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

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.

Download full-text


Available from: Brian D Strahl, Sep 27, 2015
15 Reads
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    Nucleic Acids Research 01/2013; DOI:10.1093/nar/gks1472 · 9.11 Impact Factor
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Understanding the mechanisms by which chromatin structure controls eukaryotic transcription has been an intense area of investigation for the past 25 years. Many of the key discoveries that created the foundation for this field came from studies of Saccharomyces cerevisiae, including the discovery of the role of chromatin in transcriptional silencing, as well as the discovery of chromatin-remodeling factors and histone modification activities. Since that time, studies in yeast have continued to contribute in leading ways. This review article summarizes the large body of yeast studies in this field.
    Genetics 02/2012; 190(2):351-87. DOI:10.1534/genetics.111.132266 · 5.96 Impact Factor
  • Source
    • "the results obtained through the use of this reporter plasmid could be due to events of cryptic initiation arising from within the reporter. No evidence for cryptic initiation has been found at HIS3 in previous genomic studies (Lickwar et al. 2009). However, to be certain that cryptic initiation was not occurring in the reporter, we examined HIS3 transcripts arising from within the reporter plasmids by Northern blot analysis. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The conserved eukaryotic Paf1 complex regulates RNA synthesis by RNA polymerase II at multiple levels, including transcript elongation, transcript termination, and chromatin modifications. To better understand the contributions of the Paf1 complex to transcriptional regulation, we generated mutations that alter conserved residues within the Rtf1 subunit of the Saccharomyces cerevisiae Paf1 complex. Importantly, single amino acid substitutions within a region of Rtf1 that is conserved from yeast to humans, which we termed the histone modification domain, resulted in the loss of histone H2B ubiquitylation and impaired histone H3 methylation. Phenotypic analysis of these mutations revealed additional defects in telomeric silencing, transcription elongation, and prevention of cryptic initiation. We also demonstrated that amino acid substitutions within the Rtf1 histone modification domain disrupt 3'-end formation of snoRNA transcripts and identify a previously uncharacterized regulatory role for the histone H2B K123 ubiquitylation mark in this process. Cumulatively, our results reveal functionally important residues in Rtf1, better define the roles of Rtf1 in transcription and histone modification, and provide strong genetic support for the participation of histone modification marks in the termination of noncoding RNAs.
    Genetics 03/2011; 188(2):273-89. DOI:10.1534/genetics.111.128645 · 5.96 Impact Factor
Show more