RESUMO
Multiple studies in Saccharomyces cerevisiae have measured the levels of gene silencing by inserting the URA3 gene at various loci and selecting against URA3-expressing cells by 5-flouroorotic acid (5-FOA). However, 5-FOA affects the cellular pools of dNTPs and can produce side effects. To circumvent this issue, we and others have introduced drug-free techniques to detect silent and active gene states. In this study, we compared three drug-free methods based on the expression of fluorescent reporters in the VIIL telomere of S. cerevisiae. Our results point out that only one of these methods is suitable for large-scale drug-free analyses of gene silencing.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Inativação Gênica , Regulação Fúngica da Expressão GênicaRESUMO
Eukaryotic DNA replication is accompanied by the disassembly and reassembly of nucleosomes and the transmission of epigenetic marks to the newly assembled chromatids. Several histone chaperones, including CAF-1 and Asf1p, are central to these processes. On the other hand, replication forks pause at numerous positions throughout the genome, but it is not known if and how this pausing affects the reassembly and maintenance of chromatin structures. Here, we applied drug-free gene silencing assays to analyze the genetic interactions between CAC1, ASF1, and two genes that regulate the stability of the paused replisome (TOF1) and the resumption of elongation (RRM3). Our results show that TOF1 and RRM3 differentially interact with CAF-1 and ASF1 and that the deletions of TOF1 and RRM3 lead to reduced silencing and increased frequency of epigenetic conversions at three loci in the genome of S. cerevisiae. Our study adds details to the known activities of CAF-1 and Asf1p and suggests that the pausing of the replication fork can lead to epigenetic instability.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/genética , Replicação do DNA/genética , Inativação GênicaRESUMO
Gene silencing by the SIR (Silent Information Region) family of proteins in S. cerevisiae has been extensively studied and has served as a founding paradigm for our general understanding of gene repression and its links to histone deacetylation and chromatin structure. In recent years, our understanding of other mechanisms of gene repression in S.cerevisiae was significantly advanced. In this review, we focus on such Sir-independent mechanisms of gene repression executed by various Histone Deacetylases (HDACs) and Histone Methyl Transferases (HMTs). We focus on the genes regulated by these enzymes and their known mechanisms of action. We describe the cooperation and redundancy between HDACs and HMTs, and their involvement in gene repression by non-coding RNAs or by their non-histone substrates. We also propose models of epigenetic transmission of the chromatin structures produced by these enzymes and discuss these in the context of gene repression phenomena in other organisms. These include the recycling of the epigenetic marks imposed by HMTs or the recycling of the complexes harboring HDACs.
Assuntos
Inativação Gênica , Histona Desacetilases/genética , Histona Metiltransferases/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Transcrição Gênica , Cromatina/genética , Histonas/genética , Saccharomyces cerevisiae/genéticaRESUMO
Position-effect variegation (PEV) phenotypes are characterized by the robust multigenerational repression of a gene located at a certain locus (often called gene silencing) and occasional conversions to fully active state. Consequently, the active state then persists with occasional conversions to the repressed state. These effects are mediated by the establishment and maintenance of heterochromatin or euchromatin structures, respectively. In this study, we have addressed an important but often neglected aspect of PEV: the frequency of conversions at such loci. We have developed a model and have projected various PEV scenarios based on various rates of conversions. We have also enhanced two existing assays for gene silencing in Saccharomyces cerevisiae to measure the rate of switches from repressed to active state and vice versa. We tested the validity of our methodology in Δsir1 cells and in several mutants with defects in gene silencing. The assays have revealed that the histone chaperone Chromatin Assembly Factor I is involved in the control of epigenetic conversions. Together, our model and assays provide a comprehensive methodology for further investigation of epigenetic stability and position effects.
