<p>(A) The cellular expression level of H3 was analyzed by Western blotting in wt and <i... more <p>(A) The cellular expression level of H3 was analyzed by Western blotting in wt and <i>hht1</i>Δ <i>hhf1</i>Δ cells. (B) Correlation of the effects, as measured by log base 2 ratios, of low nucleosomes levels (<i>hht1</i>Δ <i>hhf1</i>Δ) and <i>SET1</i> deletion (<i>set1</i>Δ) on the COMPASS-repressed genes versus wt cells. <i>LYS2</i> is excluded as it is used as an auxotrophic marker for the <i>hht1</i>Δ <i>hhf1</i>Δ strain. <i>PHO84</i> is marked, as it is significantly repressed in the low nucleosome strain. The two histone genes are also depicted verifying the expected 2-fold reduction in their mRNA levels. (C) Strand-specific qPCR analysis, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002952#pgen-1002952-g004" target="_blank">Figure 4B</a>, for the indicated backgrounds.</p
Accumulation of senescent cells has been causally linked to the development of age-related pathol... more Accumulation of senescent cells has been causally linked to the development of age-related pathologies. Here, we characterized a new mouse model (p21+/Tert) whose telomerase (TERT) is expressed from the p21 promoter that can be activated in response to telomere dysfunction. Lung parenchyma from p21+/Tert old mice accumulated fewer senescent cells with age and this correlated with a reduction in age-related alveolar space enlargement, a feature of pulmonary emphysema. This protection against emphysema depends on TERT catalytic activity and is associated with increased proliferation of pulmonary endothelial cells (EC) and capillary density. Single-cell RNA sequencing of lung cells revealed that TERT expression was associated with the enrichment of ECs expressing genes involved in vessel regeneration and in AT2 cells overexpressing S/G2M markers. These findings indicate that p21-promoter-dependent expression of catalytically active telomerase prevents emphysema by sustaining the prolif...
Multiciliated Ependymal Cells and Adult Neural Stem Cells are components of the adult neurogenic ... more Multiciliated Ependymal Cells and Adult Neural Stem Cells are components of the adult neurogenic niche, essential for brain homeostasis. These cells share a common glial cell lineage regulated by the Geminin family members Geminin and GemC1/Mcidas. Ependymal precursors require GemC1/Mcidas expression to massively amplify centrioles and become multiciliated cells. Here we show that GemC1-dependent differentiation occurs mostly in cycling Radial Glial Cells, in which a DNA damage response, including replicative stress and dysfunctional telomeres, arrests the cell cycle after the G1/S restriction point due to the activation of the p53-p21 pathway, which contributes to centriole amplification. Telomerase expression in Radial Glial Cells impairs ependymal differentiation and favors the Neural Stem Cell fate.
Ploidy is stably maintained in most human somatic cells by a sequential and tight coordination of... more Ploidy is stably maintained in most human somatic cells by a sequential and tight coordination of cell cycle events. Undesired whole genome doublings or duplications are frequent in tumours and have been quite recently described as macro-evolutionary events associated to poor prognosis. In vitro and in vivo studies suggest that polyploidy can favour genome instability, facilitate the formation and progression of tumours, and modify their sensitivity to chemotherapeutic agents. Stress is strongly related to changes in ploidy and whole genome doublings. In this review we summarize different mechanisms that promote polyploidization, describe a new type of stress able to trigger WGDs in S. cerevisiae, histone stress, and provide some examples and theoretical scenarios that support that cancer cells might suffer from this type of stress. We finally highlight some results showing that the kinase Swe1 (Wee1 in humans) has a role in sensing histone levels before cells enter mitosis thereby avoiding their undesired consequences on chromosome segregation and ploidy control.
Transcription is a source of genetic instability that can notably result from the formation of ge... more Transcription is a source of genetic instability that can notably result from the formation of genotoxic DNA:RNA hybrids, or R-loops, between the nascent mRNA and its template. Here we report an unexpected function for introns in counteracting R-loop accumulation in eukaryotic genomes. Deletion of endogenous introns increases R-loop formation, while insertion of an intron into an intronless gene suppresses R-loop accumulation and its deleterious impact on transcription and recombination in yeast. Recruitment of the spliceosome onto the mRNA, but not splicing per se, is shown to be critical to attenuate R-loop formation and transcription-associated genetic instability. Genome-wide analyses in a number of distant species differing in their intron content, including human, further revealed that intron-containing genes and the intron-richest genomes are best protected against R-loop accumulation and subsequent genetic instability. Our results thereby provide a possible rationale for the...
