Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
PLoS One
2012 Jan 01;710:e47162. doi: 10.1371/journal.pone.0047162.
Show Gene links
Show Anatomy links
Chromatin modification by PSC occurs at one PSC per nucleosome and does not require the acidic patch of histone H2A.
Lo SM
,
McElroy KA
,
Francis NJ
.
???displayArticle.abstract???
Chromatin architecture is regulated through both enzymatic and non-enzymatic activities. For example, the Polycomb Group (PcG) proteins maintain developmental gene silencing using an array of chromatin-based mechanisms. The essential Drosophila PcG protein, Posterior Sex Combs (PSC), compacts chromatin and inhibits chromatin remodeling and transcription through a non-enzymatic mechanism involving nucleosome bridging. Nucleosome bridging is achieved through a combination of nucleosome binding and self-interaction. Precisely how PSC interacts with chromatin to bridge nucleosomes is not known and is the subject of this work. We determine the stoichiometry of PSC-chromatin interactions in compact chromatin (in which nucleosomes are bridged) using Scanning Transmission Electron Microscopy (STEM). We find that full compaction occurs with one PSC per nucleosome. In addition to compacting chromatin, we show that PSC oligomerizes nucleosome arrays. PSC-mediated oligomerization of chromatin occurs at similar stoichiometry as compaction suggesting it may also involve nucleosome bridging. Interactions between the tail of histone H4 and the acidic patch of histone H2A are important for chromatin folding and oligomerization, and several chromatin proteins bind the histone H2A acidic patch. However, mutation of the acidic patch of histone H2A does not affect PSC's ability to inhibit chromatin remodeling or bridge nucleosomes. In fact, PSC does not require nucleosomes for bridging activity but can bridge naked DNA segments. PSC clusters nucleosomes on sparsely assembled templates, suggesting it interacts preferentially with nucleosomes over bare DNA. This may be due to the ability of PSC to bind free histones. Our data are consistent with a model in which each PSC binds a nucleosome and at least one other PSC to directly bridge nucleosomes and compact chromatin, but also suggest that naked DNA can be included in compacted structures. We discuss how our data highlight the diversity of mechanisms used to modify chromatin architecture.
???displayArticle.pubmedLink???
23071745
???displayArticle.pmcLink???PMC3469540 ???displayArticle.link???PLoS One ???displayArticle.grants???[+]
Figure 2. Centrifugation assay demonstrates oligomerization of 12-nucleosome arrays by PSC.(a) Nucleosomal arrays were mixed with PSC at the indicated ratios and reactions pelleted by centrifugation in a microfuge. Proteinase K digested samples were separated by agarose gel electrophoresis, stained with SYBR gold and quantified. Two different examples of the assay are shown; considerable variability was observed in this assay although the trend was constant. (b) Summary of PSC-induced oligomerization. Error bars are standard deviation in this and all other figures. n = 5.
Figure 3. PSC compacts chromatin at a ratio of 1∶1 with nucleosomes.(a) Representative EM images of negatively stained PSC. (b) Distribution of diameters of negatively stained PSC (n = 235). (c) Mass distributions of STEM analysis of PSC alone (n = 515). (d) Summary of measured masses of PSC. (e) Mass distributions of STEM analyses. Measured masses for 4N and 4N + PSC are 0.97±0.01 MDa (n = 130) and 1.67±0.03 MDa (n = 86) respectively.
Figure 4. PSC does not require histone modifications or the acidic patch of H2A to inhibit chromatin remodeling.(a) Amino acid sequences for the wild-type H2A acidic patch (WT) and uncharged mutant (STT). Acidic residues are highlighted, and mutated residues are underlined. (b) MgCl2 dependent oligomerization of wild type and H2A-STT containing chromatin. Chromatin was incubated with the indicated concentrations of MgCl2 and centrifuged in a microfuge. Supernatants were electrophoresed on agarose gels and stained with SYBR gold; the % of the template remaining in the supernatant was determined by comparison with the 0 mM MgCl2 control. (c) Summary of chromatin oligomerization assays. (d) Restriction enzyme accessibility (REA) assays on chromatin templates with 12 nucleosomes. The chromatin template contains a unique restriction site (HhaI) that is normally occluded by nucleosomes but is exposed upon Swi/Snf-mediated chromatin remodeling. The first two lanes are negative and positive controls (with or without Swi/Snf, no PSC) demonstrating that the HhaI site becomes more accessible in the presence of Swi/Snf. (e) Summary of REA assay on chromatin templates assembled with rec-WT and H2A-STT histones. Percent inhibition is calculated as .
