RESUMEN
Cohesin is a central architectural element of chromosomes that regulates numerous DNA-based events. The complex holds sister chromatids together until anaphase onset and organizes individual chromosomal DNAs into loops and self-associating domains. Purified cohesin diffuses along DNA in an ATP-independent manner but can be propelled by transcribing RNA polymerase. In conjunction with a cofactor, the complex also extrudes DNA loops in an ATP-dependent manner. In this study we examine transcription-driven translocation of cohesin under various conditions in yeast. To this end, obstacles of increasing size were tethered to DNA to act as roadblocks to complexes mobilized by an inducible gene. The obstacles were built from a GFP-lacI core fused to one or more mCherries. A chimera with four mCherries blocked cohesin passage in late G1. During M phase, the threshold barrier depended on the state of cohesion: non-cohesive complexes were also blocked by four mCherries whereas cohesive complexes were blocked by as few as three mCherries. Furthermore cohesive complexes that were stalled at obstacles, in turn, blocked the passage of non-cohesive complexes. That synthetic barriers capture mobilized cohesin demonstrates that transcription-driven complexes translocate processively in vivo. Together, this study reveals unexplored limitations to cohesin movement on chromosomes.
Asunto(s)
Proteínas Cromosómicas no Histona , Proteínas de Saccharomyces cerevisiae , Proteínas Cromosómicas no Histona/genética , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Proteínas de Saccharomyces cerevisiae/genética , Cromátides , ADN , Adenosina Trifosfato , CohesinasRESUMEN
The robust proliferation of cancer cells requires vastly elevated levels of protein synthesis, which relies on a steady supply of aminoacylated tRNAs. Delivery of tRNAs to the cytoplasm is a highly regulated process, but the machinery for tRNA nuclear export is not fully elucidated. In this study, using a live cell imaging strategy that visualizes nascent transcripts from a specific tRNA gene in yeast, we identified the nuclear basket proteins Mlp1 and Mlp2, two homologs of the human TPR protein, as regulators of tRNA export. TPR expression is significantly increased in lung cancer tissues and correlated with poor prognosis. Consistently, knockdown of TPR inhibits tRNA nuclear export, protein synthesis and cell growth in lung cancer cell lines. We further show that NXF1, a well-known mRNA nuclear export factor, associates with tRNAs and mediates their transport through nuclear pores. Collectively, our findings uncover a conserved mechanism that regulates nuclear export of tRNAs, which is a limiting step in protein synthesis in eukaryotes.
Asunto(s)
Núcleo Celular/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/biosíntesis , Proteínas de Complejo Poro Nuclear/fisiología , Proteínas Proto-Oncogénicas/fisiología , Transporte de ARN , ARN de Transferencia/metabolismo , Humanos , Neoplasias Pulmonares/patología , Proteínas de Complejo Poro Nuclear/genética , Pronóstico , Proteínas Proto-Oncogénicas/genética , Células Tumorales CultivadasRESUMEN
The ring-shaped cohesin complex orchestrates long-range DNA interactions to mediate sister chromatid cohesion and other aspects of chromosome structure and function. In the yeast Saccharomyces cerevisiae, the complex binds discrete sites along chromosomes, including positions within and around genes. Transcriptional activity redistributes the complex to the 3' ends of convergently oriented gene pairs. Despite the wealth of information about where cohesin binds, little is known about cohesion at individual chromosomal binding sites and how transcription affects cohesion when cohesin complexes redistribute. In this study, we generated extrachromosomal DNA circles to study cohesion in response to transcriptional induction of a model gene, URA3. Functional cohesin complexes loaded onto the locus via a poly(dA:dT) tract in the gene promoter and mediated cohesion before induction. Upon transcription, the fate of these complexes depended on whether the DNA was circular or not. When gene activation occurred before DNA circularization, cohesion was lost. When activation occurred after DNA circularization, cohesion persisted. The presence of a convergently oriented gene also prevented transcription-driven loss of functional cohesin complexes, at least in M phase-arrested cells. The results are consistent with cohesin binding chromatin in a topological embrace and with transcription mobilizing functional complexes by sliding them along DNA.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Fúngicos/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/metabolismo , Activación Transcripcional/fisiología , Adenosina Trifosfatasas/metabolismo , Sitios de Unión , Cromosomas Fúngicos/ultraestructura , ADN Circular/metabolismo , ADN de Hongos/genética , Proteínas de Unión al ADN/metabolismo , Herencia Extracromosómica , Genes Fúngicos , Genes Reporteros , Genes Sintéticos , Metafase , Complejos Multiproteicos/metabolismo , Poli dA-dT/farmacología , Regiones Promotoras Genéticas/genética , Unión Proteica , Secuencias Reguladoras de Ácidos Nucleicos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , CohesinasRESUMEN
