SWI/SNF-Like Chromatin Remodeling Factor Fun30 Supports Point Centromere Function in S. cerevisiae

Budding yeast centromeres are sequence-defined point centromeres and are, unlike in many other organisms, not embedded in heterochromatin. Here we show that Fun30, a poorly understood SWI/SNF-like chromatin remodeling factor conserved in humans, promotes point centromere function through the formation of correct chromatin architecture at centromeres. Our determination of the genome-wide binding and nucleosome positioning properties of Fun30 shows that this enzyme is consistently enriched over centromeres and that a majority of CENs show Fun30-dependent changes in flanking nucleosome position and/or CEN core micrococcal nuclease accessibility. Fun30 deletion leads to defects in histone variant Htz1 occupancy genome-wide, including at and around most centromeres. FUN30 genetically interacts with CSE4, coding for the centromere-specific variant of histone H3, and counteracts the detrimental effect of transcription through centromeres on chromosome segregation and suppresses transcriptional noise over centromere CEN3. Previous work has shown a requirement for fission yeast and mammalian homologs of Fun30 in heterochromatin assembly. As centromeres in budding yeast are not embedded in heterochromatin, our findings indicate a direct role of Fun30 in centromere chromatin by promoting correct chromatin architecture.

[1]  D. Oxley,et al.  Maintenance of silent chromatin through replication requires SWI/SNF-like chromatin remodeler SMARCAD1. , 2011, Molecular cell.

[2]  J. Peters.  The anaphase promoting complex/cyclosome: a machine designed to destroy , 2006, Nature Reviews Molecular Cell Biology.

[3]  M. Christman,et al.  A novel family of TRF (DNA topoisomerase I-related function) genes required for proper nuclear segregation. , 1996, Nucleic acids research.

[4]  Geoffrey J. Barton,et al.  Identification of multiple distinct Snf2 subfamilies with conserved structural motifs , 2006, Nucleic acids research.

[5]  Kerry Bloom,et al.  Centromeres: unique chromatin structures that drive chromosome segregation , 2011, Nature Reviews Molecular Cell Biology.

[6]  T. Itoh,et al.  Identification of cis-acting sites for condensin loading onto budding yeast chromosomes. , 2008, Genes & development.

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

[8]  Conrad A. Nieduszynski,et al.  Genome-wide identification of replication origins in yeast by comparative genomics. , 2006, Genes & development.

[9]  Xin Wang,et al.  A RSC/Nucleosome Complex Determines Chromatin Architecture and Facilitates Activator Binding , 2010, Cell.

[10]  Raymond K. Auerbach,et al.  Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing , 2009 .

[11]  Oliver J. Rando,et al.  Chromatin remodelling at promoters suppresses antisense transcription , 2007, Nature.

[12]  S. Elledge,et al.  New yeast genes important for chromosome integrity and segregation identified by dosage effects on genome stability. , 1999, Nucleic acids research.

[13]  Wei-Hua Wu,et al.  ATP-Driven Exchange of Histone H2AZ Variant Catalyzed by SWR1 Chromatin Remodeling Complex , 2004, Science.

[14]  M. Ashburner,et al.  Gene Ontology: tool for the unification of biology , 2000, Nature Genetics.

[15]  P. Sorger,et al.  Binding of the essential Saccharomyces cerevisiae kinetochore protein Ndc10p to CDEII. , 2003, Molecular biology of the cell.

[16]  O. Rando,et al.  Global Regulation of H2A.Z Localization by the INO80 Chromatin-Remodeling Enzyme Is Essential for Genome Integrity , 2011, Cell.

[17]  W. Liang,et al.  9) TM4 Microarray Software Suite , 2006 .

[18]  Andrew W. Murray,et al.  Recruiting a microtubule-binding complex to DNA directs chromosome segregation in budding yeast , 2009, Nature Cell Biology.

[19]  M. Schoor,et al.  Skeletal dysplasias, growth retardation, reduced postnatal survival, and impaired fertility in mice lacking the SNF2/SWI2 family member ETL1 , 1999, Mechanisms of Development.

[20]  O. Stemmann,et al.  A putative protein complex consisting of Ctf19, Mcm21, and Okp1 represents a missing link in the budding yeast kinetochore. , 1999, Genes & development.

