Publications by authors named "Miriam Sansó"

24 Publications

  • Page 1 of 1

Distinct Cdk9-phosphatase switches act at the beginning and end of elongation by RNA polymerase II.

Nat Commun 2020 08 28;11(1):4338. Epub 2020 Aug 28.

Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, 10029-6574, USA.

Reversible phosphorylation of Pol II and accessory factors helps order the transcription cycle. Here, we define two kinase-phosphatase switches that operate at different points in human transcription. Cdk9/cyclin T1 (P-TEFb) catalyzes inhibitory phosphorylation of PP1 and PP4 complexes that localize to 3' and 5' ends of genes, respectively, and have overlapping but distinct specificities for Cdk9-dependent phosphorylations of Spt5, a factor instrumental in promoter-proximal pausing and elongation-rate control. PP1 dephosphorylates an Spt5 carboxy-terminal repeat (CTR), but not Spt5-Ser666, a site between Kyrpides-Ouzounis-Woese (KOW) motifs 4 and 5, whereas PP4 can target both sites. In vivo, Spt5-CTR phosphorylation decreases as transcription complexes pass the cleavage and polyadenylation signal (CPS) and increases upon PP1 depletion, consistent with a PP1 function in termination first uncovered in yeast. Depletion of PP4-complex subunits increases phosphorylation of both Ser666 and the CTR, and promotes redistribution of promoter-proximally paused Pol II into gene bodies. These results suggest that switches comprising Cdk9 and either PP4 or PP1 govern pause release and the elongation-termination transition, respectively.
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http://dx.doi.org/10.1038/s41467-020-18173-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7455706PMC
August 2020

Cdk9 and H2Bub1 signal to Clr6-CII/Rpd3S to suppress aberrant antisense transcription.

Nucleic Acids Res 2020 07;48(13):7154-7168

Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Mono-ubiquitylation of histone H2B (H2Bub1) and phosphorylation of elongation factor Spt5 by cyclin-dependent kinase 9 (Cdk9) occur during transcription by RNA polymerase II (RNAPII), and are mutually dependent in fission yeast. It remained unclear whether Cdk9 and H2Bub1 cooperate to regulate the expression of individual genes. Here, we show that Cdk9 inhibition or H2Bub1 loss induces intragenic antisense transcription of ∼10% of fission yeast genes, with each perturbation affecting largely distinct subsets; ablation of both pathways de-represses antisense transcription of over half the genome. H2Bub1 and phospho-Spt5 have similar genome-wide distributions; both modifications are enriched, and directly proportional to each other, in coding regions, and decrease abruptly around the cleavage and polyadenylation signal (CPS). Cdk9-dependence of antisense suppression at specific genes correlates with high H2Bub1 occupancy, and with promoter-proximal RNAPII pausing. Genetic interactions link Cdk9, H2Bub1 and the histone deacetylase Clr6-CII, while combined Cdk9 inhibition and H2Bub1 loss impair Clr6-CII recruitment to chromatin and lead to decreased occupancy and increased acetylation of histones within gene coding regions. These results uncover novel interactions between co-transcriptional histone modification pathways, which link regulation of RNAPII transcription elongation to suppression of aberrant initiation.
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http://dx.doi.org/10.1093/nar/gkaa474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367204PMC
July 2020

Clinical value of next generation sequencing of plasma cell-free DNA in gastrointestinal stromal tumors.

BMC Cancer 2020 Feb 5;20(1):99. Epub 2020 Feb 5.

Preclinical Research Program, Vall d'Hebron Institute of Oncology, Barcelona, Spain.

Background: Gastrointestinal stromal tumor (GIST) initiation and evolution is commonly framed by KIT/PDGFRA oncogenic activation, and in later stages by the polyclonal expansion of resistant subpopulations harboring KIT secondary mutations after the onset of imatinib resistance. Thus, circulating tumor (ct)DNA determination is expected to be an informative non-invasive dynamic biomarker in GIST patients.

Methods: We performed amplicon-based next-generation sequencing (NGS) across 60 clinically relevant genes in 37 plasma samples from 18 GIST patients collected prospectively. ctDNA alterations were compared with NGS of matched tumor tissue samples (obtained either simultaneously or at the time of diagnosis) and cross-validated with droplet digital PCR (ddPCR).

Results: We were able to identify cfDNA mutations in five out of 18 patients had detectable in at least one timepoint. Overall, NGS sensitivity for detection of cell-free (cf)DNA mutations in plasma was 28.6%, showing high concordance with ddPCR confirmation. We found that GIST had relatively low ctDNA shedding, and mutations were at low allele frequencies. ctDNA was detected only in GIST patients with advanced disease after imatinib failure, predicting tumor dynamics in serial monitoring. KIT secondary mutations were the only mechanism of resistance found across 10 imatinib-resistant GIST patients progressing to sunitinib or regorafenib.

