Publications by authors named "Nehme Saksouk"

20 Publications

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The mouse HP1 proteins are essential for preventing liver tumorigenesis.

Oncogene 2020 03 4;39(13):2676-2691. Epub 2020 Feb 4.

IRCM, Institut de Recherche en Cancérologie de Montpellier, F-34298, Montpellier, France.

Chromatin organization is essential for appropriate interpretation of the genetic information. Here, we demonstrated that the chromatin-associated proteins HP1 are dispensable for hepatocytes survival but are essential within hepatocytes to prevent liver tumor development in mice with HP1β being pivotal in these functions. Yet, we found that the loss of HP1 per se is not sufficient to induce cell transformation but renders cells more resistant to specific stress such as the expression of oncogenes and thus in fine, more prone to cell transformation. Molecular characterization of HP1-Triple KO premalignant livers and BMEL cells revealed that HP1 are essential for the maintenance of heterochromatin organization and for the regulation of specific genes with most of them having well characterized functions in liver functions and homeostasis. We further showed that some specific retrotransposons get reactivated upon loss of HP1, correlating with overexpression of genes in their neighborhood. Interestingly, we found that, although HP1-dependent genes are characterized by enrichment H3K9me3, this mark does not require HP1 for its maintenance and is not sufficient to maintain gene repression in absence of HP1. Finally, we demonstrated that the loss of TRIM28 association with HP1 recapitulated several phenotypes induced by the loss of HP1 including the reactivation of some retrotransposons and the increased incidence of liver cancer development. Altogether, our findings indicate that HP1 proteins act as guardians of liver homeostasis to prevent tumor development by modulating multiple chromatin-associated events within both the heterochromatic and euchromatic compartments, partly through regulation of the corepressor TRIM28 activity.
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http://dx.doi.org/10.1038/s41388-020-1177-8DOI Listing
March 2020

SETDB1-dependent heterochromatin stimulates alternative lengthening of telomeres.

Sci Adv 2019 05 8;5(5):eaav3673. Epub 2019 May 8.

Institute of Human Genetics CNRS-Université de Montpellier UMR 9002, 141 rue de la Cardonille, Montpellier 34000, France.

Alternative lengthening of telomeres, or ALT, is a recombination-based process that maintains telomeres to render some cancer cells immortal. The prevailing view is that ALT is inhibited by heterochromatin because heterochromatin prevents recombination. To test this model, we used telomere-specific quantitative proteomics on cells with heterochromatin deficiencies. In contrast to expectations, we found that ALT does not result from a lack of heterochromatin; rather, ALT is a consequence of heterochromatin formation at telomeres, which is seeded by the histone methyltransferase SETDB1. Heterochromatin stimulates transcriptional elongation at telomeres together with the recruitment of recombination factors, while disrupting heterochromatin had the opposite effect. Consistently, loss of SETDB1, disrupts telomeric heterochromatin and abrogates ALT. Thus, inhibiting telomeric heterochromatin formation in ALT cells might offer a new therapeutic approach to cancer treatment.
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http://dx.doi.org/10.1126/sciadv.aav3673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6506250PMC
May 2019

Integrative Proteomic Profiling Reveals PRC2-Dependent Epigenetic Crosstalk Maintains Ground-State Pluripotency.

Cell Stem Cell 2019 01 21;24(1):123-137.e8. Epub 2018 Nov 21.

Department of Molecular Biology, Faculty of Science, Radboud University, Radboud Institute for Molecular Life Sciences (RIMLS), 6525GA Nijmegen, the Netherlands. Electronic address:

The pluripotent ground state is defined as a basal state free of epigenetic restrictions, which influence lineage specification. While naive embryonic stem cells (ESCs) can be maintained in a hypomethylated state with open chromatin when grown using two small-molecule inhibitors (2i)/leukemia inhibitory factor (LIF), in contrast to serum/LIF-grown ESCs that resemble early post-implantation embryos, broader features of the ground-state pluripotent epigenome are not well understood. We identified epigenetic features of mouse ESCs cultured using 2i/LIF or serum/LIF by proteomic profiling of chromatin-associated complexes and histone modifications. Polycomb-repressive complex 2 (PRC2) and its product H3K27me3 are highly abundant in 2i/LIF ESCs, and H3K27me3 is distributed genome-wide in a CpG-dependent fashion. Consistently, PRC2-deficient ESCs showed increased DNA methylation at sites normally occupied by H3K27me3 and increased H4 acetylation. Inhibiting DNA methylation in PRC2-deficient ESCs did not affect their viability or transcriptome. Our findings suggest a unique H3K27me3 configuration protects naive ESCs from lineage priming, and they reveal widespread epigenetic crosstalk in ground-state pluripotency.
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http://dx.doi.org/10.1016/j.stem.2018.10.017DOI Listing
January 2019

Proteome Characterization of a Chromatin Locus Using the Proteomics of Isolated Chromatin Segments Approach.

Methods Mol Biol 2017 ;1550:19-33

INSERM AVENIR, Institute of Human Genetics CNRS UPR1142, 141 rue de la Cardonille, 34000, Montpellier, France.

The biological functions of given genomic regions are ruled by the local chromatin composition. The Proteomics of Isolated Chromatin segments approach (PICh) is a powerful and unbiased method to analyze the composition of chosen chromatin segments, provided they are abundant (repeated) or that the organism studied has a small genome. PICh can be used to identify novel and unexpected regulatory factors, or when combined with quantitative mass spectrometric approaches, to characterize the function of a defined factor at the chosen locus, by quantifying composition changes at the locus upon removal/addition of that factor.
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http://dx.doi.org/10.1007/978-1-4939-6747-6_3DOI Listing
February 2018

Constitutive heterochromatin formation and transcription in mammals.

Epigenetics Chromatin 2015 15;8. Epub 2015 Jan 15.

INSERM AVENIR Team, Institute of Human Genetics, CNRS UPR 1142, Montpellier, France.

Constitutive heterochromatin, mainly formed at the gene-poor regions of pericentromeres, is believed to ensure a condensed and transcriptionally inert chromatin conformation. Pericentromeres consist of repetitive tandem satellite repeats and are crucial chromosomal elements that are responsible for accurate chromosome segregation in mitosis. The repeat sequences are not conserved and can greatly vary between different organisms, suggesting that pericentromeric functions might be controlled epigenetically. In this review, we will discuss how constitutive heterochromatin is formed and maintained at pericentromeres in order to ensure their integrity. We will describe the biogenesis and the function of main epigenetic pathways that are involved and how they are interconnected. Interestingly, recent findings suggest that alternative pathways could substitute for well-established pathways when disrupted, suggesting that constitutive heterochromatin harbors much more plasticity than previously assumed. In addition, despite of the heterochromatic nature of pericentromeres, there is increasing evidence for active and regulated transcription at these loci, in a multitude of organisms and under various biological contexts. Thus, in the second part of this review, we will address this relatively new aspect and discuss putative functions of pericentromeric expression.
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http://dx.doi.org/10.1186/1756-8935-8-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4363358PMC
March 2015

Redundant mechanisms to form silent chromatin at pericentromeric regions rely on BEND3 and DNA methylation.

Mol Cell 2014 Nov 6;56(4):580-94. Epub 2014 Nov 6.

INSERM AVENIR, Institute of Human Genetics CNRS UPR1142, 141 rue de la Cardonille, 34000 Montpellier, France. Electronic address:

Constitutive heterochromatin is typically defined by high levels of DNA methylation and H3 lysine 9 trimethylation (H3K9Me3), whereas facultative heterochromatin displays DNA hypomethylation and high H3 lysine 27 trimethylation (H3K27Me3). The two chromatin types generally do not coexist at the same loci, suggesting mutual exclusivity. During development or in cancer, pericentromeric regions can adopt either epigenetic state, but the switching mechanism is unknown. We used a quantitative locus purification method to characterize changes in pericentromeric chromatin-associated proteins in mouse embryonic stem cells deficient for either the methyltransferases required for DNA methylation or H3K9Me3. DNA methylation controls heterochromatin architecture and inhibits Polycomb recruitment. BEND3, a protein enriched on pericentromeric chromatin in the absence of DNA methylation or H3K9Me3, allows Polycomb recruitment and H3K27Me3, resulting in a redundant pathway to generate repressive chromatin. This suggests that BEND3 is a key factor in mediating a switch from constitutive to facultative heterochromatin.
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http://dx.doi.org/10.1016/j.molcel.2014.10.001DOI Listing
November 2014

Exchange of associated factors directs a switch in HBO1 acetyltransferase histone tail specificity.