Methylation of histone H3 lysine 4 (H3K4) by Set1 complex/COMPASS is a hallmark of eukaryotic chr... more Methylation of histone H3 lysine 4 (H3K4) by Set1 complex/COMPASS is a hallmark of eukaryotic chromatin, but it remains poorly understood how this post-translational modification contributes to the regulation of biological processes like the cell cycle. Here, we report a H3K4 methylation-dependent pathway in Saccharomyces cerevisiae that governs toxicity toward benomyl, a microtubule destabilizing drug. Benomyl-sensitive growth of wild-type cells required mono- and dimethylation of H3K4 and Pho23, a PHD-containing subunit of the Rpd3L complex. Δset1 and Δpho23 deletions suppressed defects associated with ipl1-2 aurora kinase mutant, an integral component of the spindle assembly checkpoint during mitosis. Benomyl resistance of Δset1 strains was accompanied by deregulation of all four tubulin genes and the phenotype was suppressed by tub2-423 and Δtub3 mutations, establishing a genetic link between H3K4 methylation and microtubule function. Most interestingly, sine wave fitting and cl...
The yeast protein Set1p, inactivation of which alleviates telomeric position effect (TPE), contai... more The yeast protein Set1p, inactivation of which alleviates telomeric position effect (TPE), contains a conserved SET domain present in chromosomal proteins involved in epigenetic control of transcription. Mec3p is required for efficient DNA-damage-dependent checkpoints at G1/S, intra-S and G2/M (refs 3-7). We show here that the SET domain of Set1p interacts with Mec3p. Deletion of SET1 increases the viability of mec3delta mutants after DNA damage (in a process that is mostly independent of Rad53p kinase, which has a central role in checkpoint control) but does not significantly affect cell-cycle progression. Deletion of MEC3 enhances TPE and attenuates the Set1delta-induced silencing defect. Furthermore, restoration of TPE in a Set1delta mutant by overexpression of the isolated SET domain requires Mec3p. Finally, deletion of MEC3 results in telomere elongation, whereas cells with deletions of both SET1 and MEC3 do not have elongated telomeres. Our findings indicate that interactions between SET1 and MEC3 have a role in DNA repair and telomere function.
The Set1 family of histone H3 lysine 4 (H3K4) methyltransferases is highly conserved from yeast t... more The Set1 family of histone H3 lysine 4 (H3K4) methyltransferases is highly conserved from yeast to human. Here we show that the Set1 complex (Set1C) directly binds RNA in vitro through the regions that comprise the double RNA recognition motifs (dRRM) and N-SET domain within Set1 and its subunit Spp1. To investigate the functional relevance of RNA binding, we performed UV RNA crosslinking (CRAC) for Set1 and RNA polymerase II in parallel with ChIP-seq experiments. Set1 binds nascent transcripts through its dRRM. RNA binding is important to define the appropriate topology of Set1C distribution along transcription units and correlates with the efficient deposition of the H3K4me3 mark. In addition, we uncovered that Set1 binds to different classes of RNAs to levels that largely exceed the levels of binding to the general population of transcripts, suggesting the Set1 persists on these RNAs after transcription. This class includes RNAs derived from SET1, Ty1 retrotransposons, specific t...
