RESUMO
The conserved transcription elongation factor Spt6 makes several contacts with the RNA Polymerase II (RNAPII) complex, including a high-affinity interaction between the Spt6 tandem SH2 domain (Spt6-tSH2) and phosphorylated residues of the Rpb1 subunit in the linker between the catalytic core and the C-terminal domain (CTD) heptad repeats. This interaction contributes to generic localization of Spt6, but we show here that it also has gene-specific roles. Disrupting the interface affected transcription start site selection at a subset of genes whose expression is regulated by this choice, and this was accompanied by changes in a distinct pattern of Spt6 accumulation at these sites. Splicing efficiency was also diminished, as was apparent progression through introns that encode snoRNAs. Chromatin-mediated repression was impaired, and a distinct role in maintaining +1 nucleosomes was identified, especially at ribosomal protein genes. The Spt6-tSH2:Rpb1 interface therefore has both genome-wide functions and local roles at subsets of genes where dynamic decisions regarding initiation, transcript processing, or termination are made. We propose that the interaction modulates the availability or activity of the core elongation and histone chaperone functions of Spt6, contributing to coordination between RNAPII and its accessory factors as varying local conditions call for dynamic responses.
Assuntos
Chaperonas de Histonas/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Domínios de Homologia de src , Sítios de Ligação , Regulação da Expressão Gênica , Chaperonas de Histonas/química , Chaperonas de Histonas/genética , Humanos , IMP Desidrogenase/metabolismo , Modelos Biológicos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , RNA Polimerase II/química , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade , Sítio de Iniciação de Transcrição , Transcrição Gênica , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/genéticaRESUMO
FACT, a heterodimer of Spt16 and Pob3, is an essential histone chaperone. We show that the H2A-H2B binding activity that is central to FACT function resides in short acidic regions near the C termini of each subunit. Mutations throughout these regions affect binding and cause correlated phenotypes that range from mild to lethal, with the largest individual contributions unexpectedly coming from an aromatic residue and a nearby carboxylate residue within each domain. Spt16 and Pob3 bind overlapping sites on H2A-H2B, and Spt16-Pob3 heterodimers simultaneously bind two H2A-H2B dimers, the same stoichiometry as the components of a nucleosome. An Spt16:H2A-H2B crystal structure explains the biochemical and genetic data, provides a model for Pob3 binding, and implies a mechanism for FACT reorganization that we confirm biochemically. Moreover, unexpected similarity to binding of ANP32E and Swr1 with H2A.Z-H2B reveals that diverse H2A-H2B chaperones use common mechanisms of histone binding and regulating nucleosome functions.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Histonas/metabolismo , Nucleossomos/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Motivos de Aminoácidos , Sequência Conservada , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/genética , Histonas/química , Histonas/genética , Modelos Moleculares , Dados de Sequência Molecular , Nucleossomos/metabolismo , Ligação Proteica , Multimerização Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/genéticaRESUMO
FACT (FAcilitates Chromatin Transcription) has long been considered to be a transcription elongation factor whose ability to destabilize nucleosomes promotes RNAPII progression on chromatin templates. However, this is just one function of this histone chaperone, as FACT also functions in DNA replication. While broadly conserved among eukaryotes and essential for viability in many organisms, dependence on FACT varies widely, with some differentiated cells proliferating normally in its absence. It is therefore unclear what the core functions of FACT are, whether they differ in different circumstances, and what makes FACT essential in some situations but not others. Here, we review recent advances and propose a unifying model for FACT activity. By analogy to DNA repair, we propose that the ability of FACT to both destabilize and assemble nucleosomes allows it to monitor and restore nucleosome integrity as part of a system of chromatin repair, in which disruptions in the packaging of DNA are sensed and returned to their normal state. The requirement for FACT then depends on the level of chromatin disruption occurring in the cell, and the cell's ability to tolerate packaging defects. The role of FACT in transcription would then be just one facet of a broader system for maintaining chromatin integrity.