Genes Dev 2013 Sep;27(18):2009-24

Laval University Cancer Research Center, Hôtel-Dieu de Québec (CHUQ), Quebec City, Québec G1R 2J6, Canada;

Histone acetyltransferases (HATs) assemble into multisubunit complexes in order to target distinct lysine residues on nucleosomal histones. Here, we characterize native HAT complexes assembled by the BRPF family of scaffold proteins. Their plant homeodomain (PHD)-Zn knuckle-PHD domain is essential for binding chromatin and is restricted to unmethylated H3K4, a specificity that is reversed by the associated ING subunit. Native BRPF1 complexes can contain either MOZ/MORF or HBO1 as catalytic acetyltransferase subunit. Interestingly, while the previously reported HBO1 complexes containing JADE scaffold proteins target histone H4, the HBO1-BRPF1 complex acetylates only H3 in chromatin. We mapped a small region to the N terminus of scaffold proteins responsible for histone tail selection on chromatin. Thus, alternate choice of subunits associated with HBO1 can switch its specificity between H4 and H3 tails. These results uncover a crucial new role for associated proteins within HAT complexes, previously thought to be intrinsic to the catalytic subunit.
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http://dx.doi.org/10.1101/gad.223396.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3792477PMC
September 2013

Conserved molecular interactions within the HBO1 acetyltransferase complexes regulate cell proliferation.

Mol Cell Biol 2012 Feb 5;32(3):689-703. Epub 2011 Dec 5.

Laval University Cancer Research Center, Hôtel-Dieu de Québec, Quebec City, Quebec, Canada.

Acetyltransferase complexes of the MYST family with distinct substrate specificities and functions maintain a conserved association with different ING tumor suppressor proteins. ING complexes containing the HBO1 acetylase are a major source of histone H3 and H4 acetylation in vivo and play critical roles in gene regulation and DNA replication. Here, our molecular dissection of HBO1/ING complexes unravels the protein domains required for their assembly and function. Multiple PHD finger domains present in different subunits bind the histone H3 N-terminal tail with a distinct specificity toward lysine 4 methylation status. We show that natively regulated association of the ING4/5 PHD domain with HBO1-JADE determines the growth inhibitory function of the complex, linked to its tumor suppressor activity. Functional genomic analyses indicate that the p53 pathway is a main target of the complex, at least in part through direct transcription regulation at the initiation site of p21/CDKN1A. These results demonstrate the importance of ING association with MYST acetyltransferases in controlling cell proliferation, a regulated link that accounts for the reported tumor suppressor activities of these complexes.
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http://dx.doi.org/10.1128/MCB.06455-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3266594PMC
February 2012

Signal transducers and activators of transcription-3/pim1 axis plays a critical role in the pathogenesis of human pulmonary arterial hypertension.

Circulation 2011 Mar 7;123(11):1205-15. Epub 2011 Mar 7.

Centre de recherche de L'Hôtel-Dieu de Québec, 10 Rue McMahon, Québec, QC G1R 2J6, Canada.

Background: Pulmonary artery hypertension (PAH) is a proliferative disorder associated with enhanced pulmonary artery smooth muscle cell proliferation and suppressed apoptosis. The sustainability of this phenotype required the activation of a prosurvival transcription factor like signal transducers and activators of transcription-3 (STAT3) and nuclear factor of activated T cell (NFAT). Because these factors are implicated in several physiological processes, their inhibition in PAH patients could be associated with detrimental effects. Therefore, a better understanding of the mechanism accounting for their expression/activation in PAH pulmonary artery smooth muscle cells is of great therapeutic interest.