Transcriptional silencing in Saccharomyces cerevisiae occurs at several genomic sites including the silent mating-type loci, telomeres, and the ribosomal DNA (rDNA) tandem array. Epigenetic silencing at each of these domains is characterized by the absence of nearly all histone modifications, including most prominently the lack of histone H4 lysine 16 acetylation. In all cases, silencing requires Sir2, a highly-conserved NAD(+)-dependent histone deacetylase. At locations other than the rDNA, silencing also requires additional Sir proteins, Sir1, Sir3, and Sir4 that together form a repressive heterochromatin-like structure termed silent chromatin. The mechanisms of silent chromatin establishment, maintenance, and inheritance have been investigated extensively over the last 25 years, and these studies have revealed numerous paradigms for transcriptional repression, chromatin organization, and epigenetic gene regulation. Studies of Sir2-dependent silencing at the rDNA have also contributed to understanding the mechanisms for maintaining the stability of repetitive DNA and regulating replicative cell aging. The goal of this comprehensive review is to distill a wide array of biochemical, molecular genetic, cell biological, and genomics studies down to the "nuts and bolts" of silent chromatin and the processes that yield transcriptional silencing.
Asunto(s)
Cromatina/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Sirtuina 2/genética , Transcripción Genética , Epigénesis Genética/genética , Regulación Fúngica de la Expresión Génica , Silenciador del Gen , Heterocromatina/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Cohesin associates with distinct sites on chromosomes to mediate sister chromatid cohesion. Single cohesin complexes are thought to bind by encircling both sister chromatids in a topological embrace. Transcriptionally repressed chromosomal domains in the yeast Saccharomyces cerevisiae represent specialized sites of cohesion where cohesin binds silent chromatin in a Sir2-dependent fashion. In this study, we investigated the molecular basis for Sir2-mediated cohesion. We identified a cluster of charged surface residues of Sir2, collectively termed the EKDK motif, that are required for cohesin function. In addition, we demonstrated that Esc8, a Sir2-interacting factor, is also required for silent chromatin cohesion. Esc8 was previously shown to associate with Isw1, the enzymatic core of ISW1 chromatin remodelers, to form a variant of the ISW1a chromatin remodeling complex. When ESC8 was deleted or the EKDK motif was mutated, cohesin binding at silenced chromatin domains persisted but cohesion of the domains was abolished. The data are not consistent with cohesin embracing both sister chromatids within silent chromatin domains. Transcriptional silencing remains largely intact in strains lacking ESC8 or bearing EKDK mutations, indicating that silencing and cohesion are separable functions of Sir2 and silent chromatin.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuina 2/metabolismo , Adenosina Trifosfatasas/metabolismo , Secuencias de Aminoácidos , Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Mutación , Proteínas Nucleares/genética , Conformación Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/química , Sirtuina 2/química , CohesinasRESUMEN
The capacity of Saccharomyces cerevisiae to repair exposed DNA ends by homologous recombination has long been used by experimentalists to assemble plasmids from DNA fragments in vivo. While this approach works well for engineering extrachromosomal vectors, it is not well suited to the generation, recovery and reuse of integrative vectors. Here, we describe the creation of a series of conditional centromeric shuttle vectors, termed pXR vectors, that can be used for both plasmid assembly in vivo and targeted genomic integration. The defining feature of pXR vectors is that the DNA segment bearing the centromere and origin of replication, termed CEN/ARS, is flanked by a pair of loxP sites. Passaging the vectors through bacteria that express Cre recombinase reduces the loxP-CEN/ARS-loxP module to a single loxP site, thereby eliminating the ability to replicate autonomously in yeast. Each vector also contains a selectable marker gene, as well as a fragment of the HO locus, which permits targeted integration at a neutral genomic site. The pXR vectors provide a convenient and robust method to assemble DNAs for targeted genomic modifications.
