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1.  Structural Basis of Transcriptional Gene Silencing Mediated by Arabidopsis MOM1 
PLoS Genetics  2012;8(2):e1002484.
Shifts between epigenetic states of transcriptional activity are typically correlated with changes in epigenetic marks. However, exceptions to this rule suggest the existence of additional, as yet uncharacterized, layers of epigenetic regulation. MOM1, a protein of 2,001 amino acids that acts as a transcriptional silencer, represents such an exception. Here we define the 82 amino acid domain called CMM2 (Conserved MOM1 Motif 2) as a minimal MOM1 fragment capable of transcriptional regulation. As determined by X-ray crystallography, this motif folds into an unusual hendecad-based coiled-coil. Structure-based mutagenesis followed by transgenic complementation tests in plants demonstrate that CMM2 and its dimerization are effective for transcriptional suppression at chromosomal loci co-regulated by MOM1 and the siRNA pathway but not at loci controlled by MOM1 in an siRNA–independent fashion. These results reveal a surprising separation of epigenetic activities that enable the single, large MOM1 protein to coordinate cooperating mechanisms of epigenetic regulation.
Author Summary
Epigenetic shifts in transcriptional activities are usually correlated with changes in chromatin properties and covalent modification of DNA and/or histones. There are, however, exceptional regulators that are able to switch epigenetic states without the apparent involvement of changes in chromatin or DNA modifications. MOM1 protein, derived from CHD3 chromatin remodelers, belongs to this group. Here we defined a very small domain of MOM1 (less than 5% of its total sequence) that is sufficient for epigenetic regulation. We solved the structure of this domain and found that it forms a dimer with each monomer consisting of unusual consecutive 11 amino-acid hendecad repeats folding into an antiparallel coiled-coil. In vivo experiments demonstrated that the formation of this coiled-coil is essential for silencing activity; however, it is effective only at loci co-silenced by MOM1 and small RNAs. At loci not controlled by small RNAs, the entire MOM1 protein is required. Our results demonstrate that a single epigenetic regulator is able to differentially use its domains to control diverse chromosomal targets. The acquisition of the coiled-coil domain of MOM1 reflects a neofunctionalization of CHD3 proteins, which allowed MOM1 to broaden its activity and to provide input into multiple epigenetic pathways.
PMCID: PMC3276543  PMID: 22346760
2.  Expression, crystallization and preliminary X-ray diffraction analysis of the CMM2 region of the Arabidopsis thaliana Morpheus’ molecule 1 protein 
In order to investigate its function in transcriptional gene silencing, the highly conserved motif 2 from A. thaliana Morpheus’ molecule 1 protein was expressed, purified and crystallized. X-ray diffraction analysis is reported to a resolution of 3.2 Å.
Of the known epigenetic control regulators found in plants, the Morpheus’ molecule 1 (MOM1) protein is atypical in that the deletion of MOM1 does not affect the level of epigenetic marks controlling the transcriptional status of the genome. A short 197-amino-acid fragment of the MOM1 protein sequence can complement MOM1 deletion when coupled to a nuclear localization signal, suggesting that this region contains a functional domain that compensates for the loss of the full-length protein. Numerous constructs centred on the highly conserved MOM1 motif 2 (CMM2) present in these 197 residues have been generated and expressed in Escherichia coli. Following purification and crystallization screening, diamond-shaped single crystals were obtained that diffracted to ∼3.2 Å resolution. They belonged to the trigonal space group P3121 (or P3221), with unit-cell parameters a = 85.64, c = 292.74 Å. Structure determination is ongoing.
PMCID: PMC2917290  PMID: 20693667
Morpheus’ molecule 1; conserved MOM1 motif 2; coiled-coil domain; epigenetic; transcriptional gene silencing
3.  Stress-Induced Activation of Heterochromatic Transcription 
PLoS Genetics  2010;6(10):e1001175.
Constitutive heterochromatin comprising the centromeric and telomeric parts of chromosomes includes DNA marked by high levels of methylation associated with histones modified by repressive marks. These epigenetic modifications silence transcription and ensure stable inheritance of this inert state. Although environmental cues can alter epigenetic marks and lead to modulation of the transcription of genes located in euchromatic parts of the chromosomes, there is no evidence that external stimuli can globally destabilize silencing of constitutive heterochromatin. We have found that heterochromatin-associated silencing in Arabidopsis plants subjected to a particular temperature regime is released in a genome-wide manner. This occurs without alteration of repressive epigenetic modifications and does not involve common epigenetic mechanisms. Such induced release of silencing is mostly transient, and rapid restoration of the silent state occurs without the involvement of factors known to be required for silencing initiation. Thus, our results reveal new regulatory aspects of transcriptional repression in constitutive heterochromatin and open up possibilities to identify the molecular mechanisms involved.
Author Summary
In eukaryotic cells, DNA is packaged into chromatin that is present in two different forms named euchromatin and heterochromatin. Gene-rich euchromatin is relaxed and permissive to transcription compared with heterochromatin that essentially contains transcriptionally inert non-coding repeated DNA. The silent state associated with heterochromatin correlates with the presence of distinctive repressive epigenetic modifications. Mutations in genes required for maintenance of these epigenetic marks reactivate heterochromatin transcription, which is otherwise maintained silent in a highly stable manner. In this paper, we defined a specific temperature stress that leads to genome-wide transcriptional activation of sequences located within heterochromatin of Arabidopsis thaliana. Unexpectedly, release of silencing occurs in spite of conservation of the repressive epigenetic marks and independently of common epigenetic regulators. In addition, we provide evidence that stress-induced transcriptional activation is mostly transient, and silencing is rapidly restored upon return to optimal growth conditions. These results are important in that they disclose the dynamics of silencing associated with heterochromatin as well as the existence of a new level of transcriptional control that might play a role in plant acclimation to changing environmental conditions.
PMCID: PMC2965753  PMID: 21060865
4.  A crucial role of the mitochondrial protein import receptor MOM19 for the biogenesis of mitochondria 
The Journal of Cell Biology  1994;124(5):637-648.
The novel genetic method of "sheltered RIP" (repeat induced point mutation) was used to generate a Neurospora crassa mutant in which MOM19, a component of the protein import machinery of the mitochondrial outer membrane, can be depleted. Deficiency in MOM19 resulted in a severe growth defect, but the cells remained viable. The number of mitochondrial profiles was not grossly changed, but mutant mitochondria were highly deficient in cristae membranes, cytochromes, and protein synthesis activity. Protein import into isolated mutant mitochondria was decreased by factors of 6 to 30 for most proteins from all suborganellar compartments. Proteins like the ADP/ATP carrier, MOM19, and cytochrome c, whose import into wild-type mitochondria occurs independently of MOM19 became imported normally showing that the reduced import activities are solely caused by a lack of MOM19. Depletion of MOM19 reveals a close functional relationship between MOM19 and MOM22, since loss of MOM19 led to decreased levels of MOM22 and reduced protein import through MOM22. Furthermore, MOM72 does not function as a general backup receptor for MOM19 suggesting that these two proteins have distinct precursor specificities. These findings demonstrate that the import receptor MOM19 fulfills an important role in the biogenesis of mitochondria and that it is essential for the formation of mitochondria competent in respiration and phosphorylation.
PMCID: PMC2119945  PMID: 8120088
5.  A genome-wide screen for modifiers of transgene variegation identifies genes with critical roles in development 
Genome Biology  2008;9(12):R182.
An extended ENU screen for modifiers of transgene variegation identified four new modifiers, MommeD7-D10.
Some years ago we established an N-ethyl-N-nitrosourea screen for modifiers of transgene variegation in the mouse and a preliminary description of the first six mutant lines, named MommeD1-D6, has been published. We have reported the underlying genes in three cases: MommeD1 is a mutation in SMC hinge domain containing 1 (Smchd1), a novel modifier of epigenetic gene silencing; MommeD2 is a mutation in DNA methyltransferase 1 (Dnmt1); and MommeD4 is a mutation in Smarca 5 (Snf2h), a known chromatin remodeler. The identification of Dnmt1 and Smarca5 attest to the effectiveness of the screen design.
We have now extended the screen and have identified four new modifiers, MommeD7-D10. Here we show that all ten MommeDs link to unique sites in the genome, that homozygosity for the mutations is associated with severe developmental abnormalities and that heterozygosity results in phenotypic abnormalities and reduced reproductive fitness in some cases. In addition, we have now identified the underlying genes for MommeD5 and MommeD10. MommeD5 is a mutation in Hdac1, which encodes histone deacetylase 1, and MommeD10 is a mutation in Baz1b (also known as Williams syndrome transcription factor), which encodes a transcription factor containing a PHD-type zinc finger and a bromodomain. We show that reduction in the level of Baz1b in the mouse results in craniofacial features reminiscent of Williams syndrome.
