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1.  The budding yeast Centromere DNA Element II wraps a stable Cse4 hemisome in either orientation in vivo 
eLife  2014;3:e01861.
In budding yeast, a single cenH3 (Cse4) nucleosome occupies the ∼120-bp functional centromere, however conflicting structural models for the particle have been proposed. To resolve this controversy, we have applied H4S47C-anchored cleavage mapping, which reveals the precise position of histone H4 in every nucleosome in the genome. We find that cleavage patterns at centromeres are unique within the genome and are incompatible with symmetrical structures, including octameric nucleosomes and (Cse4/H4)2 tetrasomes. Centromere cleavage patterns are compatible with a precisely positioned core structure, one in which each of the 16 yeast centromeres is occupied by oppositely oriented Cse4/H4/H2A/H2B hemisomes in two rotational phases within the population. Centromere-specific hemisomes are also inferred from distances observed between closely-spaced H4 cleavages, as predicted from structural modeling. Our results indicate that the orientation and rotational position of the stable hemisome at each yeast centromere is not specified by the functional centromere sequence.
DOI: http://dx.doi.org/10.7554/eLife.01861.001
eLife digest
DNA is tightly packaged in cells for a variety of reasons—to allow it to fit inside the nucleus, to protect it from damage, and to help control the production of proteins from genes. The basic unit of packaged DNA is called a nucleosome, which consists of DNA wrapped around a structure formed by two pairs of four different proteins.
These proteins, which are called histones, have a role that extends beyond providing structural support for DNA. When cells divide, for example, pairs of ‘sister chromosomes’ are pulled apart to ensure that the two daughter cells both have the same chromosomes as the original cell. The sister chromosomes are pulled apart from a single position called a centromere, and the nucleosomes at this position contain a histone that is different from the histones found everywhere else in the cell. However, until recently it was not clear if the nucleosomes that contained these special cenH3 histones had the same structure as other nucleosomes.
Now Henikoff et al. have used a method called H4S47C-anchored cleavage mapping to study every nucleosome in the genome of the yeast S. cerevisiae. This mapping technique uses DNA sequencing to measure the precise distances between fixed points on the DNA in the nucleosome. Knowing these distances tells researchers a great deal about the number and position of the histones within each nucleosome in the genome.
Using this approach, Henikoff et al. found that nucleosomes at centromeres are different from other nucleosomes in histone number and arrangement. In particular, the nucleosome at each yeast centromere contains only one each of the four different histones in an asymmetrical orientation, in contrast to all other yeast nucleosomes, which contain two sets of four histones in a symmetrical arrangement. Furthermore, each nucleosome at a centromere can adopt one of two orientations: these orientations are mirror images of each other, and they occur with equal probability. It should also be possible to use the mapping technique developed by Henikoff et al. to study the larger and more complex centromeres found in other organisms, including humans.
DOI: http://dx.doi.org/10.7554/eLife.01861.002
doi:10.7554/eLife.01861
PMCID: PMC3983907  PMID: 24737863
centromeres; nucleosome; chemical cleavage mapping; S. cerevisiae
2.  “Point” Centromeres of Saccharomyces Harbor Single Centromere-Specific Nucleosomes 
Genetics  2012;190(4):1575-1577.
The “point” centromere of budding yeast is genetically defined by an ∼125-bp sequence. Recent fluorescence measurements of kinetochore clusters have suggested that this sequence specifies multiple centromere histone 3 (CenH3) nucleosomes. However, high-resolution mapping demonstrates that there is only one CenH3 nucleosome per centromere, providing biochemical confirmation of the point centromere model.
