Biology subjects

Gambus, A.

Publications and source records attributed to Gambus, A..

8 recordsLinked to original sources

bioRxiv2024

RNA Polymerase II subunits overexpressions induce genome instability and deregulate transcription

Independently of the pathways or circuits deregulated in cancer cells, these present altered transcription patterns, often also direct consequence of deregulation of transcription factors. In this sense, also the RNA Polymerase complexes responsible for transcription can be affected in cancers. We find that upregulations of RNA Polymerase II subunits, especially the largest ones, correlates with poor cancer patients outcome across a range of tumor types, presenting increased genome instability. Overexpressing the subunits RPB1, RPB3 and RPB4 in cells we find that these induce DNA damage. However, the mechanisms behind this increased genome instability are specific for each subunit, linked to the unique transcription alterations generated by the subunit overexpression. Importantly, we find significant overlap between the genes with more DNA damage in our cell line models and those more affected in cancers with subunit upregulation, indicating that upregulations could be responsible for some of the phenotypes present in these patients.

molecular biology↗

bioRxiv2023

Profiling ubiquitin signaling with UBIMAX reveals DNA damage- and SCFβTRCP-dependent ubiquitylation of the actin-organizing protein Dbn1

Ubiquitin widely modifies proteins, thereby regulating most cellular functions. The complexity of ubiquitin signalling necessitates unbiased methods enabling global detection of dynamic protein ubiquitylation. Here, we describe UBIMAX (UBiquitin target Identification by Mass spectrometry in Xenopus egg extracts), which enriches ubiquitin-conjugated proteins and quantifies regulation of protein ubiquitylation under precise and adaptable conditions. We benchmark UBIMAX by investigating DNA double-strand break-responsive ubiquitylation events, identifying previously known targets and revealing the actin-organising protein Dbn1 as a novel major target of DNA damage-induced ubiquitylation. We find that Dbn1 is targeted for proteasomal degradation by the SCF{beta}-Trcp1 ubiquitin ligase, in a conserved mechanism driven by ATM-mediated phosphorylation of a previously uncharacterized {beta}-Trcp1 degron containing an SQ motif. We further show that this degron is sufficient to induce DNA-damage dependent protein degradation of a model substrate. Collectively, we demonstrate UBIMAXs ability to identify novel targets of stimulus-regulated ubiquitylation and reveal an SCF{beta}-Trcp1-mediated ubiquitylation mechanism controlled directly by the apical DNA damage response kinases.

molecular biology↗

bioRxiv2022

Characterising replisome disassembly in human cells

To ensure faultless duplication of the entire genome, eukaryotic replication initiates from thousands of replication origins. Replication forks emanating from origins move through the chromatin until they encounter forks from neighbouring origins, at which point they terminate. In the final stages of this process the replication machinery (replisome) is unloaded from chromatin and disassembled. Work from model organisms has elucidated that during replisome unloading, the MCM7 subunit of the terminated replicative helicase is polyubiquitylated and processed by p97/VCP segregase, leading to disassembly of the helicase and the replisome, which is built around it. In higher eukaryotes (worms, frogs, mouse embryonic stem cells), MCM7 ubiquitylation is driven by a Cullin2-based ubiquitin ligase, with LRR1 as a substrate receptor. To date, most of our knowledge of replication termination comes from model organisms and embryonic systems and little is known about how this process is executed and regulated in human somatic cells. Here we thus established methods to study replisome disassembly in human model cell lines. Using flow cytometry, immunofluorescence microscopy and chromatin isolation with western blotting, we can visualise unloading of the replisome (MCM7 and CDC45) from chromatin by the end of S-phase. We observe interaction of replicative helicase (CMG complex) with CUL2LRR1 and ubiquitylation of MCM7 on chromatin, specifically in S-phase, suggesting that this is a replication-dependent modification. Importantly, we are able to show that replisome disassembly in this system also requires Cullin2, LRR1 and p97, demonstrating conservation of the mechanism. Moreover, we present evidence that the back-up mitotic replisome disassembly pathway is also recapitulated in human somatic cells. Finally, while we find that treatment with small molecule inhibitors against cullin-based ubiquitin ligases (CULi) and p97 (p97i) does lead to phenotypes of replisome disassembly defects, they also both lead to induction of replication stress in somatic cells, which limits their usefulness as tools to specifically target replisome disassembly processes in this setting.

cell biology↗

bioRxiv2022

Ubiquitin ligase TRAIP plays an essential role during the S-phase of unperturbed cell cycle in the resolution of DNA replication - transcription conflicts

Cell division is the basis for the propagation of life and requires accurate duplication of all genetic information. DNA damage created during replication (replication stress) is a major cause of cancer, premature aging and a spectrum of other human disorders. Over the years, TRAIP E3 ubiquitin ligase has been shown to play a role in various cellular processes that govern genome integrity and faultless segregation. TRAIP is essential for cell viability, and mutations in TRAIP ubiquitin ligase activity lead to primordial dwarfism in patients. Here, for the first time, we have determined the mechanism of inhibition of cell proliferation in TRAIP-depleted cells. We have taken advantage of the auxin induced degron system to rapidly degrade TRAIP within cells and to dissect the importance of various functions of TRAIP in different stages of the cell cycle. We find that upon rapid TRAIP degradation, specifically in S-phase, cells cease to proliferate, arrest in G2 stage of the cell cycle and undergo senescence. Our findings reveal that TRAIP works in S-phase to prevent DNA damage at transcription start sites, caused by replication-transcription conflicts.