These results demonstrate the importance of dosage-dependent epigenetic reprogramming in the development of the embryo and the power of the screen to provide mouse models to study this process.
PMCID: PMC2646286  PMID: 19099580
6.  Arabidopsis HDA6 Regulates Locus-Directed Heterochromatin Silencing in Cooperation with MET1 
PLoS Genetics  2011;7(4):e1002055.
Heterochromatin silencing is pivotal for genome stability in eukaryotes. In Arabidopsis, a plant-specific mechanism called RNA–directed DNA methylation (RdDM) is involved in heterochromatin silencing. Histone deacetylase HDA6 has been identified as a component of such machineries; however, its endogenous targets and the silencing mechanisms have not been analyzed globally. In this study, we investigated the silencing mechanism mediated by HDA6. Genome-wide transcript profiling revealed that the loci silenced by HDA6 carried sequences corresponding to the RDR2-dependent 24-nt siRNAs, however their transcript levels were mostly unaffected in the rdr2 mutant. Strikingly, we observed significant overlap of genes silenced by HDA6 to those by the CG DNA methyltransferase MET1. Furthermore, regardless of dependence on RdDM pathway, HDA6 deficiency resulted in loss of heterochromatic epigenetic marks and aberrant enrichment for euchromatic marks at HDA6 direct targets, along with ectopic expression of these loci. Acetylation levels increased significantly in the hda6 mutant at all of the lysine residues in the H3 and H4 N-tails, except H4K16. Interestingly, we observed two different CG methylation statuses in the hda6 mutant. CG methylation was sustained in the hda6 mutant at some HDA6 target loci that were surrounded by flanking DNA–methylated regions. In contrast, complete loss of CG methylation occurred in the hda6 mutant at the HDA6 target loci that were isolated from flanking DNA methylation. Regardless of CG methylation status, CHG and CHH methylation were lost and transcriptional derepression occurred in the hda6 mutant. Furthermore, we show that HDA6 binds only to its target loci, not the flanking methylated DNA, indicating the profound target specificity of HDA6. We propose that HDA6 regulates locus-directed heterochromatin silencing in cooperation with MET1, possibly recruiting MET1 to specific loci, thus forming the foundation of silent chromatin structure for subsequent non-CG methylation.
Author Summary
Eukaryotes are defended from potentially harmful DNA elements, such as transposons, by forming inactive genomic structure. Chromatin, which consists of DNA and histone proteins, is densely packed in the silent structure, and chromatin chemical modifications such as DNA methylation and histone modifications are known to be essential for this packing. In plants, small RNA molecules have been thought to trigger DNA methylation and resulting silent chromatin formation. We revealed that elimination of specific histone modifications concomitant with DNA methylation is pivotal for the silent chromatin. Furthermore, the histone deacetylase was shown to have more profound target specificity than the DNA methyltransferase and is required for locus-directed DNA methylation, implying the involvement of the histone deacetylase for targeting the DNA methyltransferase to specific places on the genome. These proteins and their functions for gene silencing are evolutionarily conserved in higher eukaryotes, and several proteins involved in small RNA production are plant-specific. Thus, we present a hypothesis that the plant genome may build the protecting foundation by the conserved genome surveillance in eukaryotes, and the reinforcing machinery involving small RNAs could be evolutionarily added to the plant heterochromatin silencing system.
PMCID: PMC3084210  PMID: 21552333
7.  Locus-specific dependency of endogenous silent loci on MOM1 and non-CG methylation in Arabidopsis thaliana 
Plant Signaling & Behavior  2010;5(6):724-726.
RNA-directed modification of histones is essential for maintenance of heterochromatin in higher eukaryotes. In plants, cytosine methylation, especially in non-CG sequence contexts, is tightly related to inactive chromatin, but the mechanisms regulating the coexistence of cytosine methylation and repressive histone modification remain obscure. We recently revealed that MORPHEUS' MOLECULE1 (MOM1) of Arabidopsis thaliana silences endogenous loci related to transposons and homologous to the 24-nt siRNAs accumulated in wild type plants, and suggested that MOM1 transduces RNA-directed DNA methylation (RdDM) signals to repressive histone modification. In this addendum, we focus on the involvement of MOM1 in multiple transcriptional gene silencing (TGS) pathways.
PMCID: PMC3001571  PMID: 20404545
Arabidopsis thaliana; RNA-directed DNA methylation; histone modification; MORPHEUS' MOLECULE 1
8.  Bax Activation Initiates the Assembly of a Multimeric Catalyst that Facilitates Bax Pore Formation in Mitochondrial Outer Membranes 
PLoS Biology  2012;10(9):e1001394.
Bax promotes mitochondrial permeabilization during apoptosis via a phase-transition-like event in the membrane and oligomerization of a catalyst molecule that facilitates Bax pore formation.
Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) is essential for “intrinsic” apoptotic cell death. Published studies used synthetic liposomes to reveal an intrinsic pore-forming activity of Bax, but it is unclear how other mitochondrial outer membrane (MOM) proteins might facilitate this function. We carefully analyzed the kinetics of Bax-mediated pore formation in isolated MOMs, with some unexpected results. Native MOMs were more sensitive than liposomes to added Bax, and MOMs displayed a lag phase not observed with liposomes. Heat-labile MOM proteins were required for this enhanced response. A two-tiered mathematical model closely fit the kinetic data: first, Bax activation promotes the assembly of a multimeric complex, which then catalyzes the second reaction, Bax-dependent pore formation. Bax insertion occurred immediately upon Bax addition, prior to the end of the lag phase. Permeabilization kinetics were affected in a reciprocal manner by [cBid] and [Bax], confirming the “hit-and-run” hypothesis of cBid-induced direct Bax activation. Surprisingly, MOMP rate constants were linearly related to [Bax], implying that Bax acts non-cooperatively. Thus, the oligomeric catalyst is distinct from Bax. Moreover, contrary to common assumption, pore formation kinetics depend on Bax monomers, not oligomers. Catalyst formation exhibited a sharp transition in activation energy at ∼28°C, suggesting a role for membrane lipid packing. Furthermore, catalyst formation was strongly inhibited by chemical antagonists of the yeast mitochondrial fission protein, Dnm1. However, the mammalian ortholog, Drp1, was undetectable in mitochondrial outer membranes. Moreover, ATP and GTP were dispensable for MOMP. Thus, the data argue that oligomerization of a catalyst protein, distinct from Bax and Drp1, facilitates MOMP, possibly through a membrane-remodeling event.
Author Summary
Mitochondria are the key energy-producing structures inside cells, but are also crucial players in a common form of programmed cell death, apoptosis. A critical event in mitochondrion-driven apoptosis involves the formation of large pores in the mitochondrial outer membrane (MOM). These pores cause long-term damage to mitochondria and also allow mitochondrial proteins to escape and accelerate cell death. Previous studies have revealed that the protein Bax when activated can form pores in protein-free membranes and that it, along with Bak, is involved in the formation of mitochondrial pores, but the process remains unclear. We now show, however, that in naturally derived MOMs, Bax is assisted by another resident MOM protein, which we term the “catalyst,” and whose identity is still unknown. The mechanism involves two distinct stages. First, activated Bax activates the catalyst protein, causing multiple catalyst molecules to assemble into a larger structure (a complex). In the second stage, this catalyst complex in turn facilitates Bax-driven pore formation. Our data also reveal some unexpected details of the pore formation process; in particular, it appears that catalyst activation involves a physical change in the molecular arrangement of the membrane. Furthermore, contrary to what was previously assumed, pore formation does not require Bax molecules themselves to assemble together into larger complexes.
PMCID: PMC3457932  PMID: 23049480
9.  The mitochondrial receptor complex: Mom22 is essential for cell viability and directly interacts with preproteins. 
Molecular and Cellular Biology  1995;15(6):3382-3389.
A multisubunit complex in the mitochondrial outer membrane is responsible for targeting and membrane translocation of nuclear-encoded preproteins. This receptor complex contains two import receptors, a general insertion pore and the protein Mom22. It was unknown if Mom22 directly interacts with preproteins, and two views existed about the possible functions of Mom22: a central role in transfer of preproteins from both receptors to the general insertion pore or a more limited function dependent on the presence of the receptor Mom19. For this report, we identified and cloned Saccharomyces cerevisiae MOM22 and investigated whether it plays a direct role in targeting of preproteins. A preprotein accumulated at the mitochondrial outer membrane was cross-linked to Mom22. The cross-linking depended on the import stage of the preprotein. Overexpression of Mom22 suppressed the respiratory defect of yeast cells lacking Mom19 and increased preprotein import into mom19 delta mitochondria, demonstrating that Mom22 can function independently of Mom19. Overexpression of Mom22 even suppressed the lethal phenotype of a double deletion of the two import receptors known so far (mom19 delta mom72 delta). Deletion of the MOM22 gene was lethal for yeast cells, identifying Mom22 as one of the few mitochondrial membrane proteins essential for fermentative growth. These results suggest that Mom22 plays an essential role in the mitochondrial receptor complex. It directly interacts with preproteins in transit and can perform receptor-like activities.