Conclusions: ctDNA evaluation with amplicon-based NGS detects KIT primary and secondary mutations in metastatic GIST patients, particularly after imatinib progression. GIST exhibits low ctDNA shedding, but ctDNA monitoring, when positive, reflects tumor dynamics.
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http://dx.doi.org/10.1186/s12885-020-6597-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003348PMC
February 2020

Histone H2B Ubiquitylation Regulates Histone Gene Expression by Suppressing Antisense Transcription in Fission Yeast.

Genetics 2019 09 25;213(1):161-172. Epub 2019 Jul 25.

Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec H3G 1Y6, Canada

Histone H2B monoubiquitylation (H2Bub1) is tightly linked to RNA polymerase II transcription elongation, and is also directly implicated in DNA replication and repair. Loss of H2Bub1 is associated with defects in cell cycle progression, but how these are related to its various functions, and the underlying mechanisms involved, is not understood. Here we describe a role for H2Bub1 in the regulation of replication-dependent histone genes in the fission yeast H2Bub1 activates histone genes indirectly by suppressing antisense transcription of -a gene encoding a GATA-type transcription factor that activates histone genes and is required for assembly of centromeric chromatin. Mutants lacking the ubiquitylation site in H2B or the H2B-specific E3 ubiquitin ligase Brl2 had elevated levels of antisense transcripts and reduced Ams2 protein levels. These defects were reversed upon inhibition of Cdk9-an ortholog of the kinase component of positive transcription elongation factor b (P-TEFb)-indicating that they likely resulted from aberrant transcription elongation. Reduced Cdk9 activity also partially rescued chromosome segregation phenotypes of H2Bub1 mutants. In a genome-wide analysis, loss of H2Bub1 led to increased antisense transcripts at over 500 protein-coding genes in H2Bub1 mutants; for a subset of these, including several genes involved in chromosome segregation and chromatin assembly, antisense derepression was Cdk9-dependent. Our results highlight antisense suppression as a key feature of cell cycle-dependent gene regulation by H2Bub1, and suggest that aberrant transcription elongation may underlie the effects of H2Bub1 loss on cell cycle progression.
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http://dx.doi.org/10.1534/genetics.119.302499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727805PMC
September 2019

Promoter bivalency favors an open chromatin architecture in embryonic stem cells.

Nat Genet 2018 10 17;50(10):1452-1462. Epub 2018 Sep 17.

Gene Regulation, Stem Cells and Cancer Program, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain.

In embryonic stem cells (ESCs), developmental gene promoters are characterized by their bivalent chromatin state, with simultaneous modification by MLL2 and Polycomb complexes. Although essential for embryogenesis, bivalency is functionally not well understood. Here, we show that MLL2 plays a central role in ESC genome organization. We generate a catalog of bona fide bivalent genes in ESCs and demonstrate that loss of MLL2 leads to increased Polycomb occupancy. Consequently, promoters lose accessibility, long-range interactions are redistributed, and ESCs fail to differentiate. We pose that bivalency balances accessibility and long-range connectivity of promoters, allowing developmental gene expression to be properly modulated.
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http://dx.doi.org/10.1038/s41588-018-0218-5DOI Listing
October 2018

A Cdk9-PP1 switch regulates the elongation-termination transition of RNA polymerase II.

Nature 2018 06 13;558(7710):460-464. Epub 2018 Jun 13.

Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to prevent interference between neighbouring genes and to safeguard transcriptome integrity . The accumulation of Pol II downstream of the cleavage and polyadenylation signal can facilitate the recruitment of factors involved in mRNA 3'-end formation and termination , but how this sequence is initiated remains unclear. In a chemical-genetic screen, human protein phosphatase 1 (PP1) isoforms were identified as substrates of positive transcription elongation factor b (P-TEFb), also known as the cyclin-dependent kinase 9 (Cdk9)-cyclin T1 (CycT1) complex . Here we show that Cdk9 and PP1 govern phosphorylation of the conserved elongation factor Spt5 in the fission yeast Schizosaccharomyces pombe. Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis2 . Sites targeted by Cdk9 in the Spt5 carboxy-terminal domain can be dephosphorylated by Dis2 in vitro, and dis2 mutations retard Spt5 dephosphorylation after inhibition of Cdk9 in vivo. Chromatin immunoprecipitation and sequencing analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the cleavage and polyadenylation signal, concomitant with the accumulation of Pol II phosphorylated at residue Ser2 of the carboxy-terminal domain consensus heptad repeat . A conditionally lethal Dis2-inactivating mutation attenuates the drop in Spt5 phosphorylation on chromatin, promotes transcription beyond the normal termination zone (as detected by precision run-on transcription and sequencing ) and is genetically suppressed by the ablation of Cdk9 target sites in Spt5. These results suggest that the transition of Pol II from elongation to termination coincides with a Dis2-dependent reversal of Cdk9 signalling-a switch that is analogous to a Cdk1-PP1 circuit that controls mitotic progression .
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http://dx.doi.org/10.1038/s41586-018-0214-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021199PMC
June 2018

Cdk9 regulates a promoter-proximal checkpoint to modulate RNA polymerase II elongation rate in fission yeast.