Asunto(s)
Marcación de Gen/métodos , Vectores Genéticos , Genética Microbiana/métodos , Biología Molecular/métodos , Saccharomyces cerevisiae/genética , Centrómero , Recombinación Homóloga , Plásmidos , Origen de RéplicaRESUMEN
Silent chromatin in budding yeast is propagated from one generation to the next, even though 'silenced' genes are occasionally expressed.
Asunto(s)
Cromatina/genética , Silenciador del Gen , Saccharomyces cerevisiae/genética , Genes FúngicosRESUMEN
tRNAs are encoded by RNA polymerase III-transcribed genes that reside at seemingly random intervals along the chromosomes of budding yeast. Existing evidence suggests that the genes congregate together at the nucleolus and/or centromeres. In this study, we re-examined spatial and temporal aspects of tRNA gene (tDNA) expression. We show that tDNA transcription fluctuates during cell cycle progression. In M phase, when tRNA synthesis peaks, tDNAs localize at nuclear pore complexes (NPCs). Docking of a tDNA requires the DNA sequence of the contacted gene, nucleoporins Nup60 and Nup2, and cohesin. Characterization of mutants that block NPC localization revealed that docking is a consequence of elevated tDNA transcription. NPC-tDNA contact falters in the absence of the principal exportin of nascent tRNA, Los1, and genetic assays indicate that gating of tDNAs at NPCs favors cytoplasmic accumulation of functional tRNA. Collectively, the data suggest that tDNAs associate with NPCs to coordinate RNA polymerase III transcription with the nuclear export of pre-tRNA. The M-phase specificity of NPC contact reflects a regulatory mechanism that may have evolved, in part, to avoid collisions between DNA replication forks and transcribing RNA polymerase III machinery at NPCs.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Poro Nuclear/metabolismo , ARN de Transferencia/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcripción Genética , División Celular/fisiología , Mutación , Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Unión Proteica , Transporte de Proteínas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Many proteins are post-translationally modified by lipid moieties such as palmitoyl or prenyl (e.g., farnesyl) groups, creating functional proteolipids. Lipid modifications share the property of increasing a protein's hydrophobicity and thus the propensity of that protein to associate with a membrane. These modifications are used to control the localization and activity of membrane-associated proteins. A well-recognized paradigm is farnesylation of the Ras GTPase that helps target this critical signaling protein to the plasma membrane.
Asunto(s)
Núcleo Celular/metabolismo , Aciltransferasas/metabolismo , Heterocromatina/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Lipoilación , Prenilación de Proteína , Procesamiento Proteico-Postraduccional , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Telómero/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Proteínas ras/metabolismoRESUMEN
Site-specific recombinases have been harnessed for a variety of genetic manipulations involving the gain, loss, or rearrangement of genomic DNA in a variety of organisms. The enzymes have been further exploited in the model eukaryote Saccharomyces cerevisiae for mechanistic studies involving chromosomal context. In these cases, a chromosomal element of interest is converted into a DNA circle within living cells, thereby uncoupling the element from neighboring regulatory sequences, obligatory chromosomal events, and other context-dependent effects that could alter or mask intrinsic functions of the element. In this chapter, I discuss general considerations in using site-specific recombination to create DNA circles in yeast and the specific application of the R recombinase.
