scholarly journals Histone H3 K36 Methylation Is Associated with Transcription Elongation in Schizosaccharomyces pombe

2005 ◽  
Vol 4 (8) ◽  
pp. 1446-1454 ◽  
Author(s):  
Stephanie A. Morris ◽  
Yoichiro Shibata ◽  
Ken-ichi Noma ◽  
Yuko Tsukamoto ◽  
Erin Warren ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Rna Polymerase Ii ◽  
Budding Yeast ◽  
Transcription Elongation ◽  
Synthetic Medium ◽  
Histone H3 ◽  
Yeast Cells ◽  
Pol Ii ◽  
Elongation Process

ABSTRACT Set2 methylation of histone H3 at lysine 36 (K36) has recently been shown to be associated with RNA polymerase II (Pol II) elongation in Saccharomyces cerevisiae. However, whether this modification is conserved and associated with transcription elongation in other organisms is not known. Here we report the identification and characterization of the Set2 ortholog responsible for K36 methylation in the fission yeast Schizosaccharomyces pombe. We find that similar to the budding yeast enzyme, S. pombe Set2 is also a robust nucleosome-selective H3 methyltransferase that is specific for K36. Deletion of the S. pombe set2 + gene results in complete abolishment of K36 methylation as well as a slow-growth phenotype on plates containing synthetic medium. These results indicate that Set2 is the sole enzyme responsible for this modification in fission yeast and is important for cell growth under stressed conditions. Using the chromatin immunoprecipitation assay, we demonstrate that K36 methylation in S. pombe is associated with the transcribed regions of Pol II-regulated genes and is devoid in regions that are not transcribed by Pol II. Consistent with a role for Set2 in transcription elongation, we find that S. pombe Set2 associates with the hyperphosphorylated form of Pol II and can fully rescue K36 methylation and Pol II interaction in budding yeast cells deleted for Set2. These results, along with our finding that K36 methylation is highly conserved among eukaryotes, imply a conserved role for this modification in the transcription elongation process.

scholarly journals A machine learning-based framework for modeling transcription elongation

2021 ◽  
Vol 118 (6) ◽  
pp. e2007450118
Author(s):  
Peiyuan Feng ◽  
An Xiao ◽  
Meng Fang ◽  
Fangping Wan ◽  
Shuya Li ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
High Throughput Sequencing ◽  
Gene Expression Regulation ◽  
Transcription Elongation ◽  
Transcriptional Elongation ◽  
Sequencing Data ◽  
Pol Ii ◽  
High Throughput Sequencing Data ◽  
Pol Ii Pausing ◽  
Elongation Process

RNA polymerase II (Pol II) generally pauses at certain positions along gene bodies, thereby interrupting the transcription elongation process, which is often coupled with various important biological functions, such as precursor mRNA splicing and gene expression regulation. Characterizing the transcriptional elongation dynamics can thus help us understand many essential biological processes in eukaryotic cells. However, experimentally measuring Pol II elongation rates is generally time and resource consuming. We developed PEPMAN (polymerase II elongation pausing modeling through attention-based deep neural network), a deep learning-based model that accurately predicts Pol II pausing sites based on the native elongating transcript sequencing (NET-seq) data. Through fully taking advantage of the attention mechanism, PEPMAN is able to decipher important sequence features underlying Pol II pausing. More importantly, we demonstrated that the analyses of the PEPMAN-predicted results around various types of alternative splicing sites can provide useful clues into understanding the cotranscriptional splicing events. In addition, associating the PEPMAN prediction results with different epigenetic features can help reveal important factors related to the transcription elongation process. All these results demonstrated that PEPMAN can provide a useful and effective tool for modeling transcription elongation and understanding the related biological factors from available high-throughput sequencing data.

scholarly journals Schizosaccharomyces pombe Noc3 Is Essential for Ribosome Biogenesis and Cell Division but Not DNA Replication

2008 ◽  
Vol 7 (9) ◽  
pp. 1433-1440 ◽  
Author(s):  
Christopher R. Houchens ◽  
Audrey Perreault ◽  
François Bachand ◽  
Thomas J. Kelly
Keyword(s):  
Cell Division ◽  
Dna Replication ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Ribosome Biogenesis ◽  
Budding Yeast ◽  
Ribosomal Subunit ◽  
Dna Helicase ◽  
Yeast Cells ◽  
Direct Role