Nat Commun 2018 02 7;9(1):543. Epub 2018 Feb 7.

Department of Molecular Biology and Genetics, Cornell University, 107 Biotechnology Building, 526 Campus Road, Ithaca, NY, 14853-2703, USA.

Post-translational modifications of the transcription elongation complex provide mechanisms to fine-tune gene expression, yet their specific impacts on RNA polymerase II regulation remain difficult to ascertain. Here, in Schizosaccharomyces pombe, we examine the role of Cdk9, and related Mcs6/Cdk7 and Lsk1/Cdk12 kinases, on transcription at base-pair resolution with Precision Run-On sequencing (PRO-seq). Within a minute of Cdk9 inhibition, phosphorylation of Pol II-associated factor, Spt5 is undetectable. The effects of Cdk9 inhibition are more severe than inhibition of Cdk7 and Cdk12, resulting in a shift of Pol II toward the transcription start site (TSS). A time course of Cdk9 inhibition reveals that early transcribing Pol II can escape promoter-proximal regions, but with a severely reduced elongation rate of only ~400 bp/min. Our results in fission yeast suggest the existence of a conserved global regulatory checkpoint that requires Cdk9 kinase activity.
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http://dx.doi.org/10.1038/s41467-018-03006-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5803247PMC
February 2018

The Polycomb group protein CBX6 is an essential regulator of embryonic stem cell identity.

Nat Commun 2017 11 1;8(1):1235. Epub 2017 Nov 1.

Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona, 08003, Spain.

Polycomb group proteins (PcG) are transcriptional repressors that control cell identity and development. In mammals, five different CBX proteins associate with the core Polycomb repressive complex 1 (PRC1). In mouse embryonic stem cells (ESCs), CBX6 and CBX7 are the most highly expressed CBX family members. CBX7 has been recently characterized, but little is known regarding the function of CBX6. Here, we show that CBX6 is essential for ESC identity. Its depletion destabilizes the pluripotency network and triggers differentiation. Mechanistically, we find that CBX6 is physically and functionally associated to both canonical PRC1 (cPRC1) and non-canonical PRC1 (ncPRC1) complexes. Notably, in contrast to CBX7, CBX6 is recruited to chromatin independently of H3K27me3. Taken together, our findings reveal that CBX6 is an essential component of ESC biology that contributes to the structural and functional complexity of the PRC1 complex.
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http://dx.doi.org/10.1038/s41467-017-01464-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663739PMC
November 2017

Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition.

J Clin Invest 2017 Jun 8;127(6):2365-2377. Epub 2017 May 8.

Department of Pathology, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Hematopoietic transitions that accompany fetal development, such as erythroid globin chain switching, play important roles in normal physiology and disease development. In the megakaryocyte lineage, human fetal progenitors do not execute the adult morphogenesis program of enlargement, polyploidization, and proplatelet formation. Although these defects decline with gestational stage, they remain sufficiently severe at birth to predispose newborns to thrombocytopenia. These defects may also contribute to inferior platelet recovery after cord blood stem cell transplantation and may underlie inefficient platelet production by megakaryocytes derived from pluripotent stem cells. In this study, comparison of neonatal versus adult human progenitors has identified a blockade in the specialized positive transcription elongation factor b (P-TEFb) activation mechanism that is known to drive adult megakaryocyte morphogenesis. This blockade resulted from neonatal-specific expression of an oncofetal RNA-binding protein, IGF2BP3, which prevented the destabilization of the nuclear RNA 7SK, a process normally associated with adult megakaryocytic P-TEFb activation. Knockdown of IGF2BP3 sufficed to confer both phenotypic and molecular features of adult-type cells on neonatal megakaryocytes. Pharmacologic inhibition of IGF2BP3 expression via bromodomain and extraterminal domain (BET) inhibition also elicited adult features in neonatal megakaryocytes. These results identify IGF2BP3 as a human ontogenic master switch that restricts megakaryocyte development by modulating a lineage-specific P-TEFb activation mechanism, revealing potential strategies toward enhancing platelet production.
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http://dx.doi.org/10.1172/JCI88936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451240PMC
June 2017

3D structures of individual mammalian genomes studied by single-cell Hi-C.

Nature 2017 04 13;544(7648):59-64. Epub 2017 Mar 13.

Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom.