Assuntos
Montagem e Desmontagem da Cromatina , Reparo do DNA , Proteínas de Ligação a DNA/genética , DNA/genética , Proteínas de Grupo de Alta Mobilidade/genética , Chaperonas de Histonas/genética , Nucleossomos/genética , RNA Polimerase II/genética , Fatores de Elongação da Transcrição/genética , Sítios de Ligação , DNA/química , DNA/metabolismo , Dano ao DNA , Replicação do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/metabolismo , Chaperonas de Histonas/química , Chaperonas de Histonas/metabolismo , Humanos , Modelos Moleculares , Nucleossomos/química , Nucleossomos/metabolismo , Especificidade de Órgãos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Transcrição Gênica , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismoRESUMO
The essential histone chaperone FACT (facilitates chromatin transcription) promotes both nucleosome assembly and disassembly. FACT is a heterodimer of Spt16 with either SSRP1 or Pob3, differing primarily by the presence of a high-mobility group B (HMGB) DNA-binding domain furnished only by SSRP1. Yeast FACT lacks the intrinsic HMGB domain found in SSRP1-based homologs such as human FACT, but yeast FACT activity is supported by Nhp6, which is a freestanding, single HMGB-domain protein. The importance of histone binding by FACT domains has been established, but the roles of DNA-binding activity remain poorly understood. Here, we examined these roles by fusing single or multiple HMGB modules to Pob3 to mimic SSRP1 or to test the effects of extended DNA-binding capacity. Human FACT and a yeast mimic both required Nhp6 to support nucleosome reorganization in vitro, indicating that a single intrinsic DNA-binding HMGB module is insufficient for full FACT activity. Three fused HMGB modules supported activity without Nhp6 assistance, but this FACT variant did not efficiently release from nucleosomes and was toxic in vivo Notably, intrinsic DNA-binding HMGB modules reduced the DNA accessibility and histone H2A-H2B dimer loss normally associated with nucleosome reorganization. We propose that DNA bending by HMGB domains promotes nucleosome destabilization and reorganization by exposing FACT's histone-binding sites, but DNA bending also produces DNA curvature needed to accommodate nucleosome assembly. Intrinsic DNA-bending activity therefore favors nucleosome assembly by FACT over nucleosome reorganization, but excessive activity impairs FACT release, suggesting a quality control checkpoint during nucleosome assembly.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas HMGB/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Chaperonas de Histonas/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Sítios de Ligação , DNA/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/genética , Chaperonas de Histonas/química , Humanos , Modelos Teóricos , Conformação de Ácido Nucleico , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/genéticaRESUMO
Andrews et al. report a comprehensive thermodynamic analysis of nucleosomes in low salt, providing new insights into chromatin assembly and revealing an unexpected mechanism used by the histone chaperone Nap1 to prevent aberrant chromatin formation in vitro and in vivo.
Assuntos
Histonas/metabolismo , Proteína 1 de Modelagem do Nucleossomo/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína 1 de Modelagem do Nucleossomo/genética , Ligação Proteica , Multimerização Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Eukaryotic transcription and mRNA processing depend upon the coordinated interactions of many proteins, including Spn1 and Spt6, which are conserved across eukaryotes, are essential for viability, and associate with each other in some of their biologically important contexts. Here we report crystal structures of the Spn1 core alone and in complex with the binding determinant of Spt6. Mutating interface residues greatly diminishes binding in vitro and causes strong phenotypes in vivo, including a defect in maintaining repressive chromatin. Overexpression of Spn1 partially suppresses the defects caused by an spt6 mutation affecting the Spn1 interface, indicating that the Spn1-Spt6 interaction is important for managing chromatin. Spt6 binds nucleosomes directly in vitro, and this interaction is blocked by Spn1, providing further mechanistic insight into the function of the interaction. These data thereby reveal the structural and biochemical bases of molecular interactions that function in the maintenance of chromatin structure.
Assuntos
Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , Sequência de Aminoácidos , Chaperonas de Histonas , Modelos Moleculares , Dados de Sequência Molecular , Mutação , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismoRESUMO
The proteasome is an abundant protease that is critically important for numerous cellular pathways. Proteasomes are activated in vitro by three known classes of proteins/complexes, including Blm10/PA200. Here, we report a 3.4 A resolution crystal structure of a proteasome-Blm10 complex, which reveals that Blm10 surrounds the proteasome entry pore in the 1.2 MDa complex to form a largely closed dome that is expected to restrict access of potential substrates. This architecture and the observation that Blm10 induces a disordered proteasome gate structure challenge the assumption that Blm10 functions as an activator of proteolysis in vivo. The Blm10 C terminus binds in the same manner as seen for 11S activators and inferred for 19S/PAN activators and indicates a unified model for gate opening. We also demonstrate that Blm10 acts to maintain mitochondrial function. Consistent with the structural data, the C-terminal residues of Blm10 are needed for this activity.