[21]  J. Workman,et al.  Psh1 is an E3 ubiquitin ligase that targets the centromeric histone variant Cse4. , 2010, Molecular cell.

[22]  M. Collart,et al.  Preparation of Yeast RNA , 1993, Current protocols in molecular biology.

[23]  Mathieu Blanchette,et al.  Variant Histone H2A.Z Is Globally Localized to the Promoters of Inactive Yeast Genes and Regulates Nucleosome Positioning , 2005, PLoS biology.

[24]  K. Bloom,et al.  Tension-dependent nucleosome remodeling at the pericentromere in yeast , 2012, Molecular biology of the cell.

[25]  K. Bloom,et al.  Differential kinetochore protein requirements for establishment versus propagation of centromere activity in Saccharomyces cerevisiae , 2003, The Journal of cell biology.

[26]  Stefan Westermann,et al.  CENP-T proteins are conserved centromere receptors of the Ndc80 complex , 2012, Nature Cell Biology.

[27]  Andrew J Link,et al.  A Protein Complex Containing the Conserved Swi2/Snf2-Related ATPase Swr1p Deposits Histone Variant H2A.Z into Euchromatin , 2004, PLoS biology.

[28]  S. Biggins,et al.  Centromere identity is specified by a single centromeric nucleosome in budding yeast , 2007, Proceedings of the National Academy of Sciences.

[29]  G. Barton,et al.  The SWI/SNF complex acts to constrain distribution of the centromeric histone variant Cse4 , 2011, The EMBO journal.

[30]  Hasanuzzaman Bhuiyan,et al.  The FUN30 Chromatin Remodeler, Fft3, Protects Centromeric and Subtelomeric Domains from Euchromatin Formation , 2011, PLoS genetics.

[31]  P. Meluh,et al.  The Yeast RSC Chromatin-Remodeling Complex Is Required for Kinetochore Function in Chromosome Segregation , 2003, Molecular and Cellular Biology.

[32]  G. Mizuguchi,et al.  Nonhistone Scm3 and Histones CenH3-H4 Assemble the Core of Centromere-Specific Nucleosomes , 2007, Cell.

[33]  D. Fyodorov,et al.  CenH3/CID Incorporation Is Not Dependent on the Chromatin Assembly Factor CHD1 in Drosophila , 2010, PloS one.

[34]  Brian E Snydsman,et al.  Chl4p and iml3p are two new members of the budding yeast outer kinetochore. , 2003, Molecular biology of the cell.

[35]  H. Ogiwara,et al.  The INO80 Chromatin Remodeling Complex Functions in Sister Chromatid Cohesion , 2007, Cell cycle.

[36]  Ann E. Loraine,et al.  The Integrated Genome Browser: free software for distribution and exploration of genome-scale datasets , 2009, Bioinform..

[37]  P. Bjerling,et al.  The CHD remodeling factor Hrp1 stimulates CENP-A loading to centromeres , 2005, Nucleic acids research.

[38]  Cameron S. Osborne,et al.  Large Scale Loss of Data in Low-Diversity Illumina Sequencing Libraries Can Be Recovered by Deferred Cluster Calling , 2011, PloS one.

[39]  B. Cairns,et al.  Genome-Wide Dynamics of Htz1, a Histone H2A Variant that Poises Repressed/Basal Promoters for Activation through Histone Loss , 2005, Cell.

[40]  E. Tsuchiya,et al.  The Saccharomyces cerevisiae NPS1 gene, a novel CDC gene which encodes a 160 kDa nuclear protein involved in G2 phase control. , 1992, The EMBO journal.

[41]  Lambert C. J. Dorssers,et al.  GO-Mapper: functional analysis of gene expression data using the expression level as a score to evaluate Gene Ontology terms , 2004, Bioinform..

[42]  K. Ekwall.  Epigenetic control of centromere behavior. , 2007, Annual review of genetics.

[43]  T. Hazbun,et al.  Cnn1 inhibits the interactions between the KMN complexes of the yeast kinetochore , 2013, Nature Cell Biology.

[44]  S. Biggins,et al.  De novo kinetochore assembly requires the centromeric histone H3 variant. , 2005, Molecular biology of the cell.

[45]  A. Hyman,et al.  Identification of essential components of the S. cerevisiae kinetochore , 1993, Cell.