PMCID: PMC230572  PMID: 7760834
10.  Silencing of toxic gene expression by Fis 
Nucleic Acids Research  2012;40(10):4358-4367.
Bacteria and bacteriophages have evolved DNA modification as a strategy to protect their genomes. Mom protein of bacteriophage Mu modifies the phage DNA, rendering it refractile to numerous restriction enzymes and in turn enabling the phage to successfully invade a variety of hosts. A strong fortification, a combined activity of the phage and host factors, prevents untimely expression of mom and associated toxic effects. Here, we identify the bacterial chromatin architectural protein Fis as an additional player in this crowded regulatory cascade. Both in vivo and in vitro studies described here indicate that Fis acts as a transcriptional repressor of mom promoter. Further, our data shows that Fis mediates its repressive effect by denying access to RNA polymerase at mom promoter. We propose that a combined repressive effect of Fis and previously characterized negative regulatory factors could be responsible to keep the gene silenced most of the time. We thus present a new facet of Fis function in Mu biology. In addition to bringing about overall downregulation of Mu genome, it also ensures silencing of the advantageous but potentially lethal mom gene.
PMCID: PMC3378877  PMID: 22287621
11.  A Dominant Mutation in mediator of paramutation2, One of Three Second-Largest Subunits of a Plant-Specific RNA Polymerase, Disrupts Multiple siRNA Silencing Processes 
PLoS Genetics  2009;5(11):e1000725.
Paramutation involves homologous sequence communication that leads to meiotically heritable transcriptional silencing. We demonstrate that mop2 (mediator of paramutation2), which alters paramutation at multiple loci, encodes a gene similar to Arabidopsis NRPD2/E2, the second-largest subunit of plant-specific RNA polymerases IV and V. In Arabidopsis, Pol-IV and Pol-V play major roles in RNA–mediated silencing and a single second-largest subunit is shared between Pol-IV and Pol-V. Maize encodes three second-largest subunit genes: all three genes potentially encode full length proteins with highly conserved polymerase domains, and each are expressed in multiple overlapping tissues. The isolation of a recessive paramutation mutation in mop2 from a forward genetic screen suggests limited or no functional redundancy of these three genes. Potential alternative Pol-IV/Pol-V–like complexes could provide maize with a greater diversification of RNA–mediated transcriptional silencing machinery relative to Arabidopsis. Mop2-1 disrupts paramutation at multiple loci when heterozygous, whereas previously silenced alleles are only up-regulated when Mop2-1 is homozygous. The dramatic reduction in b1 tandem repeat siRNAs, but no disruption of silencing in Mop2-1 heterozygotes, suggests the major role for tandem repeat siRNAs is not to maintain silencing. Instead, we hypothesize the tandem repeat siRNAs mediate the establishment of the heritable silent state—a process fully disrupted in Mop2-1 heterozygotes. The dominant Mop2-1 mutation, which has a single nucleotide change in a domain highly conserved among all polymerases (E. coli to eukaryotes), disrupts both siRNA biogenesis (Pol-IV–like) and potentially processes downstream (Pol-V–like). These results suggest either the wild-type protein is a subunit in both complexes or the dominant mutant protein disrupts both complexes. Dominant mutations in the same domain in E. coli RNA polymerase suggest a model for Mop2-1 dominance: complexes containing Mop2-1 subunits are non-functional and compete with wild-type complexes.
Author Summary
How an individual's genes are activated or silenced is an essential question impacting all fields of biology. Usually gene expression patterns, i.e., which genes are on and which are off in different tissues and during development, are highly reproducible; and those patterns are efficiently reset in the next generation of progeny. Paramutation represents an exception to these genetic rules, in that for certain genes the silencing that is established in an individual is efficiently transmitted to their progeny. Importantly, in these subsequent generations, the silenced gene continues to silence active versions of that gene. Prior work has demonstrated that these heritable gene expression changes are not accompanied by changes in DNA sequence: they are epigenetic. Understanding mechanisms for heritable changes in gene expression has major implications for researchers studying complex traits, including diseases. In this manuscript we demonstrate that a subunit of a RNA polymerase is required for paramutation in maize and other gene silencing processes that also involve RNA–mediated chromatin changes. We show that the multiple, closely related, plant-specific RNA polymerases mediating gene silencing have diverged functions in maize. Results from our experiments suggest testable models for the role of these polymerases in multiple gene-silencing processes.
PMCID: PMC2774164  PMID: 19936058
12.  Human Genome Replication Proceeds through Four Chromatin States 
PLoS Computational Biology  2013;9(10):e1003233.
Advances in genomic studies have led to significant progress in understanding the epigenetically controlled interplay between chromatin structure and nuclear functions. Epigenetic modifications were shown to play a key role in transcription regulation and genome activity during development and differentiation or in response to the environment. Paradoxically, the molecular mechanisms that regulate the initiation and the maintenance of the spatio-temporal replication program in higher eukaryotes, and in particular their links to epigenetic modifications, still remain elusive. By integrative analysis of the genome-wide distributions of thirteen epigenetic marks in the human cell line K562, at the 100 kb resolution of corresponding mean replication timing (MRT) data, we identify four major groups of chromatin marks with shared features. These states have different MRT, namely from early to late replicating, replication proceeds though a transcriptionally active euchromatin state (C1), a repressive type of chromatin (C2) associated with polycomb complexes, a silent state (C3) not enriched in any available marks, and a gene poor HP1-associated heterochromatin state (C4). When mapping these chromatin states inside the megabase-sized U-domains (U-shaped MRT profile) covering about 50% of the human genome, we reveal that the associated replication fork polarity gradient corresponds to a directional path across the four chromatin states, from C1 at U-domains borders followed by C2, C3 and C4 at centers. Analysis of the other genome half is consistent with early and late replication loci occurring in separate compartments, the former correspond to gene-rich, high-GC domains of intermingled chromatin states C1 and C2, whereas the latter correspond to gene-poor, low-GC domains of alternating chromatin states C3 and C4 or long C4 domains. This new segmentation sheds a new light on the epigenetic regulation of the spatio-temporal replication program in human and provides a framework for further studies in different cell types, in both health and disease.
Author Summary
Previous studies revealed spatially coherent and biological-meaningful chromatin mark combinations in human cells. Here, we analyze thirteen epigenetic mark maps in the human cell line K562 at 100 kb resolution of MRT data. The complexity of epigenetic data is reduced to four chromatin states that display remarkable similarities with those reported in fly, worm and plants. These states have different MRT: (C1) is transcriptionally active, early replicating, enriched in CTCF; (C2) is Polycomb repressed, mid-S replicating; (C3) lacks of marks and replicates late and (C4) is a late-replicating gene-poor HP1 repressed heterochromatin state. When mapping these states inside the 876 replication U-domains of K562, the replication fork polarity gradient observed in these U-domains comes along with a remarkable epigenetic organization from C1 at U-domain borders to C2, C3 and ultimately C4 at centers. The remaining genome half displays early replicating, gene rich and high GC domains of intermingled C1 and C2 states segregating from late replicating, gene poor and low GC domains of concatenated C3 and/or C4 states. This constitutes the first evidence of epigenetic compartmentalization of the human genome into replication domains likely corresponding to autonomous units in the 3D chromatin architecture.
PMCID: PMC3794905  PMID: 24130466
13.  The Drosophila melanogaster CHD1 Chromatin Remodeling Factor Modulates Global Chromosome Structure and Counteracts HP1a and H3K9me2 
PLoS ONE  2013;8(3):e59496.
CHD1 is a conserved chromatin remodeling factor that localizes to active genes and functions in nucleosome assembly and positioning as well as histone turnover. Mouse CHD1 is required for the maintenance of stem cell pluripotency while human CHD1 may function as a tumor suppressor. To investigate the action of CHD1 on higher order chromatin structure in differentiated cells, we examined the consequences of loss of CHD1 and over-expression of CHD1 on polytene chromosomes from salivary glands of third instar Drosophila melanogaster larvae. We observed that chromosome structure is sensitive to the amount of this remodeler. Loss of CHD1 resulted in alterations of chromosome structure and an increase in the heterochromatin protein HP1a, while over-expression of CHD1 disrupted higher order chromatin structure and caused a decrease in levels of HP1a. Over-expression of an ATPase inactive form of CHD1 did not result in severe chromosomal defects, suggesting that the ATPase activity is required for this in vivo phenotype. Interestingly, changes in CHD1 protein levels did not correlate with changes in the levels of the euchromatin mark H3K4me3 or elongating RNA Polymerase II. Thus, while CHD1 is localized to transcriptionally active regions of the genome, it can function to alter the levels of HP1a, perhaps through changes in methylation of H3K9.