Assuntos
Mitocôndrias/enzimologia , Complexo de Endopeptidases do Proteassoma/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Genótipo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fenótipo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Conformação Proteica , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-AtividadeRESUMO
FACT plays important roles in both gene transcription and DNA replication. However, how this protein complex is targeted to these two distinct cellular processes remains largely unknown. Here we show that ubiquitylation of the Spt16 subunit of FACT by Rtt101, the cullin subunit of an E3 ubiquitin ligase in Saccharomyces cerevisiae, links FACT to DNA replication. We find Rtt101 interacts with and ubiquitylates Spt16 in vitro and in vivo. Deletion of RTT101 leads to reduced association of both FACT and the replicative helicase MCM with replication origins. Loss of Rtt101 also reduces binding of FACT to MCM, but not the association of FACT with Leo1 and Spt5, two proteins involved in transcription. Origin function is compromised in cells lacking Rtt101 or with an Spt16 mutation. These findings identify Spt16 as an Rtt101 substrate, and suggest that Spt16 ubiquitylation is important for FACT to function during DNA replication.
Assuntos
Proteínas Culina/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
FACT has been proposed to function by displacing H2A-H2B dimers from nucleosomes to form hexasomes. Results described here with yeast FACT (yFACT) suggest instead that nucleosomes are reorganized to a form with the original composition but a looser, more dynamic structure. First, yFACT enhances hydroxyl radical accessibility and endonuclease digestion in vitro at sites throughout the nucleosome, not just in regions contacted by H2A-H2B. Accessibility increases dramatically, but the DNA remains partially protected. Second, increased nuclease sensitivity can occur without displacement of dimers from the nucleosome. Third, yFACT is required for eviction of nucleosomes from the GAL1-10 promoter during transcriptional activation in vivo, but the preferential reduction in dimer occupancy expected for hexasome formation is not observed. We propose that yFACT promotes a reversible transition between two nucleosomal forms, and that this activity contributes to the establishment and maintenance of the chromatin barrier as well as to overcoming it.
Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Grupo de Alta Mobilidade/fisiologia , Histonas/metabolismo , Nucleossomos/química , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Fatores de Elongação da Transcrição/fisiologia , DNA Fúngico/química , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Dimerização , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Modelos Genéticos , Modelos Moleculares , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
The correct removal of 5'-flap structures by Rad27 and Dna2 during Okazaki fragment maturation is crucial for the stable maintenance of genetic materials and cell viability. In this study, we identified RAD52, a key recombination protein, as a multicopy suppressor of dna2-K1080E, a lethal helicase-negative mutant allele of DNA2 in yeasts. In contrast, the overexpression of Rad51, which works conjointly with Rad52 in canonical homologous recombination, failed to suppress the growth defect of the dna2-K1080E mutation, indicating that Rad52 plays a unique and distinct role in Okazaki fragment metabolism. We found that the recombination-defective Rad52-QDDD/AAAA mutant did not rescue dna2-K1080E, suggesting that Rad52-mediated recombination is important for suppression. The Rad52-mediated enzymatic stimulation of Dna2 or Rad27 is not a direct cause of suppression observed in vivo, as both Rad52 and Rad52-QDDD/AAAA proteins stimulated the endonuclease activities of both Dna2 and Rad27 to a similar extent. The recombination mediator activity of Rad52 was dispensable for the suppression, whereas both the DNA annealing activity and its ability to interact with Rad59 were essential. In addition, we found that several cohesion establishment factors, including Rsc2 and Elg1, were required for the Rad52-dependent suppression of dna2-K1080E. Our findings suggest a novel Rad52/Rad59-dependent, but Rad51-independent recombination pathway that could ultimately lead to the removal of faulty flaps in conjunction with cohesion establishment factors.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Recombinação Homóloga , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , DNA/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/genética , Immunoblotting , Mutação , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The histone chaperone FACT is an essential and abundant heterodimer found in all eukaryotes. Here we report a crystal structure of the middle domain of the large subunit of FACT (Spt16-M) to reveal a double pleckstrin homology architecture. This structure was found previously in the Pob3-M domain of the small subunit of FACT and in the related histone chaperone Rtt106, although Spt16-M is distinguished from these structures by the presence of an extended α-helix and a C-terminal addition. Consistent with our finding that the double pleckstrin homology structure is common to these three histone chaperones and reports that Pob3 and Rtt106 double pleckstrin homology domains bind histones H3-H4, we also find that Spt16-M binds H3-H4 with low micromolar affinity. Our structure provides a framework for interpreting a large body of genetic data regarding the physiological functions of FACT, including the identification of potential interaction surfaces for binding histones or other proteins.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Grupo de Alta Mobilidade/química , Chaperonas Moleculares/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Fatores de Elongação da Transcrição/química , Animais , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