Asunto(s)
ADN Nucleotidiltransferasas/metabolismo , ADN Circular/genética , ADN de Hongos/genética , Biología Molecular/métodos , Recombinación Genética , Saccharomyces cerevisiae/genética , Secuencia de Bases , Cromosomas Fúngicos/metabolismo , Enzimas de Restricción del ADN/metabolismo , ADN Circular/aislamiento & purificación , ADN de Hongos/aislamiento & purificación , Datos de Secuencia Molecular , Factores de TiempoRESUMEN
The organization of chromatin domains in the nucleus is an important factor in gene regulation. In eukaryotic nuclei, transcriptionally silenced chromatin clusters at the nuclear periphery while transcriptionally poised chromatin resides in the nuclear interior. Recent studies suggest that nuclear pore proteins (NUPs) recruit loci to nuclear pores to aid in insulation of genes from silencing and during gene activation. We investigated the role of NUPs at a native yeast insulator and show that while NUPs localize to the native tDNA insulator adjacent to the silenced HMR domain, loss of pore proteins does not compromise insulation. Surprisingly we find that NUPs contribute to silencing at HMR and are able to restore silencing to a silencing-defective HMR allele when tethered to the locus. We show that the perinuclear positioning of heterochromatin is important for the NUP-mediated silencing effect and find that loss of NUPs result in decreased localization of HMR to the nuclear periphery. We also show that loss of telomeric tethering pathways does not eliminate NUP localization to HMR, suggesting that NUPs may mediate an independent pathway for HMR association with the nuclear periphery. We propose that localization of NUPs to the tDNA insulator at HMR helps maintain the intranuclear position of the silent locus, which in turn contributes to the fidelity of silencing at HMR.
Asunto(s)
Núcleo Celular/metabolismo , Silenciador del Gen , Sitios Genéticos/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromosomas Fúngicos/genética , ADN de Hongos/metabolismo , Heterocromatina/metabolismo , Transporte de Proteínas , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/metabolismo , Telómero/metabolismoRESUMEN
The posttranslational addition of palmitate to cysteines occurs ubiquitously in eukaryotic cells, where it functions in anchoring target proteins to membranes and in vesicular trafficking. Here we show that the Saccharomyces cerevisiae palmitoyltransferase Pfa4 enhanced heterochromatin formation at the cryptic mating-type loci HMR and HML via Rif1, a telomere regulatory protein. Acylated Rif1 was detected in extracts from wild-type but not pfa4Δ mutant cells. In a pfa4Δ mutant, Rif1-GFP dispersed away from foci positioned at the nuclear periphery into the nucleoplasm. Sir3-GFP distribution was also perturbed, indicating a change in the nuclear dynamics of heterochromatin proteins. Genetic analyses indicated that PFA4 functioned upstream of RIF1. Surprisingly, the pfa4Δ mutation had only mild effects on telomeric regulation, suggesting Rif1's roles at HM loci and telomeres were more complexly related than previously thought. These data supported a model in which Pfa4-dependent palmitoylation of Rif1 anchored it to the inner nuclear membrane, influencing its role in heterochromatin dynamics.
Asunto(s)
Heterocromatina/metabolismo , Proteínas Represoras/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomycetales/genética , Proteínas de Unión a Telómeros/fisiología , Acilación , Aciltransferasas/fisiología , Lipoilación , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , TelómeroRESUMEN
The protein complex known as cohesin binds pericentric regions and other sites of eukaryotic genomes to mediate cohesion of sister chromatids. In budding yeast Saccharomyces cerevisiae, cohesin also binds silent chromatin, a repressive chromatin structure that functionally resembles heterochromatin of higher eukaryotes. We developed a protein-targeting assay to investigate the mechanistic basis for cohesion of silent chromatin domains. Individual silencing factors were tethered to sites where pairing of sister chromatids could be evaluated by fluorescence microscopy. We report that the evolutionarily conserved Sir2 histone deacetylase, an essential silent chromatin component, was both necessary and sufficient for cohesion. The cohesin genes were required, but the Sir2 deacetylase activity and other silencing factors were not. Binding of cohesin to silent chromatin was achieved with a small carboxyl terminal fragment of Sir2. Taken together, these data define a unique role for Sir2 in cohesion of silent chromatin that is distinct from the enzyme's role as a histone deacetylase.
