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

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

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

Physical interactions between MCM and Rad51 facilitate replication fork lesion bypass and ssDNA gap filling by non-recombinogenic functions

The minichromosome maintenance (MCM) helicase physically interacts with the recombination proteins Rad51 and Rad52 from yeast to human cells. We show, in Saccharomyces cerevisiae, that these interactions occur within a nuclease-insoluble scaffold enriched in replication/repair factors. Rad51 accumulates in a MCM- and DNA-binding-independent manner and interacts with MCMhelicases located outside of the replication origins and forks. MCM, Rad51, and Rad52 accumulate in this scaffold in G1 and are released during the S phase. In the presence of replication-blocking lesions, Cdc7 prevents their release from the scaffold, thus maintaining the interactions. We identify a rad51 mutant that is impaired in its ability to bind to MCM but not to the scaffold. This mutant is proficient in recombination but partially defective in single-stranded DNA (ssDNA) gap filling and replication fork progression through damaged DNA. Therefore, cells accumulate MCM/Rad51/Rad52 complexes at specific nuclear scaffolds in G1 to assist stressed forks through non-recombinogenic functions.Cancer Signaling networks and Molecular Therapeutic

Yeast arginine methyltransferase Hmt1p regulates transcription elongation and termination by methylating Npl3p

The heterogeneous nuclear ribonucleoprotein Npl3p of budding yeast is a substrate of arginine methyltransferase Hmt1p, but the role of Hmt1p in regulating Npl3p’s functions in transcription antitermination and elongation were unknown. We found that mutants lacking Hmt1p methyltransferase activity exhibit reduced recruitment of Npl3p, but elevated recruitment of a component of mRNA cleavage/termination factor CFI, to the activated GAL10-GAL7 locus. Consistent with this, hmt1 mutants displayed increased termination at the defective gal10-Δ56 terminator. Remarkably, hmt1Δ cells also exhibit diminished recruitment of elongation factor Tho2p and a reduced rate of transcription elongation in vivo. Importantly, the defects in Npl3p and Tho2p recruitment, antitermination and elongation in hmt1Δ cells all were mitigated by substitutions in Npl3p RGG repeats that functionally mimic arginine methylation by Hmt1p. Thus, Hmt1p promotes elongation and suppresses termination at cryptic terminators by methylating RGG repeats in Npl3p. As Hmt1p stimulates dissociation of Tho2p from an Npl3p-mRNP complex, it could act to recycle these elongation and antitermination factors back to sites of ongoing transcription

p53 Interacts with RNA Polymerase II through Its Core Domain and Impairs Pol II Processivity In Vivo

The tumor suppressor p53 principally functions as a gene-specific transcription factor. p53 triggers a variety of anti-proliferative programs by activating or repressing the transcription of effector genes in response to genotoxic stress. To date, much effort has been placed on understanding p53's ability to affect transcription in the context of its DNA-binding activity. How p53 regulates transcriptional output independent of DNA binding is less well understood. Here we provide evidence that human p53 can physically interact with the large subunit of RNA polymerase II (Pol II) both in in vitro interaction assays and in whole cell extracts, and that this interaction is mediated (at least in part) through p53's core DNA-binding domain and the Ser5-phosphorylated CTD of Pol II. Ectopic expression of p53, combined with mutations in transcription elongation factors or exposure to drugs that inhibit Pol II elongation, elicit sickness or lethality in yeast cells. These phenotypes are suppressed by oncogenic point mutations within p53's core domain. The growth phenotypes raise the possibility that p53 impairs Pol II elongation. Consistent with this, a p53-dependent increase in Pol II density is seen at constitutively expressed genes without a concomitant increase in transcript accumulation. Additionally, p53-expressing yeast strains exhibit reduced transcriptional processivity at an episomal reporter gene; this inhibitory activity is abolished by a core domain point mutation. Our results suggest a novel mechanism by which p53 can regulate gene transcription, and a new biological function for its core domain that is susceptible to inactivation by oncogenic point mutations