Stephen Buratowski

Harvard Medical School, Boston, Massachusetts, United States

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Publications (122)1618.45 Total impact

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    ABSTRACT: In addition to their annotated transcript, many eukaryotic mRNA promoters produce divergent noncoding transcripts. To define determinants of divergent promoter directionality, we used genomic replacement experiments. Sequences within noncoding transcripts specified their degradation pathways, and functional protein-coding transcripts could be produced in the divergent direction. To screen for mutants affecting the ratio of transcription in each direction, a bidirectional fluorescent protein reporter construct was introduced into the yeast nonessential gene deletion collection. We identified chromatin assembly as an important regulator of divergent transcription. Mutations in the CAF-I complex caused genome-wide derepression of nascent divergent noncoding transcription. In opposition to the CAF-I chromatin assembly pathway, H3K56 hyperacetylation, together with the nucleosome remodeler SWI/SNF, facilitated divergent transcription by promoting rapid nucleosome turnover. We propose that these chromatin-mediated effects control divergent transcription initiation, complementing downstream pathways linked to early termination and degradation of the noncoding RNAs.
    Cell 06/2014; 157(7):1712-23. · 31.96 Impact Factor
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    ABSTRACT: Methylation of histone H3 lysine 4 by the Set1 subunit of COMPASS correlates with active transcription. Here, we show that Set1 levels are regulated by protein degradation in response to multiple signals. Set1 levels are greatly reduced when COMPASS recruitment to genes, H3K4 methylation, or transcription is blocked. The degradation sequences map to N-terminal regions that overlap a previously identified autoinhibitory domain, as well as the catalytic domain. Truncation mutants of Set1 that cause under- or overexpression produce abnormal H3K4 methylation patterns on transcribed genes. Surprisingly, SAGA-dependent genes are more strongly affected than TFIID-dependent genes, reflecting differences in their chromatin dynamics. We propose that careful tuning of Set1 levels by regulated degradation is critical for the establishment and maintenance of proper H3K4 methylation patterns.
    Cell Reports 03/2014; · 7.21 Impact Factor
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    ABSTRACT: The RNA polymerase II (RNApII) C-terminal domain (CTD)-interacting domain (CID) proteins are involved in two distinct RNApII termination pathways and recognize different phosphorylated forms of CTD. To investigate the role of differential CTD-CID interactions in the choice of termination pathway, we altered the CTD-binding specificity of Nrd1 by domain swapping. Nrd1 with the CID from Rtt103 (Nrd1[CID(Rtt103)]) causes read-through transcription at many genes, but can also trigger termination where multiple Nrd1/Nab3-binding sites and Ser2P CTD co-exist. Therefore, CTD-CID interactions target specific termination complexes to help choose an RNApII termination pathway. Interactions of Nrd1 with both CTD and nascent transcripts contribute to efficient termination by the Nrd1 complex. Surprisingly, replacing the Nrd1 CID with that from Rtt103 reduces binding to Rrp6/Trf4, and RNA transcripts terminated by Nrd1[CID(Rtt103)] are predominantly processed by core exosome. Thus, the Nrd1 CID couples Ser5P CTD not only to termination, but also to RNA processing by the nuclear exosome.
    Journal of Biological Chemistry 11/2013; · 4.65 Impact Factor
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    ABSTRACT: The C-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II (RNApII), coordinates recruitment of RNA- and chromatin-modifying factors to transcription complexes. It is unclear whether the CTD communicates with the catalytic core region of Rpb1 and thus must be physically connected, or instead can function as an independent domain. To address this question, CTD was transferred to other RNApII subunits. Fusions to Rpb4 or Rpb6, two RNApII subunits located near the original position of CTD, support viability in a strain carrying a truncated Rpb1. In contrast, CTD fusion to Rpb9 on the other side of RNApII does not. Rpb4-CTD and Rpb6-CTD proteins are functional for phosphorylation and recruitment of various factors, albeit with some restrictions and minor defects. Normal CTD functions are not transferred to RNApI or RNApIII by Rbp6-CTD. These results show that, with some spatial constraints, CTD can function even when disconnected from Rpb1.