ABSTRACT The initiation of eukaryotic DNA replication is preceded by the assembly of prereplication complexes (pre-RCs) at chromosomal origins of DNA replication. Pre-RC assembly requires the essential DNA replication proteins ORC, Cdc6, and Cdt1 to load the MCM DNA helicase onto chromatin. Saccharomyces cerevisiae Noc3 (ScNoc3), an evolutionarily conserved protein originally implicated in 60S ribosomal subunit trafficking, has been proposed to be an essential regulator of DNA replication that plays a direct role during pre-RC formation in budding yeast. We have cloned Schizosaccharomyces pombe noc3 + (Spnoc3 +), the S. pombe homolog of the budding yeast ScNOC3 gene, and functionally characterized the requirement for the SpNoc3 protein during ribosome biogenesis, cell cycle progression, and DNA replication in fission yeast. We showed that fission yeast SpNoc3 is a functional homolog of budding yeast ScNoc3 that is essential for cell viability and ribosome biogenesis. We also showed that SpNoc3 is required for the normal completion of cell division in fission yeast. However, in contrast to the proposal that ScNoc3 plays an essential role during DNA replication in budding yeast, we demonstrated that fission yeast cells do enter and complete S phase in the absence of SpNoc3, suggesting that SpNoc3 is not essential for DNA replication in fission yeast.

scholarly journals CENP-A exceeds microtubule attachment sites in centromere clusters of both budding and fission yeast

2011 ◽  
Vol 195 (4) ◽  
pp. 563-572 ◽  
Author(s):  
Valerie C. Coffman ◽  
Pengcheng Wu ◽  
Mark R. Parthun ◽  
Jian-Qiu Wu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Budding Yeast ◽  
Histone H3 ◽  
Kinetochore Assembly ◽  
Microtubule Attachment ◽  
Attachment Sites ◽  
Histone H3 Variant ◽  
Architectural Models

The stoichiometries of kinetochores and their constituent proteins in yeast and vertebrate cells were determined using the histone H3 variant CENP-A, known as Cse4 in budding yeast, as a counting standard. One Cse4-containing nucleosome exists in the centromere (CEN) of each chromosome, so it has been assumed that each anaphase CEN/kinetochore cluster contains 32 Cse4 molecules. We report that anaphase CEN clusters instead contained approximately fourfold more Cse4 in Saccharomyces cerevisiae and ∼40-fold more CENP-A (Cnp1) in Schizosaccharomyces pombe than predicted. These results suggest that the number of CENP-A molecules exceeds the number of kinetochore-microtubule (MT) attachment sites on each chromosome and that CENP-A is not the sole determinant of kinetochore assembly sites in either yeast. In addition, we show that fission yeast has enough Dam1–DASH complex for ring formation around attached MTs. The results of this study suggest the need for significant revision of existing CEN/kinetochore architectural models.

scholarly journals A Novel Domain in Set2 Mediates RNA Polymerase II Interaction and Couples Histone H3 K36 Methylation with Transcript Elongation

2005 ◽  
Vol 25 (8) ◽  
pp. 3305-3316 ◽  
Author(s):  
Kelby O. Kizer ◽  
Hemali P. Phatnani ◽  
Yoichiro Shibata ◽  
Hana Hall ◽  
Arno L. Greenleaf ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Histone H3 ◽  
Specific Pattern ◽  
The Body ◽  
Yeast Strains ◽  
Elongation Process

