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At least 19 recordsLinked to original sources
PubMed2025

Heterochromatin de novo formation and maintenance in Plasmodium falciparum.

In the malaria parasite Plasmodium falciparum, the expression of many genes is regulated by heterochromatin (HC) based on the histone mark tri-methylation of histone H3 lysine 9 (H3K9me3). HC assembly involves three distinct steps: de novo nucleation, spreading and maintenance. Nucleation, which consists in formation of HC in a previously euchromatic region, determines the specific genomic locations where HC occurs. This process is not well understood in malaria parasites. Here we investigated the DNA sequence cis determinants of HC nucleation in P. falciparum, using a screening approach based on integration of fragments from different heterochromatic genes into an euchromatic locus, followed by H3K9me3 chromatin immunoprecipitation (ChIP) analysis. We found that fragments of var gene upstream regions nucleated HC efficiently, whereas fragments from the pfap2-g upstream region or from the mspdbl2 locus did not nucleate HC. Fragments from the beginning of the coding sequence (CDS) of pfap2-g nucleated HC with low efficiency, as evidenced by nucleation requiring long fragments of ~2 kb and occurring only in a fraction of the parasites. These results demonstrate that the primary DNA sequence is a main determinant of HC nucleation in P. falciparum. We also studied HC maintenance at the pfap2-g locus, which demonstrated that specific parts of the upstream region, different from the regions competent for HC nucleation, are required for maintenance. Together, our results provide initial insight into how HC is directed to specific loci and maintained in P. falciparum.

Plasmodium falciparum↗
PubMed2002

Ordered nucleation and spreading of silenced chromatin in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, silencing at the HM loci depends on Sir proteins, which are structural components of silenced chromatin. To explore the structure and assembly of silenced chromatin, the associations of Sir proteins with sequences across the HMR locus were examined by chromatin immunoprecipitation. In wild-type cells, Sir2p, Sir3p, and Sir4p were spread throughout and coincident with the silenced region at HMR. Sir1p, in contrast, associated only with the HMR-E silencer, consistent with its role in establishment but not maintenance of silencing. Sir4p was required for the association of other Sir proteins with silencers. In contrast, in the absence of Sir2p or Sir3p, partial assemblies of Sir proteins could form at silencers, where Sir protein assembly began. Spreading across HMR required Sir2p and Sir3p, as well as the deacetylase activity of Sir2p. These data support a model for the spreading of silenced chromatin involving cycles of nucleosome deacetylation by Sir2p followed by recruitment of additional Sir2p, Sir3p, and Sir4p to the newly deacetylated nucleosome. This model suggests mechanisms for boundary formation, and for maintenance and inheritance of silenced chromatin. The principles are generalizable to other types of heritable chromatin states.

Animals↗
PubMedDate not supplied

The effect of antifreeze proteins and poly(vinyl alcohol) on the nucleation of ice: a preliminary study.

Three substances have been tested for ice nucleation inhibition. These were an antifreeze protein AFP III from the fish Macrozoarces americanus, an antifreeze glycoprotein AFGP from the fish Dissostichus mawsoni, and an 80% hydrolysed poly(vinyl alcohol) with a molecular weight of 9 to 10 kD. A nucleation spectrometer was used to test nucleation inhibition at a range of concentrations against two types of ice nuclei: those present in tap water and a bacterial nucleator from Pseudomonas syringae. The PVA reduced the nucleation temperature of tap water and the bacterial dispersions at all the concentrations which were tested. The AFGP reduced the nucleation temperature of tap water but enhanced the nucleation activity of the bacterial nucleators. At low concentrations the AFP III reduced the nucleation temperature of both tap water and the bacterial nucleator. At high concentrations the AFP III enhanced the nucleation temperature of the bacterial nucleator and broadened the nucleation spectrum of the tap water to encompass the nucleation spread of the control. The possible mechanisms of nucleation suppression and enhancement are discussed.

Animals↗
PubMed2004

Multiple classes of MSL binding sites target dosage compensation to the X chromosome of Drosophila.