    Molecular cell 09/2013; · 14.61 Impact Factor
  • Luis M Soares, Stephen Buratowski
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    ABSTRACT: Two new studies in this issue of Molecular Cell (Kim et al., 2013 and Wu et al., 2013) provide new insights and reignite debate over how histone H2B ubiquitination promotes methylation of histone H3 lysine 4.
    Molecular cell 03/2013; 49(6):1019-20. · 14.61 Impact Factor
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    Stephen Buratowski
    Epigenetics & Chromatin 03/2013; 6(1). · 4.19 Impact Factor
  • Dane Z Hazelbaker, Stephen Buratowski
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    ABSTRACT: How do cells stop transcribing RNA Polymerase II to promote proper gene expression and prevent transcriptional havoc in the genome? In the case of Leishmania, a uniquely modified DNA base blocks RNA Polymerase II and suggests an interesting new model for transcription termination.
    Current biology: CB 11/2012; 22(22):R960-2. · 10.99 Impact Factor
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    ABSTRACT: The essential helicase-like protein Sen1 mediates termination of RNA Polymerase II (Pol II) transcription at snoRNAs and other noncoding RNAs in yeast. A mutation in the Pol II subunit Rpb1 that increases the elongation rate increases read-through transcription at Sen1-mediated terminators. Termination and growth defects in sen1 mutant cells are partially suppressed by a slowly transcribing Pol II mutant and are exacerbated by a faster-transcribing Pol II mutant. Deletion of the nuclear exosome subunit Rrp6 allows visualization of noncoding RNA intermediates that are terminated but not yet processed. Sen1 mutants or faster-transcribing Pol II increase the average lengths of preprocessed snoRNA, CUT, and SUT transcripts, while slowed Pol II transcription produces shorter transcripts. These connections between transcription rate and Sen1 activity support a model whereby kinetic competition between elongating Pol II and Sen1 helicase establishes the temporal and spatial window for early Pol II termination.
    Molecular cell 11/2012; · 14.61 Impact Factor
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    ABSTRACT: The Set3 histone deacetylase complex (Set3C) binds histone H3 dimethylated at lysine 4 (H3K4me2) to mediate deacetylation of histones in 5'-transcribed regions. To discern how Set3C affects gene expression, genome-wide transcription was analyzed in yeast undergoing a series of carbon source shifts. Deleting SET3 primarily caused changes during transition periods, as genes were induced or repressed. Surprisingly, a majority of Set3-affected genes are overlapped by noncoding RNA (ncRNA) transcription. Many Set3-repressed genes have H3K4me2 instead of me3 over promoter regions, due to either reduced H3K4me3 or ncRNA transcription from distal or antisense promoters. Set3C also represses internal cryptic promoters, but in different regions of genes than the Set2/Rpd3S pathway. Finally, Set3C stimulates some genes by repressing an overlapping antagonistic antisense transcript. These results show that overlapping noncoding transcription can fine-tune gene expression, not via the ncRNA but by depositing H3K4me2 to recruit the Set3C deacetylase.
    Cell 09/2012; 150(6):1158-69. · 31.96 Impact Factor
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    ABSTRACT: The cell-fate decision leading to gametogenesis is essential for sexual reproduction. In S. cerevisiae, only diploid MATa/α but not haploid MATa or MATα cells undergo gametogenesis, known as sporulation. We find that transcription of two long noncoding RNAs (lncRNAs) mediates mating-type control of sporulation. In MATa or MATα haploids, expression of IME1, the central inducer of gametogenesis, is inhibited in cis by transcription of the lncRNA IRT1, located in the IME1 promoter. IRT1 transcription recruits the Set2 histone methyltransferase and the Set3 histone deacetylase complex to establish repressive chromatin at the IME1 promoter. Inhibiting expression of IRT1 and an antisense transcript that antagonizes the expression of the meiotic regulator IME4 allows cells expressing the haploid mating type to sporulate with kinetics that are indistinguishable from that of MATa/α diploids. Conversely, expression of the two lncRNAs abolishes sporulation in MATa/α diploids. Thus, transcription of two lncRNAs governs mating-type control of gametogenesis in yeast.