Methods And Results: Using multidisciplinary and translational approaches, we demonstrated that STAT3 activation in both human and experimental models of PAH accounts for the expression of both NFATc2 and the oncoprotein kinase Pim1, which trigger NFATc2 activation. Because Pim1 expression correlates with the severity of PAH in humans and is confined to the PAH pulmonary artery smooth muscle cell, Pim1 was identified as an attractive therapeutic target for PAH. Indeed, specific Pim1 inhibition in vitro decreases pulmonary artery smooth muscle cell proliferation and promotes apoptosis, all of which are sustained by NFATc2 inhibition. In vivo, tissue-specific inhibition of Pim1 by nebulized siRNA reverses monocrotaline-induced PAH in rats, whereas Pim1 knockout mice are resistant to PAH development.

Conclusion: We demonstrated for the first time that inhibition of the inappropriate activation of STAT3/Pim1 axis is a novel, specific, and attractive therapeutic strategy to reverse PAH.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.110.963314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3545712PMC
March 2011

Role for miR-204 in human pulmonary arterial hypertension.

J Exp Med 2011 Mar 14;208(3):535-48. Epub 2011 Feb 14.

Département de médecine, Faculté de médecine, Hôtel-Dieude Québec, Canada.

Pulmonary arterial hypertension (PAH) is characterized by enhanced proliferation and reduced apoptosis of pulmonary artery smooth muscle cells (PASMCs). Because microRNAs have been recently implicated in the regulation of cell proliferation and apoptosis, we hypothesized that these regulatory molecules might be implicated in the etiology of PAH. In this study, we show that miR-204 expression in PASMCs is down-regulated in both human and rodent PAH. miR-204 down-regulation correlates with PAH severity and accounts for the proliferative and antiapoptotic phenotypes of PAH-PASMCs. STAT3 activation suppresses miR-204 expression, and miR-204 directly targets SHP2 expression, thereby SHP2 up-regulation, by miR-204 down-regulation, activates the Src kinase and nuclear factor of activated T cells (NFAT). STAT3 also directly induces NFATc2 expression. NFAT and SHP2 were needed to sustain PAH-PASMC proliferation and resistance to apoptosis. Finally, delivery of synthetic miR-204 to the lungs of animals with PAH significantly reduced disease severity. This study uncovers a new regulatory pathway involving miR-204 that is critical to the etiology of PAH and indicates that reestablishing miR-204 expression should be explored as a potential new therapy for this disease.
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http://dx.doi.org/10.1084/jem.20101812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3058572PMC
March 2011

A Toxoplasma type 2C serine-threonine phosphatase is involved in parasite growth in the mammalian host cell.

Microbes Infect 2009 Oct 27;11(12):935-45. Epub 2009 Jun 27.

Institut Cochin, Université Paris Descartes, CNRS UMR 8104, Paris, France.

Toxoplasma gondii is a human protozoan parasite that belongs to the phylum of Apicomplexa and causes toxoplasmosis. As the other members of this phylum, T. gondii obligatory multiplies within a host cell by a peculiar type of mitosis that leads to daughter cell assembly within a mother cell. Although parasite growth and virulence have been linked for years, few molecules controlling mitosis have been yet identified and they include a couple of kinases but not the counteracting phosphatases. Here, we report that in contrast to other animal cells, type 2C is by far the major type of serine threonine phosphatase activity both in extracellular and in intracellular dividing parasites. Using wild type and transgenic parasites, we characterized the 37kDa TgPP2C molecule as an abundant cytoplasmic and nuclear enzyme with activity being under tight regulation. In addition, we showed that the increase in TgPP2C activity significantly affected parasite growth by impairing cytokinesis while nuclear division still occurred. This study supports for the first time that type 2C protein phosphatase is an important regulator of cell growth in T. gondii.
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http://dx.doi.org/10.1016/j.micinf.2009.06.002DOI Listing
October 2009

HBO1 HAT complexes target chromatin throughout gene coding regions via multiple PHD finger interactions with histone H3 tail.

Mol Cell 2009 Jan;33(2):257-65

Laval University Cancer Research Center, Hôtel-Dieu de Québec (CHUQ), Quebec City, QC, Canada.