Assuntos
Fator 1 de Modelagem da Cromatina/fisiologia , Efeitos da Posição Cromossômica , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Inativação Gênica , Modelos Genéticos , Mutação , Antígeno Nuclear de Célula em Proliferação/genética , Ribonucleotídeo Redutases/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Telômero/genéticaRESUMO
Position effect variegation (PEV) refers to quasi-stable patterns of gene expression that are observed at specific loci throughout the genomes of eukaryotes. The genes subjected to PEV can be completely silenced or fully active. Stochastic conversions between these 2 states are responsible for the variegated phenotypes. Positional variegation is used by human pathogens (Trypanosoma, Plasmodium, and Candida) to evade the immune system or adapt to the host environment. In the yeasts Saccharomyces cerevisiae and Saccharomyces pombe, telomeric PEV aids the adaptation to a changing environment. In metazoans, similar epigenetic conversions are likely to accompany cell differentiation and the setting of tissue-specific gene expression programs. Surprisingly, we know very little about the mechanisms of epigenetic conversions. In this article, earlier models on the nature of PEV are revisited and recent advances on the dynamic nature of chromatin are reviewed. The normal dynamic histone turnover during transcription and DNA replication and its perturbation at transcription and replication pause sites are discussed. It is proposed that such perturbations play key roles in epigenetic conversions and in PEV.
Assuntos
Efeitos da Posição Cromossômica , Epigênese Genética , Eucromatina/química , Heterocromatina/química , Animais , Candida/genética , Replicação do DNA , Loci Gênicos , Histonas/genética , Histonas/metabolismo , Humanos , Plasmodium/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica , Trypanosoma/genéticaRESUMO
BACKGROUND: Classical studies on position effect variegation in Drosophila have demonstrated the existence of bi-modal Active/Silent state of the genes juxtaposed to heterochromatin. Later studies with irreversible methods for the detection of gene repression have revealed a similar phenomenon at the telomeres of Saccharomyces cerevisiae and other species. In this study, we used dual reporter constructs and a combination of reversible and non-reversible methods to present evidence for the different roles of PCNA and histone chaperones in the stability and the propagation of repressed states at the sub-telomeres of S. cerevisiae. RESULTS: We show position dependent transient repression or bi-modal expression of reporter genes at the VIIL sub-telomere. We also show that mutations in the replicative clamp POL30 (PCNA) or the deletion of the histone chaperone CAF1 or the RRM3 helicase lead to transient de-repression, while the deletion of the histone chaperone ASF1 causes a shift from transient de-repression to a bi-modal state of repression. We analyze the physical interaction of CAF1 and RRM3 with PCNA and discuss the implications of these findings for our understanding of the stability and transmission of the epigenetic state of the genes. CONCLUSIONS: There are distinct modes of gene silencing, bi-modal and transient, at the sub-telomeres of S. cerevisiae. We characterise the roles of CAF1, RRM3 and ASF1 in these modes of gene repression. We suggest that the interpretations of past and future studies should consider the existence of the dissimilar states of gene silencing.
Assuntos
Antígeno Nuclear de Célula em Proliferação , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Telômero , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Telômero/genética , Telômero/metabolismoRESUMO
BACKGROUND: Autonomously Replicating Sequences (ARS) in S. cerevisiae serve as origins of DNA replication or as components of cis-acting silencers, which impose positional repression at the mating type loci and at the telomeres. Both types of ARS can act as replicators or silencers, however it is not clear how these quite diverse functions are executed. It is believed that all ARS contain a core module of an essential ARS Consensus Sequence (ACS) and a non-essential B1 element. RESULTS: We have tested how the B1 elements contribute to the silencer and replicator function of ARS. We report that the ACS-B1 orientation of ARS has a profound effect on the levels of gene silencing at telomeres. We also report that the destruction of the canonical B1 elements in two silencer ARS (ARS317 and ARS319) has no effect on their silencer and replicator activity. CONCLUSIONS: The observed orientation effects on gene silencing suggest that ARSs can act as both proto-silencers and as insulator elements. In addition, the lack of B1 suggests that the ACS-B1 module could be different in silencer and replicator ARS.
Assuntos
Inativação Gênica , Genes Fúngicos , Saccharomyces cerevisiae/genética , Telômero/metabolismo , Sequência de Bases , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , Replicação do DNA , Mutação , Origem de Replicação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Telômero/genéticaRESUMO
Phenotypic heterogeneity provides growth advantages for a population upon changes of the environment. In S. cerevisiae, such heterogeneity has been observed as "on/off" states in the expression of individual genes in individual cells. These variations can persist for a limited or extended number of mitotic divisions. Such traits are known to be mediated by heritable chromatin structures, by the mitotic transmission of transcription factors involved in gene regulatory circuits or by the cytoplasmic partition of prions or other unstructured proteins. The significance of such epigenetic diversity is obvious, however, we have limited insight into the mechanisms that generate it. In this review, we summarize the current knowledge of epigenetically maintained heterogeneity of gene expression and point out similarities and converging points between different mechanisms. We discuss how the sharing of limiting repression or activation factors can contribute to cell-to-cell variations in gene expression and to the coordination between short- and long- term epigenetic strategies. Finally, we discuss the implications of such variations and strategies in adaptation and aging.
