Crosstalk between chromatin structure, cohesin activity and transcription

Background: A complex interplay between chromatin and topological machineries is critical for genome architec‑ ture and function. However, little is known about these reciprocal interactions, even for cohesin, despite its multiple roles in DNA metabolism. Results: We have used genome‑wide analyses to address how cohesins and chromatin structure impact each other in yeast. Cohesin inactivation in scc1‑73 mutants during the S and G2 phases causes specific changes in chromatin structure that preferentially take place at promoters; these changes include a significant increase in the occupancy of the − 1 and + 1 nucleosomes. In addition, cohesins play a major role in transcription regulation that is associated with specific promoter chromatin architecture. In scc1‑73 cells, downregulated genes are enriched in promoters with short or no nucleosome‑free region (NFR) and a fragile “nucleosome − 1/RSC complex” particle. These results, together with a preferential increase in the occupancy of nucleosome − 1 of these genes, suggest that cohesins promote transcription activation by helping RSC to form the NFR. In sharp contrast, the scc1‑73 upregulated genes are enriched in promoters with an “open” chromatin structure and are mostly at cohesin‑enriched regions, suggesting that a local accumulation of cohesins might help to inhibit transcription. On the other hand, a dramatic loss of chromatin integrity by histone depletion during DNA replication has a moderate effect on the accumulation and distribution of cohesin peaks along the genome. Conclusions: Our analyses of the interplay between chromatin integrity and cohesin activity suggest that cohesins play a major role in transcription regulation, which is associated with specific chromatin architecture and cohesin‑ mediated nucleosome alterations of the regulated promoters. In contrast, chromatin integrity plays only a minor role in the binding and distribution of cohesins.Spanish Ministry of Economy and Competitivenes BFU2012-38171, BFU2015-63698-PAndalusian Government P12-CTS-227

TFIIS is required for the balanced expression of the genes encoding ribosomal components under transcriptional stress

Transcription factor IIS (TFIIS) stimulates RNA cleavage by RNA polymerase II by allowing backtracked enzymes to resume transcription elongation. Yeast cells do not require TFIIS for viability, unless they suffer severe transcriptional stress due to NTP-depleting drugs like 6-azauracil or mycophenolic acid. In order to broaden our knowledge on the role of TFIIS under transcriptional stress, we carried out a genetic screening for suppressors of TFIIS-lacking cells’ sensitivity to 6-azauracil and mycophenolic acid. Five suppressors were identified, four of which were related to the transcriptional regulation of those genes encoding ribosomal components [rRNAs and ribosomal proteins (RP)], including global regulator SFP1. This led us to discover that RNA polymerase II is hypersensitive to the absence of TFIIS under NTP scarcity conditions when transcribing RP genes. The absence of Sfp1 led to a profound alteration of the transcriptional response to NTP-depletion, thus allowing the expression of RP genes to resist these stressful conditions in the absence of TFIIS. We discuss the effect of transcriptional stress on ribosome biogenesis and propose that TFIIS contributes to prevent a transcriptional imbalance between rDNA and RP genes.España Ministerio de Economía y competitividad BFU2007-67575-C03-02España Ministerio de Economía y competitividad BFU-2010-21975-C03-03Andalucía, Junta de Andalucía P07-CVI-02623Andalucía, Junta de Andalucía P08-CVI-035

Use of Arctium lappa Extract Against Acetaminophen-Induced Hepatotoxicity in Rats

AbstractBackgroundSevere destructive hepatic injuries can be induced by acetaminophen overdose and may lead to acute hepatic failure.ObjectiveTo investigate the ameliorative effects of Arctium lappa root extract on acetaminophen-induced hepatotoxicity.MethodsRats were divided into 4 groups: normal control group, Arctium lappa extract group, acetaminophen-injected group, and acetaminophen treated with Arctium lappa extract group.ResultsThe treatment with Arctium lappa extract reduced serum alanine transaminase, aspartate aminotransferase, and alkaline phosphatase in the acetaminophen group when compared with the control group. DNA fragments in the acetaminophen-injected group were also significantly increased (P < 0.05). The comet assay revealed increased detaching tail length and DNA concentration during the hepatic toxicity in the acetaminophen group. The malondialdehyde content was inhibited by Arctium lappa treatment (12.97±0.89 nmol/mg) when compared with the acetaminophen-treated-only group (12.97±0.89 nmol/mg). Histopathologic examination revealed that acetaminophen administration produced hepatic cell necrosis, infiltrate of lymphocytes, and vacuolation that were associated with the acetaminophen-treated animal group, but the degree of acetaminophen-induced hepatotoxicity was mediated by treatment with Arctium lappa extract.ConclusionsArctium lappa can prevent most of the hepatic tissue damage caused by acetaminophen overdose in rats