FACT is a roughly 180 kDa heterodimeric protein complex important for managing the properties of chromatin in eukaryotic cells. Chromatin is a repressive barrier that plays an important role in protecting genomic DNA and regulating access to it. This barrier must be temporarily removed during transcription, replication, and repair, but it also must be rapidly restored to the original state afterwards. Further, the properties of chromatin are dynamic and must be adjusted as conditions dictate. FACT was identified as a factor that destabilizes nucleosomes in vitro, but it has now also been implicated as a central factor in the deposition of histones to form nucleosomes, as an exchange factor that swaps the histones within existing nucleosomes for variant forms, and as a tether that prevents histones from being displaced by the passage of RNA polymerases during transcription. FACT therefore plays central roles in building, maintaining, adjusting. and overcoming the chromatin barrier. This review summarizes recent results that have begun to reveal how FACT can promote what appear to be contradictory goals, using a simple set of binding activities to both enhance and diminish the stability of nucleosomes. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas de Grupo de Alta Mobilidade/fisiologia , Nucleossomos/metabolismo , Fatores de Elongação da Transcrição/fisiologia , Animais , Chaperonas de Histonas/fisiologia , Histonas/fisiologia , Humanos , Modelos Moleculares , RNA Polimerase II/fisiologia , Transcrição Gênica/fisiologiaRESUMO
The 20S proteasome is an essential, 28-subunit protease that sequesters proteolytic sites within a central chamber, thereby repressing substrate degradation until proteasome activators open the entrance/exit gate. Two established activators, Blm10 and PAN/19S, induce gate opening by binding to the pockets between proteasome α-subunits using C-terminal HbYX (hydrophobic-tyrosine-any residue) motifs. Equivalent HbYX motifs have been identified in Pba1 and Pba2, which function in proteasome assembly. Here, we demonstrate that Pba1-Pba2 proteins form a stable heterodimer that utilizes its HbYX motifs to bind mature 20S proteasomes in vitro and that the Pba1-Pba2 HbYX motifs are important for a physiological function of proteasomes, the maintenance of mitochondrial function. Other factors that contribute to proteasome assembly or function also act in the maintenance of mitochondrial function and display complex genetic interactions with one another, possibly revealing an unexpected pathway of mitochondrial regulation involving the Pba1-Pba2 proteasome interaction. Our determination of a proteasome Pba1-Pba2 crystal structure reveals a Pba1 HbYX interaction that is superimposable with those of known activators, a Pba2 HbYX interaction that is different from those reported previously, and a gate structure that is disrupted but not sufficiently open to allow entry of even small peptides. These findings extend understanding of proteasome interactions with HbYX motifs and suggest multiple roles for Pba1-Pba2 interactions throughout proteasome assembly and function.
Assuntos
Complexo de Endopeptidases do Proteassoma/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Cristalografia por Raios X , Ligação de Hidrogênio , Proteínas Imobilizadas/química , Leupeptinas/química , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
FACT is a roughly 180kDa heterodimeric protein complex important for managing the properties of chromatin in eukaryotic cells. Chromatin is a repressive barrier that plays an important role in protecting genomic DNA and regulating access to it. This barrier must be temporarily removed during transcription, replication, and repair, but it also must be rapidly restored to the original state afterwards. Further, the properties of chromatin are dynamic and must be adjusted as conditions dictate. FACT was identified as a factor that destabilizes nucleosomes in vitro, but it has now also been implicated as a central factor in the deposition of histones to form nucleosomes, as an exchange factor that swaps the histones within existing nucleosomes for variant forms, and as a tether that prevents histones from being displaced by the passage of RNA polymerases during transcription. FACT therefore plays central roles in building, maintaining, adjusting, and overcoming the chromatin barrier. This review summarizes recent results that have begun to reveal how FACT can promote what appear to be contradictory goals, using a simple set of binding activities to both enhance and diminish the stability of nucleosomes. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.
