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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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Cryptic initiation events occur without regard to gene length.(A) The number of cryptic initiation events detected per base increases with gene length. Shown is the detection rate for five gene-length bins at conservative (supF 9, 801 transitions, blue) and liberal (supF 3, 1757 transitions, red) cutoffs. (B–C) Reducing the number of probes flanking a transition event causes statistical significance to decrease. Plotted along the length of YDR104C are z scores of set2Δ RNA/Wt RNA. The solid horizontal lines represent the average z score for the set2Δ/Wt values (red) before and after the cryptic initiation event. The black solid and dashed vertical lines correspond to the number of probes required to achieve the supF 9 and supF 3 cutoffs respectively. Panel C plots the calculated supF values (y-axis) as a window centered on the detected transition shrinks, causing fewer probes to be available for transition detection (x-axis). The solid and dashed black lines call attention to the supF 9 and supF 3 cutoffs. (D–E) Same as B and C, but for YAL026C. (F) Detection of cryptic initiation sites using subgenes of uniform length eliminates the relationship between gene length and cryptic initiation frequency. Cryptic initiation rates for subgene analysis (Methods) using liberal (green; supF = 0.58, 2417 transitions), moderate (red; supF = 0.97, 1135 transitions), and conservative (blue; supF = 1.8, 364 transitions) cutoffs are plotted for different gene-length bins. (G) Random distribution of cryptic initiation events mimics the real distribution discovered after controlling ascertainment bias. Plot is the same as panel F, but transitions were randomly assigned to ORF probes (liberal; 2417 transitions, moderate; 1135 transitions, conservative; 364 transitions). Compare G and F. The slight downward trend in both plots may result from a slightly lower probe density in longer genes, which was not corrected for.
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pone-0004886-g002: Cryptic initiation events occur without regard to gene length.(A) The number of cryptic initiation events detected per base increases with gene length. Shown is the detection rate for five gene-length bins at conservative (supF 9, 801 transitions, blue) and liberal (supF 3, 1757 transitions, red) cutoffs. (B–C) Reducing the number of probes flanking a transition event causes statistical significance to decrease. Plotted along the length of YDR104C are z scores of set2Δ RNA/Wt RNA. The solid horizontal lines represent the average z score for the set2Δ/Wt values (red) before and after the cryptic initiation event. The black solid and dashed vertical lines correspond to the number of probes required to achieve the supF 9 and supF 3 cutoffs respectively. Panel C plots the calculated supF values (y-axis) as a window centered on the detected transition shrinks, causing fewer probes to be available for transition detection (x-axis). The solid and dashed black lines call attention to the supF 9 and supF 3 cutoffs. (D–E) Same as B and C, but for YAL026C. (F) Detection of cryptic initiation sites using subgenes of uniform length eliminates the relationship between gene length and cryptic initiation frequency. Cryptic initiation rates for subgene analysis (Methods) using liberal (green; supF = 0.58, 2417 transitions), moderate (red; supF = 0.97, 1135 transitions), and conservative (blue; supF = 1.8, 364 transitions) cutoffs are plotted for different gene-length bins. (G) Random distribution of cryptic initiation events mimics the real distribution discovered after controlling ascertainment bias. Plot is the same as panel F, but transitions were randomly assigned to ORF probes (liberal; 2417 transitions, moderate; 1135 transitions, conservative; 364 transitions). Compare G and F. The slight downward trend in both plots may result from a slightly lower probe density in longer genes, which was not corrected for.

Mentions: Previous studies have concluded that longer genes are especially prone to cryptic transcription upon deletion of SET2 [12]. This conclusion was based on the observation that long genes were more likely to be identified as containing a cryptic promoter than short genes. To determine if this observation could be explained solely by the fact that longer genes afford more opportunity for a cryptic event to occur, we grouped all genes according to size. For each group, we then calculated the rate of cryptic initiation (measured in transitions per base pair, Figure 2A). Measuring the rate of transitions, rather than the absolute number of transitions, for genes binned by length is a simple way to correct for gene length. The positive correlation between gene length and the rate of transitions per base shows that transitions are indeed detected more often in the context of longer genes, and that the additional transitions detected in long genes cannot be accounted for simply by correcting for gene length.