tique et only as part of a multiprotein complex [6]; and genetic Physiologie du Dé veloppement ev... more tique et only as part of a multiprotein complex [6]; and genetic Physiologie du Dé veloppement evidence indicates that Sas2 is required for H4 lysine Institut de Biologie du Dé veloppement de Marseille 16 acetylation in vivo [7]. Sequence comparison indi-CNRS/INSERM/Université de la Mé diterrané e cates that Chm corresponds to the fly counterpart of Parc Scientifique de Luminy HBO1 (see the Supplementary Material available with Case 907 this article online). HBO1 was shown to interact with the 13288 Marseille Cedex 9 ORC largest subunit, providing the complex with HAT 2 Institut de Gé né tique Humaine activity [6], with the androgen receptor [8], and with CNRS the replication factor MCM2 [9], suggesting chromatin-141, rue de la Cardonille mediated roles in both DNA replication and transcription 34396 Montpellier Cedex 5 regulation. 3 Laboratoire d'Ingenierie des Mutations were generated by mobilization of a P ele-Systè mes Macromolé culaires ment inserted 1.2 kb downstream of the 3Ј end of chm. CNRS chm 14 deletes the last three exons, breaking in the MYST 31 Chemin Joseph Aiguier domain; Df(2L)221 deletes most of the coding region 13402 Marseille Cedex 20 (Figure 1A). These deficiencies are recessive pupal lethal France and genetically behave as null alleles of chm. They do not affect another essential gene, since the associated lethality is rescued by a heat shock construct of chm Summary cDNA in transgenic animals raised at 25ЊC (data not shown). Reversible acetylation of histone tails plays an important role in chromatin remodelling and regulation of gene activity [1, 2]. While modification by histone acet-Chm Functions in Pericentric Heterochromatinyltransferase (HAT) is usually linked to transcriptional Mediated Gene Silencing activation, we provide here evidence for HAT function To assay for a role in modulating chromatin structure in several types of epigenetic repression. Chameau and transcription, we first analyzed PEV, a phenomenon (Chm), a new Drosophila member of the MYST HAT of epigenetic repression mediated by pericentric heterofamily, dominantly suppresses position effect variegachromatin. Genes that dominantly modify PEV are tion (PEV), is required for the maintenance of Hox gene thought to encode products that affect chromatin strucsilencing by Polycomb group (PcG) proteins, and can ture, leading when mutated to an increased (supprespartially substitute for the MYST Sas2 HAT in yeast telomeric position effect (TPE). Finally, we provide in sors of PEV) or a decreased (enhancers) transcription vivo evidence that the acetyltransferase activity of of neighboring genes [10]. We first used the w m4h inver-Chm is required in these processes, since a variant sion that leads to a mosaic pattern of eye pigment. protein mutated in the catalytic domain no longer res-Mutation of one chm copy gives significant increase cues PEV modification, telomeric silencing of SAS2of pigmented area, resulting in a 2.5-fold increase of deficient yeast cells, nor lethality of chm mutant flies. pigment levels (Figures 1B and 1C). chm mutation there-These findings emphasize the role of an acetyltransfore dominantly suppresses variegation of white in w m4h. ferase in gene silencing, which supports, according To assess whether chm also affects other variegating to the histone code hypothesis [2-4], that transcription rearrangements, we used the ␥238 minichromosome at a particular locus is determined by a precise combithat contains close to centric heterochromatin the yelnation of histone tail modifications rather than by overlow (y) gene, responsible for the dark pigmentation of all acetylation levels. bristles [11]. The number of dark bristles at the wing margin is significantly increased in animals heterozy-Results gous for chm (Figure 1D), indicating thus a suppression of y variegation. To rule out a possible effect of the chm Encodes an Essential MYST Domain Protein genetic background on PEV, we controlled that Chm Homologous to Human HBO1 overexpression, provided by a HSchm transgene at MYST proteins have been identified from yeast to human 25ЊC, can reverse the y derepression seen in chm hetero-[5]. The family includes, in yeast, Sas2 and Sas3, first zygous flies (Figures 1D and 1E). This demonstrates that chm haploinsufficiency causes PEV suppression,
Telomerase maintains chromosome ends from humans to yeasts. Recruitment of yeast telomerase to te... more Telomerase maintains chromosome ends from humans to yeasts. Recruitment of yeast telomerase to telomeres occurs through its Ku and Est1 subunits via independent interactions with telomerase RNA (TLC1) and telomeric proteins Sir4 and Cdc13, respectively. However, the structures of the molecules comprising these telomerase-recruiting pathways remain unknown. Here, we report crystal structures of the Ku heterodimer and Est1 complexed with their key binding partners. Two major findings are as follows: (1) Ku specifically binds to telomerase RNA in a distinct, yet related, manner to how it binds DNA; and (2) Est1 employs two separate pockets to bind distinct motifs of Cdc13. The N-terminal Cdc13-binding site of Est1 cooperates with the TLC1-Ku-Sir4 pathway for telomerase recruitment, whereas the C-terminal interface is dispensable for binding Est1 in vitro yet is nevertheless essential for telomere maintenance in vivo. Overall, our results integrate previous models and provide fundamenta...
La colicine a est une bacteriocine produite et secretee par certaines souches d'e. Coli. Les ... more La colicine a est une bacteriocine produite et secretee par certaines souches d'e. Coli. Les cellules productrices de colicine a, se protegent de l'action de la bacteriocine grace a l'expression d'une proteine appelee proteine d'immunite (cai). Cai est exprimee tres faiblement de facon constitutive. Une sequence hydrophile, reconnue par un anticorps monoclonal greffe a l'extremite nh2-terminale de cai a permis de caracteriser la proteine d'immunite, de la localiser dans la membrane interne des bacteries et de montrer que son extremite nh2-terminale est dirigee vers le cytoplasme. Cai possede quatre segments transmembranaires definissant deux boucles periplasmiques et une boucle cytoplasmique. La deuxieme boucle periplasmique joue un role dans l'interaction avec la solicine a. Le vecteur utilise pour la construction de la proteine vl1 a permis l'expression chez e. Coli d'une hormone humaine, le hgrf (human growth releasing factor). Le h grf a e...
