RESUMEN
DNA double-strand breaks (DSBs) are among the deleterious lesions that are both endogenous and exogenous in origin and are repaired by nonhomologous end joining or homologous recombination. However, the molecular mechanisms responsible for maintaining genome stability remain incompletely understood. Here, we investigate the role of two E3 ligases, Dma1 and Dma2 (homologs of human RNF8), in the maintenance of genome stability in budding yeast. Using yeast spotting assays, chromatin immunoprecipitation and plasmid and chromosomal repair assays, we establish that Dma1 and Dma2 act in a redundant and a catalysis-dependent manner in the maintenance of genome stability, as well as localize to transcribed regions of the genome and increase in abundance upon phleomycin treatment. In addition, Dma1 and Dma2 are required for the normal kinetics of histone H4 acetylation under DNA damage conditions, genetically interact with RAD9 and SAE2, and are in a complex with Rad53 and histones. Taken together, our results demonstrate the requirement of Dma1 and Dma2 in regulating DNA repair pathway choice, preferentially affecting homologous recombination over nonhomologous end joining, and open up the possibility of using these candidates in manipulating the repair pathways toward precision genome editing.
Asunto(s)
Biocatálisis , Proteínas de Ciclo Celular/metabolismo , Inestabilidad Genómica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Reparación del ADNRESUMEN
Determining structures of protein complexes is crucial for understanding cellular functions. Here, we describe an integrative structure determination approach that relies on in vivo measurements of genetic interactions. We construct phenotypic profiles for point mutations crossed against gene deletions or exposed to environmental perturbations, followed by converting similarities between two profiles into an upper bound on the distance between the mutated residues. We determine the structure of the yeast histone H3-H4 complex based on ~500,000 genetic interactions of 350 mutants. We then apply the method to subunits Rpb1-Rpb2 of yeast RNA polymerase II and subunits RpoB-RpoC of bacterial RNA polymerase. The accuracy is comparable to that based on chemical cross-links; using restraints from both genetic interactions and cross-links further improves model accuracy and precision. The approach provides an efficient means to augment integrative structure determination with in vivo observations.
Asunto(s)
Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Mapas de Interacción de Proteínas/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Histonas/química , Histonas/genética , Mutación , Conformación Proteica , Mapeo de Interacción de Proteínas , Saccharomyces cerevisiae/genéticaRESUMEN
A hallmark of human immunodeficiency type-1 (HIV) infection is the integration of the viral genome into host chromatin, resulting in a latent reservoir that persists despite antiviral therapy or immune response. Thus, key priorities toward eradication of HIV infection are to understand the mechanisms that allow HIV latency and to develop latency reversal agents (LRAs) that can facilitate the clearance of latently infected cells. The repressive H3K27me3 histone mark, catalyzed by the PRC2 complex, plays a pivotal role in transcriptional repression at the viral promoter in both cell line and primary CD4+ T cell models of latency. EZH2 inhibitors which block H3K27 methylation have been shown to act as LRAs, suggesting other PRC2 components could also be potential targets for latency reversal. EED, a core component of PRC2, ensures the propagation of H3K27me3 by allosterically activating EZH2 methyltransferase activity. Therefore, we sought to investigate if inhibition of EED would also reverse latency. Inhibitors of EED, EED226 and A-395, demonstrated latency reversal activity as single agents, and this activity was further enhanced when used in combination with other known LRAs. Loss of H3K27me3 following EED inhibition significantly increased the levels of H3K27 acetylation globally and at the HIV LTR. These results further confirm that PRC2 mediated repression plays a significant role in the maintenance of HIV latency and suggest that EED may serve as a promising new target for LRA development.
Asunto(s)
Infecciones por VIH , Complejo Represivo Polycomb 2 , Infecciones por VIH/tratamiento farmacológico , Histonas/metabolismo , Humanos , Metilación , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Metabolic signaling to chromatin often underlies how adaptive transcriptional responses are controlled. While intermediary metabolites serve as co-factors for histone-modifying enzymes during metabolic flux, how these modifications contribute to transcriptional responses is poorly understood. Here, we utilize the highly synchronized yeast metabolic cycle (YMC) and find that fatty acid ß-oxidation genes are periodically expressed coincident with the ß-oxidation byproduct histone crotonylation. Specifically, we found that H3K9 crotonylation peaks when H3K9 acetylation declines and energy resources become limited. During this metabolic state, pro-growth gene expression is dampened; however, mutation of the Taf14 YEATS domain, a H3K9 crotonylation reader, results in de-repression of these genes. Conversely, exogenous addition of crotonic acid results in increased histone crotonylation, constitutive repression of pro-growth genes, and disrupted YMC oscillations. Together, our findings expose an unexpected link between metabolic flux and transcription and demonstrate that histone crotonylation and Taf14 participate in the repression of energy-demanding gene expression.