PMCID: PMC3606111  PMID: 23533627
14.  Co-Evolution of Transcriptional Silencing Proteins and the DNA Elements Specifying Their Assembly 
PLoS Biology  2010;8(11):e1000550.
As shown by genetic assays in Saccharomyces interspecies hybrids, the co-evolution of heterochromatin assembly proteins with silencer elements allows transcriptional silencing functions to be maintained in rapidly evolving regions of the genome.
Co-evolution of transcriptional regulatory proteins and their sites of action has been often hypothesized but rarely demonstrated. Here we provide experimental evidence of such co-evolution in yeast silent chromatin, a finding that emerged from studies of hybrids formed between two closely related Saccharomyces species. A unidirectional silencing incompatibility between S. cerevisiae and S. bayanus led to a key discovery: asymmetrical complementation of divergent orthologs of the silent chromatin component Sir4. In S. cerevisiae/S. bayanus interspecies hybrids, ChIP-Seq analysis revealed a restriction against S. cerevisiae Sir4 associating with most S. bayanus silenced regions; in contrast, S. bayanus Sir4 associated with S. cerevisiae silenced loci to an even greater degree than did S. cerevisiae's own Sir4. Functional changes in silencer sequences paralleled changes in Sir4 sequence and a reduction in Sir1 family members in S. cerevisiae. Critically, species-specific silencing of the S. bayanus HMR locus could be reconstituted in S. cerevisiae by co-transfer of the S. bayanus Sir4 and Kos3 (the ancestral relative of Sir1) proteins. As Sir1/Kos3 and Sir4 bind conserved silencer-binding proteins, but not specific DNA sequences, these rapidly evolving proteins served to interpret differences in the two species' silencers presumably involving emergent features created by the regulatory proteins that bind sequences within silencers. The results presented here, and in particular the high resolution ChIP-Seq localization of the Sir4 protein, provided unanticipated insights into the mechanism of silent chromatin assembly in yeast.
Author Summary
As eukaryotic species evolve, transcriptionally silent portions of their genomes—termed “heterochromatin”—mutate rapidly. To maintain the “off” state of certain genes in silenced regions, regulatory DNA sequences called silencers, which reside within a rapidly mutating region, must co-evolve with the regulatory proteins that bind these sequences to turn off transcription. Although hypothesized to occur widely in nature, such “molecular co-evolution” of genetic regulators has been demonstrated in only a few cases. Unlike previous examples of gene regulatory co-evolution, we found that the transcription factors that bind silencers in two budding yeast species are, in fact, functionally interchangeable, even though the silencers are not. Surprisingly, the Sir1 and Sir4 silencing proteins, which are heterochromatin components that bind the transcription factors rather than the silencer DNA sequences per se, are the proteins engaged in rapid co-evolution with the silencers. Silencer sequences therefore contain additional, evolutionarily labile information directing the assembly of heterochromatin. As mutations in Sir1 and Sir4 over evolutionary time can compensate for changes in the silencers, this “extra information” likely involves cooperative assembly of the transcription factors with the Sir1 and Sir4 “adaptor” proteins. The localization patterns of two species' Sir4 proteins across both species' genomes in interspecies yeast hybrids illuminate unexpected features of heterochromatin structure and assembly.
PMCID: PMC2994660  PMID: 21151344
15.  NUP-1 Is a Large Coiled-Coil Nucleoskeletal Protein in Trypanosomes with Lamin-Like Functions 
PLoS Biology  2012;10(3):e1001287.
NUP1, the first example of a nuclear lamin analog in nonmetazoans, performs roles similar to those of lamins in maintaining the structure and organization of the nucleus in Trypanosoma brucei.
A unifying feature of eukaryotic nuclear organization is genome segregation into transcriptionally active euchromatin and transcriptionally repressed heterochromatin. In metazoa, lamin proteins preserve nuclear integrity and higher order heterochromatin organization at the nuclear periphery, but no non-metazoan lamin orthologues have been identified, despite the likely presence of nucleoskeletal elements in many lineages. This suggests a metazoan-specific origin for lamins, and therefore that distinct protein elements must compose the nucleoskeleton in other lineages. The trypanosomatids are highly divergent organisms and possess well-documented but remarkably distinct mechanisms for control of gene expression, including polycistronic transcription and trans-splicing. NUP-1 is a large protein localizing to the nuclear periphery of Trypanosoma brucei and a candidate nucleoskeletal component. We sought to determine if NUP-1 mediates heterochromatin organization and gene regulation at the nuclear periphery by examining the influence of NUP-1 knockdown on morphology, chromatin positioning, and transcription. We demonstrate that NUP-1 is essential and part of a stable network at the inner face of the trypanosome nuclear envelope, since knockdown cells have abnormally shaped nuclei with compromised structural integrity. NUP-1 knockdown also disrupts organization of nuclear pore complexes and chromosomes. Most significantly, we find that NUP-1 is required to maintain the silenced state of developmentally regulated genes at the nuclear periphery; NUP-1 knockdown results in highly specific mis-regulation of telomere-proximal silenced variant surface glycoprotein (VSG) expression sites and procyclin loci, indicating a disruption to normal chromatin organization essential to life-cycle progression. Further, NUP-1 depletion leads to increased VSG switching and therefore appears to have a role in control of antigenic variation. Thus, analogous to vertebrate lamins, NUP-1 is a major component of the nucleoskeleton with key roles in organization of the nuclear periphery, heterochromatin, and epigenetic control of developmentally regulated loci.
Author Summary
Eukaryotes—fungi, plants, animals, and many unicellular organisms—are defined by the presence of a cell nucleus that contains the chromosomes and is enveloped by a lipid membrane lined on the inner face with a protein network called the lamina. Among other functions, the lamina serves as an anchorage site for the ends of chromosomes. In multicellular animals (metazoa), the lamina comprises a few related proteins called lamins, which are very important for many functions related to the nucleus; abnormal lamins result in multiple nuclear defects and diseases, including inappropriate gene expression and premature aging. Until now, however, lamins had been found only in metazoa; no protein of equivalent function had been identified in plants, fungi, or unicellular organisms. Here, we describe a protein from African trypanosomes—the single-cell parasites that cause sleeping sickness—that fulfils many lamin-like roles, including maintaining nuclear structure and organizing the chromosomes of this organism. We show that this protein, which we call NUP-1 for nuclear periphery protein-1, is vital for the antigenic variation mechanisms that allow the parasite to escape the host immune response. We propose that NUP-1 is a lamin analogue that performs similar functions in trypanosomes to those of authentic lamins in metazoa. These findings, we believe, have important implications for understanding the evolution of the nucleus.
PMCID: PMC3313915  PMID: 22479148
16.  Arabidopsis thaliana Chromosome 4 Replicates in Two Phases That Correlate with Chromatin State 
PLoS Genetics  2010;6(6):e1000982.
DNA replication programs have been studied extensively in yeast and animal systems, where they have been shown to correlate with gene expression and certain epigenetic modifications. Despite the conservation of core DNA replication proteins, little is known about replication programs in plants. We used flow cytometry and tiling microarrays to profile DNA replication of Arabidopsis thaliana chromosome 4 (chr4) during early, mid, and late S phase. Replication profiles for early and mid S phase were similar and encompassed the majority of the euchromatin. Late S phase exhibited a distinctly different profile that includes the remaining euchromatin and essentially all of the heterochromatin. Termination zones were consistent between experiments, allowing us to define 163 putative replicons on chr4 that clustered into larger domains of predominately early or late replication. Early-replicating sequences, especially the initiation zones of early replicons, displayed a pattern of epigenetic modifications specifying an open chromatin conformation. Late replicons, and the termination zones of early replicons, showed an opposite pattern. Histone H3 acetylated on lysine 56 (H3K56ac) was enriched in early replicons, as well as the initiation zones of both early and late replicons. H3K56ac was also associated with expressed genes, but this effect was local whereas replication time correlated with H3K56ac over broad regions. The similarity of the replication profiles for early and mid S phase cells indicates that replication origin activation in euchromatin is stochastic. Replicon organization in Arabidopsis is strongly influenced by epigenetic modifications to histones and DNA. The domain organization of Arabidopsis is more similar to that in Drosophila than that in mammals, which may reflect genome size and complexity. The distinct patterns of association of H3K56ac with gene expression and early replication provide evidence that H3K56ac may be associated with initiation zones and replication origins.