Whole-genome duplications (WGDs) have played a central role in the evolution of genomes and const... more Whole-genome duplications (WGDs) have played a central role in the evolution of genomes and constitute an important source of genome instability in cancer. Here, we show in Saccharomyces cerevisiae that abnormal accumulations of histones are sufficient to induce WGDs. Our results link these WGDs to a reduced incorporation of the histone variant H2A.Z to chromatin. Moreover, we show that high levels of histones promote Swe1 stabilisation thereby triggering the phosphorylation and inhibition of Cdc28 through a mechanism different of the canonical DNA damage response. Our results link high levels of histones to a specific type of genome instability that is quite frequently observed in cancer and uncovers a new mechanism that might be able to respond to high levels of histones. DOI: https://doi.org/10.7554/eLife.35337.001
Methylation of histone H3 lysine 4 (H3K4) by Set1/COMPASS occurs co-transcriptionally, and is imp... more Methylation of histone H3 lysine 4 (H3K4) by Set1/COMPASS occurs co-transcriptionally, and is important for gene regulation. Set1/COMPASS associates with the RNA polymerase II C-terminal domain (CTD) to establish proper levels and distribution of H3K4 methylations. However, details of CTD association remain unclear. Here we report that the Set1 N-terminal region and the COMPASS subunit Swd2, which interact with each other, are both needed for efficient CTD binding in Saccharomyces cerevisiae. Moreover, a single point mutation in Swd2 that affects its interaction with Set1 also impairs COMPASS recruitment to chromatin and H3K4 methylation. A CTD interaction domain (CID) from the protein Nrd1 can partially substitute for the Set1 N-terminal region to restore CTD interactions and histone methylation. However, even when Set1/COMPASS is recruited via the Nrd1 CID, histone H2B ubiquitylation is still required for efficient H3K4 methylation, indicating that H2Bub acts after the initial rec...
RAD53 and MEC1 are essential Saccharomyces cerevisiae genes required for the DNA replication and ... more RAD53 and MEC1 are essential Saccharomyces cerevisiae genes required for the DNA replication and DNA damage checkpoint responses. Their lethality can be suppressed by increasing the intracellular pool of deoxynucleotide triphosphates. We report that deletion of YKU70 or YKU80 suppresses mec1⌬, but not rad53⌬, lethality. We show that suppression of mec1⌬ lethality is not due to Ku ؊-associated telomeric defects but rather results from the inability of Ku ؊ cells to efficiently repair DNA double strand breaks by nonhomologous end joining. Consistent with these results, mec1⌬ lethality is also suppressed by lif1⌬, which like yku70⌬ and yku80⌬, prevents nonhomologous end joining. The viability of yku70⌬ mec1⌬ and yku80⌬ mec1⌬ cells depends on the ATM-related Tel1 kinase, the Mre11-Rad50-Xrs2 complex, and the DNA damage checkpoint protein Rad9. We further report that this Mec1-independent pathway converges with the Rad53/Dun1-regulated checkpoint kinase cascade and leads to the degradation of the ribonucleotide reductase inhibitor Sml1.
Establishing the interactome of the cancer associated stress protein NUPR1 (NUclear PRotein 1), w... more Establishing the interactome of the cancer associated stress protein NUPR1 (NUclear PRotein 1), we found that it binds to several hundreds of proteins, including proteins involved in nuclear translocation, DNA repair and key factors of the SUMO pathway. We demonstrated that the NUPR1 inhibitor ZZW-115, an organic synthetic molecule, competes with importins for the binding to the NLS region of NUPR1 thereby inhibiting its nuclear translocation. We hypothesized, and then proved, that inhibition of NUPR1 by ZZW-115 sensitizes cancer cells to DNA damage induced by several genotoxic agents. Strikingly, we found that treatment with ZZW-115 reduced SUMOylation of several proteins involved in DNA damage response (DDR). We further reported that the presence of recombinant NUPR1 improved the SUMOylation in a cell-free system indicating NUPR1 directly stimulates the SUMOylation machinery. We propose that ZZW-115 sensitizes cancer cells to genotoxic agents by inhibiting the nuclear translocation of NUPR1 and thereby decreasing the SUMOylation dependent functions of key proteins involved in the DDR.