doi:10.1534/genetics.111.137711
PMCID: PMC3316665  PMID: 22234856
3.  Identification of Functional Elements and Regulatory Circuits by Drosophila modENCODE 
Roy, Sushmita | Ernst, Jason | Kharchenko, Peter V. | Kheradpour, Pouya | Negre, Nicolas | Eaton, Matthew L. | Landolin, Jane M. | Bristow, Christopher A. | Ma, Lijia | Lin, Michael F. | Washietl, Stefan | Arshinoff, Bradley I. | Ay, Ferhat | Meyer, Patrick E. | Robine, Nicolas | Washington, Nicole L. | Di Stefano, Luisa | Berezikov, Eugene | Brown, Christopher D. | Candeias, Rogerio | Carlson, Joseph W. | Carr, Adrian | Jungreis, Irwin | Marbach, Daniel | Sealfon, Rachel | Tolstorukov, Michael Y. | Will, Sebastian | Alekseyenko, Artyom A. | Artieri, Carlo | Booth, Benjamin W. | Brooks, Angela N. | Dai, Qi | Davis, Carrie A. | Duff, Michael O. | Feng, Xin | Gorchakov, Andrey A. | Gu, Tingting | Henikoff, Jorja G. | Kapranov, Philipp | Li, Renhua | MacAlpine, Heather K. | Malone, John | Minoda, Aki | Nordman, Jared | Okamura, Katsutomo | Perry, Marc | Powell, Sara K. | Riddle, Nicole C. | Sakai, Akiko | Samsonova, Anastasia | Sandler, Jeremy E. | Schwartz, Yuri B. | Sher, Noa | Spokony, Rebecca | Sturgill, David | van Baren, Marijke | Wan, Kenneth H. | Yang, Li | Yu, Charles | Feingold, Elise | Good, Peter | Guyer, Mark | Lowdon, Rebecca | Ahmad, Kami | Andrews, Justen | Berger, Bonnie | Brenner, Steven E. | Brent, Michael R. | Cherbas, Lucy | Elgin, Sarah C. R. | Gingeras, Thomas R. | Grossman, Robert | Hoskins, Roger A. | Kaufman, Thomas C. | Kent, William | Kuroda, Mitzi I. | Orr-Weaver, Terry | Perrimon, Norbert | Pirrotta, Vincenzo | Posakony, James W. | Ren, Bing | Russell, Steven | Cherbas, Peter | Graveley, Brenton R. | Lewis, Suzanna | Micklem, Gos | Oliver, Brian | Park, Peter J. | Celniker, Susan E. | Henikoff, Steven | Karpen, Gary H. | Lai, Eric C. | MacAlpine, David M. | Stein, Lincoln D. | White, Kevin P. | Kellis, Manolis
Science (New York, N.Y.)  2010;330(6012):1787-1797.
To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.
doi:10.1126/science.1198374
PMCID: PMC3192495  PMID: 21177974
4.  Integrative Analysis of the Caenorhabditis elegans Genome by the modENCODE Project 
Gerstein, Mark B. | Lu, Zhi John | Van Nostrand, Eric L. | Cheng, Chao | Arshinoff, Bradley I. | Liu, Tao | Yip, Kevin Y. | Robilotto, Rebecca | Rechtsteiner, Andreas | Ikegami, Kohta | Alves, Pedro | Chateigner, Aurelien | Perry, Marc | Morris, Mitzi | Auerbach, Raymond K. | Feng, Xin | Leng, Jing | Vielle, Anne | Niu, Wei | Rhrissorrakrai, Kahn | Agarwal, Ashish | Alexander, Roger P. | Barber, Galt | Brdlik, Cathleen M. | Brennan, Jennifer | Brouillet, Jeremy Jean | Carr, Adrian | Cheung, Ming-Sin | Clawson, Hiram | Contrino, Sergio | Dannenberg, Luke O. | Dernburg, Abby F. | Desai, Arshad | Dick, Lindsay | Dosé, Andréa C. | Du, Jiang | Egelhofer, Thea | Ercan, Sevinc | Euskirchen, Ghia | Ewing, Brent | Feingold, Elise A. | Gassmann, Reto | Good, Peter J. | Green, Phil | Gullier, Francois | Gutwein, Michelle | Guyer, Mark S. | Habegger, Lukas | Han, Ting | Henikoff, Jorja G. | Henz, Stefan R. | Hinrichs, Angie | Holster, Heather | Hyman, Tony | Iniguez, A. Leo | Janette, Judith | Jensen, Morten | Kato, Masaomi | Kent, W. James | Kephart, Ellen | Khivansara, Vishal | Khurana, Ekta | Kim, John K. | Kolasinska-Zwierz, Paulina | Lai, Eric C. | Latorre, Isabel | Leahey, Amber | Lewis, Suzanna | Lloyd, Paul | Lochovsky, Lucas | Lowdon, Rebecca F. | Lubling, Yaniv | Lyne, Rachel | MacCoss, Michael | Mackowiak, Sebastian D. | Mangone, Marco | McKay, Sheldon | Mecenas, Desirea | Merrihew, Gennifer | Miller, David M. | Muroyama, Andrew | Murray, John I. | Ooi, Siew-Loon | Pham, Hoang | Phippen, Taryn | Preston, Elicia A. | Rajewsky, Nikolaus | Rätsch, Gunnar | Rosenbaum, Heidi | Rozowsky, Joel | Rutherford, Kim | Ruzanov, Peter | Sarov, Mihail | Sasidharan, Rajkumar | Sboner, Andrea | Scheid, Paul | Segal, Eran | Shin, Hyunjin | Shou, Chong | Slack, Frank J. | Slightam, Cindie | Smith, Richard | Spencer, William C. | Stinson, E. O. | Taing, Scott | Takasaki, Teruaki | Vafeados, Dionne | Voronina, Ksenia | Wang, Guilin | Washington, Nicole L. | Whittle, Christina M. | Wu, Beijing | Yan, Koon-Kiu | Zeller, Georg | Zha, Zheng | Zhong, Mei | Zhou, Xingliang | Ahringer, Julie | Strome, Susan | Gunsalus, Kristin C. | Micklem, Gos | Liu, X. Shirley | Reinke, Valerie | Kim, Stuart K. | Hillier, LaDeana W. | Henikoff, Steven | Piano, Fabio | Snyder, Michael | Stein, Lincoln | Lieb, Jason D. | Waterston, Robert H.
Science (New York, N.Y.)  2010;330(6012):1775-1787.
We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor–binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription factor–binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers, with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions to most of the conserved genome.
doi:10.1126/science.1196914
PMCID: PMC3142569  PMID: 21177976
5.  H2A.Z nucleosomes enriched over active genes are homotypic 
Nature structural & molecular biology  2010;17(12):1500-1507.
Nucleosomes that contain the histone variant H2A.Z are enriched around transcriptional start sites, but the mechanistic basis for enrichment is unknown. A single octameric nucleosome can contain two H2A.Z histones (homotypic) or one H2A.Z and one canonical H2A (heterotypic). To elucidate H2A.Z function, we generated high-resolution maps of homotypic and heterotypic Drosophila H2A.Z (H2Av) nucleosomes. Although homotypic and heterotypic H2A.Z nucleosomes map throughout most of the genome, homotypic nucleosomes are enriched and heterotypic nucleosomes are depleted downstream of active promoters and intron/exon junctions. The distribution of homotypic H2A.Z nucleosomes resembles that of classical active chromatin and shows evidence of disruption during transcriptional elongation. Both homotypic H2A.Z nucleosomes and classical active chromatin are depleted downstream of paused polymerases. Our results suggest that H2A.Z enrichment patterns result from intrinsic structural differences between heterotypic and homotypic H2A.Z nucleosomes following disruption during transcriptional elongation.
doi:10.1038/nsmb.1926
PMCID: PMC3051840  PMID: 21057526
6.  Genome-wide kinetics of nucleosome turnover determined by metabolic labeling of histones 
Science (New York, N.Y.)  2010;328(5982):1161-1164.