Author Summary
During growth and development, all plants and animals must replicate their DNA. This process is regulated to ensure that all sequences are completely and accurately replicated and is limited to S phase of the cell cycle. In the cell, DNA is packaged with histone proteins into chromatin, and both DNA and histones are subject to epigenetic modifications that affect chromatin state. Euchromatin and heterochromatin are chromatin states marked by epigenetic modifications specifying open and closed conformations, respectively. Using the model plant Arabidopsis thaliana, we show that the time at which a DNA sequence replicates is influenced by the epigenetic modifications to the surrounding chromatin. DNA replication occurs in two phases, with euchromatin replicating in early and mid S phase and heterochromatin replicating late. DNA replication time has been linked to gene expression in other organisms, and this is also true in Arabidopsis because more genes are active in euchromatin when compared to heterochromatin. The earliest replicating DNA sequences are associated with acetylation of histone H3 on lysine 56 (H3K56ac). H3K56ac is also abundant in active genes, but the patterns of association of H3K56ac with gene expression and DNA replication are distinct, suggesting that H3K56ac is independently linked to both processes.
PMCID: PMC2883604  PMID: 20548960
17.  A Position Effect on the Heritability of Epigenetic Silencing 
PLoS Genetics  2008;4(10):e1000216.
In animals and yeast, position effects have been well documented. In animals, the best example of this process is Position Effect Variegation (PEV) in Drosophila melanogaster. In PEV, when genes are moved into close proximity to constitutive heterochromatin, their expression can become unstable, resulting in variegated patches of gene expression. This process is regulated by a variety of proteins implicated in both chromatin remodeling and RNAi-based silencing. A similar phenomenon is observed when transgenes are inserted into heterochromatic regions in fission yeast. In contrast, there are few examples of position effects in plants, and there are no documented examples in either plants or animals for positions that are associated with the reversal of previously established silenced states. MuDR transposons in maize can be heritably silenced by a naturally occurring rearranged version of MuDR. This element, Muk, produces a long hairpin RNA molecule that can trigger DNA methylation and heritable silencing of one or many MuDR elements. In most cases, MuDR elements remain inactive even after Muk segregates away. Thus, Muk-induced silencing involves a directed and heritable change in gene activity in the absence of changes in DNA sequence. Using classical genetic analysis, we have identified an exceptional position at which MuDR element silencing is unstable. Muk effectively silences the MuDR element at this position. However, after Muk is segregated away, element activity is restored. This restoration is accompanied by a reversal of DNA methylation. To our knowledge, this is the first documented example of a position effect that is associated with the reversal of epigenetic silencing. This observation suggests that there are cis-acting sequences that alter the propensity of an epigenetically silenced gene to remain inactive. This raises the interesting possibility that an important feature of local chromatin environments may be the capacity to erase previously established epigenetic marks.
Author Summary
Epigenetics involves the heritable alteration of gene activity without changes in DNA sequence. Although clearly a repository for heritable information, what makes epigenetic states distinct is that they are far more labile than those associated with DNA sequence. The epigenetic landscape of eukaryotic genomes is far from uniform. Vast stretches of them are effectively epigenetically silenced, while other regions are largely active. The experiments described here suggest that the propensity to maintain heritable epigenetic states can vary depending on position within the genome. Because transposable elements, or transposons, move from place to place within the genome, they make an ideal probe for differences in epigenetic states at various positions. Our model system uses a single transposon, MuDR in maize, and a variant of MuDR, Mu killer (Muk). When MuDR and Muk are combined genetically, MuDR elements become epigenetically silenced, and they generally remain so even after Muk is lost in subsequent generations. However, we have identified a particular position at which the MuDR element reactivates after Muk is lost. These data show that there are some parts of the maize genome that are either competent to erase epigenetic silencing or are incapable of maintaining it. These results suggest that erasure of heritable information may be an important component of epigenetic regulation.
PMCID: PMC2563033  PMID: 18846225
18.  Metal-on-Metal Total Hip Resurfacing Arthroplasty 
Executive Summary
The objective of this review was to assess the safety and effectiveness of metal on metal (MOM) hip resurfacing arthroplasty for young patients compared with that of total hip replacement (THR) in the same population.
Clinical Need
Total hip replacement has proved to be very effective for late middle-aged and elderly patients with severe degenerative diseases of the hips. As indications for THR began to include younger patients and those with a more active life style, the longevity of the implant became a concern. Evidence suggests that these patients experience relatively higher rates of early implant failure and the need for revision. The Swedish hip registry, for example, has demonstrated a survival rate in excess of 80% at 20 years for those aged over 65 years, whereas this figure was 33% by 16 years in those aged under 55 years.
Hip resurfacing arthroplasty is a bone-conserving alternative to THR that restores normal joint biomechanics and load transfer. The technique has been used around the world for more than 10 years, specifically in the United Kingdom and other European countries.
The Technology
Metal-on-metal hip resurfacing arthroplasty is an alternative procedure to conventional THR in younger patients. Hip resurfacing arthroplasty is less invasive than THR and addresses the problem of preserving femoral bone stock at the initial operation. This means that future hip revisions are possible with THR if the initial MOM arthroplasty becomes less effective with time in these younger patients. The procedure involves the removal and replacement of the surface of the femoral head with a hollow metal hemisphere, which fits into a metal acetabular cup.
Hip resurfacing arthroplasty is a technically more demanding procedure than is conventional THR. In hip resurfacing, the femoral head is retained, which makes it much more difficult to access the acetabular cup. However, hip resurfacing arthroplasty has several advantages over a conventional THR with a small (28 mm) ball. First, the large femoral head reduces the chance of dislocation, so that rates of dislocation are less than those with conventional THR. Second, the range of motion with hip resurfacing arthroplasty is higher than that achieved with conventional THR.
A variety of MOM hip resurfacing implants are used in clinical practice. Six MOM hip resurfacing implants have been issued licences in Canada.
Review Strategy
A search of electronic bibliographies (OVID Medline, Medline In-Process and Other Non-Indexed Citations, Embase, Cochrane CENTRAL and DSR, INAHTA) was undertaken to identify evidence published from Jan 1, 1997 to October 27, 2005. The search was limited to English-language articles and human studies. The literature search yielded 245 citations. Of these, 11 met inclusion criteria (9 for effectiveness, 2 for safety).
The result of the only reported randomized controlled trial on MOM hip resurfacing arthroplasty could not be included in this assessment, because it used a cemented acetabular component, whereas in the new generation of implants, a cementless acetabular component is used. After omitting this publication, only case series remained.
Summary of Findings
Health Outcomes
The Harris hip score and SF-12 are 2 measures commonly used to report health outcomes in MOM hip resurfacing arthroplasty studies. Other scales used are the Oxford hip score and the University of California Los Angeles hip score.
The case series showed that the mean revision rate of MOM hip resurfacing arthroplasty is 1.5% and the incidence of femoral neck fracture is 0.67%. Across all studies, 2 cases of osteonecrosis were reported. Four studies reported improvement in Harris hip scores. However, only 1 study reported a statistically significant improvement. Three studies reported improvement in SF-12 scores, of which 2 reported a significant improvement. One study reported significant improvement in UCLA hip score. Two studies reported postoperative Oxford hip scores, but no preoperative values were reported.
None of the reviewed studies reported procedure-related deaths. Four studies reported implant survival rates ranging from 94.4% to 99.7% for a follow-up period of 2.8 to 3.5 years. Three studies reported on the range of motion. One reported improvement in all motions including flexion, extension, abduction-adduction, and rotation, and another reported improvement in flexion. Yet another reported improvement in range of motion for flexion abduction-adduction and rotation arc. However, the author reported a decrease in the range of motion in the arc of flexion in patients with Brooker class III or IV heterotopic bone (all patients were men).
Safety of Metal-on-Metal Hip Resurfacing Arthroplasty
There is a concern about metal wear debris and its systemic distribution throughout the body. Detectable metal concentrations in the serum and urine of patients with metal hip implants have been described as early as the 1970s, and this issue is still controversial after 35 years.
Several studies have reported high concentration of cobalt and chromium in serum and/or urine of the patients with metal hip implants. Potential toxicological effects of the elevated metal ions have heightened concerns about safety of MOM bearings. This is of particular concern in young and active patients in whom life expectancy after implantation is long.
Since 1997, 15 studies, including 1 randomized clinical trial, have reported high levels of metal ions after THR with metal implants. Some of these studies have reported higher metal levels in patients with loose implants.
Adverse Biological Effects of Cobalt and Chromium
Because patients who receive a MOM hip arthroplasty are shown to be exposed to high concentrations of metallic ions, the Medical Advisory Secretariat searched the literature for reports of adverse biological effects of cobalt and chromium. Cobalt and chromium make up the major part of the metal articulations; therefore, they are a focus of concern.