<p>(A) The cellular expression level of H3 was analyzed by Western blotting in wt and <i... more <p>(A) The cellular expression level of H3 was analyzed by Western blotting in wt and <i>hht1</i>Δ <i>hhf1</i>Δ cells. (B) Correlation of the effects, as measured by log base 2 ratios, of low nucleosomes levels (<i>hht1</i>Δ <i>hhf1</i>Δ) and <i>SET1</i> deletion (<i>set1</i>Δ) on the COMPASS-repressed genes versus wt cells. <i>LYS2</i> is excluded as it is used as an auxotrophic marker for the <i>hht1</i>Δ <i>hhf1</i>Δ strain. <i>PHO84</i> is marked, as it is significantly repressed in the low nucleosome strain. The two histone genes are also depicted verifying the expected 2-fold reduction in their mRNA levels. (C) Strand-specific qPCR analysis, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002952#pgen-1002952-g004" target="_blank">Figure 4B</a>, for the indicated backgrounds.</p
Accumulation of senescent cells has been causally linked to the development of age-related pathol... more Accumulation of senescent cells has been causally linked to the development of age-related pathologies. Here, we characterized a new mouse model (p21+/Tert) whose telomerase (TERT) is expressed from the p21 promoter that can be activated in response to telomere dysfunction. Lung parenchyma from p21+/Tert old mice accumulated fewer senescent cells with age and this correlated with a reduction in age-related alveolar space enlargement, a feature of pulmonary emphysema. This protection against emphysema depends on TERT catalytic activity and is associated with increased proliferation of pulmonary endothelial cells (EC) and capillary density. Single-cell RNA sequencing of lung cells revealed that TERT expression was associated with the enrichment of ECs expressing genes involved in vessel regeneration and in AT2 cells overexpressing S/G2M markers. These findings indicate that p21-promoter-dependent expression of catalytically active telomerase prevents emphysema by sustaining the prolif...
Multiciliated Ependymal Cells and Adult Neural Stem Cells are components of the adult neurogenic ... more Multiciliated Ependymal Cells and Adult Neural Stem Cells are components of the adult neurogenic niche, essential for brain homeostasis. These cells share a common glial cell lineage regulated by the Geminin family members Geminin and GemC1/Mcidas. Ependymal precursors require GemC1/Mcidas expression to massively amplify centrioles and become multiciliated cells. Here we show that GemC1-dependent differentiation occurs mostly in cycling Radial Glial Cells, in which a DNA damage response, including replicative stress and dysfunctional telomeres, arrests the cell cycle after the G1/S restriction point due to the activation of the p53-p21 pathway, which contributes to centriole amplification. Telomerase expression in Radial Glial Cells impairs ependymal differentiation and favors the Neural Stem Cell fate.
Ploidy is stably maintained in most human somatic cells by a sequential and tight coordination of... more Ploidy is stably maintained in most human somatic cells by a sequential and tight coordination of cell cycle events. Undesired whole genome doublings or duplications are frequent in tumours and have been quite recently described as macro-evolutionary events associated to poor prognosis. In vitro and in vivo studies suggest that polyploidy can favour genome instability, facilitate the formation and progression of tumours, and modify their sensitivity to chemotherapeutic agents. Stress is strongly related to changes in ploidy and whole genome doublings. In this review we summarize different mechanisms that promote polyploidization, describe a new type of stress able to trigger WGDs in S. cerevisiae, histone stress, and provide some examples and theoretical scenarios that support that cancer cells might suffer from this type of stress. We finally highlight some results showing that the kinase Swe1 (Wee1 in humans) has a role in sensing histone levels before cells enter mitosis thereby avoiding their undesired consequences on chromosome segregation and ploidy control.
Transcription is a source of genetic instability that can notably result from the formation of ge... more Transcription is a source of genetic instability that can notably result from the formation of genotoxic DNA:RNA hybrids, or R-loops, between the nascent mRNA and its template. Here we report an unexpected function for introns in counteracting R-loop accumulation in eukaryotic genomes. Deletion of endogenous introns increases R-loop formation, while insertion of an intron into an intronless gene suppresses R-loop accumulation and its deleterious impact on transcription and recombination in yeast. Recruitment of the spliceosome onto the mRNA, but not splicing per se, is shown to be critical to attenuate R-loop formation and transcription-associated genetic instability. Genome-wide analyses in a number of distant species differing in their intron content, including human, further revealed that intron-containing genes and the intron-richest genomes are best protected against R-loop accumulation and subsequent genetic instability. Our results thereby provide a possible rationale for the...