Asunto(s)
Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica/genética , Saccharomyces cerevisiae/fisiología , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Sirtuina 2/fisiología , Cromatina/genética , Microscopía Fluorescente , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Sirtuina 2/genética , CohesinasRESUMEN
Persistent DNA double-strand breaks and telomeres represent genomic hazards, as they can instigate inappropriate repair reactions. Two recent papers by Oza and colleagues (pp. 912-917) and Schober and colleagues (pp. 928-938) show that both types of DNA ends are sequestered from bulk DNA by Mps3, a SUN domain protein that spans the inner nuclear membrane. Anchorage maintains telomere integrity and steers double-strand breaks toward specialized repair pathways. This work defines the nuclear periphery as a subcompartment where dangerous DNA elements can be handled with care.
Asunto(s)
Núcleo Celular/fisiología , Roturas del ADN de Doble Cadena , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Telómero/metabolismo , Inestabilidad Cromosómica , Replicación del ADN , Proteínas de la Membrana/metabolismo , Membrana Nuclear/metabolismo , Telómero/genéticaRESUMEN
Condensin and cohesin are loaded onto yeast chromosomes by a common mechanism at RNA polymerase III transcribed genes. Whereas cohesin translocates from these loading sites to mediate cohesion at secondary locations, condensin remains, bringing distant sites together into clusters.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Complejos Multiproteicos/metabolismo , Adenosina Trifosfatasas/genética , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Fúngicos , Proteínas de Unión al ADN/genética , Modelos Biológicos , Complejos Multiproteicos/genética , ARN Polimerasa III , Saccharomyces cerevisiae/metabolismo , CohesinasRESUMEN
The yeast Sir2/3/4 complex forms a heterochromatin-like structure that represses transcription. The proteins nucleate at silencers and spread distally, utilizing the Sir2 NAD(+)-dependent histone deacetylase activity and the affinity of Sir3/4 for deacetylated histone tails. A by-product of the Sir2 reaction, O-acetyl-ADP-ribose (OAADPr), is thought to aid spreading by binding one of the Sir proteins. We developed a protein chimera approach to reexamine the contributions of Sir2. We show that a Sir3 chimera-bearing Hos3, an unrelated NAD(+)-independent histone deacetylase, substitutes for Sir2 in silencing. Sir3-Hos3 operates within the Sir pathway, spreading while deacetylating histones. Moreover, the chimera represses HM loci in strains lacking all five OAADPr-producing deacetylases, indicating that OAADPr is not necessary for silencing. Repression by a Hos3 hybrid bearing the targeting motifs of Sir2 shows that targeting doesn't require the Sir2 reaction. Together, these data demonstrate that protein deacetylation is the only essential function of Sir2 in creating silenced chromatin.
Asunto(s)
Silenciador del Gen , Histona Desacetilasas/metabolismo , O-Acetil-ADP-Ribosa/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuinas/metabolismo , Transcripción Genética , Histona Desacetilasas/genética , Modelos Moleculares , O-Acetil-ADP-Ribosa/genética , Proteínas Recombinantes de Fusión/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Sirtuina 2 , Sirtuinas/genéticaRESUMEN
Gene regulation involves long-range communication between silencers, enhancers, and promoters. In Saccharomyces cerevisiae, silencers flank transcriptionally repressed genes to mediate regional silencing. Silencers recruit the Sir proteins, which then spread along chromatin to encompass the entire silenced domain. In this report we have employed a boundary trap assay, an enhancer activity assay, chromatin immunoprecipitations, and chromosome conformation capture analyses to demonstrate that the two HMR silencer elements are in close proximity and functionally communicate with one another in vivo. We further show that silencing is necessary for these long-range interactions, and we present models for Sir-mediated silencing based upon these results.