Asunto(s)
Acilcoenzima A/metabolismo , Metabolismo Energético , Regulación Fúngica de la Expresión Génica , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factor de Transcripción TFIID/metabolismo , Metabolismo Energético/genética , Ácidos Grasos/metabolismo , Histonas/genética , Homeostasis , Lisina , Oxidación-Reducción , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Factor de Transcripción TFIID/genética , Transcripción GenéticaRESUMEN
Spt6 is a histone chaperone that associates with RNA polymerase II and deposits nucleosomes in the wake of transcription. Although Spt6 has an essential function in nucleosome deposition, it is not known whether this function is influenced by post-translational modification. Here, we report that casein kinase II (CKII) phosphorylation of Spt6 is required for nucleosome occupancy at the 5' ends of genes to prevent aberrant antisense transcription and enforce transcriptional directionality. Mechanistically, we show that CKII phosphorylation of Spt6 promotes the interaction of Spt6 with Spn1, a binding partner required for chromatin reassembly and full recruitment of Spt6 to genes. Our study defines a function for CKII phosphorylation in transcription and highlights the importance of post-translational modification in histone chaperone function.
Asunto(s)
Quinasa de la Caseína II/metabolismo , Chaperonas de Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transcripción Genética , Factores de Elongación Transcripcional/metabolismo , Cromatina/metabolismo , Genoma Fúngico , Chaperonas de Histonas/química , Modelos Biológicos , Nucleosomas/metabolismo , Fosforilación , Unión Proteica , Proteínas de Saccharomyces cerevisiae/química , Factores de Elongación Transcripcional/químicaRESUMEN
Spt6 is an essential histone chaperone that mediates nucleosome reassembly during gene transcription. Spt6 also associates with RNA polymerase II (RNAPII) via a tandem Src2 homology domain. However, the significance of Spt6-RNAPII interaction is not well understood. Here, we show that Spt6 recruitment to genes and the nucleosome reassembly functions of Spt6 can still occur in the absence of its association with RNAPII. Surprisingly, we found that Spt6-RNAPII association is required for efficient recruitment of the Ccr4-Not de-adenylation complex to transcribed genes for essential degradation of a range of mRNAs, including mRNAs required for cell-cycle progression. These findings reveal an unexpected control mechanism for mRNA turnover during transcription facilitated by a histone chaperone.
Asunto(s)
Chaperonas de Histonas/metabolismo , ARN Polimerasa II/metabolismo , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Elongación Transcripcional/metabolismo , Chaperonas de Histonas/genética , Histonas/genética , Histonas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , ARN Polimerasa II/genética , Estabilidad del ARN , ARN Mensajero/genética , Elementos Reguladores de la Transcripción , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genética , Transcripción Genética , Factores de Elongación Transcripcional/genéticaRESUMEN
Methylation of histone H3 lysine 36 (H3K36me) by yeast Set2 is critical for the maintenance of chromatin structure and transcriptional fidelity. However, we do not know the full range of Set2/H3K36me functions or the scope of mechanisms that regulate Set2-dependent H3K36 methylation. Here, we show that the APC/CCDC20 complex regulates Set2 protein abundance during the cell cycle. Significantly, absence of Set2-mediated H3K36me causes a loss of cell cycle control and pronounced defects in the transcriptional fidelity of cell cycle regulatory genes, a class of genes that are generally long, hence highly dependent on Set2/H3K36me for their transcriptional fidelity. Because APC/C also controls human SETD2, and SETD2 likewise regulates cell cycle progression, our data imply an evolutionarily conserved cell cycle function for Set2/SETD2 that may explain why recurrent mutations of SETD2 contribute to human disease.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/genética , Ciclo Celular/genética , Regulación Fúngica de la Expresión Génica , Metiltransferasas/genética , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transcripción Genética , Evolución Biológica , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Ciclo Celular/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilación , Metiltransferasas/metabolismo , Nocodazol/farmacología , Proteolisis , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Moduladores de Tubulina/farmacologíaRESUMEN
H2A.Z is a histone H2A variant that contributes to transcriptional regulation, DNA damage response and limits heterochromatin spreading. In Saccharomyces cerevisiae, H2A.Z is deposited by the SWR-C complex, which relies on several histone chaperones including Nap1 and Chz1 to deliver H2A.Z-H2B dimers to SWR-C. However, the mechanisms by which Nap1 and Chz1 cooperate to bind H2A.Z and their contribution to H2A.Z deposition in chromatin is not well understood. Using structural modeling and molecular dynamics simulations, we identify a series of H2A.Z residues that form a chaperone-specific binding surface. Mutation of these residues revealed different surface requirements for Nap1 and Chz1 interaction with H2A.Z. Consistent with this result, we found that loss of Nap1 or Chz1 individually resulted in mild defects in H2A.Z deposition, but that deletion of both Nap1 and Chz1 resulted in a significant reduction of H2A.Z deposition at promoters and led to heterochromatin spreading. Together, our findings reveal unique H2A.Z surface dependences for Nap1 and Chz1 and a redundant role for these chaperones in H2A.Z deposition.