ABSTRACT Histone methylation and the enzymes that mediate it are important regulators of chromatin structure and gene transcription. In particular, the histone H3 lysine 36 (K36) methyltransferase Set2 has recently been shown to associate with the phosphorylated C-terminal domain (CTD) of RNA polymerase II (RNAPII), implying that this enzyme has an important role in the transcription elongation process. Here we show that a novel domain in the C terminus of Set2 is responsible for interaction between Set2 and RNAPII. This domain, termed the Set2 Rpb1 interacting (SRI) domain, is encompassed by amino acid residues 619 to 718 in Set2 and is found to occur in a number of putative Set2 homologs from Schizosaccharomyces pombe to humans. Unexpectedly, BIACORE analysis reveals that the SRI domain binds specifically, and with high affinity, to CTD repeats that are doubly modified (serine 2 and serine 5 phosphorylated), indicating that Set2 association across the body of genes requires a specific pattern of phosphorylated RNAPII. Deletion of the SRI domain not only abolishes Set2-RNAPII interaction but also abolishes K36 methylation in vivo, indicating that this interaction is required for establishing K36 methylation on chromatin. Using 6-azauracil (6AU) as an indicator of transcription elongation defects, we found that deletion of the SRI domain conferred a strong resistance to this compound, which was identical to that observed with set2 deletion mutants. Furthermore, yeast strains carrying set2 alleles that are catalytically inactive or yeast strains bearing point mutations at K36 were also found to be resistant to 6AU. These data suggest that it is the methylation by Set2 that affects transcription elongation. In agreement with this, we have determined that deletion of SET2, its SRI domain, or amino acid substitutions at K36 result in an alteration of RNAPII occupancy levels over transcribing genes. Taken together, these data indicate K36 methylation, established by the SRI domain-mediated association of Set2 with RNAPII, plays an important role in the transcription elongation process.

scholarly journals Fission Yeast Cdk7 Controls Gene Expression through both Its CAK and C-Terminal Domain Kinase Activities

2015 ◽  
Vol 35 (9) ◽  
pp. 1480-1490 ◽  
Author(s):  
Maxime Devos ◽  
Elise Mommaerts ◽  
Valerie Migeot ◽  
Harm van Bakel ◽  
Damien Hermand
Keyword(s):  
Fission Yeast ◽  
Rna Polymerase Ii ◽  
Ribosome Biogenesis ◽  
Transcriptional Control ◽  
Kinase Activity ◽  
Budding Yeast ◽  
Transcriptome Profiling ◽  
Pol Ii ◽  
Physiological Importance ◽  
Terminal Domain

Cyclin-dependent kinase (Cdk) activation and RNA polymerase II transcription are linked by the Cdk7 kinase, which phosphorylates Cdks as a trimeric Cdk-activating kinase (CAK) complex, and serine 5 within the polymerase II (Pol II) C-terminal domain (CTD) as transcription factor TFIIH-bound CAK. However, the physiological importance of integrating these processes is not understood. Besides the Cdk7 ortholog Mcs6, fission yeast possesses a second CAK, Csk1. The two enzymes have been proposed to act redundantly to activate Cdc2. Using an improved analogue-sensitive Mcs6-as kinase, we show that Csk1 is not a relevant CAK for Cdc2. Further analyses revealed that Csk1 lacks a 20-amino-acid sequence required for its budding yeast counterpart, Cak1, to bind Cdc2. Transcriptome profiling of the Mcs6-as mutant in the presence or absence of the budding yeast Cak1 kinase, in order to uncouple the CTD kinase and CAK activities of Mcs6, revealed an unanticipated role of the CAK branch in the transcriptional control of the cluster of genes implicated in ribosome biogenesis and cell growth. The analysis of a Cdc2 CAK site mutant confirmed these data. Our data show that the Cdk7 kinase modulates transcription through its well-described RNA Pol II CTD kinase activity and also through the Cdc2-activating kinase activity.

scholarly journals Separable Functions of the Fission Yeast Spt5 Carboxyl-Terminal Domain (CTD) in Capping Enzyme Binding and Transcription Elongation Overlap with Those of the RNA Polymerase II CTD

2010 ◽  
Vol 30 (10) ◽  
pp. 2353-2364 ◽  
Author(s):  
Susanne Schneider ◽  
Yi Pei ◽  
Stewart Shuman ◽  
Beate Schwer
Keyword(s):  
Rna Polymerase ◽  
Fission Yeast ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Pol Ii ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Capping Enzyme ◽  
Carboxyl Terminal ◽  
Capping Enzymes

