Search

The development of next-generation sequencing (NGS) technologies paved the way for studying the spatiotemporal coordination of cellular processes along the genome. However, data sets are commonly limited to a few time points, and missing information needs to be interpolated. Most models assume that the studied dynamics are similar between individual cells, so that a homogeneous cell culture can be represented by a population-wide average. Here, we demonstrate that this understanding can be inappropriate. We developed a thought experiment-which we call the NGS chess problem-in which we compare the temporal sequencing data analysis to observing a superimposed picture of many independent games of chess at a time. The analysis of the spatiotemporal kinetics advocates for a new methodology that considers DNA-particle interactions in each cell independently even for a homogeneous cell population., (© The Author(s) 2024. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

The Mediator complex is an essential coregulator of RNA polymerase II transcription. More recent developments suggest Mediator functions as a link between transcription regulation, genome organisation and DNA repair mechanisms including nucleotide excision repair, base excision repair, and homologous recombination. Dysfunctions of these processes are frequently associated with human pathologies, and growing evidence shows Mediator involvement in cancers, neurological, metabolic and infectious diseases. The detailed deciphering of molecular mechanisms of Mediator functions, using interdisciplinary approaches in different biological models and considering all functions of this complex, will contribute to our understanding of relevant human diseases., Competing Interests: Declaration of Competing Interest We hereby declare that we do not have any conflicts of interests regarding this manuscript., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

In eukaryotic cells, the one-dimensional DNA molecules need to be tightly packaged into the spatially constraining nucleus. Folding is achieved on its lowest level by wrapping the DNA around nucleosomes. Their arrangement regulates other nuclear processes, such as transcription and DNA repair. Despite strong efforts to study nucleosome positioning using Next Generation Sequencing (NGS) data, the mechanism of their collective arrangement along the gene body remains poorly understood. Here, we classify nucleosome distributions of protein-coding genes in Saccharomyces cerevisiae according to their profile similarity and analyse their differences using functional Principal Component Analysis. By decomposing the NGS signals into their main descriptive functions, we compared wild type and chromatin remodeler-deficient strains, keeping position-specific details preserved whilst considering the nucleosome arrangement as a whole. A correlation analysis with other genomic properties, such as gene size and length of the upstream Nucleosome Depleted Region (NDR), identified key factors that influence the nucleosome distribution. We reveal that the RSC chromatin remodeler-which is responsible for NDR maintenance-is indispensable for decoupling nucleosome arrangement within the gene from positioning outside, which interfere in rsc8-depleted conditions. Moreover, nucleosome profiles in chd1Δ strains displayed a clear correlation with RNA polymerase II presence, whereas wild type cells did not indicate a noticeable interdependence. We propose that RSC is pivotal for global nucleosome organisation, whilst Chd1 plays a key role for maintaining local arrangement., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zeitler et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Chromatin organization is crucial for transcriptional regulation in eukaryotes. Mediator is an essential and conserved co-activator thought to act in concert with chromatin regulators. However, it remains largely unknown how their functions are coordinated. Here, we provide evidence in the yeast Saccharomyces cerevisiae that Mediator establishes physical contact with RSC (Remodels the Structure of Chromatin), a conserved and essential chromatin remodeling complex that is crucial for nucleosome-depleted region (NDR) formation. We determine the role of Mediator-RSC interaction in their chromatin binding, nucleosome occupancy, and transcription on a genomic scale. Mediator and RSC co-localize on wide NDRs of promoter regions, and specific Mediator mutations affect nucleosome eviction and TSS-associated +1 nucleosome stability. This work shows that Mediator contributes to RSC remodeling function to shape NDRs and maintain chromatin organization on promoter regions. It will help in our understanding of transcriptional regulation in the chromatin context relevant for severe diseases., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The great advances of sequencing technologies allow the in vivo measurement of nuclear processes-such as DNA repair after UV exposure-over entire cell populations. However, data sets usually contain only a few samples over several hours, missing possibly important information in between time points. We developed a data-driven approach to analyse CPD repair kinetics over time in Saccharomyces cerevisiae. In contrast to other studies that consider sequencing signals as an average behaviour, we understand them as the superposition of signals from independent cells. By motivating repair as a stochastic process, we derive a minimal model for which the parameters can be conveniently estimated. We correlate repair parameters to a variety of genomic features that are assumed to influence repair, including transcription rate and nucleosome density. The clearest link was found for the transcription unit length, which has been unreported for budding yeast to our knowledge. The framework hence allows a comprehensive analysis of nuclear processes on a population scale., Competing Interests: The authors have declared that no competing interests exist.