[46]  F. Thoma,et al.  Kinetochores Prevent Repair of UV Damage in Saccharomyces cerevisiae Centromeres , 2004, Molecular and Cellular Biology.

[47]  D. Tremethick,et al.  RNA interference demonstrates a novel role for H2A.Z in chromosome segregation , 2004, Nature Structural &Molecular Biology.

[48]  M. Grunstein,et al.  Specific functions for the fission yeast Sirtuins Hst2 and Hst4 in gene regulation and retrotransposon silencing , 2007, The EMBO journal.

[49]  K. Bloom,et al.  Genetic manipulation of centromere function , 1987, Molecular and cellular biology.

[50]  F. Azorín,et al.  Focus on the centre: the role of chromatin on the regulation of centromere identity and function , 2009, The EMBO journal.

[51]  D. Koshland,et al.  Budding yeast centromere composition and assembly as revealed by in vivo cross-linking. , 1997, Genes & development.

[52]  D. Reinberg,et al.  Histone variants meet their match , 2005, Nature Reviews Molecular Cell Biology.

[53]  A I Saeed,et al.  TM4: a free, open-source system for microarray data management and analysis. , 2003, BioTechniques.

[54]  Janina Maier,et al.  Guide to yeast genetics and molecular biology. , 1991, Methods in enzymology.

[55]  J. Ausió,et al.  H2A.Z-Mediated Genome-Wide Chromatin Specialization. , 2007, Current genomics.

[56]  T. Itoh,et al.  Cohesin relocation from sites of chromosomal loading to places of convergent transcription , 2004, Nature.

[57]  J. Workman,et al.  Preferential occupancy of histone variant H2AZ at inactive promoters influences local histone modifications and chromatin remodeling. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[58]  M. Washburn,et al.  Scm3 is essential to recruit the histone h3 variant cse4 to centromeres and to maintain a functional kinetochore. , 2007, Molecular cell.

[59]  T. Fukagawa,et al.  CENP-H-containing complex facilitates centromere deposition of CENP-A in cooperation with FACT and CHD1. , 2009, Molecular biology of the cell.

[60]  M. Gerstein,et al.  The Transcriptional Landscape of the Yeast Genome Defined by RNA Sequencing , 2008, Science.

[61]  K. Sanyal,et al.  The IML3/MCM19 gene of Saccharomyces cerevisiae is required for a kinetochore-related process during chromosome segregation , 2001, Molecular Genetics and Genomics.

[62]  J. Hegemann,et al.  A fast method to diagnose chromosome and plasmid loss in Saccharomyces cerevisiae strains , 1999, Yeast.

[63]  Sheng Zhong,et al.  Dissecting Early Differentially Expressed Genes in a Mixture of Differentiating Embryonic Stem Cells , 2009, PLoS Comput. Biol..

[64]  S. Schreiber,et al.  Histone Variant H2A.Z Marks the 5′ Ends of Both Active and Inactive Genes in Euchromatin , 2006, Cell.

[65]  Eiko Tsuchiya,et al.  A mutation in NPS1/STH1, an essential gene encoding a component of a novel chromatin-remodeling complex RSC, alters the chromatin structure of Saccharomyces cerevisiae centromeres , 1998, Nucleic Acids Res..

[66]  D. Tollervey,et al.  The nuclear RNA surveillance machinery: the link between ncRNAs and genome structure in budding yeast? , 2008, Biochimica et biophysica acta.

[67]  Gerald R. Fink,et al.  Guide to yeast genetics and molecular biology , 1993 .

[68]  Huiming Ding,et al.  A Snf2 family ATPase complex required for recruitment of the histone H2A variant Htz1. , 2003, Molecular cell.

[69]  T. Owen-Hughes,et al.  The Snf2 Homolog Fun30 Acts as a Homodimeric ATP-dependent Chromatin-remodeling Enzyme* , 2010, The Journal of Biological Chemistry.

[70]  J. Miller,et al.  The SNF2-Family Member Fun30 Promotes Gene Silencing in Heterochromatic Loci , 2009, PloS one.

[71]  M. MacCoss,et al.  An E3 ubiquitin ligase prevents ectopic localization of the centromeric histone H3 variant via the centromere targeting domain. , 2010, Molecular cell.