    Cell 09/2012; 150(6):1170-81. · 31.96 Impact Factor
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    ABSTRACT: In-depth characterization of RNA polymerase II preinitiation complexes remains an important and challenging goal. We used quantitative mass spectrometry to explore context-dependent Saccharomyces cerevisiae preinitiation complex formation at the HIS4 promoter reconstituted on naked and chromatinized DNA templates. The transcription activators Gal4-VP16 and Gal4-Gcn4 recruited a limited set of chromatin-related coactivator complexes, namely the chromatin remodeler Swi/Snf and histone acetyltransferases SAGA and NuA4, suggesting that transcription stimulation is mediated through these factors. Moreover, the two activators differentially recruited the coactivator complexes, consistent with specific activator-coactivator interactions. Chromatinized templates suppressed recruitment of basal transcription factors, thereby amplifying the effect of activators, compared with naked DNA templates. This system is sensitive, highly reproducible, and easily applicable to mapping the repertoire of proteins found at any promoter.
    Journal of Biological Chemistry 08/2012; 287(42):35397-408. · 4.65 Impact Factor
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    ABSTRACT: Chd proteins are ATP-dependent chromatin remodeling enzymes implicated in biological functions from transcriptional elongation to control of pluripotency. Previous studies of the Chd1 subclass of these proteins have implicated them in diverse roles in gene expression including functions during initiation, elongation, and termination. Furthermore, some evidence has suggested a role for Chd1 in replication-independent histone exchange or assembly. Here, we examine roles of Chd1 in replication-independent dynamics of histone H3 in both Drosophila and yeast. We find evidence of a role for Chd1 in H3 dynamics in both organisms. Using genome-wide ChIP-on-chip analysis, we find that Chd1 influences histone turnover at the 5' and 3' ends of genes, accelerating H3 replacement at the 5' ends of genes while protecting the 3' ends of genes from excessive H3 turnover. Although consistent with a direct role for Chd1 in exchange, these results may indicate that Chd1 stabilizes nucleosomes perturbed by transcription. Curiously, we observe a strong effect of gene length on Chd1's effects on H3 turnover. Finally, we show that Chd1 also affects histone modification patterns over genes, likely as a consequence of its effects on histone replacement. Taken together, our results emphasize a role for Chd1 in histone replacement in both budding yeast and Drosophila melanogaster, and surprisingly they show that the major effects of Chd1 on turnover occur at the 3' ends of genes.
    PLoS Genetics 07/2012; 8(7):e1002811. · 8.52 Impact Factor
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    ABSTRACT: Packaging of eukaryotic genomes into chromatin has wide-ranging effects on gene transcription. Curiously, it is commonly observed that deletion of a global chromatin regulator affects expression of only a limited subset of genes bound to or modified by the regulator in question. However, in many single-gene studies it has become clear that chromatin regulators often do not affect steady-state transcription, but instead are required for normal transcriptional reprogramming by environmental cues. We therefore have systematically investigated the effects of 83 histone mutants, and 119 gene deletion mutants, on induction/repression dynamics of 170 transcripts in response to diamide stress in yeast. Importantly, we find that chromatin regulators play far more pronounced roles during gene induction/repression than they do in steady-state expression. Furthermore, by jointly analyzing the substrates (histone mutants) and enzymes (chromatin modifier deletions) we identify specific interactions between histone modifications and their regulators. Combining these functional results with genome-wide mapping of several histone marks in the same time course, we systematically investigated the correspondence between histone modification occurrence and function. We followed up on one pathway, finding that Set1-dependent H3K4 methylation primarily acts as a gene repressor during multiple stresses, specifically at genes involved in ribosome biosynthesis. Set1-dependent repression of ribosomal genes occurs via distinct pathways for ribosomal protein genes and ribosomal biogenesis genes, which can be separated based on genetic requirements for repression and based on chromatin changes during gene repression. Together, our dynamic studies provide a rich resource for investigating chromatin regulation, and identify a significant role for the "activating" mark H3K4me3 in gene repression.