The HBO1 HAT protein is the major source of histone H4 acetylation in vivo and has been shown to play critical roles in gene regulation and DNA replication. A distinctive characteristic of HBO1 HAT complexes is the presence of three PHD finger domains in two different subunits: tumor suppressor proteins ING4/5 and JADE1/2/3. Biochemical and functional analyses indicate that these domains interact with histone H3 N-terminal tail region, but with a different specificity toward its methylation status. Their combinatorial action is essential in regulating chromatin binding and substrate specificity of HBO1 complexes, as well as cell growth. Importantly, localization analyses on the human genome indicate that HBO1 complexes are enriched throughout the coding regions of genes, supporting a role in transcription elongation. These results underline the importance and versatility of PHD finger domains in regulating chromatin association and histone modification crosstalk within a single protein complex.
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http://dx.doi.org/10.1016/j.molcel.2009.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2677731PMC
January 2009

Histone H3 tail clipping regulates gene expression.

Nat Struct Mol Biol 2009 Jan 14;16(1):17-22. Epub 2008 Dec 14.

Gurdon Institute and Department of Pathology, Tennis Court Road, Cambridge CB2 1QN, UK.

Induction of gene expression in yeast and human cells involves changes in the histone modifications associated with promoters. Here we identify a histone H3 endopeptidase activity in Saccharomyces cerevisiae that may regulate these events. The endopeptidase cleaves H3 after Ala21, generating a histone that lacks the first 21 residues and shows a preference for H3 tails carrying repressive modifications. In vivo, the H3 N terminus is clipped, specifically within the promoters of genes following the induction of transcription. H3 clipping precedes the process of histone eviction seen when genes become fully active. A truncated H3 product is not generated in yeast carrying a mutation of the endopeptidase recognition site (H3 Q19A L20A) and gene induction is defective in these cells. These findings identify clipping of H3 tails as a previously uncharacterized modification of promoter-bound nucleosomes, which may result in the localized clearing of repressive signals during the induction of gene expression.
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http://dx.doi.org/10.1038/nsmb.1534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3350865PMC
January 2009

The histone methylase KMTox interacts with the redox-sensor peroxiredoxin-1 and targets genes involved in Toxoplasma gondii antioxidant defences.

Mol Microbiol 2009 Jan 5;71(1):212-26. Epub 2008 Nov 5.

Laboratoire Adaptation et Pathogénie des Micro-organismes, Université Joseph Fourier GRENOBLE 1, BP 170, F-38042 Grenoble cedex 9, France.

The ability of living cells to alter their gene expression patterns in response to environmental changes is essential for viability. Oxidative stress represents a common threat for all aerobic life. In normally growing cells, in which hydrogen peroxide generation is transient or pulsed, the antioxidant systems efficiently control its concentration. Intracellular parasites must also protect themselves against the oxidative burst imposed by the host. In this work, we have investigated the role of KMTox, a new histone lysine methyltransferase, in the obligate intracellular parasite Toxoplasma gondii. KMTox is a nuclear protein that holds a High Mobility Group domain, which is thought to recognize bent DNA. The enzyme methylates both histones H4 and H2A in vitro with a great preference for the substrate in reduced conditions. Importantly, KMTox interacts specifically with the typical 2-cys peroxiredoxin-1 and the binding is to some extent enhanced upon oxidation. It appears that the cellular functions that are primarily regulated by the KMTox are antioxidant defences and maintenance of cellular homeostasis. KMTox may regulate gene expression in T. gondii by providing the rapid re-arrangement of chromatin domains and by interacting with the redox-sensor TgPrx1 contribute to establish the antioxidant 'firewall' in T. gondii.
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http://dx.doi.org/10.1111/j.1365-2958.2008.06519.xDOI Listing
January 2009

Role of Jade-1 in the histone acetyltransferase (HAT) HBO1 complex.

J Biol Chem 2008 Oct 6;283(43):28817-26. Epub 2008 Aug 6.

Department of Medicine, Section of Nephrology, Boston University School of Medicine and Medical Center, Evans Biomedical Research Center, Boston, Massachusetts 02118, USA.