The folding of genomic DNA from the beads-on-a-string-like structure of nucleosomes into higher-order assemblies is crucially linked to nuclear processes. Here we calculate 3D structures of entire mammalian genomes using data from a new chromosome conformation capture procedure that allows us to first image and then process single cells. The technique enables genome folding to be examined at a scale of less than 100 kb, and chromosome structures to be validated. The structures of individual topological-associated domains and loops vary substantially from cell to cell. By contrast, A and B compartments, lamina-associated domains and active enhancers and promoters are organized in a consistent way on a genome-wide basis in every cell, suggesting that they could drive chromosome and genome folding. By studying genes regulated by pluripotency factor and nucleosome remodelling deacetylase (NuRD), we illustrate how the determination of single-cell genome structure provides a new approach for investigating biological processes.
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http://dx.doi.org/10.1038/nature21429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5385134PMC
April 2017

P-TEFb regulation of transcription termination factor Xrn2 revealed by a chemical genetic screen for Cdk9 substrates.

Genes Dev 2016 Jan;30(1):117-31

Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA;

The transcription cycle of RNA polymerase II (Pol II) is regulated at discrete transition points by cyclin-dependent kinases (CDKs). Positive transcription elongation factor b (P-TEFb), a complex of Cdk9 and cyclin T1, promotes release of paused Pol II into elongation, but the precise mechanisms and targets of Cdk9 action remain largely unknown. Here, by a chemical genetic strategy, we identified ∼ 100 putative substrates of human P-TEFb, which were enriched for proteins implicated in transcription and RNA catabolism. Among the RNA processing factors phosphorylated by Cdk9 was the 5'-to-3' "torpedo" exoribonuclease Xrn2, required in transcription termination by Pol II, which we validated as a bona fide P-TEFb substrate in vivo and in vitro. Phosphorylation by Cdk9 or phosphomimetic substitution of its target residue, Thr439, enhanced enzymatic activity of Xrn2 on synthetic substrates in vitro. Conversely, inhibition or depletion of Cdk9 or mutation of Xrn2-Thr439 to a nonphosphorylatable Ala residue caused phenotypes consistent with inefficient termination in human cells: impaired Xrn2 chromatin localization and increased readthrough transcription of endogenous genes. Therefore, in addition to its role in elongation, P-TEFb regulates termination by promoting chromatin recruitment and activation of a cotranscriptional RNA processing enzyme, Xrn2.
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http://dx.doi.org/10.1101/gad.269589.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4701974PMC
January 2016

Modelling the CDK-dependent transcription cycle in fission yeast.

Biochem Soc Trans 2013 Dec;41(6):1660-5

*Department of Structural and Chemical Biology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1677, New York, NY 10029-6574, U.S.A.

CDKs (cyclin-dependent kinases) ensure directionality and fidelity of the eukaryotic cell division cycle. In a similar fashion, the transcription cycle is governed by a conserved subfamily of CDKs that phosphorylate Pol II (RNA polymerase II) and other substrates. A genetic model organism, the fission yeast Schizosaccharomyces pombe, has yielded robust models of cell-cycle control, applicable to higher eukaryotes. From a similar approach combining classical and chemical genetics, fundamental principles of transcriptional regulation by CDKs are now emerging. In the present paper, we review the current knowledge of each transcriptional CDK with respect to its substrate specificity, function in transcription and effects on chromatin modifications, highlighting the important roles of CDKs in ensuring quantity and quality control over gene expression in eukaryotes.
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http://dx.doi.org/10.1042/BST20130238DOI Listing
December 2013

Modification of tRNA(Lys) UUU by elongator is essential for efficient translation of stress mRNAs.

PLoS Genet 2013 18;9(7):e1003647. Epub 2013 Jul 18.

Oxidative Stress and Cell Cycle Group, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain.

The Elongator complex, including the histone acetyl transferase Sin3/Elp3, was isolated as an RNA polymerase II-interacting complex, and cells deficient in Elongator subunits display transcriptional defects. However, it has also been shown that Elongator mediates the modification of some tRNAs, modulating translation efficiency. We show here that the fission yeast Sin3/Elp3 is important for oxidative stress survival. The stress transcriptional program, governed by the Sty1-Atf1-Pcr1 pathway, is affected in mutant cells, but not severely. On the contrary, cells lacking Sin3/Elp3 cannot modify the uridine wobble nucleoside of certain tRNAs, and other tRNA modifying activities such as Ctu1-Ctu2 are also essential for normal tolerance to H2O2. In particular, a plasmid over-expressing the tRNA(Lys) UUU complements the stress-related phenotypes of Sin3/Elp3 mutant cells. We have determined that the main H2O2-dependent genes, including those coding for the transcription factors Atf1 and Pcr1, are highly expressed mRNAs containing a biased number of lysine-coding codons AAA versus AAG. Thus, their mRNAs are poorly translated after stress in cells lacking Sin3/Elp3 or Ctu2, whereas a mutated atf1 transcript with AAA-to-AAG lysine codons is efficiently translated in all strain backgrounds. Our study demonstrates that the lack of a functional Elongator complex results in stress phenotypes due to its contribution to tRNA modification and subsequent translation inefficiency of certain stress-induced, highly expressed mRNAs. These results suggest that the transcriptional defects of these strain backgrounds may be a secondary consequence of the deficient expression of a transcription factor, Atf1-Pcr1, and other components of the transcriptional machinery.
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http://dx.doi.org/10.1371/journal.pgen.1003647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3715433PMC
January 2014