RESUMO
Dbf4-Dependent Kinase (DDK) has a well-established essential role at origins of DNA replication, where it phosphorylates and activates the replicative MCM helicase. It also acts in the response to mutagens and in DNA repair as well as in key steps during meiosis. Recent studies have indicated that, in addition to the MCM helicase, DDK phosphorylates several substrates during the elongation stage of DNA replication or upon replication stress. However, these activities of DDK are not essential for viability. Dbf4-Dependent Kinase is also emerging as a key factor in the regulation of genome-wide origin firing and in replication-coupled chromatin assembly. In this review, we summarize recent progress in our understanding of the diverse roles of DDK.
Assuntos
Proteínas de Ciclo Celular , Replicação do DNA , Proteínas de Saccharomyces cerevisiae , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases , Origem de Replicação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Autonomously replicating sequences (ARSs) in Saccharomyces cerevisiae have been extensively characterized as both origins of DNA replication and as chromatin repressors/silencers. It has been conclusively shown that the origin and the silencer activities of ARS are substantially, but not entirely interchangeable and that they are modulated by position effects and chromatin environment. It remains unclear how these two quite divergent functions of ARS co-exist. This perspective focuses on recent advances, which have shown that slight differences in ARSs can modulate their affinity for origin recognition complex and their activity as silencers or origins.
Assuntos
Replicação do DNA , Inativação Gênica , Genes Fúngicos , Origem de Replicação , Sequência de Bases , Cromatina/genética , Sequência Consenso , DNA Fúngico/genética , DNA Fúngico/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
Chromatin Assembly Factor I (CAF-I) plays a central role in the reassembly of H3/H4 histones during DNA replication. In S. cerevisiae CAF-I is not essential and its loss is associated with reduced gene silencing at telomeres and increased sensitivity to DNA damage. Two kinases, Cyclin Dependent Kinase (CDK) and Dbf4-Dependent Kinase (DDK), are known to phosphorylate the Cac1p subunit of CAF-I, but their role in the regulation of CAF-I activity is not well understood. In this study we systematically mutated the phosphorylation target sites of these kinases. We show that concomitant mutations of the CDK and DDK target sites of Cac1p lead to growth retardation and significant cell cycle defects, altered cell morphology and increased sensitivity to DNA damage. Surprisingly, some mutations also produced flocculation, a phenotype that is lost in most laboratory strains, and displayed elevated expression of FLO genes. None of these effects is observed upon the destruction of CAF-I. In contrast, the mutations that caused flocculation did not affect gene silencing at the mating type and subtelomeric loci. We conclude that dysfunctional CAF-I produces severe phenotypes, which reveal a possible role of CAF-I in the coordination of DNA replication, chromatin reassembly and cell cycle progression. Our study highlights the role of phosphorylation of Cac1p by CDK and a putative role for DDK in the transmission and re-assembly of chromatin during DNA replication.