The ribosome assembly gene network is controlled by the feedback regulation of transcription elongation

Ribosome assembly requires the concerted expression of hundreds of genes, which are transcribed by all three nuclear RNA polymerases. Transcription elongation involves dynamic interactions between RNA polymerases and chromatin. We performed a synthetic lethal screening in Saccharomyces cerevisiae with a conditional allele of SPT6, which encodes one of the factors that facilitates this process. Some of these synthetic mutants corresponded to factors that facilitate pre-rRNA processing and ribosome biogenesis. We found that the in vivo depletion of one of these factors, Arb1, activated transcription elongation in the set of genes involved directly in ribosome assembly. Under these depletion conditions, Spt6 was physically targeted to the upregulated genes, where it helped maintain their chromatin integrity and the synthesis of properly stable mRNAs. The mRNA profiles of a large set of ribosome biogenesismutants confirmed the existence of a feedback regulatory network among ribosome assembly genes. The transcriptional response in this network depended on both the specific malfunction and the role of the regulated gene. In accordance with our screening, Spt6 positively contributed to the optimal operation of this global network. On the whole, this work uncovers a feedback control of ribosome biogenesis by fine-tuning transcription elongation in ribosome assembly factor-coding genes.Ministerio de Economía y Competitividad BFU2013-48643-C3-1-P, BFU2016-77728-C3-1-P, BFU2013-48643-C3- 3-P, BFU2013-42958-PJunta de Andalucía P12-BIO1938MO, P08-CVI-03508Comunidad Valenciana 2015/00

The SWR1 Histone Replacement Complex Causes Genetic Instability and Genome-Wide Transcription Misregulation in the Absence of H2A.Z

The SWR1 complex replaces the canonical histone H2A with the variant H2A.Z (Htz1 in yeast) at specific chromatin regions. This dynamic alteration in nucleosome structure provides a molecular mechanism to regulate transcription, gene silencing, chromosome segregation and DNA repair. Here we show that genetic instability, sensitivity to drugs impairing different cellular processes and genome-wide transcriptional misregulation in htz1Δ can be partially or totally suppressed if SWR1 is not formed (swr1Δ), if it forms but cannot bind to chromatin (swc2Δ) or if it binds to chromatin but lacks histone replacement activity (swc5Δ and the ATPase-dead swr1-K727G). These results suggest that in htz1Δ the nucleosome remodelling activity of SWR1 affects chromatin integrity because of an attempt to replace H2A with Htz1 in the absence of the latter. This would impair transcription and, either directly or indirectly, other cellular processes. Specifically, we show that in htz1Δ, the SWR1 complex causes an accumulation of recombinogenic DNA damage by a mechanism dependent on phosphorylation of H2A at Ser129, a modification that occurs in response to DNA damage, suggesting that the SWR1 complex impairs the repair of spontaneous DNA damage in htz1Δ. In addition, SWR1 causes DSBs sensitivity in htz1Δ; consistently, in the absence of Htz1 the SWR1 complex bound near an endonuclease HO-induced DSB at the mating-type (MAT) locus impairs DSB-induced checkpoint activation. Our results support a stepwise mechanism for the replacement of H2A with Htz1 and demonstrate that a tight control of this mechanism is essential to regulate chromatin dynamics but also to prevent the deleterious consequences of an incomplete nucleosome remodelling

FACT Prevents the Accumulation of Free Histones Evicted from Transcribed Chromatin and a Subsequent Cell Cycle Delay in G1

The FACT complex participates in chromatin assembly and disassembly during transcription elongation. The yeast mutants affected in the SPT16 gene, which encodes one of the FACT subunits, alter the expression of G1 cyclins and exhibit defects in the G1/S transition. Here we show that the dysfunction of chromatin reassembly factors, like FACT or Spt6, down-regulates the expression of the gene encoding the cyclin that modulates the G1 length (CLN3) in START by specifically triggering the repression of its promoter. The G1 delay undergone by spt16 mutants is not mediated by the DNA–damage checkpoint, although the mutation of RAD53, which is otherwise involved in histone degradation, enhances the cell-cycle defects of spt16-197. We reveal how FACT dysfunction triggers an accumulation of free histones evicted from transcribed chromatin. This accumulation is enhanced in a rad53 background and leads to a delay in G1. Consistently, we show that the overexpression of histones in wild-type cells down-regulates CLN3 in START and causes a delay in G1. Our work shows that chromatin reassembly factors are essential players in controlling the free histones potentially released from transcribed chromatin and describes a new cell cycle phenomenon that allows cells to respond to excess histones before starting DNA replication