RESUMO
PROBLEM: Using pass/fail (P/F) course grades may motivate students to perform well enough to earn a passing grade, giving them a false sense of competence and not motivating them to remediate deficiencies. The authors explored whether adding a not yet pass (NYP) grade to a P/F scale would promote students' mastery orientation toward learning. APPROACH: The authors captured student outcomes and data on time and cost of implementing the NYP grade in 2021 at the University of Utah School of Medicine. One cohort of medical students, who had experienced both P/F and P/NYP/F scales in years 1 and 2, completed an adapted Achievement Goal Questionnaire-Revised (AGQ-R) in fall 2021 to measure how well the P/NYP/F grading scale compared with the P/F scale promoted mastery orientation and performance orientation goals. Students who received an NYP grade provided feedback on the NYP process. OUTCOMES: Students reported that the P/NYP/F scale increased their achievement of both mastery and performance orientation goals, with significantly higher ratings for mastery orientation goals than for performance orientation goals on the AGQ-R (response rate = 124/125 [99%], P ≤ .001, effect size = 0.31). Thirty-eight students received 48 NYP grades in 7 courses during 2021, and 3 (2%) failed a subsequent course after receiving an NYP grade. Most NYP students reported the NYP process enabled them to identify and correct a deficiency (32/36 [89%]) and made them feel supported (28/36 [78%]). The process was time intensive (897 hours total for 48 NYP grades), but no extra funding was budgeted. NEXT STEPS: The findings suggest mastery orientation can be increased with an NYP grade. Implementing a P/NYP/F grading scale for years 1 and/or 2 may help students transition to programmatic assessment or no grading later in medical school, which may better prepare graduates for lifelong learning.
Assuntos
Objetivos , Estudantes de Medicina , Humanos , Faculdades de Medicina , Aprendizagem , MotivaçãoRESUMO
Yeast Hmo1 is a high mobility group B (HMGB) protein that participates in the transcription of ribosomal protein genes and rDNA, and also stimulates the activities of some ATP-dependent remodelers. Hmo1 binds both DNA and nucleosomes and has been proposed to be a functional yeast analog of mammalian linker histones. We used EMSA and single particle Förster resonance energy transfer (spFRET) microscopy to characterize the effects of Hmo1 on nucleosomes alone and with the histone chaperone FACT. Hmo1 induced a significant increase in the distance between the DNA gyres across the nucleosomal core, and also caused the separation of linker segments. This was opposite to the effect of the linker histone H1, which enhanced the proximity of linkers. Similar to Nhp6, another HMGB factor, Hmo1, was able to support large-scale, ATP-independent, reversible unfolding of nucleosomes by FACT in the spFRET assay and partially support FACT function in vivo. However, unlike Hmo1, Nhp6 alone does not affect nucleosome structure. These results suggest physiological roles for Hmo1 that are distinct from Nhp6 and possibly from other HMGB factors and linker histones, such as H1.
Assuntos
Nucleossomos , Proteínas de Saccharomyces cerevisiae , Trifosfato de Adenosina/metabolismo , Animais , DNA Ribossômico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Mamíferos/metabolismo , Nucleossomos/metabolismo , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Elongação da TranscriçãoRESUMO
Introduction: Assessment for learning has many benefits, but learners will still encounter high-stakes decisions about their performance throughout training. It is unknown if assessment for learning can be promoted with a combination model where scores from some assessments are factored into course grades and scores from other assessments are not used for course grading. Methods: At the University of Utah School of Medicine, year 1-2 medical students (MS) completed multiple-choice question quiz assessments and final examinations in six systems-based science courses. Quiz and final examination performance counted toward course grades for MS2017-MS2018. Starting with the MS2020 cohort, quizzes no longer counted toward course grades. Quiz, final examination, and Step 1 scores were compared between ungraded quiz and graded quiz cohorts with independent samples t-tests. Student and faculty feedback was collected. Results: Quiz performance was not different for the ungraded and graded cohorts (p = 0.173). Ungraded cohorts scored 4% higher on final examinations than graded cohorts (p ≤ 0.001, d = 0.88). Ungraded cohorts scored above the national average and 11 points higher on Step 1 compared to graded cohorts, who had scored below the national average (p ≤ 0.001, d = 0.64). During the study period, Step 1 scores increased by 2 points nationally. Student feedback was positive, and faculty felt it improved their relationship with students. Discussion: The change to ungraded quizzes did not negatively affect final examination or Step 1 performance, suggesting a combination of ungraded and graded assessments can effectively promote assessment for learning.