Risk of Cancer
To date, only one study has examined the incidence of cancer after MOM and polyethylene on metal total hip arthroplasties. The results were compared to that of general population in Finland. The mean duration of follow-up for MOM arthroplasty was 15.7 years; for polyethylene arthroplasty, it was 12.5 years. The standardized incidence ratio for all cancers in the MOM group was 0.95 (95% CI, 0.79–1.13). In the polyethylene on metal group it was 0.76 (95% CI, 0.68–0.86). The combined standardized incidence ratio for lymphoma and leukemia in the patients who had MOM THR was 1.59 (95% CI, 0.82–2.77). It was 0.59 (95% CI, 0.29–1.05) for the patients who had polyethylene on metal THR. Patients with MOM THR had a significantly higher risk of leukemia. All patients who had leukemia were aged over than 60 years.
Cobalt Cardiotoxicity
Epidemiological Studies of Myocardiopathy of Beer Drinkers
An unusual type of myocardiopathy, characterized by pericardial effusion, elevated hemoglobin concentrations, and congestive heart failure, occurred as an epidemic affecting 48 habitual beer drinkers in Quebec City between 1965 and 1966. This epidemic was directly related the consumption of a popular beer containing cobalt sulfate. The epidemic appeared 1 month after cobalt sulfate was added to the specific brewery, and no further cases were seen a month after this specific chemical was no longer used in making this beer. A beer of the same name is made in Montreal, and the only difference at that time was that the Quebec brand of beer contained about 10 times more cobalt sulphate. Cobalt has been added to some Canadian beers since 1965 to improve the stability of the foam but it has been added in larger breweries only to draught beer. However, in small breweries, such as those in Quebec City, separate batches were not brewed for bottle and draught beer; therefore, cobalt was added to all of the beer processed in this brewery.
In March 1966, a committee was appointed under the chairmanship of the Deputy Minister of Health for Quebec that included members of the department of forensic medicine of Quebec’s Ministry of Justice, epidemiologists, members of Food and Drug Directorate of Ottawa, toxicologists, biomedical researchers, pathologists, and members of provincial police. Epidemiological studies were carried out by the Provincial Ministry of Health and the Quebec City Health Department.
The association between the development of myocardiopathy and the consumption of the particular brand of beer was proven. The mortality rate of this epidemic was 46.1% and those who survived were desperately ill, and recovered only after a struggle for their lives.
Similar cases were seen in Omaha (Nebraska). The epidemic started after a cobalt additive was used in 1 of the beers marketed in Nebraska. Sixty-four patients with the clinical diagnosis of alcoholic myocardiopathy were seen during an 18-month period (1964–1965). Thirty of these patients died. The first patient became ill within 1 month after cobalt was added to the beer, and the last patient was seen within 1 month of withdrawal of cobalt.
A similar epidemic occurred in Minneapolis, Minnesota. Between 1964 and 1967, 42 patients with acute heart failure were admitted to a hospital in Minneapolis, Minnesota. Twenty of these patients were drinking 6 to 30 bottles per day of a particular brand of beer exclusively. The other 14 patients also drank the same brand of beer, but not exclusively. The mortality rate from the acute illness was 18%, but late deaths accounted for a total mortality rate of 43%. Examination of the tissue from these patients revealed markedly abnormal changes in myofibrils (heart muscles), mitochondria, and sarcoplasmic reticulum.
In Belgium, a similar epidemic was reported in 1966, in which, cobalt was used in some Belgian beers. There was a difference in mortality between the Canadian or American epidemic and this series. Only 1 of 24 patients died, 1.5 years after the diagnosis. In March 1965, at an international meeting in Brussels, a new heart disease in chronic beer drinkers was described. This disease consists of massive pericardial effusion, low cardiac output, raised venous pressure, and polycythemia in some cases. This syndrome was thought to be different from the 2 other forms of alcoholic heart disease (beriberi and a form characterized by myocardial fibrosis).
The mystery of the above epidemics as stated by investigators is that the amount of cobalt added to the beer was below the therapeutic doses used for anemia. For example, 24 pints of Quebec brand of beer in Quebec would contain 8 mg of cobalt chloride, whereas an intake of 50 to 100 mg of cobalt as an antianemic agent has been well tolerated. Thus, greater cobalt intake alone does not explain the occurrence of myocardiopathy. It seems that there are individual differences in cobalt toxicity. Other features, like subclinical alcoholic heart disease, deficient diet, and electrolyte imbalance could have been precipitating factors that made these patients susceptible to cobalt’s toxic effects.
In the Omaha epidemic, 60% of the patients had weight loss, anorexia, and occasional vomiting and diarrhea 2 to 6 months before the onset of cardiac symptoms. In the Quebec epidemic, patients lost their appetite 3 to 6 months before the diagnosis of myocardiopathy and developed nausea in the weeks before hospital admission. In the Belgium epidemic, anorexia was one of the most predominant symptoms at the time of diagnosis, and the quality and quantity of food intake was poor. Alcohol has been shown to increase the uptake of intracoronary injected cobalt by 47%. When cobalt enters the cells, calcium exits; this shifts the cobalt to calcium ratio. The increased uptake of cobalt in alcoholic patients may explain the high incidence of cardiomyopathies in beer drinkers’ epidemics.
As all of the above suggest, it may be that prior chronic exposure to alcohol and/or a nutritionally deficient diet may have a marked synergistic effect with the cardiotoxicity of cobalt.
MOM hip resurfacing arthroplasty has been shown to be an effective arthroplasty procedure as tested in younger patients.
However, evidence for effectiveness is based only on 7 case series with short duration of follow-up (2.8–3.5 years). There are no RCTs or other well-controlled studies that compare MOM hip resurfacing with THR.
Revision rates reported in the MOM studies using implants currently licensed in Canada (hybrid systems, uncemented acetabular, and cemented femoral) range from 0.3% to 3.6% for a mean follow-up ranging from 2.8 to 3.5 years.
Fracture of femoral neck is not very common; it occurs in 0.4% to 2.2% of cases (as observed in a short follow-up period).
All the studies that measured health outcomes have reported improvement in Harris Hip and SF-12 scores; 1 study reported significant reduction in pain and improvement in function, and 2 studies reported significant improvement in SF-12 scores. One study reported significant improvement in UCLA Hip scores.
Concerns remain on the potential adverse effects of metal ions. Longer-term follow-up data will help to resolve the inconsistency of findings on adverse effects, including toxicity and carcinogenicity.
Ontario-Based Economic Analysis
The device cost for MOM ranges from $4,300 to $6,000 (Cdn). Traditional hip replacement devices cost about $2,000 (Cdn). Using Ontario Case Costing Initiative data, the total estimated costs for hip resurfacing surgery including physician fees, device fees, follow-up consultation, and postsurgery rehabilitation is about $15,000 (Cdn).
Cost of Total Hip Replacement Surgery in Ontario
MOM hip arthroplasty is generally recommended for patients aged under 55 years because its bone-conserving advantage enables patients to “buy time” and hence helps THRs to last over the lifetime of the patient. In 2004/2005, 15.9% of patients who received THRs were aged 55 years and younger. It is estimated that there are from 600 to 1,000 annual MOM hip arthroplasty surgeries in Canada with an estimated 100 to 150 surgeries in Ontario. Given the increased public awareness of this device, it is forecasted that demand for MOM hip arthroplasty will steadily increase with a conservative estimate of demand rising to 1,400 cases by 2010 (Figure 10). The net budget impact over a 5-year period could be $500,000 to $4.7 million, mainly because of the increasing cost of the device.
Projected Number of Metal-on-Metal Hip Arthroplasty Surgeries in Ontario: to 2010
PMCID: PMC3379532  PMID: 23074495
19.  ATP-dependent chromatin assembly is functionally distinct from chromatin remodeling 
eLife  2013;2:e00863.
Chromatin assembly involves the combined action of ATP-dependent motor proteins and histone chaperones. Because motor proteins in chromatin assembly also function as chromatin remodeling factors, we investigated the relationship between ATP-driven chromatin assembly and chromatin remodeling in the generation of periodic nucleosome arrays. We found that chromatin remodeling-defective Chd1 motor proteins are able to catalyze ATP-dependent chromatin assembly. The resulting nucleosomes are not, however, spaced in periodic arrays. Wild-type Chd1, but not chromatin remodeling-defective Chd1, can catalyze the conversion of randomly-distributed nucleosomes into periodic arrays. These results reveal a functional distinction between ATP-dependent nucleosome assembly and chromatin remodeling, and suggest a model for chromatin assembly in which randomly-distributed nucleosomes are formed by the nucleosome assembly function of Chd1, and then regularly-spaced nucleosome arrays are generated by the chromatin remodeling activity of Chd1. These findings uncover an unforeseen level of specificity in the role of motor proteins in chromatin assembly.
eLife digest
In many cells, genomic DNA is wrapped around proteins known as histones to produce particles called nucleosomes. These particles then join together—like beads on a string—to form a highly periodic structure called chromatin. In the nucleus, chromatin is further folded and condensed into chromosomes. However, many important processes, including the replication of DNA and the transcription of genes, require access to the DNA. The cell must therefore be able to disassemble chromatin and remove the histones, and then, once these processes are complete, to reassemble the chromatin. Enzymes known as chromatin assembly factors are responsible for the disassembly and reassembly of chromatin.
