Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning

BackgroundTFIIS stimulates RNA cleavage by RNA polymerase II and promotes the resolution of backtracking events. TFIIS acts in the chromatin context, but its contribution to the chromatin landscape has not yet been investigated. Co-transcriptional chromatin alterations include subtle changes in nucleosome positioning, like those expected to be elicited by TFIIS, which are elusive to detect. The most popular method to map nucleosomes involves intensive chromatin digestion by micrococcal nuclease (MNase). Maps based on these exhaustively digested samples miss any MNase-sensitive nucleosomes caused by transcription. In contrast, partial digestion approaches preserve such nucleosomes, but introduce noise due to MNase sequence preferences. A systematic way of correcting this bias for massively parallel sequencing experiments is still missing.ResultsTo investigate the contribution of TFIIS to the chromatin landscape, we developed a refined nucleosome-mapping method in Saccharomyces cerevisiae. Based on partial MNase digestion and a sequence-bias correction derived from naked DNA cleavage, the refined method efficiently mapped nucleosomes in promoter regions rich in MNase-sensitive structures. The naked DNA correction was also important for mapping gene body nucleosomes, particularly in those genes whose core promoters contain a canonical TATA element. With this improved method, we analyzed the global nucleosomal changes caused by lack of TFIIS. We detected a general increase in nucleosomal fuzziness and more restricted changes in nucleosome occupancy, which concentrated in some gene categories. The TATA-containing genes were preferentially associated with decreased occupancy in gene bodies, whereas the TATA-like genes did so with increased fuzziness. The detected chromatin alterations correlated with functional defects in nascent transcription, as revealed by genomic run-on experiments.ConclusionsThe combination of partial MNase digestion and naked DNA correction of the sequence bias is a precise nucleosomal mapping method that does not exclude MNase-sensitive nucleosomes. This method is useful for detecting subtle alterations in nucleosome positioning produced by lack of TFIIS. Their analysis revealed that TFIIS generally contributed to nucleosome positioning in both gene promoters and bodies. The independent effect of lack of TFIIS on nucleosome occupancy and fuzziness supports the existence of alternative chromatin dynamics during transcription elongation.

[1]  Roderic Guigó,et al.  The GEM mapper: fast, accurate and versatile alignment by filtration , 2012, Nature Methods.

[2]  E. Trifonov,et al.  The pitch of chromatin DNA is reflected in its nucleotide sequence. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[3]  Bryan J Venters,et al.  A barrier nucleosome model for statistical positioning of nucleosomes throughout the yeast genome. , 2008, Genome research.

[4]  Martin Vingron,et al.  The Effect of Micrococcal Nuclease Digestion on Nucleosome Positioning Data , 2010, PloS one.

[5]  Kevin Struhl,et al.  Evidence for Eviction and Rapid Deposition of Histones upon Transcriptional Elongation by RNA Polymerase II , 2004, Molecular and Cellular Biology.

[6]  Nicholas Proudfoot,et al.  Isw1 Chromatin Remodeling ATPase Coordinates Transcription Elongation and Termination by RNA Polymerase II , 2003, Cell.

[7]  Cizhong Jiang,et al.  A compiled and systematic reference map of nucleosome positions across the Saccharomyces cerevisiae genome , 2009, Genome Biology.

[8]  I. Albert,et al.  Translational and rotational settings of H2A.Z nucleosomes across the Saccharomyces cerevisiae genome , 2007, Nature.

[9]  Geoffrey J. Barton,et al.  A Role for Snf2-Related Nucleosome-Spacing Enzymes in Genome-Wide Nucleosome Organization , 2011, Science.

[10]  Marcel Geertz,et al.  Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription , 2014, Genes & development.

[11]  Ø. Hammer,et al.  PAST: PALEONTOLOGICAL STATISTICAL SOFTWARE PACKAGE FOR EDUCATION AND DATA ANALYSIS , 2001 .

[12]  Srinivas Ramachandran,et al.  Asymmetric nucleosomes flank promoters in the budding yeast genome , 2015, Genome research.

[13]  Kevin Struhl,et al.  Nucleosome depletion at yeast terminators is not intrinsic and can occur by a transcriptional mechanism linked to 3’-end formation , 2010, Proceedings of the National Academy of Sciences.