Figure 5. Bridging of nucleosomes by PSC does not require the acidic patch on histone H2A.(a) Schematic diagram of nucleosome bridging assay. (b) Representative control reactions for nucleosome bridging (mock MNase digested) showing that PSC binds both rec-WT and H2A-STT chromatin. Arrows point to the main plasmid forms (nicked and supercoiled); the array of minor isoforms observed here is atypical but the isoforms behave similarly. (c) Representative MNase digested nucleosome bridging reactions demonstrating that PSC bridges both rec-WT and H2A-STT nucleosomes. (d) Summary of nucleosome bridging assays on chromatin templates assembled with recombinant wild-type (rec-WT) and H2A acidic patch mutant histones (H2A-STT). Values from fractions 5–7 (bottom fractions) of sucrose gradients were summed and plotted.
Figure 6. PSC can bridge bare DNA.(a) Schematic diagram of DNA bridging assay. (b) Representative analysis of bridging of naked DNA by PSC. Top panels show sucrose gradient fractions that were pooled for streptavidin pull-down. Bottom panels show streptavidin pull-down results. The per cent bound refers to how much of the unbiotinylated fragment is present in the pellet as a fraction of the total (pellet + supernatant). Asterisks indicate position of biotinylated fragment; note that in pellet fractions, biotinylated fragment is incompletely recovered by Proteinase K treatment of streptavidin coated beads, and migrates slowly likely due to bound streptavidin. (c) Summary of streptavidin pull-down experiments. Graphs show average per cent of the unbiotinylated fragment associated with streptavidin beads.
Figure 7. PSC clusters nucleosomes and DNA on sparsely assembled plasmids.(a) Representative EM images of plasmids with two 601 nucleosome positioning sequences assembled at low ratio of histones to DNA. Note that fully assembled plasmids would contain 17 nucleosomes and 601 sequences are separated by 385 base pairs. Plasmids were assembled in the presence of E.coli Topoisomerse I so that plasmids are relaxed. Arrows point to nucleosomes. Note that template 2 was the only observed example of more than 2 nucleosomes on the plasmid (3), out of the 103 molecules that were analyzed (Table 1). (b) Sparsely assembled plasmids with PSC. Class 1 molecules (see text) are more extended, and likely have fewer copies of PSC bound than Class 2 molecules (c), which are highly compacted. Arrows point to particles (likely to be PSC bound nucleosomes) that have come together and may represent the bridged configuration. Note that in some cases, more than one template may be clustered (such as molecule 3). Asterisks indicate particles that may be unbound nucleosomes (based on their size), although they could also be bound PSC. (c) Sparsely assembled plasmids with PSC with Class 2 configurations. Note that the molecules represent a series between the most extended Class 1 molecules and the most highly compacted Class 2 molecules. (d) Summary of the number of particles per template. The finding that Class 1 molecules frequently have more than 2 particles indicates that PSC must bind to naked DNA (as well as nucleosomes) on some templates. See Table 1 for summary of measurements from this and a similar experiment.
Figure 8. PSC can bind histone proteins.(a) Representative assay of PSC binding to histone octamers. PSC was mixed with histone octamers that contain one biotinylated and one fluorescent copy of either H3 (H3 labeled octamers) or H2B (H2B labeled octamers) (see Methods for detailed description). Mixtures were incubated with streptavidin coated beads and the amount of captured PSC determined by Western blotting. Fluorescence (Cy5) was used to monitor octamer capture. Similar results were observed in two additional assays. (b) Representative assay of PSC binding to H2A/H2B dimers or H3/H4 tetramers. Dimers and tetramers were fluorophore labeled on the indicated (asterisk) subunit. High levels of background binding to beads was observed for both dimers and tetramers, as shown, but in each of three assays, more H2A/H2B and H3/H4 were eluted from Flag beads that have immobilized PSC than control beads with no immobilized protein. PSC in the elution is detected by Western blotting.
Figure 9. Model for nucleosome bridging and chromatin oligomerization by PSC.See discussion for details.
Figure 1. PSC compacts and oligomerizes 4-nucleosome arrays.(a) Representative glycerol gradient purification of nucleosomal templates with and without PSC for EM and STEM. Boxes indicate fractions selected for analysis. (b) Representative EM images from indicated fractions. Photographs were taken in dark field and are inverted to enhance contrast. (c) Distribution of maximum diameters of particles determined from micrographs like those in (b). Note that less than 10% of the 4N alone arrays have diameters larger than 95 nm and are not shown on the graph. (d) Summary of diameter measurements. Note that all fractions of 4N arrays incubated with PSC were significantly smaller than 4N arrays alone, and fractions 4 and 5 are different from 3. Table shows p-values for student’s t-test (unpaired, assuming equal variance in samples).
Adkins,
Role of nucleic acid binding in Sir3p-dependent interactions with chromatin fibers.