There are two main types of chromatin assembly factors in eukaryotic cells (i.e., cells with nuclei)—histone chaperones and motor proteins. The histone chaperones escort histones from the cytoplasm, where they are made, to the nucleus. The motor proteins—using energy supplied by ATP molecules—then catalyze the formation of nucleosomes. This involves two activities: the motor proteins assemble nucleosomes by helping the DNA to wrap around the histones, and they also remodel chromatin by altering the positions of nucleosomes along the DNA to ensure that they are periodic—that is, regularly spaced.
A conserved motor protein called Chd1 performs chromatin assembly and remodeling in eukaryotic cells. Chd1 works in conjunction with histone chaperones—both are needed for chromatin assembly, and so are DNA, histones and ATP. However, whether or not chromatin assembly and chromatin remodeling by Chd1 are identical or distinct processes is not well understood.
Torigoe et al. have now discovered a mutant Chd1 protein that has nucleosome assembly activity (i.e., it can make nucleosomes) but cannot remodel chromatin (i.e., it is unable to move nucleosomes), and thus have demonstrated that these two processes are functionally distinct. Torigoe et al. additionally have found that the mutant Chd1 proteins produce randomly distributed nucleosomes rather than the periodic arrays normally found in chromatin. Further analysis then revealed that the wild-type Chd1 protein, which can remodel chromatin, is able to convert randomly distributed nucleosomes into periodic arrays.
These findings have led to a new model for chromatin assembly in which Chd1 initially generates randomly distributed nucleosomes (via its assembly function), and then converts them into periodic arrays of nucleosomes (via its remodeling function). Together, these studies shed light on the mechanisms by which chromatin is created and manipulated in cells.
PMCID: PMC3748710  PMID: 23986862
chromatin assembly; chromatin remodeling; Chd1; D. melanogaster; S. cerevisiae
20.  The Role of Multiple Marks in Epigenetic Silencing and the Emergence of a Stable Bivalent Chromatin State 
PLoS Computational Biology  2013;9(7):e1003121.
We introduce and analyze a minimal model of epigenetic silencing in budding yeast, built upon known biomolecular interactions in the system. Doing so, we identify the epigenetic marks essential for the bistability of epigenetic states. The model explicitly incorporates two key chromatin marks, namely H4K16 acetylation and H3K79 methylation, and explores whether the presence of multiple marks lead to a qualitatively different systems behavior. We find that having both modifications is important for the robustness of epigenetic silencing. Besides the silenced and transcriptionally active fate of chromatin, our model leads to a novel state with bivalent (i.e., both active and silencing) marks under certain perturbations (knock-out mutations, inhibition or enhancement of enzymatic activity). The bivalent state appears under several perturbations and is shown to result in patchy silencing. We also show that the titration effect, owing to a limited supply of silencing proteins, can result in counter-intuitive responses. The design principles of the silencing system is systematically investigated and disparate experimental observations are assessed within a single theoretical framework. Specifically, we discuss the behavior of Sir protein recruitment, spreading and stability of silenced regions in commonly-studied mutants (e.g., sas2, dot1) illuminating the controversial role of Dot1 in the systems biology of yeast silencing.
Author Summary
Epigenetics is the study of heritable phenotypic variations that are not caused by changes in the genotype. Silent Information Regulator (SIR) silencing in budding yeast is an important model system for epigenetics. The standard model of silencing relies on feedback, mediated by chromatin modifications (for example, deacetylation of histone residues) which lead to enhanced recruitment of chromatin modifiers. However, the SIR mechanism is not completely understood and it is important to investigate whether as-yet-undiscovered components alter the systems design in a fundamental way. We address this question using minimal models constructed from experimentally known interactions. Rather than building a detailed network model with parameters to fit for quantitative predictions, we build an effective model and study its bifurcation diagram which leads to robust qualitative predictions on the nature of mutants. This minimal modeling delineates a phase space with qualitatively different epigenetic mechanisms and states; some of which arise from drug/genetic perturbations and exhibit large cell-to-cell variation in chromatin marks. Our methodology can be applied to the study of epigenetic chromatin silencing in other model systems, especially Polycomb silencing, and reveals engineering principles that may be of broad relevance.
PMCID: PMC3715441  PMID: 23874171
21.  The first mouse mutants of D14Abb1e (Fam208a) show that it is critical for early development 
Mammalian Genome  2014;25(7-8):293-303.
An ENU mutagenesis screen to identify novel epigenetic modifiers was established in mice carrying a multi-copy GFP transgene, which is expressed in a variegated manner in erythrocytes and is highly sensitive to epigenetic silencing. The screen has produced mouse mutants of both known modifiers of epigenetic state, such as Dnmt1 and Smarca5, and novel modifiers, such as Smchd1 and Rlf. Here we report two mouse lines generated from the screen, MommeD6 and MommeD20, with point mutations in D14Abb1e. These are the first mouse mutants of D14Abb1e (alsoknownasFam208a), a gene about which little is known. Heterozygous intercrosses show that homozygous mutants from both the MommeD6 and MommeD20 lines are not viable beyond gastrulation, demonstrating an important role for D14Abb1e in development. We demonstrate that haploinsufficiency for D14Abb1e effects transgene expression at the RNA level. Analysis of the predicted D14Abb1e protein sequence reveals that it contains putative nuclear localisation signals and a domain of unknown function, DUF3715. Our studies reveal that D14Abb1e is localised to the nucleus and is expressed in skin and testes.
PMCID: PMC4105592  PMID: 24781204
22.  Genomewide Analysis of PRC1 and PRC2 Occupancy Identifies Two Classes of Bivalent Domains 
PLoS Genetics  2008;4(10):e1000242.
In embryonic stem (ES) cells, bivalent chromatin domains with overlapping repressive (H3 lysine 27 tri-methylation) and activating (H3 lysine 4 tri-methylation) histone modifications mark the promoters of more than 2,000 genes. To gain insight into the structure and function of bivalent domains, we mapped key histone modifications and subunits of Polycomb-repressive complexes 1 and 2 (PRC1 and PRC2) genomewide in human and mouse ES cells by chromatin immunoprecipitation, followed by ultra high-throughput sequencing. We find that bivalent domains can be segregated into two classes—the first occupied by both PRC2 and PRC1 (PRC1-positive) and the second specifically bound by PRC2 (PRC2-only). PRC1-positive bivalent domains appear functionally distinct as they more efficiently retain lysine 27 tri-methylation upon differentiation, show stringent conservation of chromatin state, and associate with an overwhelming number of developmental regulator gene promoters. We also used computational genomics to search for sequence determinants of Polycomb binding. This analysis revealed that the genomewide locations of PRC2 and PRC1 can be largely predicted from the locations, sizes, and underlying motif contents of CpG islands. We propose that large CpG islands depleted of activating motifs confer epigenetic memory by recruiting the full repertoire of Polycomb complexes in pluripotent cells.
Author Summary
Polycomb-group (PcG) proteins play essential roles in the epigenetic regulation of gene expression during development. PcG proteins are repressors that catalyze lysine 27 tri-methylation on histone H3. They are antagonized by trithorax-group proteins that catalyze lysine 4 tri-methylation. Recent studies of ES cells revealed a novel chromatin pattern consisting of overlapping lysine 27 and lysine 4 tri-methylation. Genomic regions with these opposing modifications were termed “bivalent domains” and proposed to silence developmental regulators while keeping them “poised” for alternate fates. However, our understanding of PcG regulation and bivalent domains remains limited. For instance, bivalent domains affect over 2,000 promoters with diverse functions, which suggests that they may function in diverse cellular processes. Moreover, the mechanisms that underlie the targeting of PcG complexes to specific genomic regions remain completely unknown. To gain insight into these issues, we used ultra high-throughput sequencing to map PcG complexes and related modifications genomewide in human and mouse ES cells. The data identify two classes of bivalent domains with distinct regulatory properties. They also reveal striking relationships between genome sequence and chromatin state that suggest a prominent role for the DNA sequence in dictating the genomewide localization of PcG complexes and, consequently, bivalent domains in ES cells.
PMCID: PMC2567431  PMID: 18974828
23.  The Epigenome of Evolving Drosophila Neo-Sex Chromosomes: Dosage Compensation and Heterochromatin Formation 
PLoS Biology  2013;11(11):e1001711.
This study shows how young sex chromosomes have altered their chromatin structure in Drosophila, and what genomic changes have led to silencing of the Y, and hyper-transcription of the X.