MSL complexes bind hundreds of sites along the single male X chromosome to achieve dosage compensation in Drosophila. Previously, we proposed that approximately 35 "high-affinity" or "chromatin entry" sites (CES) might nucleate spreading of MSL complexes in cis to paint the X chromosome. This was based on analysis of the first characterized sites roX1 and roX2. roX transgenes attract MSL complex to autosomal locations where it can spread long distances into flanking chromatin. roX1 and roX2 also produce noncoding RNA components of the complex. Here we identify a third site from the 18D10 region of the X chromosome. Like roX genes, 18D binds full and partial MSL complexes in vivo and encompasses a male-specific DNase I hypersensitive site (DHS). Unlike roX genes, the 510 bp 18D site is apparently not transcribed and shows high affinity for MSL complex and spreading only as a multimer. While mapping 18D, we discovered MSL binding to X cosmids that do not carry one of the approximately 35 high-affinity sites. Based on additional analyses of chromosomal transpositions, we conclude that spreading in cis from the roX genes or the approximately 35 originally proposed "entry sites" cannot be the sole mechanism for MSL targeting to the X chromosome.

Animals↗
PubMed2006

Direct visualization of saposin remodelling of lipid bilayers.

Saposins A, B, C and D are soluble, non-enzymatic proteins that interact with lysosomal membranes to activate the breakdown and transfer of glycosphingolipids. The mechanisms of hydrolase activation and lipid transfer by saposins remain unknown. We have used in situ atomic force microscopy (AFM) with simultaneous confocal fluorescence microscopy to investigate the interactions of saposins with lipid membranes. AFM images of the effect of saposins A, B and C on supported lipid bilayers showed a time and concentration-dependent nucleated spread of membrane transformation. Saposin B produced deep gaps that ultimately filled with granular material, while saposins A and C lead to localized areas of membrane that were reduced in height by approximately 1.5 nm. Fluorescence-labeled saposin C co-localized with the transformed areas of the bilayer, indicating stable binding to the membrane. Fluorescence resonance energy transfer confirmed a direct interaction between saposin C and lipid. Under certain conditions of membrane lipid composition and saposin concentration, extensive bilayer lipid removal was observed. We propose a multi-step mechanism that integrates the structural features and amphipathic properties of the saposin proteins.

Fluorescence Resonance Energy Transfer↗
PubMed2004

Sequence-specific targeting of MSL complex regulates transcription of the roX RNA genes.

In Drosophila, dosage compensation is controlled by the male-specific lethal (MSL) complex consisting of at least five proteins and two noncoding RNAs, roX1 and roX2. The roX RNAs function in targeting MSL complex to the X chromosome, and roX transgenes can nucleate spreading of the MSL complex into flanking chromatin when inserted on an autosome. An MSL-binding site (DHS, DNaseI hypersensitive site) has been identified in each roX gene. Here, we investigate the functions of the DHS using transgenic deletion analyses and reporter assays. We find that MSL interaction with the DHS counteracts constitutive repression at roX1, resulting in male-specific expression of roX1 RNA. Surprisingly, the DHS is not required for initiation of cis spreading of MSL complex, instead local transcription of roX RNAs correlates with extensive spreading.

Animals↗
PubMed1998

Corrective gene transfer of keratinocytes from patients with junctional epidermolysis bullosa restores assembly of hemidesmosomes in reconstructed epithelia.

Herlitz junctional epidermolysis bullosa (H-JEB) provides a promising model for somatic gene therapy of heritable mechano-bullous disorders. This genodermatosis is caused by the lack of laminin-5 that results in absence of hemidesmosomes (HD) and defective adhesion of squamous epithelia. To establish whether re-expression of laminin-5 can restore assembly of the dermal-epidermal attachment structures lacking in the H-JEB skin, we corrected the genetic mutation hindering expression of the beta 3 chain of laminin-5 in human H-JEB keratinocytes by transfer of a laminin beta 3 transgene. The transduced keratinocytes synthesized a recombinant beta 3 polypeptide that assembled with the endogenous laminin alpha 3 and gamma 2 chains into a biologically active laminin-5 that was secreted, processed and deposited into the extracellular matrix. Re-expression of laminin-5 induced cell spreading, nucleation of hemidesmosomal-like structures and enhanced adhesion to the culture substrate. Organotypic cultures performed with the transduced keratinocytes, reconstituted epidermis closely adhering to the mesenchyme and presenting mature hemidesmosomes, bridging the cytoplasmic intermediate filaments of the basal cells to the anchoring filaments of the basement membrane. Our results provide the first evidence of phenotypic reversion of JEB keratinocytes by somatic gene therapy and demonstrate that genetic treatment of the mild forms of skin blistering diseases and other inherited extracellular matrix pathologies is a realistic goal.