RESUMO
Chromatin-modifying factors regulate both transcription and DNA replication. The yFACT chromatin-reorganizing complex is involved in both processes, and the sensitivity of some yFACT mutants to the replication inhibitor hydroxyurea (HU) is one indication of a replication role. This HU sensitivity can be suppressed by disruptions of the SET2 or CHD1 genes, encoding a histone H3(K36) methyltransferase and a chromatin remodeling factor, respectively. The additive effect of set2 and chd1 mutations in suppressing the HU sensitivity of yFACT mutants suggests that these two factors function in separate pathways. The HU suppression is not an indirect effect of altered regulation of ribonucleotide reductase induced by HU. set2 and chd1 mutations also suppress the HU sensitivity of mutations in other genes involved in DNA replication, including CDC2, CTF4, ORC2, and MEC1. Additionally, a chd1 mutation can suppress the lethality normally caused by disruption of either MEC1 or RAD53 DNA damage checkpoint genes, as well as the lethality seen when a mec1 sml1 mutant is exposed to low levels of HU. The pob3 defect in S-phase progression is suppressed by set2 or chd1 mutations, suggesting that Set2 and Chd1 have specific roles in negatively regulating DNA replication.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/genética , Metiltransferases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Cromatina/genética , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Hidroxiureia/farmacologia , Metiltransferases/metabolismo , Mutagênese , Mutação , Ribonucleosídeo Difosfato Redutase/genética , Fase S , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição GênicaRESUMO
Monoubiquitination of histone H2B (H2B-Ub) plays a role in transcription and DNA replication, and is required for normal localization of the histone chaperone, FACT. In yeast, H2B-Ub is deubiquitinated by Ubp8, a subunit of SAGA, and Ubp10. Although they target the same substrate, loss of Ubp8 and Ubp10 cause different phenotypes and alter the transcription of different genes. We show that Ubp10 has poor activity on yeast nucleosomes, but that the addition of FACT stimulates Ubp10 activity on nucleosomes and not on other substrates. Consistent with a role for FACT in deubiquitinating H2B in vivo, a FACT mutant strain shows elevated levels of H2B-Ub. Combination of FACT mutants with deletion of Ubp10, but not Ubp8, confers increased sensitivity to hydroxyurea and activates a cryptic transcription reporter, suggesting that FACT and Ubp10 may coordinate nucleosome assembly during DNA replication and transcription. Our findings reveal unexpected interplay between H2B deubiquitination and nucleosome dynamics.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Alelos , Replicação do DNA/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Hidroxiureia/farmacologia , Mutação/genética , Nucleossomos/efeitos dos fármacos , Fenótipo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Transcrição Gênica/efeitos dos fármacos , Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
FACT (FAcilitates Chromatin Transcription/Transactions) is a histone chaperone that can destabilize or assemble nucleosomes. Acetylation of histone H3-K56 weakens a histone-DNA contact that is central to FACT activity, suggesting that this modification could affect FACT functions. We tested this by asking how mutations of H3-K56 and FACT affect nucleosome reorganization activity in vitro, and chromatin integrity and transcript output in vivo Mimics of unacetylated or permanently acetylated H3-K56 had different effects on FACT activity as expected, but the same mutations had surprisingly similar effects on global transcript levels. The results are consistent with emerging models that emphasize FACT's importance in establishing global chromatin architecture prior to transcription, promoting transitions among different states as transcription profiles change, and restoring chromatin integrity after it is disturbed. Optimal FACT activity required the availability of both modified and unmodified states of H3-K56. Perturbing this balance was especially detrimental for maintaining repression of genes with high nucleosome occupancy over their promoters and for blocking antisense transcription at the +1 nucleosome. The results reveal a complex collaboration between H3-K56 modification status and multiple FACT functions, and support roles for nucleosome reorganization by FACT before, during, and after transcription.