Methylation of histone H3 lysine 4 (H3K4) by Set1 complex/COMPASS is a hallmark of eukaryotic chr... more Methylation of histone H3 lysine 4 (H3K4) by Set1 complex/COMPASS is a hallmark of eukaryotic chromatin, but it remains poorly understood how this post-translational modification contributes to the regulation of biological processes like the cell cycle. Here, we report a H3K4 methylation-dependent pathway in Saccharomyces cerevisiae that governs toxicity toward benomyl, a microtubule destabilizing drug. Benomyl-sensitive growth of wild-type cells required mono- and dimethylation of H3K4 and Pho23, a PHD-containing subunit of the Rpd3L complex. Δset1 and Δpho23 deletions suppressed defects associated with ipl1-2 aurora kinase mutant, an integral component of the spindle assembly checkpoint during mitosis. Benomyl resistance of Δset1 strains was accompanied by deregulation of all four tubulin genes and the phenotype was suppressed by tub2-423 and Δtub3 mutations, establishing a genetic link between H3K4 methylation and microtubule function. Most interestingly, sine wave fitting and cl...
The yeast protein Set1p, inactivation of which alleviates telomeric position effect (TPE), contai... more The yeast protein Set1p, inactivation of which alleviates telomeric position effect (TPE), contains a conserved SET domain present in chromosomal proteins involved in epigenetic control of transcription. Mec3p is required for efficient DNA-damage-dependent checkpoints at G1/S, intra-S and G2/M (refs 3-7). We show here that the SET domain of Set1p interacts with Mec3p. Deletion of SET1 increases the viability of mec3delta mutants after DNA damage (in a process that is mostly independent of Rad53p kinase, which has a central role in checkpoint control) but does not significantly affect cell-cycle progression. Deletion of MEC3 enhances TPE and attenuates the Set1delta-induced silencing defect. Furthermore, restoration of TPE in a Set1delta mutant by overexpression of the isolated SET domain requires Mec3p. Finally, deletion of MEC3 results in telomere elongation, whereas cells with deletions of both SET1 and MEC3 do not have elongated telomeres. Our findings indicate that interactions between SET1 and MEC3 have a role in DNA repair and telomere function.
The Set1 family of histone H3 lysine 4 (H3K4) methyltransferases is highly conserved from yeast t... more The Set1 family of histone H3 lysine 4 (H3K4) methyltransferases is highly conserved from yeast to human. Here we show that the Set1 complex (Set1C) directly binds RNA in vitro through the regions that comprise the double RNA recognition motifs (dRRM) and N-SET domain within Set1 and its subunit Spp1. To investigate the functional relevance of RNA binding, we performed UV RNA crosslinking (CRAC) for Set1 and RNA polymerase II in parallel with ChIP-seq experiments. Set1 binds nascent transcripts through its dRRM. RNA binding is important to define the appropriate topology of Set1C distribution along transcription units and correlates with the efficient deposition of the H3K4me3 mark. In addition, we uncovered that Set1 binds to different classes of RNAs to levels that largely exceed the levels of binding to the general population of transcripts, suggesting the Set1 persists on these RNAs after transcription. This class includes RNAs derived from SET1, Ty1 retrotransposons, specific t...