Pause, play, repeat: CDKs push RNAP II's buttons.

Transcription 2013 Jul-Aug;4(4):146-52. Epub 2013 Jun 11.

Department of Structural and Chemical Biology; Icahn School of Medicine at Mount Sinai; New York, NY USA.

Cyclin-dependent kinases (CDKs) play a central role in governing eukaryotic cell division. It is becoming clear that the transcription cycle of RNA polymerase II (RNAP II) is also regulated by CDKs; in metazoans, the cell cycle and transcriptional CDK networks even share an upstream activating kinase, which is itself a CDK. From recent chemical-genetic analyses we know that CDKs and their substrates control events both early in transcription (the transition from initiation to elongation) and late (3' end formation and transcription termination). Moreover, mutual dependence on CDK activity might couple the "beginning" and "end" of the cycle, to ensure the fidelity of mRNA maturation and the efficient recycling of RNAP II from sites of termination to the transcription start site (TSS). As is the case for CDKs involved in cell cycle regulation, different transcriptional CDKs act in defined sequence on multiple substrates. These phosphorylations are likely to influence gene expression by several mechanisms, including direct, allosteric effects on the transcription machinery, co-transcriptional recruitment of proteins needed for mRNA-capping, splicing and 3' end maturation, dependent on multisite phosphorylation of the RNAP II C-terminal domain (CTD) and, perhaps, direct regulation of RNA-processing or histone-modifying machinery. Here we review these recent advances, and preview the emerging challenges for transcription-cycle research.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3977912PMC
http://dx.doi.org/10.4161/trns.25146DOI Listing
March 2015

Cyclin-dependent kinase control of the initiation-to-elongation switch of RNA polymerase II.

Nat Struct Mol Biol 2012 Nov 14;19(11):1108-15. Epub 2012 Oct 14.

Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, New York, USA.

Promoter-proximal pausing by RNA polymerase II (Pol II) ensures gene-specific regulation and RNA quality control. Structural considerations suggested a requirement for initiation-factor eviction in elongation-factor engagement and pausing of transcription complexes. Here we show that selective inhibition of Cdk7--part of TFIIH--increases TFIIE retention, prevents DRB sensitivity-inducing factor (DSIF) recruitment and attenuates pausing in human cells. Pause release depends on Cdk9-cyclin T1 (P-TEFb); Cdk7 is also required for Cdk9-activating phosphorylation and Cdk9-dependent downstream events--Pol II C-terminal domain Ser2 phosphorylation and histone H2B ubiquitylation--in vivo. Cdk7 inhibition, moreover, impairs Pol II transcript 3'-end formation. Cdk7 thus acts through TFIIE and DSIF to establish, and through P-TEFb to relieve, barriers to elongation: incoherent feedforward that might create a window to recruit RNA-processing machinery. Therefore, cyclin-dependent kinases govern Pol II handoff from initiation to elongation factors and cotranscriptional RNA maturation.
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http://dx.doi.org/10.1038/nsmb.2399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3746743PMC
November 2012

A positive feedback loop links opposing functions of P-TEFb/Cdk9 and histone H2B ubiquitylation to regulate transcript elongation in fission yeast.

PLoS Genet 2012 2;8(8):e1002822. Epub 2012 Aug 2.

Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, New York, United States of America.