[72]  Ming Zhou,et al.  Histone H2A.Z cooperates with RNAi and heterochromatin factors to suppress antisense RNAs , 2009, Nature.

[73]  Stefan Westermann,et al.  Structures and functions of yeast kinetochore complexes. , 2007, Annual review of biochemistry.

[74]  Michael A Quail,et al.  Improved Protocols for the Illumina Genome Analyzer Sequencing System , 2009, Current protocols in human genetics.

[75]  O. Homann,et al.  MochiView: versatile software for genome browsing and DNA motif analysis , 2010, BMC Biology.

[76]  D. Tollervey,et al.  Trf4 targets ncRNAs from telomeric and rDNA spacer regions and functions in rDNA copy number control , 2007, The EMBO journal.

[77]  Cole Trapnell,et al.  Ultrafast and memory-efficient alignment of short DNA sequences to the human genome , 2009, Genome Biology.

[78]  S. Stoler,et al.  A mutation in CSE4, an essential gene encoding a novel chromatin-associated protein in yeast, causes chromosome nondisjunction and cell cycle arrest at mitosis. , 1995, Genes & development.

[79]  W. Liang,et al.  TM4 microarray software suite. , 2006, Methods in enzymology.

[80]  J. McIntosh,et al.  The Dam1 ring binds microtubules strongly enough to be a processive as well as energy-efficient coupler for chromosome motion , 2008, Proceedings of the National Academy of Sciences.

[81]  Nicholas A. Kent,et al.  Chromatin particle spectrum analysis: a method for comparative chromatin structure analysis using paired-end mode next-generation DNA sequencing , 2010, Nucleic acids research.

[82]  R. Baker,et al.  Analysis of Primary Structural Determinants That Distinguish the Centromere-Specific Function of Histone Variant Cse4p from Histone H3 , 1999, Molecular and Cellular Biology.

[83]  P. Ridgway,et al.  H2A.Z contributes to the unique 3D structure of the centromere , 2007, Proceedings of the National Academy of Sciences.

[84]  Trisha N Davis,et al.  Ctf3p, the Mis6 budding yeast homolog, interacts with Mcm22p and Mcm16p at the yeast outer kinetochore. , 2002, Genes & development.

[85]  K. Kitagawa,et al.  Endogenous Transcription at the Centromere Facilitates Centromere Activity in Budding Yeast , 2011, Current Biology.

[86]  S. Grewal.  RNAi-dependent formation of heterochromatin and its diverse functions. , 2010, Current opinion in genetics & development.

[87]  Xin Bi,et al.  Roles of Chromatin Remodeling Factors in the Formation and Maintenance of Heterochromatin Structure* , 2011, The Journal of Biological Chemistry.

[88]  Gary D Bader,et al.  The Genetic Landscape of a Cell , 2010, Science.

[89]  R. Baker,et al.  The N Terminus of the Centromere H3-Like Protein Cse4p Performs an Essential Function Distinct from That of the Histone Fold Domain , 2000, Molecular and Cellular Biology.

[90]  E. Petfalski,et al.  RNA Degradation by the Exosome Is Promoted by a Nuclear Polyadenylation Complex , 2005, Cell.

[91]  P. Bjerling,et al.  Fission yeast hrp1, a chromodomain ATPase, is required for proper chromosome segregation and its overexpression interferes with chromatin condensation. , 2000, Nucleic acids research.

[92]  S. Elledge,et al.  The mitotic spindle is required for loading of the DASH complex onto the kinetochore. , 2002, Genes & development.

[93]  D. Tollervey,et al.  The Many Pathways of RNA Degradation , 2009, Cell.

[94]  L. Clarke,et al.  Nucleotide sequence comparisons and functional analysis of yeast centromere DNAs , 1982, Cell.

[95]  M. Snyder,et al.  Spindle Checkpoint Maintenance Requires Ame1 and Okp1 , 2005, Cell cycle.

[96]  G. Karpen,et al.  Epigenetic regulation of centromeric chromatin: old dogs, new tricks? , 2008, Nature Reviews Genetics.

[97]  M. Stephens,et al.  RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. , 2008, Genome research.

[98]  S. Grewal,et al.  Heterochromatin revisited , 2007, Nature Reviews Genetics.