    PLoS Biology 07/2012; 10(7):e1001369. · 12.69 Impact Factor
  • Luis M Soares, Stephen Buratowski
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    ABSTRACT: The Set1 complex (also known as complex associated with Set1 or COMPASS) methylates histone H3 on lysine 4, with different levels of methylation affecting transcription by recruiting various factors to distinct regions of active genes. Neither Set1 nor its associated proteins are essential for viability with the notable exception of Swd2, a WD repeat protein that is also a subunit of the essential transcription termination factor APT (associated with Pta1). Cells lacking Set1 lose COMPASS recruitment but show increased promoter cross-linking of TFIIE large subunit and the serine 5 phosphorylated form of the Rpb1 C-terminal domain. Although Swd2 is normally required for bringing APT to genes, deletion of SET1 restores both viability and APT recruitment to a strain lacking Swd2. We propose a model in which Swd2 is required for APT to overcome antagonism by COMPASS.
    Journal of Biological Chemistry 03/2012; 287(19):15219-31. · 4.65 Impact Factor
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    Stephen Buratowski
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    ABSTRACT: A genome-wide, high-resolution study of DNA-binding sites for proteins that transcribe DNA into RNA reveals details about how this process occurs in vivo. See Article p.295
    Nature 03/2012; 483(7389):286-7. · 38.60 Impact Factor
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    ABSTRACT: Single-stranded DNA-binding proteins play many roles in nucleic acid metabolism, but their importance during transcription remains unclear. Quantitative proteomic analysis of RNA polymerase II (RNApII) preinitiation complexes (PICs) identified Sub1 and the replication protein A complex (RPA), both of which bind single-stranded DNA (ssDNA). Sub1, homolog of mammalian coactivator PC4, exhibits strong genetic interactions with factors necessary for promoter melting. Sub1 localizes near the transcription bubble in vitro and binds to promoters in vivo dependent upon PIC assembly. In contrast, RPA localizes to transcribed regions of active genes, strongly correlated with transcribing RNApII but independently of replication. RFA1 interacts genetically with transcription elongation factor genes. Interestingly, RPA levels increase at active promoters in cells carrying a Sub1 deletion or ssDNA-binding mutant, suggesting competition for a common binding site. We propose that Sub1 and RPA interact with the nontemplate strand of RNApII complexes during initiation and elongation, respectively.
    Molecular cell 11/2011; 44(3):397-409. · 14.61 Impact Factor
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    John Holik, TaeSoo Kim, Stephen Buratowski
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    ABSTRACT: Graphical Abstract Figure optionsView in workspaceDownload full-size imageDownload high-quality image (176 K)Download as PowerPoint slide Highlights ► A quantitative proteomics screen finds Sub1 and RPA in transcription complexes ► Sub1 localizes near the transcription bubble for open complex formation ► RPA associates with transcribed regions of the genome ► Mutations in Sub1 ssDNA binding result in relocalization of RPA to promoters
    Molecular Cell. 11/2011; 44(3):397–409.
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    ABSTRACT: In Saccharomyces cerevisiae, the Nrd1-Nab3-Sen1 pathway mediates the termination of snoRNAs and cryptic unstable transcripts (CUTs). Both Nrd1 and the Set1 histone H3K4 methyltransferase complex interact with RNA polymerase II (Pol II) during early elongation, leading us to test whether these two processes are functionally linked. The deletion of SET1 exacerbates the growth rate and termination defects of nrd1 mutants. Set1 is important for the appropriate recruitment of Nrd1. Additionally, Set1 modulates histone acetylation levels in the promoter-proximal region via the Rpd3L deacetylase and NuA3 acetyltransferase complexes, both of which contain PHD finger proteins that bind methylated H3K4. Increased levels of histone acetylation reduce the efficiency of Nrd1-dependent termination. We speculate that Set1 promotes proper early termination by the Nrd1-Nab3-Sen1 complex by affecting the kinetics of Pol II transcription in early elongation.