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)

Cryptic initiation events occur without regard to gene length.(A) The number of cryptic initiation events detected per base increases with gene length. Shown is the detection rate for five gene-length bins at conservative (supF 9, 801 transitions, blue) and liberal (supF 3, 1757 transitions, red) cutoffs. (B–C) Reducing the number of probes flanking a transition event causes statistical significance to decrease. Plotted along the length of YDR104C are z scores of set2Δ RNA/Wt RNA. The solid horizontal lines represent the average z score for the set2Δ/Wt values (red) before and after the cryptic initiation event. The black solid and dashed vertical lines correspond to the number of probes required to achieve the supF 9 and supF 3 cutoffs respectively. Panel C plots the calculated supF values (y-axis) as a window centered on the detected transition shrinks, causing fewer probes to be available for transition detection (x-axis). The solid and dashed black lines call attention to the supF 9 and supF 3 cutoffs. (D–E) Same as B and C, but for YAL026C. (F) Detection of cryptic initiation sites using subgenes of uniform length eliminates the relationship between gene length and cryptic initiation frequency. Cryptic initiation rates for subgene analysis (Methods) using liberal (green; supF = 0.58, 2417 transitions), moderate (red; supF = 0.97, 1135 transitions), and conservative (blue; supF = 1.8, 364 transitions) cutoffs are plotted for different gene-length bins. (G) Random distribution of cryptic initiation events mimics the real distribution discovered after controlling ascertainment bias. Plot is the same as panel F, but transitions were randomly assigned to ORF probes (liberal; 2417 transitions, moderate; 1135 transitions, conservative; 364 transitions). Compare G and F. The slight downward trend in both plots may result from a slightly lower probe density in longer genes, which was not corrected for.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004886-g002: Cryptic initiation events occur without regard to gene length.(A) The number of cryptic initiation events detected per base increases with gene length. Shown is the detection rate for five gene-length bins at conservative (supF 9, 801 transitions, blue) and liberal (supF 3, 1757 transitions, red) cutoffs. (B–C) Reducing the number of probes flanking a transition event causes statistical significance to decrease. Plotted along the length of YDR104C are z scores of set2Δ RNA/Wt RNA. The solid horizontal lines represent the average z score for the set2Δ/Wt values (red) before and after the cryptic initiation event. The black solid and dashed vertical lines correspond to the number of probes required to achieve the supF 9 and supF 3 cutoffs respectively. Panel C plots the calculated supF values (y-axis) as a window centered on the detected transition shrinks, causing fewer probes to be available for transition detection (x-axis). The solid and dashed black lines call attention to the supF 9 and supF 3 cutoffs. (D–E) Same as B and C, but for YAL026C. (F) Detection of cryptic initiation sites using subgenes of uniform length eliminates the relationship between gene length and cryptic initiation frequency. Cryptic initiation rates for subgene analysis (Methods) using liberal (green; supF = 0.58, 2417 transitions), moderate (red; supF = 0.97, 1135 transitions), and conservative (blue; supF = 1.8, 364 transitions) cutoffs are plotted for different gene-length bins. (G) Random distribution of cryptic initiation events mimics the real distribution discovered after controlling ascertainment bias. Plot is the same as panel F, but transitions were randomly assigned to ORF probes (liberal; 2417 transitions, moderate; 1135 transitions, conservative; 364 transitions). Compare G and F. The slight downward trend in both plots may result from a slightly lower probe density in longer genes, which was not corrected for.
Mentions: Previous studies have concluded that longer genes are especially prone to cryptic transcription upon deletion of SET2 [12]. This conclusion was based on the observation that long genes were more likely to be identified as containing a cryptic promoter than short genes. To determine if this observation could be explained solely by the fact that longer genes afford more opportunity for a cryptic event to occur, we grouped all genes according to size. For each group, we then calculated the rate of cryptic initiation (measured in transitions per base pair, Figure 2A). Measuring the rate of transitions, rather than the absolute number of transitions, for genes binned by length is a simple way to correct for gene length. The positive correlation between gene length and the rate of transitions per base shows that transitions are indeed detected more often in the context of longer genes, and that the additional transitions detected in long genes cannot be accounted for simply by correcting for gene length.

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