Asunto(s)
Chaperonas de Histonas/metabolismo , Proteína 1 de Ensamblaje de Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Chaperonas de Histonas/química , Chaperonas de Histonas/genética , Modelos Moleculares , Proteína 1 de Ensamblaje de Nucleosomas/química , Proteína 1 de Ensamblaje de Nucleosomas/genética , Unión Proteica , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Relación Estructura-ActividadRESUMEN
Set2-mediated histone methylation at H3K36 regulates diverse activities, including DNA repair, mRNA splicing, and suppression of inappropriate (cryptic) transcription. Although failure of Set2 to suppress cryptic transcription has been linked to decreased lifespan, the extent to which cryptic transcription influences other cellular functions is poorly understood. Here, we uncover a role for H3K36 methylation in the regulation of the nutrient stress response pathway. We found that the transcriptional response to nutrient stress was dysregulated in SET2-deleted (set2Δ) cells and was correlated with genome-wide bi-directional cryptic transcription that originated from within gene bodies. Antisense transcripts arising from these cryptic events extended into the promoters of the genes from which they arose and were associated with decreased sense transcription under nutrient stress conditions. These results suggest that Set2-enforced transcriptional fidelity is critical to the proper regulation of inducible and highly regulated transcription programs.
Asunto(s)
Procesamiento Proteico-Postraduccional/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcripción Genética/genética , MetilaciónRESUMEN
Using affinity purification MS approaches, we have identified a novel role for casein kinase II (CKII) in the modification of the polymerase associated factor complex (PAF-C). Our data indicate that the facilitates chromatin transcription complex (FACT) interacts with CKII and may facilitate PAF complex phosphorylation. Posttranslational modification analysis of affinity-isolated PAF-C shows extensive CKII phosphorylation of all five subunits of PAF-C, although CKII subunits were not detected as interacting partners. Consistent with this, recombinant CKII or FACT-associated CKII isolated from cells can phosphorylate PAF-C in vitro, whereas no intrinsic kinase activity was detected in PAF-C samples. Significantly, PAF-C purifications combined with stable isotope labeling in cells (SILAC) quantitation for PAF-C phosphorylation from wild-type and CKII temperature-sensitive strains (cka1Δ cka2-8) showed that PAF-C phosphorylation at consensus CKII sites is significantly reduced in cka1Δ cka2-8 strains. Consistent with a role of CKII in FACT and PAF-C function, we show that decreased CKII function in vivo results in decreased levels of histone H2B lysine 123 monoubiquitylation, a modification dependent on FACT and PAF-C. Taken together, our results define a coordinated role of CKII and FACT in the regulation of RNA polymerase II transcription through chromatin via phosphorylation of PAF-C.
Asunto(s)
Quinasa de la Caseína II/metabolismo , Histonas/metabolismo , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcripción Genética/fisiología , Ubiquitinación/fisiología , Quinasa de la Caseína II/genética , Cromatina/genética , Cromatina/metabolismo , Histonas/genética , Fosforilación/fisiología , ARN Polimerasa II/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiaeRESUMEN
The YEATS domain, found in a number of chromatin-associated proteins, has recently been shown to have the capacity to bind histone lysine acetylation. Here, we show that the YEATS domain of Taf14, a member of key transcriptional and chromatin-modifying complexes in yeast, is a selective reader of histone H3 Lys9 acetylation (H3K9ac). Structural analysis reveals that acetylated Lys9 is sandwiched in an aromatic cage formed by F62 and W81. Disruption of this binding in cells impairs gene transcription and the DNA damage response. Our findings establish a highly conserved acetyllysine reader function for the YEATS domain protein family and highlight the significance of this interaction for Taf14.