ABSTRACT An interaction network connecting mRNA capping enzymes, the RNA polymerase II (Pol II) carboxyl-terminal domain (CTD), elongation factor Spt5, and the Cdk7 and Cdk9 protein kinases is thought to comprise a transcription elongation checkpoint. A crux of this network is Spt5, which regulates early transcription elongation and has an imputed role in pre-mRNA processing via its physical association with capping enzymes. Schizosaccharomyces pombe Spt5 has a distinctive CTD composed of tandem nonapeptide repeats of the consensus sequence 1TPAWNSGSK9. The Spt5 CTD binds the capping enzymes and is a substrate for threonine phosphorylation by the Cdk9 kinase. Here we report that deletion of the S. pombe Spt5 CTD results in slow growth and aberrant cell morphology. The severity of the spt5-ΔCTD phenotype is exacerbated by truncation of the Pol II CTD and ameliorated by overexpression of the capping enzymes RNA triphosphatase and RNA guanylyltransferase. These results suggest that the Spt5 and Pol II CTDs play functionally overlapping roles in capping enzyme recruitment. We probed structure-activity relations of the Spt5 CTD by alanine scanning of the consensus nonapeptide. The T1A change abolished CTD phosphorylation by Cdk9 but did not affect CTD binding to the capping enzymes. The T1A and P2A mutations elicited cold-sensitive (cs) and temperature-sensitive (ts) growth defects and conferred sensitivity to growth inhibition by 6-azauracil that was exacerbated by partial truncations of the Pol II CTD. The T1A phenotypes were rescued by a phosphomimetic T1E change but not by capping enzyme overexpression. These results imply a positive role for Spt5 CTD phosphorylation in Pol Il transcription elongation in fission yeast, distinct from its capping enzyme interactions. Viability of yeast cells bearing both Spt5 CTD T1A and Pol II CTD S2A mutations heralds that the Cdk9 kinase has an essential target other than Spt5 and Pol II CTD-Ser2.

scholarly journals Cockayne syndrome B protein acts as an ATP-dependent processivity factor that helps RNA polymerase II overcome nucleosome barriers

2020 ◽  
Vol 117 (41) ◽  
pp. 25486-25493 ◽  
Author(s):  
Jun Xu ◽  
Wei Wang ◽  
Liang Xu ◽  
Jia-Yu Chen ◽  
Jenny Chong ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Neurological Diseases ◽  
Transcription Elongation ◽  
Cockayne Syndrome ◽  
Stable Complex ◽  
Loss Of Function ◽  
Pol Ii ◽  
Chromatin Remodelers ◽  
Processivity Factor

While loss-of-function mutations in Cockayne syndrome group B protein (CSB) cause neurological diseases, this unique member of the SWI2/SNF2 family of chromatin remodelers has been broadly implicated in transcription elongation and transcription-coupled DNA damage repair, yet its mechanism remains largely elusive. Here, we use a reconstituted in vitro transcription system with purified polymerase II (Pol II) and Rad26, a yeast ortholog of CSB, to study the role of CSB in transcription elongation through nucleosome barriers. We show that CSB forms a stable complex with Pol II and acts as an ATP-dependent processivity factor that helps Pol II across a nucleosome barrier. This noncanonical mechanism is distinct from the canonical modes of chromatin remodelers that directly engage and remodel nucleosomes or transcription elongation factors that facilitate Pol II nucleosome bypass without hydrolyzing ATP. We propose a model where CSB facilitates gene expression by helping Pol II bypass chromatin obstacles while maintaining their structures.

scholarly journals DIPG-03. THERAPEUTIC TARGETING OF TRANSCRIPTIONAL ELONGATION IN DIFFUSE INTRINSIC PONTINE GLIOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii287-iii287
Author(s):  
Hiroaki Katagi ◽  
Nozomu Takata ◽  
Yuki Aoi ◽  
Yongzhan Zhang ◽  
Emily J Rendleman ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Xenograft Model ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Transcriptional Elongation ◽  
Pontine Glioma ◽  
Elongation Complex ◽  
Pol Ii ◽  
Repair Genes ◽  
Novel Therapeutic

Abstract Diffuse intrinsic pontine glioma (DIPG) is highly aggressive brain stem tumor and needed to develop novel therapeutic agents for the treatment. The super elongation complex (SEC) is essential for transcription elongation through release of RNA polymerase II (Pol II). We found that AFF4, a scaffold protein of the SEC, is required for the growth of H3K27M-mutant DIPG cells. In addition, the small molecule SEC inhibitor, KL-1, increased promoter-proximal pausing of Pol II, and reduced transcription elongation, resulting in down-regulate cell cycle, transcription and DNA repair genes. KL-1 treatment decreased cell growth and increased apoptosis in H3K27M-mutant DIPG cells, and prolonged animal survival in our human H3K27M-mutant DIPG xenograft model. Our results demonstrate that the SEC disruption by KL-1 is a novel therapeutic strategy for H3K27M-mutant DIPG.