Mediator is a conserved coregulator playing a key role in RNA polymerase (Pol) II transcription. Mediator also links transcription and nucleotide excision repair (NER) via a direct contact with Rad2/ERCC5(XPG) endonuclease. In this work, we analyzed the genome-wide distribution of Rad26/ERCC6(CSB) and Rad1-Rad10/ERCC4(XPF)-ERCC1, addressing the question of a potential link of these proteins with Mediator and Pol II in yeast Saccharomyces cerevisiae Our genomic analyses reveal that Rad1-Rad10 and Rad26 are present on the yeast genome in the absence of genotoxic stress, especially at highly transcribed regions, with Rad26 binding strongly correlating with that of Pol II. Moreover, we show that Rad1-Rad10 and Rad26 colocalize with Mediator at intergenic regions and physically interact with this complex. Using kin28 TFIIH mutant, we found that Mediator stabilization on core promoters leads to an increase in Rad1-Rad10 chromatin binding, whereas Rad26 occupancy follows mainly a decrease in Pol II transcription. Combined with multivariate analyses, our results show the relationships between Rad1-Rad10, Rad26, Mediator, and Pol II, modulated by the changes in binding dynamics of Mediator and Pol II transcription. In conclusion, we extend the Mediator link to Rad1-Rad10 and Rad26 NER proteins and reveal important differences in their dependence on Mediator and Pol II. Rad2 is the most dependent on Mediator, followed by Rad1-Rad10, whereas Rad26 is the most closely related to Pol II. Our work thus contributes to new concepts of the functional interplay between transcription and DNA repair machineries, which are relevant for human diseases including cancer and XP/CS syndromes., (© 2022 Gopaul et al.; Published by Cold Spring Harbor Laboratory Press.)

Mutations in DNA have large-ranging consequences, from evolution to disease. Many mechanisms contribute to mutational processes such as dysfunctions in DNA repair pathways and exogenous or endogenous mutagen exposures. Model organisms and mutation accumulation (MA) experiments are indispensable to study mutagenesis. Classical MA is, however, time consuming and laborious. To fill the need for more efficient approaches to characterize mutational profiles, we have developed an innovative microfluidic-based system that automatizes MA culturing over many generations in budding yeast. This unique experimental tool, coupled with high-throughput sequencing, reduces by one order of magnitude the time required for genome-wide measurements of mutational profiles, while also parallelizing and simplifying the cell culture. To validate our approach, we performed microfluidic MA experiments on two different genetic backgrounds, a wild-type strain and a base-excision DNA repair ung1 mutant characterized by a well-defined mutational profile. We show that the microfluidic device allows for mutation accumulation comparable to the traditional method on plate. Our approach thus paves the way to massively-parallel MA experiments with minimal human intervention that can be used to investigate mutational processes at the origin of human diseases and to identify mutagenic compounds relevant for medical and environmental research.