Transcript elongation by RNA polymerase II (RNAPII) is accompanied by conserved patterns of histone modification. Whereas histone modifications have established roles in transcription initiation, their functions during elongation are not understood. Mono-ubiquitylation of histone H2B (H2Bub1) plays a key role in coordinating co-transcriptional histone modification by promoting site-specific methylation of histone H3. H2Bub1 also regulates gene expression through an unidentified, methylation-independent mechanism. Here we reveal bidirectional communication between H2Bub1 and Cdk9, the ortholog of metazoan positive transcription elongation factor b (P-TEFb), in the fission yeast Schizosaccharomyces pombe. Chemical and classical genetic analyses indicate that lowering Cdk9 activity or preventing phosphorylation of its substrate, the transcription processivity factor Spt5, reduces H2Bub1 in vivo. Conversely, mutations in the H2Bub1 pathway impair Cdk9 recruitment to chromatin and decrease Spt5 phosphorylation. Moreover, an Spt5 phosphorylation-site mutation, combined with deletion of the histone H3 Lys4 methyltransferase Set1, phenocopies morphologic and growth defects due to H2Bub1 loss, suggesting independent, partially redundant roles for Cdk9 and Set1 downstream of H2Bub1. Surprisingly, mutation of the histone H2B ubiquitin-acceptor residue relaxes the Cdk9 activity requirement in vivo, and cdk9 mutations suppress cell-morphology defects in H2Bub1-deficient strains. Genome-wide analyses by chromatin immunoprecipitation also demonstrate opposing effects of Cdk9 and H2Bub1 on distribution of transcribing RNAPII. Therefore, whereas mutual dependence of H2Bub1 and Spt5 phosphorylation indicates positive feedback, mutual suppression by cdk9 and H2Bub1-pathway mutations suggests antagonistic functions that must be kept in balance to regulate elongation. Loss of H2Bub1 disrupts that balance and leads to deranged gene expression and aberrant cell morphologies, revealing a novel function of a conserved, co-transcriptional histone modification.
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http://dx.doi.org/10.1371/journal.pgen.1002822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3410854PMC
December 2012

Separate domains of fission yeast Cdk9 (P-TEFb) are required for capping enzyme recruitment and primed (Ser7-phosphorylated) Rpb1 carboxyl-terminal domain substrate recognition.

Mol Cell Biol 2012 Jul 16;32(13):2372-83. Epub 2012 Apr 16.

Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, New York, USA.

In fission yeast, discrete steps in mRNA maturation and synthesis depend on a complex containing the 5'-cap methyltransferase Pcm1 and Cdk9, which phosphorylates the RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) and the processivity factor Spt5 to promote transcript elongation. Here we show that a Cdk9 carboxyl-terminal extension, distinct from the catalytic domain, mediates binding to both Pcm1 and the Pol II CTD. Removal of this segment diminishes Cdk9/Pcm1 chromatin recruitment and Spt5 phosphorylation in vivo and leads to slow growth and hypersensitivity to cold temperature, nutrient limitation, and the IMP dehydrogenase inhibitor mycophenolic acid (MPA). These phenotypes, and the Spt5 phosphorylation defect, are suppressed by Pcm1 overproduction, suggesting that normal transcript elongation and gene expression depend on physical linkage between Cdk9 and Pcm1. The extension is dispensable, however, for recognition of CTD substrates "primed" by Mcs6 (Cdk7). On defined peptide substrates in vitro, Cdk9 prefers CTD repeats phosphorylated at Ser7 over unmodified repeats. In vivo, Ser7 phosphorylation depends on Mcs6 activity, suggesting a conserved mechanism, independent of chromatin recruitment, to order transcriptional CDK functions. Therefore, fission yeast Cdk9 comprises a catalytic domain sufficient for primed substrate recognition and a multivalent recruitment module that couples transcription with capping.
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http://dx.doi.org/10.1128/MCB.06657-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3434489PMC
July 2012

Nuclear roles and regulation of chromatin structure by the stress-dependent MAP kinase Sty1 of Schizosaccharomyces pombe.

Mol Microbiol 2011 Nov 13;82(3):542-54. Epub 2011 Oct 13.

Oxidative Stress and Cell Cycle Group, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, C/ Dr Aiguader 88, 08003 Barcelona, Spain.

Microorganisms are invariably exposed to abrupt changes in their environment, and consequently display robust, high plasticity gene programmes to respond to stresses. In fission yeast, the Sty1 pathway is activated in response to diverse stress conditions, such as osmotic and oxidative stress, heat shock or nitrogen deprivation. The MAP kinase Sty1 and its substrate, the transcription factor Atf1, regulate diverse processes mainly at the nucleus. For instance, Sty1, Atf1 and its heterodimeric partner Pcr1 participate in promoting recombination at some hot spots, and in the assembly of heterochromatin at the mating locus. Their main role, however, is to engage a wide gene expression programme aimed to allow cellular survival by decreasing and repairing the damage exerted. Once Sty1 and Atf1 are activated by stress, they are recruited to promoters of up to 5-10% of the coding genes and regulate their transcription. Even though there is no simple, global relationship establishing RNA polymerase II occupancy, nucleosome architecture and transcriptional activity in eukaryotes, we discuss within this review the current knowledge and future perspectives of how activation of Sty1 and Atf1 affect chromatin architecture of a large fraction of the Schizosaccharomyces pombe genome to trigger the cellular response to environmental stress.
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http://dx.doi.org/10.1111/j.1365-2958.2011.07851.xDOI Listing
November 2011

Gcn5 facilitates Pol II progression, rather than recruitment to nucleosome-depleted stress promoters, in Schizosaccharomyces pombe.