Regulation of global chromatin acetylation is important for chromatin remodeling. A small family of Jade proteins includes Jade-1L, Jade-2, and Jade-3, each bearing two mid-molecule tandem plant homology domain (PHD) zinc fingers. We previously demonstrated that the short isoform of Jade-1L protein, Jade-1, is associated with endogenous histone acetyltransferase (HAT) activity. It has been found that Jade-1L/2/3 proteins co-purify with a novel HAT complex, consisting of HBO1, ING4/5, and Eaf6. We investigated a role for Jade-1/1L in the HBO1 complex. When overexpressed individually, neither Jade-1/1L nor HBO1 affected histone acetylation. However, co-expression of Jade-1/1L and HBO1 increased acetylation of the bulk of endogenous histone H4 in epithelial cells in a synergistic manner, suggesting that Jade1/1L positively regulates HBO1 HAT activity. Conversely, small interfering RNA-mediated depletion of endogenous Jade resulted in reduced levels of H4 acetylation. Moreover, HBO1-mediated H4 acetylation activity was enhanced severalfold by the presence of Jade-1/1L in vitro. The removal of PHD fingers affected neither binding nor mutual Jade-1-HBO1 stabilization but completely abrogated the synergistic Jade-1/1L- and HBO1-mediated histone H4 acetylation in live cells and in vitro with reconstituted oligonucleosome substrates. Therefore, PHDs are necessary for Jade-1/1L-induced acetylation of nucleosomal histones by HBO1. In contrast to Jade-1/1L, the PHD zinc finger protein ING4/5 failed to synergize with HBO1 to promote histone acetylation. The physical interaction of ING4/5 with HBO1 occurred in the presence of Jade-1L or Jade-3 but not with the Jade-1 short isoform. In summary, this study demonstrates that Jade-1/1L are crucial co-factors for HBO1-mediated histone H4 acetylation.
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http://dx.doi.org/10.1074/jbc.M801407200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2570895PMC
October 2008

The crystal structure of the ING5 PHD finger in complex with an H3K4me3 histone peptide.

Proteins 2008 Sep;72(4):1371-6

Department of Pharmacology, University of Colorado at Denver, Aurora, Colorado 80045, USA.

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http://dx.doi.org/10.1002/prot.22140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2756976PMC
September 2008

Histone modifications in response to DNA damage.

Mutat Res 2007 May 21;618(1-2):81-90. Epub 2007 Jan 21.

Laval University Cancer Research Center, Hôtel-Dieu de Québec, Quebec City, Quebec, Canada.

The packaging of the eukaryotic genome into highly condensed chromatin makes it inaccessible to the factors required for gene transcription, DNA replication, recombination and repair. Eukaryotes have developed intricate mechanisms to overcome this repressive barrier imposed by chromatin. Histone modifying enzymes and ATP-dependent chromatin remodeling complexes play key roles here as they regulate many nuclear processes by altering the chromatin structure. Significantly, these activities are integral to the process of DNA repair where histone modifications act as signals and landing platforms for various repair proteins. This review summarizes the recent developments in our understanding of histone modifications and their role in the maintenance of genome integrity.
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http://dx.doi.org/10.1016/j.mrfmmm.2006.09.009DOI Listing
May 2007

Histone-modifying complexes regulate gene expression pertinent to the differentiation of the protozoan parasite Toxoplasma gondii.

Mol Cell Biol 2005 Dec;25(23):10301-14

ATIP-UMR5163-CNRS, Jean-Roget Institute, Domaine de la Merci, 38700 Grenoble, France.

Pathogenic apicomplexan parasites like Toxoplasma and Plasmodium (malaria) have complex life cycles consisting of multiple stages. The ability to differentiate from one stage to another requires dramatic transcriptional changes, yet there is a paucity of transcription factors in these protozoa. In contrast, we show here that Toxoplasma possesses extensive chromatin remodeling machinery that modulates gene expression relevant to differentiation. We find that, as in other eukaryotes, histone acetylation and arginine methylation are marks of gene activation in Toxoplasma. We have identified mediators of these histone modifications, as well as a histone deacetylase (HDAC), and correlate their presence at target promoters in a stage-specific manner. We purified the first HDAC complex from apicomplexans, which contains novel components in addition to others previously reported in eukaryotes. A Toxoplasma orthologue of the arginine methyltransferase CARM1 appears to work in concert with the acetylase TgGCN5, which exhibits an unusual bias for H3 [K18] in vitro. Inhibition of TgCARM1 induces differentiation, showing that the parasite life cycle can be manipulated by interfering with epigenetic machinery. This may lead to new approaches for therapy against protozoal diseases and highlights Toxoplasma as an informative model to study the evolution of epigenetics in eukaryotic cells.
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http://dx.doi.org/10.1128/MCB.25.23.10301-10314.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1291236PMC
December 2005