Animals↗
PubMed2006

The bacterial chromosome segregation protein Spo0J spreads along DNA from parS nucleation sites.

Regulation of chromosome inheritance is essential to ensure proper transmission of genetic information. To accomplish accurate genome segregation, cells organize their chromosomes and actively separate them prior to cytokinesis. In Bacillus subtilis the Spo0J protein is required for accurate chromosome segregation and it regulates the developmental switch from vegetative growth to sporulation. Spo0J is a DNA-binding protein that recognizes at least eight identified parS sites located near the origin of replication. As judged by fluorescence microscopy, Spo0J forms discrete foci associated with the oriC region of the chromosome throughout the cell cycle. In an attempt to determine the mechanisms utilized by Spo0J to facilitate productive chromosome segregation, we have investigated the DNA binding activity of Spo0J. In vivo we find Spo0J associates with several kilobases of DNA flanking its specific binding sites (parS) through a parS-dependent nucleation event that promotes lateral spreading of Spo0J along the chromosome. Using purified components we find that Spo0J has the ability to coat non-specific DNA substrates. These 'Spo0J domains' provide large structures near oriC that could potentially demark, organize or localize the origin region of the chromosome.

Bacillus subtilis↗
PubMed2003

Local spreading of MSL complexes from roX genes on the Drosophila X chromosome.

MSL proteins and noncoding roX RNAs form complexes to up-regulate hundreds of genes on the Drosophila male X chromosome, and make X-linked gene expression equal in males and females. Altering the ratio of MSL proteins to roX RNA dramatically changes X-chromosome morphology. In protein excess, the MSL complex concentrates near sites of roX transcription and is depleted elsewhere. These results support a model for distribution of MSL complexes, in which local spreading in cis from roX genes is balanced with diffusion of soluble complexes in trans. When overexpressed, MSL proteins can recognize the X chromosome, modify histones, and partially restore male viability even in the absence of roX RNAs. Thus, the protein components can carry out all essential functions of dosage compensation, but roX RNAs facilitate the correct targeting of MSL complexes, in part by nucleation of spreading from their sites of synthesis.

Animals↗
PubMed1998

Mechanisms of silencing in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, heterochromatin-like regions are formed at the silent mating type loci and at telomeres. The past year of investigations has led to a clearer understanding of the nature of nucleation and spreading of heterochromatin, as well as uncovering a fascinating link between silencing, the nucleolus and aging.

Animals↗
PubMed2006

Incorporation of alpha-amylase enzyme and a bioactive filler into hydrophilic, partially degradable, and bioactive cements (HDBCs) as a new approach to tailor simultaneously their degradation and bioactive behavior.

Hydrophilic, partially degradable, and bioactive cements (HDBCs) are starch-containing cements intended to degrade partially in the human body and, in so doing, allow for bone ingrowth inside the pores formed during degradation. Therefore, the study of degradation and bioactivity behavior was performed to assess the suitability of the current HDBCs formulations to achieve those aims. The degradation profile of HDBCs was studied under different conditions, including incubation in phosphate-buffered saline (PBS) and PBS supplemented with alpha-amylase at different concentrations. Thermostable alpha-amylase was also added to some formulations to allow control of the degradation rate and its extent. In a second stage the simultaneous phenomena of enzymatic degradation and bioactivity (both in vitro) was studied. We observed that the degradation of starch present in HDBCs can be easily controlled by the amount of alpha-amylase added to the cement and high values of degradation may be achieved if high enough quantities of enzyme are incorporated. However, the maximum degradation extent is much more dependent on the total amount of starch present in the formulation than on the amount of enzyme added to it: for full pore connectivity, the amount of starch should be higher than the percolation threshold for a 3D specimen. Nonetheless, calcium phosphate was able to nucleate and spread in inner pores of the cement, formed due to degradation, if they were interconnected. For a more thorough covering of the pores with calcium phosphates the amount of starch present in HDBCs should be increased to be higher than the percolation threshold.

Apatites↗
PubMed2005

A var gene promoter controls allelic exclusion of virulence genes in Plasmodium falciparum malaria.