Sex chromosomes originated from autosomes but have evolved a highly specialized chromatin structure. Drosophila Y chromosomes are composed entirely of silent heterochromatin, while male X chromosomes have highly accessible chromatin and are hypertranscribed as a result of dosage compensation. Here, we dissect the molecular mechanisms and functional pressures driving heterochromatin formation and dosage compensation of the recently formed neo-sex chromosomes of Drosophila miranda. We show that the onset of heterochromatin formation on the neo-Y is triggered by an accumulation of repetitive DNA. The neo-X has evolved partial dosage compensation and we find that diverse mutational paths have been utilized to establish several dozen novel binding consensus motifs for the dosage compensation complex on the neo-X, including simple point mutations at pre-binding sites, insertion and deletion mutations, microsatellite expansions, or tandem amplification of weak binding sites. Spreading of these silencing or activating chromatin modifications to adjacent regions results in massive mis-expression of neo-sex linked genes, and little correspondence between functionality of genes and their silencing on the neo-Y or dosage compensation on the neo-X. Intriguingly, the genomic regions being targeted by the dosage compensation complex on the neo-X and those becoming heterochromatic on the neo-Y show little overlap, possibly reflecting different propensities along the ancestral chromosome that formed the sex chromosome to adopt active or repressive chromatin configurations. Our findings have broad implications for current models of sex chromosome evolution, and demonstrate how mechanistic constraints can limit evolutionary adaptations. Our study also highlights how evolution can follow predictable genetic trajectories, by repeatedly acquiring the same 21-bp consensus motif for recruitment of the dosage compensation complex, yet utilizing a diverse array of random mutational changes to attain the same phenotypic outcome.
Author Summary
Sex chromosomes differ from non-sex chromosomes (“autosomes”) at the genomic, transcriptomic, and epigenomic level, yet the X and Y share a common evolutionary origin. The Drosophila Y chromosome is gene-poor and associated with a compact and transcriptionally inactive form of genetic material called heterochromatin. The X, in contrast, is enriched for activating chromatin marks and is consequently hyper-transcribed, a process thought to be an adaptation to decay and silencing of genes on the Y, resulting in “dosage compensation.” How sex chromosomes have altered their chromatin structure, and what genomic changes led to this dramatically different epigenetic makeup, however, has remained a mystery. By studying the genome, epigenome, and transcriptome of a species with a very recently evolved pair of sex chromosomes (the neo-X and neo-Y of a fruit fly, Drosophila miranda), we here recapitulate how both dosage compensation and heterochromatin formation evolve in Drosophila and establish several novel and important principles governing the evolution of chromatin structure. We dissect the evolutionary history of over 60 novel binding sites for the dosage compensation complex that evolved by natural selection on the neo-X within the last one million years. We show that the 21-bp consensus motifs for recruiting the dosage compensation complex were acquired by diverse molecular mechanisms along the neo-X, while the onset of heterochromatin formation is triggered by the accumulation of transposable elements, leading to silencing of adjacent neo-Y genes. We find that spreading of these chromatin modifications results in massive mis-expression of neo-sex linked genes, and that little correspondence exists between functional activity of genes on the neo-Y and whether they are dosage-compensated on the neo-X. Intriguingly, the genomic regions being targeted by the dosage compensation complex on the neo-X and those that are heterochromatic on the neo-Y show little overlap, possibly reflecting different propensities of the ancestral chromosome that formed the sex chromosome to evolve active versus repressive chromatin configurations. These findings have broad implications for current models of sex chromosome evolution.
PMCID: PMC3825665  PMID: 24265597
24.  Deposition of Histone Variant H2A.Z within Gene Bodies Regulates Responsive Genes 
PLoS Genetics  2012;8(10):e1002988.
The regulation of eukaryotic chromatin relies on interactions between many epigenetic factors, including histone modifications, DNA methylation, and the incorporation of histone variants. H2A.Z, one of the most conserved but enigmatic histone variants that is enriched at the transcriptional start sites of genes, has been implicated in a variety of chromosomal processes. Recently, we reported a genome-wide anticorrelation between H2A.Z and DNA methylation, an epigenetic hallmark of heterochromatin that has also been found in the bodies of active genes in plants and animals. Here, we investigate the basis of this anticorrelation using a novel h2a.z loss-of-function line in Arabidopsis thaliana. Through genome-wide bisulfite sequencing, we demonstrate that loss of H2A.Z in Arabidopsis has only a minor effect on the level or profile of DNA methylation in genes, and we propose that the global anticorrelation between DNA methylation and H2A.Z is primarily caused by the exclusion of H2A.Z from methylated DNA. RNA sequencing and genomic mapping of H2A.Z show that H2A.Z enrichment across gene bodies, rather than at the TSS, is correlated with lower transcription levels and higher measures of gene responsiveness. Loss of H2A.Z causes misregulation of many genes that are disproportionately associated with response to environmental and developmental stimuli. We propose that H2A.Z deposition in gene bodies promotes variability in levels and patterns of gene expression, and that a major function of genic DNA methylation is to exclude H2A.Z from constitutively expressed genes.
Author Summary
Eukaryotes package their DNA to fit within the nucleus using well-conserved proteins, called histones, that form the building blocks of nucleosomes, the fundamental units of chromatin. Histone variants are specialized versions of these proteins that change the chromatin landscape by altering the biochemical properties and interacting partners of the nucleosome. H2A.Z, a conserved eukaryotic histone variant, is preferentially enriched at the beginnings of genes, though the significance of this enrichment remains unknown. We and others have shown that H2A.Z is conspicuously absent from methylated DNA across the genome in plants and animals. Typically considered a mark of epigenetic silencing, DNA methylation has more recently been discovered in the bodies of many genes. Here, we present evidence that the genome-wide anticorrelation between DNA methylation and H2A.Z enrichment in Arabidopsis is the result of DNA methylation acting to prevent H2A.Z incorporation. We demonstrate that the presence of H2A.Z within gene bodies is correlated with lower transcription levels and higher variability in expression patterns across tissue types and environmental conditions, and we propose that a major function of gene-body DNA methylation is to exclude H2A.Z from the bodies of highly and constitutively expressed genes.
PMCID: PMC3469445  PMID: 23071449
25.  The Role of the Arabidopsis Exosome in siRNA–Independent Silencing of Heterochromatic Loci 
PLoS Genetics  2013;9(3):e1003411.
The exosome functions throughout eukaryotic RNA metabolism and has a prominent role in gene silencing in yeast. In Arabidopsis, exosome regulates expression of a “hidden” transcriptome layer from centromeric, pericentromeric, and other heterochromatic loci that are also controlled by small (sm)RNA-based de novo DNA methylation (RdDM). However, the relationship between exosome and smRNAs in gene silencing in Arabidopsis remains unexplored. To investigate whether exosome interacts with RdDM, we profiled Arabidopsis smRNAs by deep sequencing in exosome and RdDM mutants and also analyzed RdDM-controlled loci. We found that exosome loss had a very minor effect on global smRNA populations, suggesting that, in contrast to fission yeast, in Arabidopsis the exosome does not control the spurious entry of RNAs into smRNA pathways. Exosome defects resulted in decreased histone H3K9 dimethylation at RdDM-controlled loci, without affecting smRNAs or DNA methylation. Exosome also exhibits a strong genetic interaction with RNA Pol V, but not Pol IV, and physically associates with transcripts produced from the scaffold RNAs generating region. We also show that two Arabidopsis rrp6 homologues act in gene silencing. Our data suggest that Arabidopsis exosome may act in parallel with RdDM in gene silencing, by epigenetic effects on chromatin structure, not through siRNAs or DNA methylation.
Author Summary
To maintain genomic stability and prevent expansion of invasive genomic sequences such as transposable elements (TEs), eukaryotes have evolved defensive mechanisms to control them. Here, we examine the role of the Arabidopsis exosome complex in such mechanisms. Evolutionarily conserved from archaea to humans, the exosome is a stable complex of RNase-like and RNA binding proteins that plays a central role in RNA metabolism in eukaryotes. Depletion of the exosome allows some repetitive sequences to escape from silencing. Most of these transcripts emanate from centromeric and pericentromeric chromosomal regions and other heterochromatic loci, and many derive from repetitive and transposable elements. In plants, TEs are targeted for de novo DNA methylation by smRNA–mediated pathways. However, we found that exosome depletion has only minor effects on smRNA populations that are acting in the main silencing mechanism in Arabidopsis, siRNAs–dependent DNA methylation RdDM. Instead, exosome depletion affects histone H3K9 dimethylation, an epigenetic mark that affects chromatin structure and thus alters transcription. Our data suggest that the exosome collaborates in gene silencing, likely acting in a parallel pathway to other mechanisms. We also propose that the Arabidopsis exosome may coordinate the transcriptional interplay of different RNA polymerases to modulate repression of some repetitive sequences.
PMCID: PMC3610620  PMID: 23555312

Results 1-25 (1053554)