The Set2/Rpd3S Pathway Suppresses Cryptic Transcription without Regard to Gene Length or Transcription Frequency

Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
PLoS ONE (Impact Factor: 3.23). 02/2009; 4(3):e4886. DOI: 10.1371/journal.pone.0004886
Source: PubMed


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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    • "Previous studies have shown that depletion of Set2 in yeast (11–14) leads to increased levels of spurious transcripts initiated from cryptic promoter-like sequences within genes. These cryptic alternative start sites are widespread in eukaryotic genomes and can be found on virtually every single gene (15). Thus, H3K36me3 is thought to be required for repression of cryptic transcriptional initiation, but the mechanisms involved are not yet completely understood. "
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    ABSTRACT: Histone H3 of nucleosomes positioned on active genes is trimethylated at Lys36 (H3K36me3) by the SETD2 (also termed KMT3A/SET2 or HYPB) methyltransferase. Previous studies in yeast indicated that H3K36me3 prevents spurious intragenic transcription initiation through recruitment of a histone deacetylase complex, a mechanism that is not conserved in mammals. Here, we report that downregulation of SETD2 in human cells leads to intragenic transcription initiation in at least 11% of active genes. Reduction of SETD2 prevents normal loading of the FACT (FAcilitates Chromatin Transcription) complex subunits SPT16 and SSRP1, and decreases nucleosome occupancy in active genes. Moreover, co-immunoprecipitation experiments suggest that SPT16 is recruited to active chromatin templates, which contain H3K36me3-modified nucleosomes. Our results further show that within minutes after transcriptional activation, there is a SETD2-dependent reduction in gene body occupancy of histone H2B, but not of histone H3, suggesting that SETD2 coordinates FACT-mediated exchange of histone H2B during transcription-coupled nucleosome displacement. After inhibition of transcription, we observe a SETD2-dependent recruitment of FACT and increased histone H2B occupancy. These data suggest that SETD2 activity modulates FACT recruitment and nucleosome dynamics, thereby repressing cryptic transcription initiation.
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    • "Antagonism of kanMX Expression Depends on the Set2-Rpd3 Pathway H3K36me3, a modification catalyzed by the Set2 methyltransferase (Krogan et al., 2003), is a known element contributing to the repression of spurious transcription initiation via recruitment of the Rpd3 histone deacetylase complex (Carrozza et al., 2005; Lickwar et al., 2009). To test the hypothesis that this mechanism of regulation may also play a role in relation to kanMX, we measured kanMX expression in strains without SET2 or RPD3. "
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    • "In spt6 mutants, a genome-wide assay revealed that cryptic initiation occurs at 1000 genes (Cheung et al. 2008). This level of cryptic initiation is likely an underestimate of the true level, as this study looked only at coding strands, and the method of detection would have found cryptic initiation only in genes transcribed at low levels (Cheung et al. 2008; Lickwar et al. 2009). Cryptic initiation has also been observed in several other mutants, including spt16 and set2 (Kaplan et al. 2003; Mason and Struhl 2003; Carrozza et al. 2005; Prather et al. 2005; Nourani et al. 2006; Li et al. 2007b; Xiao et al. 2007; Cheung et al. 2008; Imbeault et al. 2008), with spt6 and spt16 mutants having the strongest effects (Cheung et al. 2008). "
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