Asunto(s)
Reparación del ADN/genética , Regulación Fúngica de la Expresión Génica/genética , Histonas/metabolismo , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Factor de Transcripción TFIID/metabolismo , Acetilación , Daño del ADN , Histonas/química , Histonas/genética , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismoRESUMEN
The C-terminal domain (CTD) of RNA polymerase II is sequentially modified for recruitment of numerous accessory factors during transcription. One such factor is Spt6, which couples transcription elongation with histone chaperone activity and the regulation of H3 lysine 36 methylation. Here, we show that CTD association of Spt6 is required for Ser2 CTD phosphorylation and for the protein stability of Ctk1 (the major Ser2 CTD kinase). We also find that Spt6 associates with Ctk1, and, unexpectedly, Ctk1 and Ser2 CTD phosphorylation are required for the stability of Spt6-thus revealing a Spt6-Ctk1 feed-forward loop that robustly maintains Ser2 phosphorylation during transcription. In addition, we find that the BUR kinase and the polymerase associated factor transcription complex function upstream of the Spt6-Ctk1 loop, most likely by recruiting Spt6 to the CTD at the onset of transcription. Consistent with requirement of Spt6 in histone gene expression and nucleosome deposition, mutation or deletion of members of the Spt6-Ctk1 loop leads to global loss of histone H3 and sensitivity to hydroxyurea. In sum, these results elucidate a new control mechanism for the regulation of RNAPII CTD phosphorylation during transcription elongation that is likely to be highly conserved.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Proteínas Nucleares/metabolismo , Proteínas Quinasas/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Elongación de la Transcripción Genética , Factores de Elongación Transcripcional/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Chaperonas de Histonas , Fosforilación , Estabilidad Proteica , Estructura Terciaria de Proteína , ARN Polimerasa II/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Chromatin structure regulates the dynamics of the recognition and repair of DNA double strand breaks; open chromatin enhances the recruitment of DNA damage response factors, while compact chromatin is refractory to the assembly of radiation-induced repair foci. MU2, an orthologue of human MDC1, a scaffold for ionizing radiation-induced repair foci, is a widely distributed chromosomal protein in Drosophila melanogaster that moves to DNA repair foci after irradiation. Here we show using yeast 2 hybrid screens and co-immunoprecipitation that MU2 binds the chromoshadow domain of the heterochromatin protein HP1 in untreated cells. We asked what role HP1 plays in the formation of repair foci and cell cycle control in response to DNA damage. After irradiation repair foci form in heterochromatin but are shunted to the edge of heterochromatic regions an HP1-dependent manner, suggesting compartmentalized repair. Hydroxyurea-induced repair foci that form at collapsed replication forks, however, remain in the heterochromatic compartment. HP1a depletion in irradiated imaginal disc cells increases apoptosis and disrupts G2/M arrest. Further, cells irradiated in mitosis produced more and brighter repair foci than to cells irradiated during interphase. Thus, the interplay between MU2 and HP1a is dynamic and may be different in euchromatin and heterochromatin during DNA break recognition and repair.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Ciclo Celular/genética , Ciclo Celular/fisiología , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Reparación del ADN/genética , Reparación del ADN/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Inmunoprecipitación , Técnicas del Sistema de Dos HíbridosRESUMEN
Telomere capture, a rare event that stabilizes chromosome breaks, is associated with certain genetic abnormalities in humans. Studies pertaining to the generation, maintenance, and biological effects of telomere formation are limited in metazoans. A mutation, mu2(a), in Drosophila melanogaster decreases the rate of repair of double strand DNA breaks in oocytes, thus leading to chromosomes that have lost a natural telomere and gained a new telomere. Amino acid sequence, domain architecture, and protein interactions suggest that MU2 is an ortholog of human MDC1. The MU2 protein is a component of meiotic recombination foci and localizes to repair foci in S2 cells after irradiation in a manner similar to that of phosphorylated histone variant H2Av. Domain searches indicated that the protein contains an N-terminal FHA domain and a C-terminal tandem BRCT domain. Peptide pull-down studies showed that the BRCT domain interacts with phosphorylated H2Av, while the FHA domain interacts with the complex of MRE11, RAD50, and NBS. A frameshift mutation that eliminates the MU2 BRCT domain decreases the number and size of meiotic phospho-H2Av foci. MU2 is also required for the intra-S checkpoint in eye-antennal imaginal discs. MU2 participates at an early stage in the recognition of DNA damage at a step that is prerequisite for both DNA repair and cell cycle checkpoint control. We propose a model suggesting that neotelomeres may arise when radiation-induced chromosome breaks fail to be repaired, fail to arrest progression through meiosis, and are deposited in the zygote, where cell cycle control is absent and rapid rounds of replication and telomere formation ensue.