Assuntos
Ciclo Celular/genética , Fator 1 de Modelagem da Cromatina/genética , Cromatina/metabolismo , Inativação Gênica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/genética , Cromatina/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Replicação do DNA/genética , Genes Fúngicos Tipo Acasalamento , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Mutação , Fenótipo , Fosforilação , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/genética , Sirtuína 2/metabolismo , Telômero/metabolismoRESUMO
BACKGROUND: Biofilm formation or flocculation is a major phenotype in wild type budding yeasts but rarely seen in laboratory yeast strains. Here, we analysed flocculation phenotypes and the expression of FLO genes in laboratory strains with various genetic backgrounds. RESULTS: We show that mutations in histone chaperones, the helicase RRM3 and the Histone Deacetylase HDA1 de-repress the FLO genes and partially reconstitute flocculation. We demonstrate that the loss of repression correlates to elevated expression of several FLO genes, to increased acetylation of histones at the promoter of FLO1 and to variegated expression of FLO11. We show that these effects are related to the activity of CAF-1 at the replication forks. We also demonstrate that nitrogen starvation or inhibition of histone deacetylases do not produce flocculation in W303 and BY4742 strains but do so in strains compromised for chromatin maintenance. Finally, we correlate the de-repression of FLO genes to the loss of silencing at the subtelomeric and mating type gene loci. CONCLUSIONS: We conclude that the deregulation of chromatin maintenance and transmission is sufficient to reconstitute flocculation in laboratory yeast strains. Consequently, we propose that a gain in epigenetic silencing is a major contributing factor for the loss of flocculation phenotypes in these strains. We suggest that flocculation in yeasts provides an excellent model for addressing the challenging issue of how epigenetic mechanisms contribute to evolution.
Assuntos
DNA Helicases/metabolismo , Chaperonas de Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilação , Cromatina/metabolismo , Floculação/efeitos dos fármacos , Inativação Gênica , Chaperonas de Histonas/genética , Histona Desacetilases/metabolismo , Lectinas de Ligação a Manose/genética , Glicoproteínas de Membrana/metabolismo , Mutação , Niacinamida/farmacologia , Nitrogênio/deficiência , Nitrogênio/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Ribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The establishment of silent chromatin requires passage through S-phase, but not DNA replication per se. Nevertheless, many proteins that affect silencing are bona fide DNA replication factors. It is not clear if mutations in these replication factors affect silencing directly or indirectly via deregulation of S-phase or DNA replication. Consequently, the relationship between DNA replication and silencing remains an issue of debate. Here we analyze the effect of mutations in DNA replication factors (mcm5-461, mcm5-1, orc2-1, orc5-1, cdc45-1, cdc6-1, and cdc7-1) on the silencing of a group of reporter constructs, which contain different combinations of "natural" subtelomeric elements. We show that the mcm5-461, mcm5-1, and orc2-1 mutations affect silencing through subtelomeric ARS consensus sequences (ACS), while cdc6-1 affects silencing independently of ACS. orc5-1, cdc45-1, and cdc7-1 affect silencing through ACS, but also show ACS-independent effects. We also demonstrate that isolated nontelomeric ACS do not recapitulate the same effects when inserted in the telomere. We propose a model that defines the modes of action of MCM5 and CDC6 in silencing.
Assuntos
Sequência Consenso , Replicação do DNA , Inativação Gênica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Telômero , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fase S , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Built of DNA polymerases and multiple associated factors, the replication fork steadily progresses along the DNA template and faithfully replicates DNA. This model can be found in practically every textbook of genetics, with the more complex situation of chromatinized DNA in eukaryotes often viewed as a variation. However, the replication-coupled disassembly/reassembly of chromatin adds significant complexity to the whole replication process. During the course of eukaryotic DNA replication the forks encounter various conditions and numerous impediments. These include nucleosomes with a variety of post-translational modifications, euchromatin and heterochromatin, differentially methylated DNA, tightly bound proteins, active gene promoters and DNA loops. At such positions the forks slow down or even stall. Dedicated factors stabilize the fork and prevent its rotation or collapse, while other factors resolve the replication block and facilitate the resumption of elongation. The fate of histones during replication stalling and resumption is not well understood. In this review we briefly describe recent advances in our understanding of histone turnover during DNA replication and focus on the possible mechanisms of nucleosome disassembly/reassembly at paused replication forks. We propose that replication pausing provides opportunities for an epigenetic change of the associated locus.
RESUMO
Cyclin-dependent kinases have established roles in the regulation of cell cycle, in gene expression and in cell differentiation. Many of these kinases have been considered as drug targets and numerous efforts have been made to develop specific and potent inhibitors against them. The first step in all of these attempts and in many other biochemical analyses is the production of highly purified and reliable kinase, most frequently in a recombinant form. In this chapter we describe our experience in the cloning, expression, and purification of CDKs using CDK7/CycH, CDK8/CycC, and CDK9/CycT1 as an example.