tique et only as part of a multiprotein complex [6]; and genetic Physiologie du Dé veloppement ev... more tique et only as part of a multiprotein complex [6]; and genetic Physiologie du Dé veloppement evidence indicates that Sas2 is required for H4 lysine Institut de Biologie du Dé veloppement de Marseille 16 acetylation in vivo [7]. Sequence comparison indi-CNRS/INSERM/Université de la Mé diterrané e cates that Chm corresponds to the fly counterpart of Parc Scientifique de Luminy HBO1 (see the Supplementary Material available with Case 907 this article online). HBO1 was shown to interact with the 13288 Marseille Cedex 9 ORC largest subunit, providing the complex with HAT 2 Institut de Gé né tique Humaine activity [6], with the androgen receptor [8], and with CNRS the replication factor MCM2 [9], suggesting chromatin-141, rue de la Cardonille mediated roles in both DNA replication and transcription 34396 Montpellier Cedex 5 regulation. 3 Laboratoire d'Ingenierie des Mutations were generated by mobilization of a P ele-Systè mes Macromolé culaires ment inserted 1.2 kb downstream of the 3Ј end of chm. CNRS chm 14 deletes the last three exons, breaking in the MYST 31 Chemin Joseph Aiguier domain; Df(2L)221 deletes most of the coding region 13402 Marseille Cedex 20 (Figure 1A). These deficiencies are recessive pupal lethal France and genetically behave as null alleles of chm. They do not affect another essential gene, since the associated lethality is rescued by a heat shock construct of chm Summary cDNA in transgenic animals raised at 25ЊC (data not shown). Reversible acetylation of histone tails plays an important role in chromatin remodelling and regulation of gene activity [1, 2]. While modification by histone acet-Chm Functions in Pericentric Heterochromatinyltransferase (HAT) is usually linked to transcriptional Mediated Gene Silencing activation, we provide here evidence for HAT function To assay for a role in modulating chromatin structure in several types of epigenetic repression. Chameau and transcription, we first analyzed PEV, a phenomenon (Chm), a new Drosophila member of the MYST HAT of epigenetic repression mediated by pericentric heterofamily, dominantly suppresses position effect variegachromatin. Genes that dominantly modify PEV are tion (PEV), is required for the maintenance of Hox gene thought to encode products that affect chromatin strucsilencing by Polycomb group (PcG) proteins, and can ture, leading when mutated to an increased (supprespartially substitute for the MYST Sas2 HAT in yeast telomeric position effect (TPE). Finally, we provide in sors of PEV) or a decreased (enhancers) transcription vivo evidence that the acetyltransferase activity of of neighboring genes [10]. We first used the w m4h inver-Chm is required in these processes, since a variant sion that leads to a mosaic pattern of eye pigment. protein mutated in the catalytic domain no longer res-Mutation of one chm copy gives significant increase cues PEV modification, telomeric silencing of SAS2of pigmented area, resulting in a 2.5-fold increase of deficient yeast cells, nor lethality of chm mutant flies. pigment levels (Figures 1B and 1C). chm mutation there-These findings emphasize the role of an acetyltransfore dominantly suppresses variegation of white in w m4h. ferase in gene silencing, which supports, according To assess whether chm also affects other variegating to the histone code hypothesis [2-4], that transcription rearrangements, we used the ␥238 minichromosome at a particular locus is determined by a precise combithat contains close to centric heterochromatin the yelnation of histone tail modifications rather than by overlow (y) gene, responsible for the dark pigmentation of all acetylation levels. bristles [11]. The number of dark bristles at the wing margin is significantly increased in animals heterozy-Results gous for chm (Figure 1D), indicating thus a suppression of y variegation. To rule out a possible effect of the chm Encodes an Essential MYST Domain Protein genetic background on PEV, we controlled that Chm Homologous to Human HBO1 overexpression, provided by a HSchm transgene at MYST proteins have been identified from yeast to human 25ЊC, can reverse the y derepression seen in chm hetero-[5]. The family includes, in yeast, Sas2 and Sas3, first zygous flies (Figures 1D and 1E). This demonstrates that chm haploinsufficiency causes PEV suppression,
Telomerase maintains chromosome ends from humans to yeasts. Recruitment of yeast telomerase to te... more Telomerase maintains chromosome ends from humans to yeasts. Recruitment of yeast telomerase to telomeres occurs through its Ku and Est1 subunits via independent interactions with telomerase RNA (TLC1) and telomeric proteins Sir4 and Cdc13, respectively. However, the structures of the molecules comprising these telomerase-recruiting pathways remain unknown. Here, we report crystal structures of the Ku heterodimer and Est1 complexed with their key binding partners. Two major findings are as follows: (1) Ku specifically binds to telomerase RNA in a distinct, yet related, manner to how it binds DNA; and (2) Est1 employs two separate pockets to bind distinct motifs of Cdc13. The N-terminal Cdc13-binding site of Est1 cooperates with the TLC1-Ku-Sir4 pathway for telomerase recruitment, whereas the C-terminal interface is dispensable for binding Est1 in vitro yet is nevertheless essential for telomere maintenance in vivo. Overall, our results integrate previous models and provide fundamenta...
La colicine a est une bacteriocine produite et secretee par certaines souches d'e. Coli. Les ... more La colicine a est une bacteriocine produite et secretee par certaines souches d'e. Coli. Les cellules productrices de colicine a, se protegent de l'action de la bacteriocine grace a l'expression d'une proteine appelee proteine d'immunite (cai). Cai est exprimee tres faiblement de facon constitutive. Une sequence hydrophile, reconnue par un anticorps monoclonal greffe a l'extremite nh2-terminale de cai a permis de caracteriser la proteine d'immunite, de la localiser dans la membrane interne des bacteries et de montrer que son extremite nh2-terminale est dirigee vers le cytoplasme. Cai possede quatre segments transmembranaires definissant deux boucles periplasmiques et une boucle cytoplasmique. La deuxieme boucle periplasmique joue un role dans l'interaction avec la solicine a. Le vecteur utilise pour la construction de la proteine vl1 a permis l'expression chez e. Coli d'une hormone humaine, le hgrf (human growth releasing factor). Le h grf a e...