Elongación transcripcional en Saccharomyces Cerevisiae influencia en el control del ciclo celular y nuevas herramientas para su estudio "in vivo"

La transcripción es el proceso mediante el cual la información genética, contenida en el DNA, es expresada en forma de RNA. La mayor parte de los genes eucarióticos, tanto aquellos que codifican proteínas como la mayoría de los RNA nucleares peque&ntild e;os (snRNA), son transcritos por una maquinaria cuya pieza fundamental es la RNA polimerasa II (RNAPII).La transcripción por parte de la RNAPII comprende varias fases: iniciación, elongación y terminación. La producción de un RNA mensajero (mRNA) maduro requiere además el procesamientod el RNA transcrito para ser finalmente transportado al citoplasma, donde se llevará a cabo su traducción a proteína. Todos estos procesos: transcripción, procesamiento y transporte al citoplasma no son independientes, sino que existe una coordinación entre ellos (Burckin et al., 2005), de forma que desde los primeros estadíos de la transcripción factores implicados en el procesamiento y transporte del RNa al citoplasma se asocian a la maquinaria transcripcional (Aguilera, 2005; Maniatis and Reed, 2002).La maquinaria transcripcional está bastante conservada en todos los eucariotas desde levaduras hasta humanos. La RNAPII está formada por doce subunidades codificadas por los genes RPB1 a RPB12. Todos ellos son esenciales excepto RPB4 y RPB9 (Woychik and Young, 1989).Las subunidades Rpb1 y Rpb2 son las de mayor tamaño y las más conservadas evolutivamente. La subunidad Rpb1 posee un dominio carboxilo terminal (CTD, del inglés "carboxi-terminal-domain" ), exclusivo de la RNAPII. Este CTD es esencial y desempeña un papel fundamental tanto en la regulación de la transcripción como en la coordinación entre la transcripción y procesos postranscripcionales. Consiste en una serie de repeticiones en tándem de heptapéptido Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Dicha secuencia está conservada en todos los eucariotas, aunque varia el número de repeticiones (26 en Saccharomyces cerevisiaey 52 en humanos). Dos de las serinas del heptapéptido (ser2 y Ser5) están sujetas a fosforilaciones reversibles durante el ciclo de la transcripción (Dahmus, 1996).Rpb9 desempeña un importante papel en la regulación de la elongación transcirpcional "in vivo" , así se han encontrado mutantes de RPB9 sensibles a 6-azauracilo (Hemming et al., 2000), droga que inhibe la síntesis de nucleótidos, produce un descenso del nivel de nucleótidos disponibles (Exinger and Lacroute, 1992) y en consecuencia afecta a la elongación transcripcional, de forma que la sensibilidad a la misma indica frecuentemente defecto en elongación transcripcional. La subunidad Rpb9 parece mediar el papel del factor TFIIS en la superación de las situacones de bloqueo que sufre la RNAPII. Además, la deleción de RPB9 presenta letalidad sintética con la deleción del gen que codifica la subunidad acetiltransferasa del elongador (Elp3) y la subunidad desacetilasa del complejo SAGA (Gcn5) (Van Mullem et al., 2002). También se ha descrito que Rpb9 juega un papel fundamental en la fidelidad de la RNAPII (Nesser et al., 2006).En esta Tenis nos hemos planteado dos objetivos principales:1. La construcción y puesta a punto de una herramienta para la medida de la eficiencia de la elongación transcripcional "in vivo" .2. El estudio de la influencia del estado de la elongación transcripcional sobre la progresión del ciclo celular en la transición G1-S. este segundo objetivo se ha abordado desde dos puntos de vista: 2.1. El estudio del mecanismo responsable del bloqueo en G1 experimentado por el mutante spt16-197, afectado en una subunidad del complejo FACT, a temperatura restrictiva. 2.2. El estudio del efecto sobre la progresión del ciclo celular en la transición G1-S de drogas inhibidoras de la elongación transcripcional