Mono-allelic expression of gene families is used by many organisms to mediate phenotypic variation of surface proteins. In the apicomplexan parasite Plasmodium falciparum, responsible for the severe form of malaria in humans, this is exemplified by antigenic variation of the highly polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1, encoded by the 60-member var gene family, represents a major virulence factor due to its central role in immune evasion and intravascular parasite sequestration. Mutually exclusive expression of PfEMP1 is controlled by epigenetic mechanisms involving chromatin modification and perinuclear var locus repositioning. Here we show that a var promoter mediates the nucleation and spreading of stably inherited silenced chromatin. Transcriptional activation of this promoter occurs at the nuclear periphery in association with chromosome-end clusters. Additionally, the var promoter sequence is sufficient to infiltrate a transgene into the allelic exclusion programme of var gene expression, as transcriptional activation of this transgene results in silencing of endogenous var gene transcription. These results show that a var promoter is sufficient for epigenetic silencing and mono-allelic transcription of this virulence gene family, and are fundamental for our understanding of antigenic variation in P. falciparum. Furthermore, the PfEMP1 knockdown parasites obtained in this study will be important tools to increase our understanding of P. falciparum-mediated virulence and immune evasion.

Alleles↗
PubMed2004

Counting of Rif1p and Rif2p on Saccharomyces cerevisiae telomeres regulates telomere length.

Telomere length is negatively regulated by proteins of the telomeric DNA-protein complex. Rap1p in Saccharomyces cerevisiae binds the telomeric TG(1-3) repeat DNA, and the Rap1p C terminus interacts with Rif1p and Rif2p. We investigated how these three proteins negatively regulate telomere length. We show that direct tethering of each Rif protein to a telomere shortens that telomere proportionally to the number of tethered molecules, similar to previously reported counting of Rap1p. Surprisingly, Rif proteins could also regulate telomere length even when the Rap1p C terminus was absent, and tethered Rap1p counting was completely dependent on the Rif proteins. Thus, Rap1p counting is in fact Rif protein counting. In genetic settings that cause telomeres to be abnormally long, tethering even a single Rif2p molecule was sufficient for maximal effectiveness in preventing the telomere overelongation. We show that a heterologous protein oligomerization domain, the mammalian PDZ domain, when fused to Rap1p can confer telomere length control. We propose that a nucleation and spreading mechanism is involved in forming the higher-order telomere structure that regulates telomere length.

Binding Sites↗
PubMed2006

Sir3 C-terminal domain involvement in the initiation and spreading of heterochromatin.

Heterochromatin is nucleated at a specific site and subsequently spreads into distal sequences through multiple interactions between modified histones and nonhistone proteins. In the yeast Saccharomyces cerevisiae, these nonhistone proteins include Sir2, Sir3, and Sir4. We have previously shown that loss of the C-terminal Rap1 domain containing Sir3 and Sir4 association sites can be overcome by tethering a 144-amino-acid C-terminal domain (CTD) of Sir3 adjacent to the telomere. Here, we explore the substructure and functions of the CTD. We demonstrate that the CTD is the minimum domain for Sir3 homodimerization, a function that is conserved in related yeasts. However, CTD heterodimers associate at only low efficiencies and correspondingly have low levels of tethered silencing, consistent with an essential role for dimerization in tethered silencing. Six missense alleles were generated, with ctd-Y964A producing the most extreme phenotypes when tethered to the LexA binding sites. Although ctd-Y964A is capable of dimerization, telomere silencing is abrogated, indicating that the CTD serves a second essential function in silencing. Chromatin immunoprecipitation analyses of wild-type and ctd-Y964A mutant cells indicate an association of the CTD with the deacetylated histone tails of H3 and H4 that is necessary for the recruitment of Sir3. The efficiency of spreading depends upon the apparent stoichiometry and stability during the initiation event. The predicted Cdc6 domain III winged-helix structure may well be responsible for dimerization.

Binding Sites↗
PubMed2001

Phase-field modeling of microstructural pattern formation during directional solidification of peritectic alloys without morphological instability.