Assuntos
Quinase 8 Dependente de Ciclina/isolamento & purificação , Quinase 9 Dependente de Ciclina/isolamento & purificação , Quinases Ciclina-Dependentes/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Animais , Catálise , Diferenciação Celular , Cromatografia , Quinase 8 Dependente de Ciclina/química , Quinase 9 Dependente de Ciclina/química , Quinases Ciclina-Dependentes/química , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Glutationa Transferase/metabolismo , Insetos , Biologia Molecular/métodos , Níquel/química , Peptídeos/química , Proteínas Recombinantes/químicaRESUMO
Chromatin structures are transmitted to daughter cells through a complex system of nucleosome disassembly and re-assembly at the advancing replication forks. However, the role of replication pausing in the transmission and perturbation of chromatin structures has not been addressed. RRM3 encodes a DNA helicase, which facilitates replication at sites covered with non-histone protein complexes (tRNA genes, active gene promoters, telomeres) in Saccharomyces cerevisiae. In this report we show that the deletion of RRM3 reduces the frequency of epigenetic conversions in the subtelomeric regions of the chromosomes. This phenotype is strongly dependent on 2 histone chaperones, CAF-I and ASF1, which are involved in the reassembly of nucleosomes behind replication forks, but not on the histone chaperone HIR1. We also show that the deletion of RRM3 increases the spontaneous mutation rates in conjunction with CAF-I and ASF1, but not HIR1. Finally, we demonstrate that Rrm3p and CAF-I compete for the binding to the DNA replication clamp PCNA (Proliferating Cell Nuclear Antigen). We propose that the stalling of DNA replication predisposes to epigenetic conversions and that RRM3 and CAF-I play key roles in this process.
Assuntos
Fator 1 de Modelagem da Cromatina/metabolismo , DNA Helicases/metabolismo , Epigênese Genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ligação Competitiva , Fator 1 de Modelagem da Cromatina/deficiência , Fator 1 de Modelagem da Cromatina/genética , Deleção de Genes , Antígeno Nuclear de Célula em Proliferação/metabolismoRESUMO
MCM (minichromosome maintenance) proteins function as a replication licensing factor (RLF-M), which contributes to limiting initiation of DNA replication to once per cell cycle. In the present study we show that a truncation of the pol II CTD in a S. cerevisiae strain harboring a mutation in mcm5 partially reverses its ts phenotype and improves maintenance of CEN/ARS minichromosomes. We correlate this phenotype to effects on DNA replication rather than to effects on transcription or specific gene expression. We also demonstrate that a similar truncation of the CTD reduces minichromosome stability and impairs stimulation of DNA replication by trans-activators and that tethering of recombinant pol II CTD to an origin of replication has a significant stimulatory effect on minichromosome stability. Furthermore, we show that pol II is recruited to ARS1. We propose that in S. cerevisiae a mechanism of coordinating pol II transcription and DNA replication is mediated by the CTD of pol II.
Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , RNA Polimerase II/metabolismo , Origem de Replicação , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Mutação , Estrutura Terciária de Proteína , RNA Polimerase II/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Embryonal totipotent cells can produce both embryonic and extraembryonic tissues and can generate whole organisms. In mice this level of genome plasticity is preserved in the 2-cell embryos, but is absent in embryonic cells from later stages of development. Recently it has been demonstrated that totipotent-like cells spontaneously appear in embryonic stem cell cultures and that the depletion of the histone chaperone Chromatin Assembly Factor I (CAF-I) increases the abundance of 2cell-like cells. On the other hand, earlier studies have demonstrated that CAF-I is necessary for epigenetic conversions at the telomeres of S. cerevisiae. This commentary proposes that the absence of CAF-I confers totipotency of embryonic cells and that its activation triggers chromatin changes that reset the epigenome toward cell differentiation.
Assuntos
Fator 1 de Modelagem da Cromatina/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Camundongos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Telômero/metabolismo , Células-Tronco Totipotentes/citologia , Células-Tronco Totipotentes/metabolismoRESUMO
Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase CDC7. Here we show that another kinase, CDC28, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of CDC28 but not of CDC7 substantially reduce the in vivo phosphorylation of Cac1p. However, mutations in the putative CDC7 target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a cdc28-1 mutant and to a lesser extent in a cdc7-1 mutant. In addition, mutations in the Cac1p-phosphorylation sites by both CDC28 and CDC7 reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate CDC28 in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly.