scholarly journals Active transcription and Orc1 drive chromatin association of the AAA+ ATPase Pch2 during meiotic G2/prophase

2019 ◽  
Author(s):  
Richard Cardoso da Silva ◽  
María Ascensión Villar-Fernández ◽  
Gerben Vader
Keyword(s):  
Rna Polymerase Ii ◽  
Origin Recognition Complex ◽  
Budding Yeast ◽  
Yeast Genome ◽  
Checkpoint Signaling ◽  
Aaa Atpase ◽  
Meiotic Chromosomes ◽  
Chromosomal Association ◽  
Active Transcription ◽  
And Function

AbstractPch2 is an AAA+ protein that controls DNA break formation, recombination and checkpoint signaling during meiotic G2/prophase. Chromosomal association of Pch2 is linked to these processes, and several factors influence the association of Pch2 to euchromatin and the specialized chromatin of the ribosomal (r)DNA array of budding yeast. Here, we describe a comprehensive mapping of Pch2 localization across the budding yeast genome during meiotic G2/prophase. Within non-rDNA chromatin, Pch2 associates with a subset of actively RNA Polymerase II (RNAPII)-dependent transcribed genes. Chromatin immunoprecipitation (ChIP)- and microscopy-based analysis reveals that active transcription is required for chromosomal recruitment of Pch2. Similar to what was previously established for association of Pch2 with rDNA chromatin, we find that Orc1, a component of the Origin Recognition Complex (ORC), is required for the association of Pch2 to these euchromatic, transcribed regions, revealing a broad connection between chromosomal association of Pch2 and Orc1/ORC function. Ectopic mitotic expression is insufficient to drive recruitment of Pch2, despite the presence of active transcription and Orc1/ORC in mitotic cells. This suggests meiosis-specific ‘licensing’ of Pch2 recruitment to sites of transcription, and accordingly, we find that the synaptonemal complex (SC) component Zip1 is required for the recruitment of Pch2 to transcription-associated binding regions. Interestingly, Pch2 binding patterns are distinct from meiotic axis enrichment sites (as defined by Red1, Hop1 and Rec8). This suggests that although Pch2 is linked to axis/SC-directed recruitment and function, the chromosomal population of Pch2 described here is not directly associated with chromosomal axis sites. In line with this observation, interfering with the pool of Pch2 that associates with active RNAPII transcription does not lead to effects on the chromosomal abundance of Hop1, a known axial client of Pch2. We thus report characteristics and dependencies for Pch2 recruitment to meiotic chromosomes, and reveal an unexpected link between Pch2, SC formation, chromatin and active transcription.

scholarly journals Getting there: understanding the chromosomal recruitment of the AAA+ ATPase Pch2/TRIP13 during meiosis

2021 ◽  
Author(s):  
Richard Cardoso da Silva ◽  
Gerben Vader
Keyword(s):  
Rna Polymerase Ii ◽  
Meiotic Recombination ◽  
Origin Recognition Complex ◽  
Comprehensive Overview ◽  
Homologous Chromosomes ◽  
Functional Roles ◽  
Aaa Atpase ◽  
Meiotic Chromosomes ◽  
Chromosomal Association ◽  
Dna Break

AbstractThe generally conserved AAA+ ATPase Pch2/TRIP13 is involved in diverse aspects of meiosis, such as prophase checkpoint function, DNA break regulation, and meiotic recombination. The controlled recruitment of Pch2 to meiotic chromosomes allows it to use its ATPase activity to influence HORMA protein-dependent signaling. Because of the connection between Pch2 chromosomal recruitment and its functional roles in meiosis, it is important to reveal the molecular details that govern Pch2 localization. Here, we review the current understanding of the different factors that control the recruitment of Pch2 to meiotic chromosomes, with a focus on research performed in budding yeast. During meiosis in this organism, Pch2 is enriched within the nucleolus, where it likely associates with the specialized chromatin of the ribosomal (r)DNA. Pch2 is also found on non-rDNA euchromatin, where its recruitment is contingent on Zip1, a component of the synaptonemal complex (SC) that assembles between homologous chromosomes. We discuss recent findings connecting the recruitment of Pch2 with its association with the Origin Recognition Complex (ORC) and reliance on RNA Polymerase II-dependent transcription. In total, we provide a comprehensive overview of the pathways that control the chromosomal association of an important meiotic regulator.