Nucleosome disruption and replacement are crucial activities that maintain epigenomes, but these highly dynamic processes have been difficult to study. Here, we describe a direct method for measuring nucleosome dynamics genome-wide. We found that nucleosome turnover is most rapid over active gene bodies, epigenetic regulatory elements, and replication origins in Drosophila cells. Nucleosomes turn over faster at sites for trithorax-group than Polycomb-group protein binding, suggesting that nucleosome turnover differences underlie their opposing activities and challenging models for epigenetic inheritance that rely on stability of histone marks. Our results establish a general strategy for studying nucleosome dynamics and uncover nucleosome turnover differences across the genome that are likely to have functional significance for epigenome maintenance, gene regulation, and control of DNA replication.
doi:10.1126/science.1186777
PMCID: PMC2879085  PMID: 20508129
7.  A Comprehensive Map of Insulator Elements for the Drosophila Genome 
PLoS Genetics  2010;6(1):e1000814.
Insulators are DNA sequences that control the interactions among genomic regulatory elements and act as chromatin boundaries. A thorough understanding of their location and function is necessary to address the complexities of metazoan gene regulation. We studied by ChIP–chip the genome-wide binding sites of 6 insulator-associated proteins—dCTCF, CP190, BEAF-32, Su(Hw), Mod(mdg4), and GAF—to obtain the first comprehensive map of insulator elements in Drosophila embryos. We identify over 14,000 putative insulators, including all classically defined insulators. We find two major classes of insulators defined by dCTCF/CP190/BEAF-32 and Su(Hw), respectively. Distributional analyses of insulators revealed that particular sub-classes of insulator elements are excluded between cis-regulatory elements and their target promoters; divide differentially expressed, alternative, and divergent promoters; act as chromatin boundaries; are associated with chromosomal breakpoints among species; and are embedded within active chromatin domains. Together, these results provide a map demarcating the boundaries of gene regulatory units and a framework for understanding insulator function during the development and evolution of Drosophila.
Author Summary
The spatiotemporal specificity of gene expression is controlled by interactions among regulatory proteins, cis-regulatory elements, chromatin modifications, and genes. These interactions can occur over large distances, and the mechanisms by which they are controlled are poorly understood. Insulators are DNA sequences that can both block the interaction between regulatory elements and genes, as well as block the spread of regions of modified chromatin. To date, relatively few insulators have been identified in developing Drosophila embryos. We here present the genome wide identification of over 14,000 binding sites for 6 insulator-associated proteins. We demonstrate the existence of two broad classes of insulators. Insulators of both classes are enriched at the boundaries of a particular chromatin modification. However, only insulators bound by BEAF-32, CP190, and dCTCF are enriched in regions of open chromatin or demarcate gene boundaries, with a particular enrichment between differentially expressed promoters. Furthermore, insulators of this class are enriched at points of chromosomal rearrangement among the 12 species of sequenced Drosophila, suggesting that insulator defined regulatory boundaries are evolutionarily conserved.
doi:10.1371/journal.pgen.1000814
PMCID: PMC2797089  PMID: 20084099
8.  A native chromatin purification system for epigenomic profiling in Caenorhabditis elegans 
Nucleic Acids Research  2009;38(4):e26.
High-resolution mapping of chromatin features has emerged as an important strategy for understanding gene regulation and epigenetic inheritance. We describe an in vivo tagging system coupled to chromatin purification for genome-wide epigenetic profiling in Caenorhabditis elegans. In this system, we coexpressed the Escherichia coli biotin ligase enzyme (BirA), together with the C. elegans H3.3 gene fused to BioTag, a 23-amino-acid peptide serving as a biotinylation substrate for BirA, in vivo in worms. We found that the fusion BioTag::H3.3 was efficiently biotinylated in vivo. We developed methods to isolate chromatin under different salt extraction conditions, followed by affinity purification of biotinylated chromatin with streptavidin and genome-wide profiling with microarrays. We found that embryonic chromatin is differentially extracted with increasing salt concentrations. Interestingly, chromatin that remains insoluble after washing in 600 mM salt is enriched at 5′ and 3′ ends, suggesting the presence of large protein complexes that render chromatin insoluble at transcriptional initiation and termination sites. We also found that H3.3 landscapes from these salt fractions display consistent features that correlate with gene activity: the most highly expressed genes contain the most H3.3. This versatile two-component approach has the potential of facilitating genome-wide chromatin dynamics and regulatory site identification in C. elegans.
doi:10.1093/nar/gkp1090
PMCID: PMC2831312  PMID: 19966274
9.  Chromatin and siRNA pathways cooperate to maintain DNA methylation of small transposable elements in Arabidopsis 
Genome Biology  2005;6(11):R90.