Asunto(s)
Cromosomas Fúngicos/genética , ADN de Hongos/genética , Elementos de Facilitación Genéticos/genética , Regulación Fúngica de la Expresión Génica , Silenciador del Gen/fisiología , Genes del Tipo Sexual de los Hongos/genética , Región de Control de Posición/genética , Modelos Genéticos , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Inmunoprecipitación de Cromatina , Cromosomas Fúngicos/ultraestructura , ADN de Hongos/ultraestructura , Proteínas de Saccharomyces cerevisiae/fisiología , Complejo Shelterina , Proteínas de Unión a Telómeros/fisiología , Factores de Transcripción/fisiologíaRESUMEN
The 32 telomeres in the budding yeast genome cluster in three to seven perinuclear foci. Although individual telomeres and telomeric foci are in constant motion, preferential juxtaposition of some telomeres has been scored. To examine the principles that guide such long-range interactions, we differentially tagged pairs of chromosome ends and developed an automated three-dimensional measuring tool that determines distances between two telomeres. In yeast, all chromosomal ends terminate in TG(1-3) and middle repetitive elements, yet subgroups of telomeres also share extensive homology in subtelomeric coding domains. We find that up to 21 kb of >90% sequence identity does not promote telomere pairing in interphase cells. To test whether unique sequence elements, arm length, or chromosome territories influence juxtaposition, we reciprocally swapped terminal domains or entire chromosomal arms from one chromosome to another. We find that the distal 10 kb of Tel6R promotes interaction with Tel6L, yet only when the two telomeres are present on the same chromosome. By manipulating the length and sequence composition of the right arm of chr 5, we confirm that contact between telomeres on opposite chromatid arms of equal length is favored. These results can be explained by the polarized Rabl arrangement of yeast centromeres and telomeres, which promote to telomere pairing by allowing contact between chromosome arms of equal length in anaphase.
Asunto(s)
Cromosomas Fúngicos/genética , Intercambio Genético/genética , Regulación Fúngica de la Expresión Génica/genética , Saccharomyces cerevisiae/genética , Telómero/metabolismo , Southern Blotting , Electroforesis en Gel de Agar , Microscopía Fluorescente , Telómero/genéticaRESUMEN
DNA replication generates sister chromatid pairs that are bound to one another until anaphase onset. The process, termed sister chromatid cohesion, requires the multisubunit cohesin complex that resides at centromeres and sites where genes converge. At the HMR mating-type locus of budding yeast, cohesin associates with a heterochromatin-like structure known as silent chromatin. In this report, we show that silent chromatin is necessary but not sufficient for cohesion of the replicating locus. A tRNA gene (tDNA) that delimits the silent chromatin domain is also required, as are subunits of the TFIIIB and RSC complexes that bind the gene. Non-tDNA boundary elements do not substitute for tDNAs in cohesion, suggesting that barrier activity is not responsible for the phenomenon. The results reveal an unexpected role for tDNAs and RNA polymerase III-associated proteins in establishment of sister chromatid cohesion.
Asunto(s)
Cromatina/fisiología , Genes del Tipo Sexual de los Hongos , ARN Polimerasa III/fisiología , ARN de Transferencia/genética , Proteínas de Ciclo Celular/fisiología , Proteínas Cromosómicas no Histona/fisiología , Replicación del ADN , Proteínas de Unión al ADN/fisiología , Silenciador del Gen , Modelos Genéticos , Proteínas Nucleares/fisiología , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/fisiología , Factor de Transcripción TFIIB/fisiología , Factores de Transcripción/fisiología , CohesinasRESUMEN
Sir2 and Hst1 are NAD(+)-dependent histone deacetylases of budding yeast that are related by strong sequence similarity. Nevertheless, the two proteins promote two mechanistically distinct forms of gene repression. Hst1 interacts with Rfm1 and Sum1 to repress the transcription of specific middle-sporulation genes. Sir2 interacts with Sir3 and Sir4 to silence genes contained within the silent-mating-type loci and telomere chromosomal regions. To identify the determinants of gene-specific versus regional repression, we created a series of Hst1::Sir2 hybrids. Our analysis yielded two dual-specificity chimeras that were able to perform both regional and gene-specific repression. Regional silencing by the chimeras required Sir3 and Sir4, whereas gene-specific repression required Rfm1 and Sum1. Our findings demonstrate that the nonconserved N-terminal region and two amino acids within the enzymatic core domain account for cofactor specificity and proper targeting of these proteins. These results suggest that the differences in the silencing and repression functions of Sir2 and Hst1 may not be due to differences in enzymatic activities of the proteins but rather may be the result of distinct cofactor specificities.