scholarly journals A high-resolution protein architecture of the budding yeast genome

Nature ◽  
2021 ◽  
Author(s):  
Matthew J. Rossi ◽  
Prashant K. Kuntala ◽  
William K. M. Lai ◽  
Naomi Yamada ◽  
Nitika Badjatia ◽  
...  
Keyword(s):  
High Resolution ◽  
Budding Yeast ◽  
Yeast Genome ◽  
Protein Architecture

The Budding Yeast Msh4 Protein Functions in Chromosome Synapsis and the Regulation of Crossover Distribution

Genetics ◽  
2001 ◽  
Vol 158 (3) ◽  
pp. 1013-1025 ◽  
Author(s):  
Janet E Novak ◽  
Petra B Ross-Macdonald ◽  
G Shirleen Roeder
Keyword(s):  
Mismatch Repair ◽  
Budding Yeast ◽  
Null Mutation ◽  
Crossing Over ◽  
Wild Type ◽  
Crossover Interference ◽  
Meiotic Chromosomes ◽  
Chromosome Synapsis ◽  
Protein Functions ◽  
Or Gene

AbstractThe budding yeast MSH4 gene encodes a MutS homolog produced specifically in meiotic cells. Msh4 is not required for meiotic mismatch repair or gene conversion, but it is required for wild-type levels of crossing over. Here, we show that a msh4 null mutation substantially decreases crossover interference. With respect to the defect in interference and the level of crossing over, msh4 is similar to the zip1 mutant, which lacks a structural component of the synaptonemal complex (SC). Furthermore, epistasis tests indicate that msh4 and zip1 affect the same subset of meiotic crossovers. In the msh4 mutant, SC formation is delayed compared to wild type, and full synapsis is achieved in only about half of all nuclei. The simultaneous defects in synapsis and interference observed in msh4 (and also zip1 and ndj1/tam1) suggest a role for the SC in mediating interference. The Msh4 protein localizes to discrete foci on meiotic chromosomes and colocalizes with Zip2, a protein involved in the initiation of chromosome synapsis. Both Zip2 and Zip1 are required for the normal localization of Msh4 to chromosomes, raising the possibility that the zip1 and zip2 defects in crossing over are indirect, resulting from the failure to localize Msh4 properly.

scholarly journals Bisection of the X chromosome disrupts the initiation of chromosome silencing during meiosis in Caenorhabditis elegans

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yisrael Rappaport ◽  
Hanna Achache ◽  
Roni Falk ◽  
Omer Murik ◽  
Oren Ram ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Rna Polymerase Ii ◽  
X Chromosome ◽  
Sex Chromosomes ◽  
Sex Chromosome ◽  
Transcription Analysis ◽  
X Chromosomes ◽  
Unique Character ◽  
Active Transcription ◽  
Heterogametic Sex

AbstractDuring meiosis, gene expression is silenced in aberrantly unsynapsed chromatin and in heterogametic sex chromosomes. Initiation of sex chromosome silencing is disrupted in meiocytes with sex chromosome-autosome translocations. To determine whether this is due to aberrant synapsis or loss of continuity of sex chromosomes, we engineered Caenorhabditis elegans nematodes with non-translocated, bisected X chromosomes. In early meiocytes of mutant males and hermaphrodites, X segments are enriched with euchromatin assembly markers and active RNA polymerase II staining, indicating active transcription. Analysis of RNA-seq data showed that genes from the X chromosome are upregulated in gonads of mutant worms. Contrary to previous models, which predicted that any unsynapsed chromatin is silenced during meiosis, our data indicate that unsynapsed X segments are transcribed. Therefore, our results suggest that sex chromosome chromatin has a unique character that facilitates its meiotic expression when its continuity is lost, regardless of whether or not it is synapsed.

Form and function of eukaryotic unstable non-coding RNAs

2012 ◽  
Vol 40 (4) ◽  
pp. 836-841 ◽  
Author(s):  
Jonathan Houseley
Keyword(s):  
Unknown Function ◽  
Budding Yeast ◽  
Rna Degradation ◽  
Present Review ◽  
Form And Function ◽  
Non Coding Rna ◽  
Non Coding Rnas ◽  
Higher Eukaryotes ◽  
And Function

Unstable non-coding RNAs are produced from thousands of loci in all studied eukaryotes (and also prokaryotes), but remain of largely unknown function. The present review summarizes the mechanisms of eukaryotic non-coding RNA degradation and highlights recent findings regarding function. The focus is primarily on budding yeast where the bulk of this research has been performed, but includes results from higher eukaryotes where available.

scholarly journals Commuting to Work: Nucleolar Long Non-Coding RNA Control Ribosome Biogenesis from Near and Far

2021 ◽  
Vol 7 (3) ◽  
pp. 42
Author(s):  
Victoria Mamontova ◽  
Barbara Trifault ◽  
Lea Boten ◽  
Kaspar Burger
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Ribosome Biogenesis ◽  
Intergenic Spacer ◽  
Cellular Growth ◽  
Rrna Synthesis ◽  
Protein Coding ◽  
Non Coding Rna ◽  
And Function ◽  
In Cis

Gene expression is an essential process for cellular growth, proliferation, and differentiation. The transcription of protein-coding genes and non-coding loci depends on RNA polymerases. Interestingly, numerous loci encode long non-coding (lnc)RNA transcripts that are transcribed by RNA polymerase II (RNAPII) and fine-tune the RNA metabolism. The nucleolus is a prime example of how different lncRNA species concomitantly regulate gene expression by facilitating the production and processing of ribosomal (r)RNA for ribosome biogenesis. Here, we summarise the current findings on how RNAPII influences nucleolar structure and function. We describe how RNAPII-dependent lncRNA can both promote nucleolar integrity and inhibit ribosomal (r)RNA synthesis by modulating the availability of rRNA synthesis factors in trans. Surprisingly, some lncRNA transcripts can directly originate from nucleolar loci and function in cis. The nucleolar intergenic spacer (IGS), for example, encodes nucleolar transcripts that counteract spurious rRNA synthesis in unperturbed cells. In response to DNA damage, RNAPII-dependent lncRNA originates directly at broken ribosomal (r)DNA loci and is processed into small ncRNA, possibly to modulate DNA repair. Thus, lncRNA-mediated regulation of nucleolar biology occurs by several modes of action and is more direct than anticipated, pointing to an intimate crosstalk of RNA metabolic events.

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.

scholarly journals Intergenic RNA mainly derives from nascent transcripts of known genes

2020 ◽  
Author(s):  
Agostini Federico ◽  
Zagalak Julian ◽  
Attig Jan ◽  
Ule Jernej ◽  
Nicholas M. Luscombe
Keyword(s):  
Rna Polymerase Ii ◽  
Human Genome ◽  
Genomic Context ◽  
Alternative Processing ◽  
Intergenic Regions ◽  
Transcriptional Units ◽  
And Function ◽  
Cellular Compartments ◽  
Nascent Transcripts ◽  
Transcriptional Start Sites

AbstractBackgroundEukaryotic genomes undergo pervasive transcription, leading to the production of many types of stable and unstable RNAs. Transcription is not restricted to regions with annotated gene features but includes almost any genomic context. Currently, the source and function of most RNAs originating from intergenic regions in the human genome remains unclear.ResultsWe hypothesised that many intergenic RNA can be ascribed to the presence of as-yet unannotated genes or the ‘fuzzy’ transcription of known genes that extends beyond the annotated boundaries. To elucidate the contributions of these two sources, we assembled a dataset of >2.5 billion publicly available RNA-seq reads across 5 human cell lines and multiple cellular compartments to annotate transcriptional units in the human genome. About 80% of transcripts from unannotated intergenic regions can be attributed to the fuzzy transcription of existing genes; the remaining transcripts originate mainly from putative long non-coding RNA loci that are rarely spliced. We validated the transcriptional activity of these intergenic RNA using independent measurements, including transcriptional start sites, chromatin signatures, and genomic occupancies of RNA polymerase II in various phosphorylation states. We also analysed the nuclear localisation and sensitivities of intergenic transcripts to nucleases to illustrate that they tend to be rapidly degraded either ‘on-chromatin’ by XRN2 or ‘off-chromatin’ by the exosome.ConclusionsWe provide a curated atlas of intergenic RNAs that distinguishes between alternative processing of well annotated genes from independent transcriptional units based on the combined analysis of chromatin signatures, nuclear RNA localisation and degradation pathways.