Microarray-based profiling of the involvement of two DNA methyltransferases (CMT3 and DRM), a histone H3 lysine-9 methyltransferase (KYP) and an Argonaute-related siRNA silencing component (AGO4) in methylating target loci in Arabidopsis reveals that transposable elements are the targets of both DNA methylation and histone H3K9 methylation pathways, irrespective of element type and position.
Background
DNA methylation occurs at preferred sites in eukaryotes. In Arabidopsis, DNA cytosine methylation is maintained by three subfamilies of methyltransferases with distinct substrate specificities and different modes of action. Targeting of cytosine methylation at selected loci has been found to sometimes involve histone H3 methylation and small interfering (si)RNAs. However, the relationship between different cytosine methylation pathways and their preferred targets is not known.
Results
We used a microarray-based profiling method to explore the involvement of Arabidopsis CMT3 and DRM DNA methyltransferases, a histone H3 lysine-9 methyltransferase (KYP) and an Argonaute-related siRNA silencing component (AGO4) in methylating target loci. We found that KYP targets are also CMT3 targets, suggesting that histone methylation maintains CNG methylation genome-wide. CMT3 and KYP targets show similar proximal distributions that correspond to the overall distribution of transposable elements of all types, whereas DRM targets are distributed more distally along the chromosome. We find an inverse relationship between element size and loss of methylation in ago4 and drm mutants.
Conclusion
We conclude that the targets of both DNA methylation and histone H3K9 methylation pathways are transposable elements genome-wide, irrespective of element type and position. Our findings also suggest that RNA-directed DNA methylation is required to silence isolated elements that may be too small to be maintained in a silent state by a chromatin-based mechanism alone. Thus, parallel pathways would be needed to maintain silencing of transposable elements.
doi:10.1186/gb-2005-6-11-r90
PMCID: PMC1297646  PMID: 16277745
10.  CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design 
Nucleic Acids Research  2003;31(13):3763-3766.
We have developed a new primer design strategy for PCR amplification of distantly related gene sequences based on consensus-degenerate hybrid oligonucleotide primers (CODEHOPs). An interactive program has been written to design CODEHOP PCR primers from conserved blocks of amino acids within multiply-aligned protein sequences. Each CODEHOP consists of a pool of related primers containing all possible nucleotide sequences encoding 3–4 highly conserved amino acids within a 3′ degenerate core. A longer 5′ non-degenerate clamp region contains the most probable nucleotide predicted for each flanking codon. CODEHOPs are used in PCR amplification to isolate distantly related sequences encoding the conserved amino acid sequence. The primer design software and the CODEHOP PCR strategy have been utilized for the identification and characterization of new gene orthologs and paralogs in different plant, animal and bacterial species. In addition, this approach has been successful in identifying new pathogen species. The CODEHOP designer (http://blocks.fhcrc.org/codehop.html) is linked to BlockMaker and the Multiple Alignment Processor within the Blocks Database World Wide Web (http://blocks.fhcrc.org).
PMCID: PMC168931  PMID: 12824413
11.  Increased coverage of protein families with the Blocks Database servers 
Nucleic Acids Research  2000;28(1):228-230.
The Blocks Database WWW (http://blocks.fhcrc.org ) and Email (blocks@blocks.fhcrc.org ) servers provide tools to search DNA and protein queries against the Blocks+ Database of multiple alignments, which represent conserved protein regions. Blocks+ nearly doubles the number of protein families included in the database by adding families from the Pfam-A, ProDom and Domo databases to those from PROSITE and PRINTS. Other new features include improved Block Searcher statistics, searching with NCBI’s IMPALA program and 3D display of blocks on PDB structures.
PMCID: PMC102407  PMID: 10592233

Results 1-11 (11)