Dysfunctions of nuclear processes including transcription and DNA repair lead to severe human diseases. Gaining an understanding of how these processes operate in the crowded context of chromatin can be particularly challenging. Mediator is a large multiprotein complex conserved in eukaryotes with a key coactivator role in the regulation of RNA polymerase (Pol) II transcription. Despite intensive studies, the molecular mechanisms underlying Mediator function remain to be fully understood. Novel findings have provided insights into the relationship between Mediator and chromatin architecture, revealed its role in connecting transcription with DNA repair and proposed an emerging mechanism of phase separation involving Mediator condensates. Recent developments in the field suggest multiple functions of Mediator going beyond transcriptional processes per se that would explain its involvement in various human pathologies., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Mre11-Rad50-Xrs2 (MRX) is a highly conserved complex with key roles in various aspects of DNA repair. Here, we report a new function for MRX in limiting transcription in budding yeast. We show that MRX interacts physically and colocalizes on chromatin with the transcriptional co-regulator Mediator. MRX restricts transcription of coding and noncoding DNA by a mechanism that does not require the nuclease activity of Mre11. MRX is required to tether transcriptionally active loci to the nuclear pore complex (NPC), and it also promotes large-scale gene-NPC interactions. Moreover, MRX-mediated chromatin anchoring to the NPC contributes to chromosome folding and helps to control gene expression. Together, these findings indicate that MRX has a role in transcription and chromosome organization that is distinct from its known function in DNA repair., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Transcription and maintenance of genome integrity are fundamental cellular functions. Deregulation of transcription and defects in DNA repair lead to serious pathologies. The Mediator complex links RNA polymerase (Pol) II transcription and nucleotide excision repair via Rad2/XPG endonuclease. However, the functional interplay between Rad2/XPG, Mediator and Pol II remains to be determined. In this study, we investigated their functional dynamics using genomic and genetic approaches. In a mutant affected in Pol II phosphorylation leading to Mediator stabilization on core promoters, Rad2 genome-wide occupancy shifts towards core promoters following that of Mediator, but decreases on transcribed regions together with Pol II. Specific Mediator mutations increase UV sensitivity, reduce Rad2 recruitment to transcribed regions, lead to uncoupling of Rad2, Mediator and Pol II and to colethality with deletion of Rpb9 Pol II subunit involved in transcription-coupled repair. We provide new insights into the functional interplay between Rad2, Mediator and Pol II and propose that dynamic interactions with Mediator and Pol II are involved in Rad2 loading to the chromatin. Our work contributes to the understanding of the complex link between transcription and DNA repair machineries, dysfunction of which leads to severe diseases., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In this issue of Cell, Casellas and colleagues provide insights into the structural and functional aspects of the mammalian multi-subunit Mediator complex, a conserved and essential transcriptional coregulator. Combining cryo-EM, genetic, and genomic analyses, the work sheds light on Mediator's mode of action as a functional bridge between enhancers and promoters., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Alterations in the regulation of gene expression are frequently associated with developmental diseases or cancer. Transcription activation is a key phenomenon in the regulation of gene expression. In all eukaryotes, mediator of RNA polymerase II transcription (Mediator), a large complex with modular organization, is generally required for transcription by RNA polymerase II, and it regulates various steps of this process. The main function of Mediator is to transduce signals from the transcription activators bound to enhancer regions to the transcription machinery, which is assembled at promoters as the preinitiation complex (PIC) to control transcription initiation. Recent functional studies of Mediator with the use of structural biology approaches and functional genomics have revealed new insights into Mediator activity and its regulation during transcription initiation, including how Mediator is recruited to transcription regulatory regions and how it interacts and cooperates with PIC components to assist in PIC assembly. Novel roles of Mediator in the control of gene expression have also been revealed by showing its connection to the nuclear pore and linking Mediator to the regulation of gene positioning in the nuclear space. Clear links between Mediator subunits and disease have also encouraged studies to explore targeting of this complex as a potential therapeutic approach in cancer and fungal infections.

Mediator is a multisubunit complex conserved in eukaryotes that plays an essential coregulator role in RNA polymerase (Pol) II transcription. Despite intensive studies of the Mediator complex, the molecular mechanisms of its function in vivo remain to be fully defined. In this review, we will discuss the different aspects of Mediator function starting with its interactions with specific transcription factors, its recruitment to chromatin and how, as a coregulator, it contributes to the assembly of transcription machinery components within the preinitiation complex (PIC) in vivo and beyond the PIC formation.

Mediator is a large coregulator complex conserved from yeast to humans and involved in many human diseases, including cancers. Together with general transcription factors, it stimulates preinitiation complex (PIC) formation and activates RNA polymerase II (Pol II) transcription. In this study, we analyzed how Mediator acts in PIC assembly using in vivo, in vitro, and in silico approaches. We revealed an essential function of the Mediator middle module exerted through its Med10 subunit, implicating a key interaction between Mediator and TFIIB. We showed that this Mediator-TFIIB link has a global role on PIC assembly genome-wide. Moreover, the amplitude of Mediator's effect on PIC formation is gene-dependent and is related to the promoter architecture in terms of TATA elements, nucleosome occupancy, and dynamics. This study thus provides mechanistic insights into the coordinated function of Mediator and TFIIB in PIC assembly in different chromatin contexts., (© 2016 Eychenne et al.; Published by Cold Spring Harbor Laboratory Press.)

Mediator is a large multiprotein complex conserved in all eukaryotes, which has a crucial coregulator function in transcription by RNA polymerase II (Pol II). However, the molecular mechanisms of its action in vivo remain to be understood. Med17 is an essential and central component of the Mediator head module. In this work, we utilised our large collection of conditional temperature-sensitive med17 mutants to investigate Mediator's role in coordinating preinitiation complex (PIC) formation in vivo at the genome level after a transfer to a non-permissive temperature for 45 minutes. The effect of a yeast mutation proposed to be equivalent to the human Med17-L371P responsible for infantile cerebral atrophy was also analyzed. The ChIP-seq results demonstrate that med17 mutations differentially affected the global presence of several PIC components including Mediator, TBP, TFIIH modules and Pol II. Our data show that Mediator stabilizes TFIIK kinase and TFIIH core modules independently, suggesting that the recruitment or the stability of TFIIH modules is regulated independently on yeast genome. We demonstrate that Mediator selectively contributes to TBP recruitment or stabilization to chromatin. This study provides an extensive genome-wide view of Mediator's role in PIC formation, suggesting that Mediator coordinates multiple steps of a PIC assembly pathway., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)