scholarly journals Programmed ER fragmentation drives selective ER inheritance and degradation in budding yeast meiosis

2021 ◽  
Author(s):  
George M. Otto ◽  
Tia Cheunkarndee ◽  
Jessica M. Leslie ◽  
Gloria A. Brar
Keyword(s):  
Plasma Membrane ◽  
Cell Differentiation ◽  
Budding Yeast ◽  
Developmentally Regulated ◽  
Selective Degradation ◽  
Cell Plasma ◽  
Membrane Tethering ◽  
Membrane Bound ◽  
Yeast Meiosis ◽  
And Function

AbstractThe endoplasmic reticulum (ER) is a membrane-bound organelle with diverse, essential functions that rely on the maintenance of membrane shape and distribution within cells. ER structure and function are remodeled in response to changes in cellular demand, such as the presence of external stressors or the onset of cell differentiation, but mechanisms controlling ER remodeling during cell differentiation are not well understood. Here, we describe a series of developmentally regulated changes in ER morphology and composition during budding yeast meiosis, a conserved differentiation program that gives rise to gametes. During meiosis, the cortical ER undergoes fragmentation before collapsing away from the plasma membrane at anaphase II. This programmed collapse depends on the meiotic transcription factor Ndt80, conserved ER membrane structuring proteins Lnp1 and reticulons, and the actin cytoskeleton. A subset of ER is retained at the mother cell plasma membrane and excluded from gamete cells via the action of ER-plasma membrane tethering proteins. ER remodeling is coupled to ER degradation by selective autophagy, which is regulated by the developmentally timed expression of the autophagy receptor Atg40. Autophagy relies on ER collapse, as artificially targeting ER proteins to the cortically retained ER pool prevents their degradation. Thus, developmentally programmed changes in ER morphology determine the selective degradation or inheritance of ER subdomains by gametes.

scholarly journals A human cancer cell line initiates DNA replication normally in the absence of ORC5 and ORC2 proteins

2020 ◽  
Vol 295 (50) ◽  
pp. 16949-16959
Author(s):  
Etsuko Shibata ◽  
Anindya Dutta
Keyword(s):  
Dna Replication ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Origin Recognition Complex ◽  
Human Cancer ◽  
Cancer Cell Line ◽  
Aaa Atpase ◽  
Normal Number ◽  
Mutant Cells

The origin recognition complex (ORC), composed of six subunits, ORC1–6, binds to origins of replication as a ring-shaped heterohexameric ATPase that is believed to be essential to recruit and load MCM2–7, the minichromosome maintenance protein complex, around DNA and initiate DNA replication. We previously reported the creation of viable cancer cell lines that lacked detectable ORC1 or ORC2 protein without a reduction in the number of origins firing. Here, using CRISPR-Cas9–mediated mutations, we report that human HCT116 colon cancer cells also survive when ORC5 protein expression is abolished via a mutation in the initiator ATG of the ORC5 gene. Even if an internal methionine is used to produce an undetectable, N terminally deleted ORC5, the protein would lack 80% of the AAA+ ATPase domain, including the Walker A motif. The ORC5-depleted cells show normal chromatin binding of MCM2–7 and initiate replication from a similar number of origins as WT cells. In addition, we introduced a second mutation in ORC2 in the ORC5 mutant cells, rendering both ORC5 and ORC2 proteins undetectable in the same cells and destabilizing the ORC1, ORC3, and ORC4 proteins. Yet the double mutant cells grow, recruit MCM2–7 normally to chromatin, and initiate DNA replication with normal number of origins. Thus, in these selected cancer cells, either a crippled ORC lacking ORC2 and ORC5 and present at minimal levels on the chromatin can recruit and load enough MCM2–7 to initiate DNA replication, or human cell lines can sometimes recruit MCM2–7 to origins independent of ORC.

Sign in / Sign up

Export Citation Format

Share Document