2009, Pubmed
Adkins,
Role of nucleic acid binding in Sir3p-dependent interactions with chromatin fibers.
2009,
Pubmed
Armache,
Structural basis of silencing: Sir3 BAH domain in complex with a nucleosome at 3.0 Å resolution.
2011,
Pubmed
Bantignies,
Polycomb-dependent regulatory contacts between distant Hox loci in Drosophila.
2011,
Pubmed
Bantignies,
Polycomb group proteins: repression in 3D.
2011,
Pubmed
Bantignies,
Inheritance of Polycomb-dependent chromosomal interactions in Drosophila.
2003,
Pubmed
Beh,
A core subunit of Polycomb repressive complex 1 is broadly conserved in function but not primary sequence.
2012,
Pubmed
Belotserkovskaya,
FACT facilitates transcription-dependent nucleosome alteration.
2003,
Pubmed
Butenko,
Polycomb-group mediated epigenetic mechanisms through plant evolution.
2011,
Pubmed
Canzio,
Chromodomain-mediated oligomerization of HP1 suggests a nucleosome-bridging mechanism for heterochromatin assembly.
2011,
Pubmed
Carruthers,
Assembly of defined nucleosomal and chromatin arrays from pure components.
1999,
Pubmed
Chambeyron,
Nuclear re-organisation of the Hoxb complex during mouse embryonic development.
2005,
Pubmed
Chambeyron,
Chromatin decondensation and nuclear reorganization of the HoxB locus upon induction of transcription.
2004,
Pubmed
Comet,
A chromatin insulator driving three-dimensional Polycomb response element (PRE) contacts and Polycomb association with the chromatin fiber.
2011,
Pubmed
Delaval,
Epigenetic regulation of mammalian genomic imprinting.
2004,
Pubmed
Dorigo,
Chromatin fiber folding: requirement for the histone H4 N-terminal tail.
2003,
Pubmed
,
Xenbase
Dyer,
Reconstitution of nucleosome core particles from recombinant histones and DNA.
2004,
Pubmed
Emmons,
Molecular genetic analysis of Suppressor 2 of zeste identifies key functional domains.
2009,
Pubmed
Eskeland,
Ring1B compacts chromatin structure and represses gene expression independent of histone ubiquitination.
2010,
Pubmed
Fauvarque,
polyhomeotic regulatory sequences induce developmental regulator-dependent variegation and targeted P-element insertions in Drosophila.
1993,
Pubmed
Fedorova,
The nuclear organization of Polycomb/Trithorax group response elements in larval tissues of Drosophila melanogaster.
2008,
Pubmed
Francis,
Chromatin compaction by a polycomb group protein complex.
2004,
Pubmed
Francis,
Polycomb proteins remain bound to chromatin and DNA during DNA replication in vitro.
2009,
Pubmed
,
Xenbase
Francis,
Reconstitution of a functional core polycomb repressive complex.
2001,
Pubmed
Gambetta,
Essential role of the glycosyltransferase sxc/Ogt in polycomb repression.
2009,
Pubmed
Georgel,
Chromatin compaction by human MeCP2. Assembly of novel secondary chromatin structures in the absence of DNA methylation.
2003,
Pubmed
Ghosh,
MeCP2 binds cooperatively to its substrate and competes with histone H1 for chromatin binding sites.
2010,
Pubmed
Gutiérrez,
The role of the histone H2A ubiquitinase Sce in Polycomb repression.
2012,
Pubmed
Heard,
Dosage compensation in mammals: fine-tuning the expression of the X chromosome.
2006,
Pubmed
Kassis,
Pairing-sensitive silencing, polycomb group response elements, and transposon homing in Drosophila.
2002,
Pubmed
King,
Analysis of a polycomb group protein defines regions that link repressive activity on nucleosomal templates to in vivo function.
2005,
Pubmed
King,
Native and recombinant polycomb group complexes establish a selective block to template accessibility to repress transcription in vitro.
2002,
Pubmed
Lagarou,
dKDM2 couples histone H2A ubiquitylation to histone H3 demethylation during Polycomb group silencing.
2008,
Pubmed
Lanzuolo,
Polycomb response elements mediate the formation of chromosome higher-order structures in the bithorax complex.
2007,
Pubmed
Lavigne,
Propagation of silencing; recruitment and repression of naive chromatin in trans by polycomb repressed chromatin.
2004,
Pubmed
Lee,
Assembly of nucleosomal templates by salt dialysis.
2001,
Pubmed
Lewis,
A gene complex controlling segmentation in Drosophila.
1978,
Pubmed
Li,
Polycomb group genes Psc and Su(z)2 restrict follicle stem cell self-renewal and extrusion by controlling canonical and noncanonical Wnt signaling.