Nucleic Acids Res 2011 Aug 22;39(15):6369-79. Epub 2011 Apr 22.

Departament de Ciències Experimentals i de la Salut, Oxidative Stress and Cell Cycle Group, Universitat Pompeu Fabra, C/ Dr. Aiguader 88, E-08003 Barcelona, Spain.

In the fission yeast, the MAP kinase Sty1 and the transcription factor Atf1 regulate up to 400 genes in response to environmental signals, and both proteins have been shown to bind to their promoters in a stress-dependent manner. In a genetic search, we have isolated the histone H3 acetyltransferase Gcn5, a component of the SAGA complex, as being essential for oxidative stress survival and activation of those genes. Upon stress, Gcn5 is recruited to promoters and coding sequences of stress genes in a Sty1- and Atf1-dependent manner, causing both an enhanced acetylation of histone H3 and nucleosome eviction. Unexpectedly, recruitment of RNA polymerase II (Pol II) is not impaired in Δgcn5 cells. We show here that stress genes display a 400-bp long nucleosome depleted region upstream of the transcription start site even prior to activation. Stress treatment does not alter promoter nucleosome architecture, but induces eviction of the downstream nucleosomes at stress genes, which is not observed in Δgcn5 cells. We conclude that, while Pol II is recruited to nucleosome-free stress promoters in a transcription factor dependent manner, Gcn5 mediates eviction of nucleosomes positioned downstream of promoters, allowing efficient Pol II progression along the genes.
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http://dx.doi.org/10.1093/nar/gkr255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3159446PMC
August 2011

Genome-wide screen of genes required for caffeine tolerance in fission yeast.

PLoS One 2009 Aug 12;4(8):e6619. Epub 2009 Aug 12.

Oxidative Stress and Cell Cycle Group, Universitat Pompeu Fabra, Barcelona, Spain.

Background: An excess of caffeine is cytotoxic to all eukaryotic cell types. We aim to study how cells become tolerant to a toxic dose of this drug, and the relationship between caffeine and oxidative stress pathways.

Methodology/principal Findings: We searched for Schizosaccharomyces pombe mutants with inhibited growth on caffeine-containing plates. We screened a collection of 2,700 haploid mutant cells, of which 98 were sensitive to caffeine. The genes mutated in these sensitive clones were involved in a number of cellular roles including the H(2)O(2)-induced Pap1 and Sty1 stress pathways, the integrity and calcineurin pathways, cell morphology and chromatin remodeling. We have investigated the role of the oxidative stress pathways in sensing and promoting survival to caffeine. The Pap1 and the Sty1 pathways are both required for normal tolerance to caffeine, but only the Sty1 pathway is activated by the drug. Cells lacking Pap1 are sensitive to caffeine due to the decreased expression of the efflux pump Hba2. Indeed, ?hba2 cells are sensitive to caffeine, and constitutive activation of the Pap1 pathway enhances resistance to caffeine in an Hba2-dependent manner.

Conclusions/significance: With our caffeine-sensitive, genome-wide screen of an S. pombe deletion collection, we have demonstrated the importance of some oxidative stress pathway components on wild-type tolerance to the drug.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0006619PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2720375PMC
August 2009

Transcription factors Pcr1 and Atf1 have distinct roles in stress- and Sty1-dependent gene regulation.

Eukaryot Cell 2008 May 28;7(5):826-35. Epub 2008 Mar 28.

Oxidative Stress and Cell Cycle Group, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, C/ Dr. Aiguader 88, E-08003 Barcelona, Spain.

The mitogen-activated protein kinase Sty1 is essential for the regulation of transcriptional responses that promote cell survival in response to different types of environmental stimuli in Schizosaccharomyces pombe. Upon stress activation, Sty1 reversibly accumulates in the nucleus, where it stimulates gene expression via the Atf1 transcription factor. The Atf1 protein forms a heterodimer with Pcr1, but the specific role of this association is controversial. We have carried out a comparative analysis of strains lacking these proteins individually. We demonstrate that Atf1 and Pcr1 have similar but not identical roles in S. pombe, since cells lacking Pcr1 do not share all the phenotypes reported for Deltaatf1 cells. Northern blot and microarray analyses demonstrate that the responses to specific stresses of cells lacking either Pcr1 or Atf1 do not fully overlap, and even though most Atf1-dependent genes induced by osmotic stress are also Pcr1 dependent, a subset of genes require only the presence of Atf1 for their induction. Whereas binding of Atf1 to most stress-dependent genes requires the presence of Pcr1, we demonstrate here that Atf1 can bind to the Pcr1-independent promoters in a Deltapcr1 strain in vivo. Furthermore, these analyses show that both proteins have a global repressive effect on stress-dependent and stress-independent genes.
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http://dx.doi.org/10.1128/EC.00465-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2394965PMC
May 2008

The peroxiredoxin Tpx1 is essential as a H2O2 scavenger during aerobic growth in fission yeast.