During the directional solidification of peritectic alloys, two stable solid phases (parent and peritectic) grow competitively into a metastable liquid phase of larger impurity content than either solid phase. When the parent or both solid phases are morphologically unstable, i.e., for a small temperature gradient/growth rate ratio (G/v(p)), one solid phase usually outgrows and covers the other phase, leading to a cellular-dendritic array structure closely analogous to the one formed during monophase solidification of a dilute binary alloy. In contrast, when G/v(p) is large enough for both phases to be morphologically stable, the formation of the microstructure becomes controlled by a subtle interplay between the nucleation and growth of the two solid phases. The structures that have been observed in this regime (in small samples where convection effects are suppressed) include alternate layers (bands) of the parent and peritectic phases perpendicular to the growth direction, which are formed by alternate nucleation and lateral spreading of one phase onto the other as proposed in a recent model [R. Trivedi, Metall. Mater. Trans. A 26, 1 (1995)], as well as partially filled bands (islands), where the peritectic phase does not fully cover the parent phase which grows continuously. We develop a phase-field model of peritectic solidification that incorporates nucleation processes in order to explore the formation of these structures. Simulations of this model shed light on the morphology transition from islands to bands, the dynamics of spreading of the peritectic phase on the parent phase following nucleation, which turns out to be characterized by a remarkably constant acceleration, and the types of growth morphology that one might expect to observe in large samples under purely diffusive growth conditions.

Journal Article↗
PubMed2005

Y chromosomes: born to be destroyed.

Suppression of recombination is the prerequisite for stable genetically determined sex systems. A consequence of suppression of recombination is the strong bias in the distribution of transposable elements (TEs), mostly retrotransposons. Our results and those from others indicate that the major force driving the degeneration of Y chromosomes are retrotransposons in remodelling former euchromatic chromosome structures into heterochromatic ones. We put forward the following hypotheses. (1) A massive accumulation of retrotransposons occurs early in non-recombining regions. (2) Heterochromatic nucleation centres are formed as a genomic defence mechanism against invasive parasitic elements. The newly established nucleation centres become epigenetically inherited. (3) Spreading of heterochromatin from the nucleation centres into flanking regions induces, in an adaptive process, transcriptional gene silencing of neighbourhood genes that could either be still intact or in an already eroded condition. (4) Constitutive silenced genes are not under the same genetic selection pressure as active genes. They are more exposed to the decay process. (5) Gene dosage balance is re-established by the parallel evolution of dosage compensation mechanisms.

Animals↗
PubMed2005

Recognition and modification of seX chromosomes.

Flies, worms and mammals employ dosage compensation complexes that alter chromatin or chromosome structure to equalize X-linked gene expression between the sexes. Recent work has improved our understanding of how dosage compensation complexes achieve X chromosome-wide association and has provided significant insight into the epigenetic modifications directed by these complexes to modulate gene expression. In flies, the prevailing view that dosage compensation complexes assemble on the X chromosome at approximately 35 chromatin-entry sites and then spread in cis to cover the chromosome has been re-evaluated in light of the evidence that these chromatin-entry sites are not required for localization of the complex. By contrast, identification of discrete recruitment elements indicates that nucleation at and spread from a limited number of sites directs dosage compensation complex localization on the worm X-chromosome. Studies in flies and mammals have extended our understanding of how ribonucleoprotein complexes are used to modify X chromatin, for either activation or repression of transcription. Finally, evidence from mammals suggests that the chromatin modifications that mediate dosage compensation are very dynamic, because they are established, reversed and re-established early in development.

Animals↗
PubMed2001

Association and spreading of the Drosophila dosage compensation complex from a discrete roX1 chromatin entry site.

In Drosophila, dosage compensation is controlled by the male-specific lethal (MSL) complex consisting of MSL proteins and roX RNAs. The MSL complex is specifically localized on the male X chromosome to increase its expression approximately 2-fold. We recently proposed a model for the targeted assembly of the MSL complex, in which initial binding occurs at approximately 35 dispersed chromatin entry sites, followed by spreading in cis into flanking regions. Here, we analyze one of the chromatin entry sites, the roX1 gene, to determine which sequences are sufficient to recruit the MSL complex. We found association and spreading of the MSL complex from roX1 transgenes in the absence of detectable roX1 RNA synthesis from the transgene. We mapped the recruitment activity to a 217 bp roX1 fragment that shows male-specific DNase hypersensitivity and can be preferentially cross-linked in vivo to the MSL complex. When inserted on autosomes, this small roX1 segment is sufficient to produce an ectopic chromatin entry site that can nucleate binding and spreading of the MSL complex hundreds of kilobases into neighboring regions.

Animals↗