2010,
Pubmed
Li,
Insulators, not Polycomb response elements, are required for long-range interactions between Polycomb targets in Drosophila melanogaster.
2011,
Pubmed
Lo,
Inhibition of chromatin remodeling by polycomb group protein posterior sex combs is mechanistically distinct from nucleosome binding.
2010,
Pubmed
Lo,
A bridging model for persistence of a polycomb group protein complex through DNA replication in vitro.
2012,
Pubmed
Logie,
Catalytic activity of the yeast SWI/SNF complex on reconstituted nucleosome arrays.
1997,
Pubmed
Luger,
Preparation of nucleosome core particle from recombinant histones.
1999,
Pubmed
,
Xenbase
Mahmoudi,
GAGA can mediate enhancer function in trans by linking two separate DNA molecules.
2002,
Pubmed
Margueron,
Ezh1 and Ezh2 maintain repressive chromatin through different mechanisms.
2008,
Pubmed
McBryant,
Chromatin architectural proteins.
2006,
Pubmed
McBryant,
The silent information regulator 3 protein, SIR3p, binds to chromatin fibers and assembles a hypercondensed chromatin architecture in the presence of salt.
2008,
Pubmed
McBryant,
Determinants of histone H4 N-terminal domain function during nucleosomal array oligomerization: roles of amino acid sequence, domain length, and charge density.
2009,
Pubmed
,
Xenbase
Mito,
Histone replacement marks the boundaries of cis-regulatory domains.
2007,
Pubmed
Mohd-Sarip,
Architecture of a polycomb nucleoprotein complex.
2006,
Pubmed
Mohd-Sarip,
Transcription-independent function of Polycomb group protein PSC in cell cycle control.
2012,
Pubmed
Muller,
The mcp element from the Drosophila melanogaster bithorax complex mediates long-distance regulatory interactions.
1999,
Pubmed
Papp,
Histone trimethylation and the maintenance of transcriptional ON and OFF states by trxG and PcG proteins.
2006,
Pubmed
Polach,
Mechanism of protein access to specific DNA sequences in chromatin: a dynamic equilibrium model for gene regulation.
1995,
Pubmed
Richly,
Roles of the Polycomb group proteins in stem cells and cancer.
2011,
Pubmed
Ringrose,
Epigenetic regulation of cellular memory by the Polycomb and Trithorax group proteins.
2004,
Pubmed
Schuettengruber,
Genome regulation by polycomb and trithorax proteins.
2007,
Pubmed
Schwartz,
Polycomb silencing mechanisms and the management of genomic programmes.
2007,
Pubmed
Schwarz,
Reversible oligonucleosome self-association: dependence on divalent cations and core histone tail domains.
1996,
Pubmed
Schwarz,
Formation and stability of higher order chromatin structures. Contributions of the histone octamer.
1994,
Pubmed
Sexton,
Three-dimensional folding and functional organization principles of the Drosophila genome.
2012,
Pubmed
Shao,
Stabilization of chromatin structure by PRC1, a Polycomb complex.
1999,
Pubmed
Shogren-Knaak,
Histone H4-K16 acetylation controls chromatin structure and protein interactions.
2006,
Pubmed
Sif,
Mitotic inactivation of a human SWI/SNF chromatin remodeling complex.
1998,
Pubmed
Sinclair,
Drosophila O-GlcNAc transferase (OGT) is encoded by the Polycomb group (PcG) gene, super sex combs (sxc).
2009,
Pubmed
Terranova,
Polycomb group proteins Ezh2 and Rnf2 direct genomic contraction and imprinted repression in early mouse embryos.
2008,
Pubmed
Tolhuis,
Interactions among Polycomb domains are guided by chromosome architecture.
2011,
Pubmed
Trojer,
L3MBTL1, a histone-methylation-dependent chromatin lock.
2007,
Pubmed
Utley,
Transcriptional activators direct histone acetyltransferase complexes to nucleosomes.
1998,
Pubmed
Wall,
Scanning transmission electron microscopy of nuclear structures.
1998,
Pubmed
Wong,
A double-filter method for nitrocellulose-filter binding: application to protein-nucleic acid interactions.
1993,
Pubmed
Woodcock,
Electron microscopic imaging of chromatin with nucleosome resolution.
1998,
Pubmed
Workman,
Control of class II gene transcription during in vitro nucleosome assembly.
1991,
Pubmed
Wyrick,
Ascending the nucleosome face: recognition and function of structured domains in the histone H2A-H2B dimer.
2012,
Pubmed
Yang,
FRET-based methods to study ATP-dependent changes in chromatin structure.
2007,
Pubmed
,
Xenbase
Zhou,
The nucleosome surface regulates chromatin compaction and couples it with transcriptional repression.
2007,
Pubmed