Mol Biol Cell 2007 Jun 4;18(6):2288-95. Epub 2007 Apr 4.

Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, E-08003 Barcelona, Spain.

Peroxiredoxins are known to interact with hydrogen peroxide (H(2)O(2)) and to participate in oxidant scavenging, redox signal transduction, and heat-shock responses. The two-cysteine peroxiredoxin Tpx1 of Schizosaccharomyces pombe has been characterized as the H(2)O(2) sensor that transduces the redox signal to the transcription factor Pap1. Here, we show that Tpx1 is essential for aerobic, but not anaerobic, growth. We demonstrate that Tpx1 has an exquisite sensitivity for its substrate, which explains its participation in maintaining low steady-state levels of H(2)O(2). We also show in vitro and in vivo that inactivation of Tpx1 by oxidation of its catalytic cysteine to a sulfinic acid is always preceded by a sulfinic acid form in a covalently linked dimer, which may be important for understanding the kinetics of Tpx1 inactivation. Furthermore, we provide evidence that a strain expressing Tpx1.C169S, lacking the resolving cysteine, can sustain aerobic growth, and we show that small reductants can modulate the activity of the mutant protein in vitro, probably by supplying a thiol group to substitute for cysteine 169.
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http://dx.doi.org/10.1091/mbc.e06-11-1039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1877099PMC
June 2007

Oxidative stress in Schizosaccharomyces pombe: different H2O2 levels, different response pathways.

Mol Genet Genomics 2006 Dec 17;276(6):495-502. Epub 2006 Oct 17.

Cell Signalling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, C/Dr. Aiguader 80, Barcelona, 08003, Spain.

Schizosaccharomyces pombe triggers different signalling pathways depending on the severity of the oxidative stress exerted, the main ones being the Pap1 and the Sty1 pathways. The Pap1 transcription factor is more sensitive to hydrogen peroxide (H(2)O(2)) than the MAP kinase Sty1 pathway, and is designed to induce adaptation, rather than survival, responses. The peroxiredoxin Tpx1 acts as a H(2)O(2) sensor and the upstream activator of the Pap1 pathway. Therefore, sensitivity to H(2)O(2) depends on this thioredoxin peroxidase. In order to achieve maximal activation of the MAP kinase pathway, the concentration of H(2)O(2) needs to be at least fivefold higher than that to fully activate Pap1. Tpx1 is a H(2)O(2) scavenger, thus its peroxidase activity is essential for aerobic growth. As described for other eukaryotic peroxiredoxins, high doses of H(2)O(2) temporarily inactivate Tpx1 and delay Pap1 activation, whereas the Sty1 pathway remains fully functional under these conditions. As part of the Sty1-dependent transcriptional response, the expression of Srx1 is induced and this reductase re-activates the over-oxidised Tpx1. Therefore, the antioxidant pathways of the fission yeast are perfectly designed so that the transcriptional programs triggered by the different signalling pathways never overlap.
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http://dx.doi.org/10.1007/s00438-006-0175-zDOI Listing
December 2006

The glycolytic metabolite methylglyoxal activates Pap1 and Sty1 stress responses in Schizosaccharomyces pombe.

J Biol Chem 2005 Nov 1;280(44):36708-13. Epub 2005 Sep 1.

Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, C/Dr. Aiguader 80, 08003 Barcelona, Spain.

Methylglyoxal, a toxic metabolite synthesized in vivo during glycolysis, inhibits cell growth. One of the mechanisms protecting eukaryotic cells against its toxicity is the glyoxalase system, composed of glyoxalase I and II (glo1 and glo2), which converts methylglyoxal into d-lactic acid in the presence of glutathione. Here we have shown that the two principal oxidative stress response pathways of Schizosaccharomyces pombe, Sty1 and Pap1, are involved in the response to methylglyoxal toxicity. The mitogen-activated protein kinase Sty1 is phosphorylated and accumulates in the nucleus following methylglyoxal treatment. Moreover, glo2 expression is induced by methylglyoxal and environmental stresses in a Sty1-dependent manner. The transcription factor Pap1 also accumulates in the nucleus, activating the expression of its target genes following methylglyoxal treatment. Our studies showed that the C-terminal cysteine-rich domain of Pap1 is sufficient for methylglyoxal sensing. Furthermore, the redox status of Pap1 is not changed by methylglyoxal. We propose that methylglyoxal treatment triggers Pap1 and Sty1 nuclear accumulation, and we describe the molecular basis of such activation mechanisms. In addition, we discuss the potential physiological significance of these responses to a natural toxic metabolite.
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http://dx.doi.org/10.1074/jbc.M508400200DOI Listing
November 2005