacking Set2.(A) YGL142C exhibits cryptic initiation in the set2Δ strain (red, supF 29.7), but not wild-type (blue). Plotted along the length of YGL142C are z scores of wild type (Wt) RNA raw probe intensity values (blue); set2Δ RNA/Wt RNA (red) and set2Δ/Wt ratios from Li et al. 2007 (green). The transition probe detected by our algorithm is marked by a dashed red line and red arrow. The solid lines represent the average z score for Wt (blue) and set2Δ/Wt values (red) before and after the cryptic initiation event. This cryptic initiation event was confirmed by Northern blotting (not shown). (B) Genes identified as containing cryptic initiation sites according to our liberal (supF 3.0, 1193 genes) or conservative (supF 9.0, 429 genes) criteria were compared to the genes identified as containing a cryptic initiation event from Li et al. 2007 (1033 genes, orange). Shown at far right is the average intersection of 429 genes chosen at random with the Li et al set.
Mentions: We selected two significance thresholds which led to family-wise error rates of roughly 10−10 and 10−26, and identified 1193 and 429 genes, respectively, with at least one cryptic initiation event (Table S1). Individual genes identified by the algorithm as containing cryptic initiation events typically exhibit internal transitions that are clear by visual inspection (Figure 1A). Furthermore, genes characterized previously as containing cryptic transcripts in set2Δ strains, including FLO8 (supF = 3.62) STE11 (supF = 9.02) and PCA1 (supF = 70.9) were identified by our algorithm. Using the more stringent cutoff, 59% of the genes we identified were identified previously as containing a cryptic initiation event [12]. We find this concordance striking, especially because the earlier study made use of a different microarray platform, a different RNA labeling method, and a different method of identifying cryptic initiation sites (Figure 1B). Indeed, examination of individual loci reveals that the raw data from this study and the previous study are consistent with each other (Figure 1A). The concordance of our raw data with the raw data from the lower-resolution study, along with the utility of our data in the identification of genes previously characterized as containing cryptic transcripts, provides support for our experimental design, analysis methods, and threshold selections.

Bottom Line: It was previously reported that this "cryptic" transcription occurs preferentially in long genes, and in genes that are infrequently transcribed.Our conclusions differ with those reported previously because we obtained a higher-resolution dataset, we accounted for the fact that gene length and transcriptional frequency are not independent variables, and we accounted for several ascertainment biases that make cryptic transcription easier to detect in long, infrequently transcribed genes.These new results and conclusions have implications for many commonly used genomic analysis approaches, and for the evolution of high-fidelity RNA polymerase II transcriptional initiation in eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

ABSTRACT
In cells lacking the histone methyltransferase Set2, initiation of RNA polymerase II transcription occurs inappropriately within the protein-coding regions of genes, rather than being restricted to the proximal promoter. It was previously reported that this "cryptic" transcription occurs preferentially in long genes, and in genes that are infrequently transcribed. Here, we mapped the transcripts produced in an S. cerevisiae strain lacking Set2, and applied rigorous statistical methods to identify sites of cryptic transcription at high resolution. We find that suppression of cryptic transcription occurs independent of gene length or transcriptional frequency. Our conclusions differ with those reported previously because we obtained a higher-resolution dataset, we accounted for the fact that gene length and transcriptional frequency are not independent variables, and we accounted for several ascertainment biases that make cryptic transcription easier to detect in long, infrequently transcribed genes. These new results and conclusions have implications for many commonly used genomic analysis approaches, and for the evolution of high-fidelity RNA polymerase II transcriptional initiation in eukaryotes.

Show MeSH
Related in: MedlinePlus