The capsular polysaccharide biosynthesis of Streptococcus pneumoniae serotype 8: functional identification of the glycosyltransferase WciS (Cap8H).

Biochem J 2005 Jul;389(Pt 1):63-72

Laboratoire Adaptation et Pathogénie des Micro-organismes, CNRS UMR 5163, Bâtiment Jean Roget, Faculté de Médecine Pharmacie, La Tronche, France.

CPS (capsular polysaccharide) is a major virulence factor in Streptococcus pneumoniae. Biosynthesis of CPS RU (repeat unit) proceeds by sequential transfer of sugar residues from the appropriate sugar donor to an activated lipid carrier by committed GTs (glycosyltransferases). While the nucleotide sequence of many cps loci is already known, the real substrate specificity of the hypothetical GTs, as well as the sequence of sugar addition is unclear. In the present paper, we report the biochemical characterization of one alpha-galactosyltransferase, WciS (Cap8H), a member of GT family 4. This enzyme is implicated in the tetrasaccharide RU biosynthetic pathway of Strep. pneumoniae CPS 8 ([-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Galp-(1-->4)-beta-D-GlcAp-(1-->4)-beta-D-Glcp-(1-->]n). Expression of WciS-His6 in Escherichia coli BL21 (DE3) strains or BL21 (DE3)/DeltagalU strain resulted in synthesis of a 39 kDa membrane-associated protein identified by N-terminal sequencing and recognized by anti-His6-tag antibody. This protein was capable of adding a galactose residue cellobiuronic acid [beta-D-GlcAp-(1-->4)-D-Glcp]-pyrophosphate-polyprenol from UDP-Gal. The newly added galactose residue is removed by alpha-galactosidase, indicating that WciS is a retaining GT. Our results suggest that WciS catalyses the addition of the third sugar residue of the CPS 8 RU. The recombinant WciS-His6 was solubilized and purified as a soluble multimer, opening the way for structural studies.
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http://dx.doi.org/10.1042/BJ20050217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1188263PMC
July 2005

The glucosyl-1-phosphate transferase WchA (Cap8E) primes the capsular polysaccharide repeat unit biosynthesis of Streptococcus pneumoniae serotype 8.

Biochem Biophys Res Commun 2005 Feb;327(3):857-65

Laboratoire Adaptation et Pathogénie des Micro-organismes, CNRS UMR 5163, Institut Jean Roget, Faculté de Médecine Pharmacie, Domaine de La Merci, 38700 La Tronche, France.

The gene wchA (cap8E) belongs to the cps8 locus that is involved in biosynthesis of the capsular polysaccharide (CPS) repeat unit (RU) of the virulent Streptococcus pneumoniae serotype 8. We report here the biochemical characterization of the membrane-associated protein WchA (Cap8E), overexpressed in Escherichia coli BL21(DE3)/pLysS. Our results demonstrate that the recombinant enzyme transfers in vitro a glucosyl-1-phosphate from UDP-glucose to an endogenous phosphoryl-polyprenol, thereby priming the RU biosynthetic pathway of S. pneumoniae CPS 8. We also show that the C-terminal half of WchA is the glycosyltransferase domain as observed for the galactosyl-1-phosphate transferase WbaP from Salmonella enterica, previously described to prime the first step of O-antigen biosynthesis. These results demonstrate that WchA plays a prominent function in the capsule biosynthesis and explain the key role it occupies in the pneumococcal capsule variation.
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http://dx.doi.org/10.1016/j.bbrc.2004.12.082DOI Listing
February 2005