[14]  K. Severinov,et al.  Analysis of the mechanism of nucleosome survival during transcription , 2013, Nucleic acids research.

[15]  Glòria Mas,et al.  Recruitment of a chromatin remodelling complex by the Hog1 MAP kinase to stress genes , 2009, The EMBO journal.

[16]  R. Chen,et al.  Nucleosome fragility reveals novel functional states of chromatin and poises genes for activation. , 2011, Genome research.

[17]  Benjamin Guglielmi,et al.  TFIIS elongation factor and Mediator act in conjunction during transcription initiation in vivo , 2007, Proceedings of the National Academy of Sciences.

[18]  Lidia Delgado-Ramos,et al.  TFIIS is required for the balanced expression of the genes encoding ribosomal components under transcriptional stress , 2012, Nucleic acids research.

[19]  J. Pérez-Ortín,et al.  Chromatin structure of yeast genes , 1989, Yeast.

[20]  Modesto Orozco,et al.  Physical properties of naked DNA influence nucleosome positioning and correlate with transcription start and termination sites in yeast , 2011, BMC Genomics.

[21]  M. Noll.  Subunit structure of chromatin , 1974, Nature.

[22]  E. O’Shea,et al.  A computational approach to map nucleosome positions and alternative chromatin states with base pair resolution , 2016, eLife.

[23]  J. Pérez-Ortín,et al.  Genomic-wide methods to evaluate transcription rates in yeast. , 2011, Methods in molecular biology.

[24]  Zhaoyu Li,et al.  DANPOS: Dynamic analysis of nucleosome position and occupancy by sequencing , 2013, Genome research.

[25]  R. Morse,et al.  Assays for nucleosome positioning in yeast. , 1999, Methods in enzymology.

[26]  T. Richmond,et al.  Crystal structure of the nucleosome core particle at 2.8 Å resolution , 1997, Nature.

[27]  Daria A. Gaykalova,et al.  Mechanism of chromatin remodeling and recovery during passage of RNA polymerase II , 2009, Nature Structural &Molecular Biology.

[28]  W Hörz,et al.  Sequence specific cleavage of DNA by micrococcal nuclease. , 1981, Nucleic acids research.

[29]  R. Roeder,et al.  Transcription of in vitro assembled chromatin templates in a highly purified RNA polymerase II system. , 2009, Methods.

[30]  C. Bustamante,et al.  The elongation rate of RNA polymerase determines the fate of transcribed nucleosomes , 2011, Nature Structural &Molecular Biology.

[31]  R. Kornberg.  Chromatin structure: a repeating unit of histones and DNA. , 1974, Science.

[32]  G. Ammerer,et al.  H3K4 monomethylation dictates nucleosome dynamics and chromatin remodeling at stress-responsive genes , 2015, Nucleic acids research.

[33]  Juan González-Vallinas,et al.  Hog1 bypasses stress-mediated down-regulation of transcription by RNA polymerase II redistribution and chromatin remodeling , 2012, Genome Biology.

[34]  S. Henikoff,et al.  Epigenome characterization at single base-pair resolution , 2011, Proceedings of the National Academy of Sciences.

[35]  K. Struhl,et al.  Determinants of nucleosome positioning , 2013, Nature Structural &Molecular Biology.

[36]  L. Steinmetz,et al.  Bidirectional promoters generate pervasive transcription in yeast , 2009, Nature.

[37]  B. Pugh,et al.  Genome-wide structure and organization of eukaryotic pre-initiation complexes , 2011, Nature.

[38]  R. Aebersold,et al.  Molecular basis of Rrn3-regulated RNA polymerase I initiation and cell growth. , 2011, Genes & development.

[39]  D. Luse,et al.  Efficient and Rapid Nucleosome Traversal by RNA Polymerase II Depends on a Combination of Transcript Elongation Factors* , 2010, The Journal of Biological Chemistry.

[40]  S. Malik,et al.  Mediator-regulated transcription through the +1 nucleosome. , 2012, Molecular cell.

[41]  Kairong Cui,et al.  Genome-wide approaches to determining nucleosome occupancy in metazoans using MNase-Seq. , 2012, Methods in molecular biology.