cell biology↗

bioRxiv2021

The p97 cofactor Ubxn7 facilitates replisome disassembly during S-phase

Complex cellular processes are driven by the regulated assembly and disassembly of large multi-protein complexes. In eukaryotic DNA replication, whilst we are beginning to understand the molecular mechanism for assembly of the replication machinery (replisome), we still know relatively little about the regulation of its disassembly at replication termination. Over recent years, the first elements of this process have emerged, revealing that the replicative helicase, at the heart of the replisome, is polyubiquitylated prior to unloading and that this unloading requires p97 segregase activity. Two different E3 ubiquitin ligases are now known to ubiquitylate the helicase under different conditions: Cul2Lrr1 and TRAIP. Here we have found two p97 cofactors, Ubxn7 and Faf1, which can interact with p97 during replisome disassembly in S-phase. Only Ubxn7 however facilitates efficient replisome disassembly through its interaction with both Cul2Lrr1 and p97. Our data therefore characterise Ubxn7 as the first substrate-specific p97 cofactor regulating replisome disassembly in vertebrates.

biochemistry↗

bioRxiv2020

An ATM-MYBL2-CDC7 axis regulates replication initiation and prevents replication stress in pluripotent stem cells

Replication stress, a major cause of genome instability in cycling cells, is mainly prevented by the ATR-dependent replication stress response pathway in somatic cells. However, the replication stress response pathway in embryonic stem cells (ESCs) may be substantially different due to alterations in cell cycle phase length. The transcription factor MYBL2, which is implicated in cell cycle regulation, is expressed between hundred to thousand-fold more highly in ESCs compared to somatic cells. Here we show that MYBL2 functions to activate ATM and suppress replication stress in ESCs. Consequently, loss of MYBL2 or inhibition of ATM or Mre11 in ESCs, results in replication fork slowing, increased fork stalling and elevated origin firing. Additionally, we demonstrate that inhibition of CDC7 activity rescues replication stress induced by MYBL2 loss and ATM inhibition, suggesting that uncontrolled new origin firing may underlie the replication stress phenotype resulting from loss/inhibition of MYBL2 and ATM. Overall, this study proposes that in addition to ATR, a MYBL2-MRN-ATM replication stress response pathway functions in ESCs to control DNA replication initiation and prevent genome instability.

cell biology↗

bioRxiv2018

Mitotic replisome disassembly in vertebrates

Recent years have brought a breakthrough in our understanding of the process of eukaryotic DNA replication termination. We have shown that the process of replication machinery (replisome) disassembly at the termination of DNA replication forks in S-phase of the cell cycle is driven through polyubiquitylation of one of the replicative helicase subunits Mcm7. Our previous work in C.elegans embryos suggested also an existence of a back-up pathway of replisome disassembly in mitosis. Here we show, that in Xenopus laevis egg extract, any replisome retained on chromatin after S-phase is indeed removed from chromatin in mitosis. This mitotic disassembly pathway depends on formation of K6 and K63 ubiquitin chains on Mcm7 by TRAIP ubiquitin ligase and activity of p97/VCP protein segregase. The mitotic replisome pathway is therefore conserved through evolution in higher eukaryotes. However, unlike in lower eukaryotes it does not require SUMO modifications. This process can also remove any helicases from chromatin, including \"active\" stalled ones, indicating a much wider application of this pathway than just a \"back-up\" for terminated helicases.

molecular biology↗

bioRxiv2018

A cell cycle-coordinated nuclear compartment for Polymerase II transcription encompasses the earliest gene expression before global genome activation

Most metazoan embryos commence development with rapid cleavages without zygotic gene expression and their genome activation is delayed until the mid-blastula transition (MBT). However, a set of genes escape global repression during the extremely fast cell cycles, which lack gap phases and their transcription is activated before the MBT. Here we describe the formation and the spatio-temporal dynamics of a distinct transcription compartment, which encompasses the earliest detectable transcription during the first wave of genome activation. Simultaneous 4D imaging of expression of pri-miR430 and zinc finger genes by a novel, native transcription imaging approach reveals a pair of shared transcription compartments regulated by homolog chromosome organisation. These nuclear compartments carry the majority of nascent RNAs and transcriptionally active Polymerase II, are depleted of compact chromatin and represent the main sites for detectable transcription before MBT. We demonstrate that transcription occurs in the S-phase of the cleavage cycles and that the gradual slowing of these cell cycles are permissive to transcription before global genome activation. We propose that the demonstrated transcription compartment is part of the regulatory architecture of nucleus organisation, and provides a transcriptionally competent, supporting environment to facilitate early escape from the general nuclear repression before global genome activation.

developmental biology↗