Use of electron microscopy to characterize the surfaces of flocculent and nonflocculent yeast cells

1989 ◽  
Vol 35 (12) ◽  
pp. 1081-1086 ◽  
Author(s):  
Byron F. Johnson ◽  
L. C. Sowden ◽  
Teena Walker ◽  
Bong Y. Yoo ◽  
Gode B. Calleja
Keyword(s):  
Electron Microscopy ◽  
Fission Yeast ◽  
Budding Yeast ◽  
The Other ◽  
Yeast Cells ◽  
Scanning Electron Micrographs ◽  
Soft Or ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Scanning Electron

The surfaces of flocculent and nonflocculent yeast cells have been examined by electron microscopy. Nonextractive preparative procedures for scanning electron microscopy allow comparison in which sharp or softened images of surface details (scars, etc.) are the criteria for relative abundance of flocculum material. Asexually flocculent budding-yeast cells cannot be distinguished from nonflocculent budding-yeast cells in scanning electron micrographs because the scar details of both are well resolved, being hard and sharp. On the other hand, flocculent fission-yeast cells are readily distinguished from nonflocculent cells because fission scars are mostly soft or obscured on flocculent cells, but sharp on nonflocculent cells. Sexually and asexually flocculent fission-yeast cells cannot be distinguished from one another as both are heavily clad in "mucilaginous" or "hairy" coverings. Examination of lightly extracted and heavily extracted flocculent fission-yeast cells by transmission electron microscopy provides micrographs consistent with the scanning electron micrographs.Key words: flocculation, budding yeast, fission yeast, scanning, transmission.

Mediator dynamics during heat shock in budding yeast

2021 ◽  
pp. gr.275750.121
Author(s):  
Debasish Sarkar ◽  
Z. Iris Zhu ◽  
Elisabeth R. Knoll ◽  
Emily Paul ◽  
David Landsman ◽  
...  
Keyword(s):  
Heat Shock ◽  
Rna Polymerase Ii ◽  
Budding Yeast ◽  
Core Promoter ◽  
Growth Conditions ◽  
Promoter Regions ◽  
Pol Ii ◽  
A Genome ◽  
Wide Scale ◽  
Stable Association

The Mediator complex is central to transcription by RNA polymerase II (Pol II) in eukaryotes. In budding yeast (Saccharomyces cerevisiae), Mediator is recruited by activators and associates with core promoter regions, where it facilitates pre-initiation complex (PIC) assembly, only transiently prior to Pol II escape. Interruption of the transcription cycle by inactivation or depletion of Kin28 inhibits Pol II escape and stabilizes this association. However, Mediator occupancy and dynamics have not been examined on a genome-wide scale in yeast grown in nonstandard conditions. Here we investigate Mediator occupancy following heat shock or CdCl2 exposure, with and without depletion of Kin28. We find that Pol II occupancy exhibits similar dependence on Mediator under normal and heat shock conditions. However, while Mediator association increases at many genes upon Kin28 depletion under standard growth conditions, little or no increase is observed at most genes upon heat shock, indicating a more stable association of Mediator after heat shock. Mediator remains associated upstream of the core promoter at genes repressed by heat shock or CdCl2 exposure whether or not Kin28 is depleted, suggesting that Mediator is recruited by activators but is unable to engage PIC components at these repressed targets. This persistent association is strongest at promoters that bind the HMGB family member Hmo1, and is reduced but not eliminated in hmo1∆ yeast. Finally, we show a reduced dependence on PIC components for Mediator occupancy at promoters after heat shock, further supporting altered dynamics or stronger engagement with activators under these conditions.

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