Whole-genome duplications (WGDs) have played a central role in the evolution of genomes and const... more Whole-genome duplications (WGDs) have played a central role in the evolution of genomes and constitute an important source of genome instability in cancer. Here, we show in Saccharomyces cerevisiae that abnormal accumulations of histones are sufficient to induce WGDs. Our results link these WGDs to a reduced incorporation of the histone variant H2A.Z to chromatin. Moreover, we show that high levels of histones promote Swe1 stabilisation thereby triggering the phosphorylation and inhibition of Cdc28 through a mechanism different of the canonical DNA damage response. Our results link high levels of histones to a specific type of genome instability that is quite frequently observed in cancer and uncovers a new mechanism that might be able to respond to high levels of histones. DOI: https://doi.org/10.7554/eLife.35337.001
Methylation of histone H3 lysine 4 (H3K4) by Set1/COMPASS occurs co-transcriptionally, and is imp... more Methylation of histone H3 lysine 4 (H3K4) by Set1/COMPASS occurs co-transcriptionally, and is important for gene regulation. Set1/COMPASS associates with the RNA polymerase II C-terminal domain (CTD) to establish proper levels and distribution of H3K4 methylations. However, details of CTD association remain unclear. Here we report that the Set1 N-terminal region and the COMPASS subunit Swd2, which interact with each other, are both needed for efficient CTD binding in Saccharomyces cerevisiae. Moreover, a single point mutation in Swd2 that affects its interaction with Set1 also impairs COMPASS recruitment to chromatin and H3K4 methylation. A CTD interaction domain (CID) from the protein Nrd1 can partially substitute for the Set1 N-terminal region to restore CTD interactions and histone methylation. However, even when Set1/COMPASS is recruited via the Nrd1 CID, histone H2B ubiquitylation is still required for efficient H3K4 methylation, indicating that H2Bub acts after the initial rec...
RAD53 and MEC1 are essential Saccharomyces cerevisiae genes required for the DNA replication and ... more RAD53 and MEC1 are essential Saccharomyces cerevisiae genes required for the DNA replication and DNA damage checkpoint responses. Their lethality can be suppressed by increasing the intracellular pool of deoxynucleotide triphosphates. We report that deletion of YKU70 or YKU80 suppresses mec1⌬, but not rad53⌬, lethality. We show that suppression of mec1⌬ lethality is not due to Ku ؊-associated telomeric defects but rather results from the inability of Ku ؊ cells to efficiently repair DNA double strand breaks by nonhomologous end joining. Consistent with these results, mec1⌬ lethality is also suppressed by lif1⌬, which like yku70⌬ and yku80⌬, prevents nonhomologous end joining. The viability of yku70⌬ mec1⌬ and yku80⌬ mec1⌬ cells depends on the ATM-related Tel1 kinase, the Mre11-Rad50-Xrs2 complex, and the DNA damage checkpoint protein Rad9. We further report that this Mec1-independent pathway converges with the Rad53/Dun1-regulated checkpoint kinase cascade and leads to the degradation of the ribonucleotide reductase inhibitor Sml1.
Establishing the interactome of the cancer associated stress protein NUPR1 (NUclear PRotein 1), w... more Establishing the interactome of the cancer associated stress protein NUPR1 (NUclear PRotein 1), we found that it binds to several hundreds of proteins, including proteins involved in nuclear translocation, DNA repair and key factors of the SUMO pathway. We demonstrated that the NUPR1 inhibitor ZZW-115, an organic synthetic molecule, competes with importins for the binding to the NLS region of NUPR1 thereby inhibiting its nuclear translocation. We hypothesized, and then proved, that inhibition of NUPR1 by ZZW-115 sensitizes cancer cells to DNA damage induced by several genotoxic agents. Strikingly, we found that treatment with ZZW-115 reduced SUMOylation of several proteins involved in DNA damage response (DDR). We further reported that the presence of recombinant NUPR1 improved the SUMOylation in a cell-free system indicating NUPR1 directly stimulates the SUMOylation machinery. We propose that ZZW-115 sensitizes cancer cells to genotoxic agents by inhibiting the nuclear translocation of NUPR1 and thereby decreasing the SUMOylation dependent functions of key proteins involved in the DDR.
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Papers by Vincent Geli