[42]  Vicent Pelechano,et al.  A Complete Set of Nascent Transcription Rates for Yeast Genes , 2010, PloS one.

[43]  J. Pérez-Ortín,et al.  Impact of high pH stress on yeast gene expression: A comprehensive analysis of mRNA turnover during stress responses. , 2015, Biochimica et biophysica acta.

[44]  D. Shore,et al.  Nucleosome Stability Distinguishes Two Different Promoter Types at All Protein-Coding Genes in Yeast. , 2015, Molecular cell.

[45]  J. Pérez-Ortín,et al.  Biotin-Genomic Run-On (Bio-GRO): A High-Resolution Method for the Analysis of Nascent Transcription in Yeast. , 2016, Methods in molecular biology.

[46]  Daria A. Gaykalova,et al.  Structural analysis of nucleosomal barrier to transcription , 2015, Proceedings of the National Academy of Sciences.

[47]  P. Cramer,et al.  Structural basis of RNA polymerase II backtracking, arrest and reactivation , 2011, Nature.

[48]  Nir Friedman,et al.  High-resolution nucleosome mapping reveals transcription-dependent promoter packaging. , 2010, Genome research.

[49]  S. Elgin,et al.  Analysis of Drosophila chromatin structure in vivo. , 1999, Methods in enzymology.

[50]  R A Laskey,et al.  High sequence specificity of micrococcal nuclease. , 1981, Nucleic acids research.

[51]  J. Mellor,et al.  Precise nucleosome positioning and the TATA box dictate requirements for the histone H4 tail and the bromodomain factor Bdf1. , 2004, Molecular cell.

[52]  Kristin R Brogaard,et al.  A base pair resolution map of nucleosome positions in yeast , 2012, Nature.

[53]  J. Reeve,et al.  Archaeal Histones, Nucleosomes, and Transcription Initiation , 1997, Cell.

[54]  D. Clark,et al.  MNase-Sensitive Complexes in Yeast: Nucleosomes and Non-histone Barriers. , 2017, Molecular cell.

[55]  Vasily M Studitsky,et al.  Nature of the nucleosomal barrier to RNA polymerase II. , 2005, Molecular cell.

[56]  Noam Kaplan,et al.  Gene expression divergence in yeast is coupled to evolution of DNA-encoded nucleosome organization , 2009, Nature Genetics.

[57]  J. Svejstrup,et al.  Evidence that Transcript Cleavage Is Essential for RNA Polymerase II Transcription and Cell Viability , 2010, Molecular cell.

[58]  L. Steinmetz,et al.  Chromatin-dependent regulation of RNA polymerases II and III activity throughout the transcription cycle , 2014, Nucleic acids research.

[59]  Sebastián Chávez,et al.  Genome-wide studies of mRNA synthesis and degradation in eukaryotes. , 2012, Biochimica et biophysica acta.

[60]  R. Aebersold,et al.  The transcription elongation factor TFIIS is a component of RNA polymerase II preinitiation complexes , 2007, Proceedings of the National Academy of Sciences.

[61]  Steven M. Johnson,et al.  A high-resolution, nucleosome position map of C. elegans reveals a lack of universal sequence-dictated positioning. , 2008, Genome research.

[62]  R. Kornberg,et al.  Upstream activation sequence-dependent alteration of chromatin structure and transcription activation of the yeast GAL1-GAL10 genes , 1989, Molecular and cellular biology.

[63]  F. Winston,et al.  Evidence that the Elongation Factor TFIIS Plays a Role in Transcription Initiation at GAL1 in Saccharomyces cerevisiae , 2005, Molecular and Cellular Biology.

[64]  J. Workman,et al.  Histone density is maintained during transcription mediated by the chromatin remodeler RSC and histone chaperone NAP1 in vitro , 2012, Proceedings of the National Academy of Sciences.

[65]  Lani F. Wu,et al.  Genome-Scale Identification of Nucleosome Positions in S. cerevisiae , 2005, Science.

[66]  Alexandre V Morozov,et al.  Ubiquitous nucleosome crowding in the yeast genome , 2014, Proceedings of the National Academy of Sciences.