    Molecular and cellular biology 06/2011; 31(17):3569-83. · 6.06 Impact Factor
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    ABSTRACT: Packaging of DNA into chromatin has a profound impact on gene expression. To understand how changes in chromatin influence transcription, we analyzed 165 mutants of chromatin machinery components in Saccharomyces cerevisiae. mRNA expression patterns change in 80% of mutants, always with specific effects, even for loss of widespread histone marks. The data are assembled into a network of chromatin interaction pathways. The network is function based, has a branched, interconnected topology, and lacks strict one-to-one relationships between complexes. Chromatin pathways are not separate entities for different gene sets, but share many components. The study evaluates which interactions are important for which genes and predicts additional interactions, for example between Paf1C and Set3C, as well as a role for Mediator in subtelomeric silencing. The results indicate the presence of gene-dependent effects that go beyond context-dependent binding of chromatin factors and provide a framework for understanding how specificity is achieved through regulating chromatin.
    Molecular cell 05/2011; 42(4):536-49. · 14.61 Impact Factor
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    S Buratowski, T Kim
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    ABSTRACT: The carboxy-terminal domain (CTD) of the RNA polymerase II subunit Rpb1 undergoes dynamic phosphorylation, with different phosphorylation sites predominating at different stages of transcription. Our laboratory studies show how various mRNA-processing and chromatin-modifying enzymes interact with the phosphorylated CTD to efficiently produce mRNAs. The H3K36 methyltransferase Set2 interacts with CTD carrying phosphorylations characteristic of downstream elongation complexes, and the resulting cotranscriptional H3K36 methylation targets the Rpd3S histone deacetylase to downstream transcribed regions. Although positively correlated with gene activity, this pathway actually inhibits transcription elongation as well as initiation from cryptic promoters within genes. During early elongation, CTD serine 5 phosphorylation helps recruit the H3K4 methyltransferase complex containing Set1. Within 5' transcribed regions, cotranscriptional H3K4 dimethylation (H3K4me2) by Set1 recruits the deacetylase complex Set3C. Finally, H3K4 trimethylation at the most promoter-proximal nucleosomes is thought to stimulate transcription by promoting histone acetylation by complexes containing the ING/Yng PHD finger proteins. Surprisingly, the Rpd3L histone deacetylase complex, normally a transcription repressor, may also recognize H3K4me3. Together, the cotranscriptional histone methylations appear to function primarily to distinguish active promoter regions, which are marked by high levels of acetylation and nucleosome turnover, from the deacetylated, downstream transcribed regions of genes.
    Cold Spring Harbor Symposia on Quantitative Biology 03/2011; 75:95-102.

Publication Stats

10k Citations
1,618.45 Total Impact Points

Institutions

  • 1995–2014
    • Harvard Medical School
      • Department of Biological Chemistry and Molecular Pharmacology
      Boston, Massachusetts, United States
  • 2012
    • Hebrew University of Jerusalem
      • Rachel and Selim Benin School of Computer Science and Engineering
      Jerusalem, Jerusalem District, Israel
  • 1999–2012
    • Harvard University
      • • FAS Center for Systems Biology
      • • Department of Chemistry and Chemical Biology
      Cambridge, Massachusetts, United States
  • 2011
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
  • 2009
    • Hanyang University
      • Department of Molecular and Life Science
      Seoul, Seoul, South Korea
  • 2002–2005
    • University of Toronto
      • Banting and Best Department of Medical Research
      Toronto, Ontario, Canada
  • 1991–1994
    • Whitehead Institute for Biomedical Research
      Cambridge, Massachusetts, United States
  • 1988–1990
    • Massachusetts Institute of Technology
      • Department of Biology
      Cambridge, MA, United States
  • 1989
    • Fred Hutchinson Cancer Research Center
      Seattle, Washington, United States