laser-micro-irradiation-to-study-dna-recruitment-during-s-phase

Laser Micro-Irradiation to Study DNA Recruitment During S Phase

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both ...

visualizing-single-stranded-dna-foci-in-the-g1-phase-of-the-cell-cycle

Visualizing Single-Stranded DNA Foci in the G1 Phase of the Cell Cycle

DNA has dedicated cellular repair pathways capable of coping with lesions that could arise from both endogenous and/or exogenous sources. DNA repair ...

New York University School of Medicine

NYU Grossman School of Medicine

NYU Langone Health

HUN-REN Research Centre for Natural Sciences

SLBP (stem-loop binding protein) is a highly conserved factor necessary for the processing, translation, and degradation of H2AFX and canonical histone mRNAs. We identified the F-box protein cyclin F, a substrate recognition subunit of an SCF (Skp1-Cul1-F-box protein) complex, as the G2 ubiquitin ligase for SLBP. SLBP interacts with cyclin F via an atypical CY motif, and mutation of this motif prevents SLBP degradation in G2. Expression of an SLBP stable mutant results in increased loading of H2AFX mRNA onto polyribosomes, resulting in increased expression of H2A.X (encoded by H2AFX). Upon genotoxic stress in G2, high levels of H2A.X lead to persistent γH2A.X signaling, high levels of H2A.X phosphorylated on Tyr142, high levels of p53, and induction of apoptosis. We propose that cyclin F co-evolved with the appearance of stem-loops in vertebrate H2AFX mRNA to mediate SLBP degradation, thereby limiting H2A.X synthesis and cell death upon genotoxic stress.

Cyclin F-Mediated Degradation of SLBP Limits H2A.X Accumulation and Apoptosis upon Genotoxic Stress in G2.

Transmembrane proteins play crucial role in signaling, ion transport, nutrient uptake, as well as in maintaining the dynamic equilibrium between the internal and external environment of cells. Despite their important biological functions and abundance, less than 2% of all determined structures are transmembrane proteins. Given the persisting technical difficulties associated with high resolution structure determination of transmembrane proteins, additional methods, including computational and experimental techniques remain vital in promoting our understanding of their topologies, 3D structures, functions and interactions. Here we report a method for the high-throughput determination of extracellular segments of transmembrane proteins based on the identification of surface labeled and biotin captured peptide fragments by LC/MS/MS. We show that reliable identification of extracellular protein segments increases the accuracy and reliability of existing topology prediction algorithms. Using the experimental topology data as constraints, our improved prediction tool provides accurate and reliable topology models for hundreds of human transmembrane proteins.

Identification of Extracellular Segments by Mass Spectrometry Improves Topology Prediction of Transmembrane Proteins.

The mammalian FBXL10-RNF68-RNF2 ubiquitin ligase complex (FRRUC) mono-ubiquitylates H2A at Lys119 to repress transcription in unstressed cells. We found that the FRRUC is rapidly and transiently recruited to sites of DNA damage in a PARP1- and TIMELESS-dependent manner to promote mono-ubiquitylation of H2A at Lys119, a local decrease of H2A levels, and an increase of H2A.Z incorporation. Both the FRRUC and H2A.Z promote transcriptional repression, double strand break signaling, and homologous recombination repair (HRR). All these events require both the presence and activity of the FRRUC. Moreover, the FRRUC and its activity are required for the proper recruitment of BMI1-RNF2 and MEL18-RNF2, two other ubiquitin ligases that mono-ubiquitylate Lys119 in H2A upon genotoxic stress. Notably, whereas H2A.Z is not required for H2A mono-ubiquitylation, impairment of the latter results in the inhibition of H2A.Z incorporation. We propose that the recruitment of the FRRUC represents an early and critical regulatory step in HRR.

PARP1-dependent recruitment of the FBXL10-RNF68-RNF2 ubiquitin ligase to sites of DNA damage controls H2A.Z loading.

Sanitization of nucleotide pools is essential for genome maintenance. Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is a key enzyme in this pathway since it catalyzes the cleavage of 2'-deoxyuridine 5'-triphosphate (dUTP) into 2'-deoxyuridine 5'-monophosphate (dUMP) and inorganic pyrophosphate. Through its action dUTPase efficiently prevents uracil misincorporation into DNA and at the same time provides dUMP, the substrate for de novo thymidylate biosynthesis. Despite its physiological significance, knock-out models of dUTPase have not yet been investigated in mammals, but only in unicellular organisms, such as bacteria and yeast. Here we generate CRISPR/Cas9-mediated dUTPase knock-out in mice. We find that heterozygous  +/- animals are viable while having decreased dUTPase levels. Importantly, we show that dUTPase is essential for embryonic development since early  -/- embryos reach the blastocyst stage, however, they die shortly after implantation. Analysis of pre-implantation embryos indicates perturbed growth of both inner cell mass (ICM) and trophectoderm (TE). We conclude that dUTPase is indispensable for post-implantation development in mice.

CRISPR/Cas9-Mediated Knock-Out of dUTPase in Mice Leads to Early Embryonic Lethality.

Epithelial to mesenchymal transition (EMT) is a multipurpose process involved in wound healing, development, and certain pathological processes, such as metastasis formation. The Tks4 scaffold protein has been implicated in cancer progression; however, its role in oncogenesis is not well defined. In this study, the function of Tks4 was investigated in HCT116 colon cancer cells by knocking the protein out using the CRISPR/Cas9 system. Surprisingly, the absence of Tks4 induced significant changes in cell morphology, motility, adhesion and expression, and localization of E-cadherin, which are all considered as hallmarks of EMT. In agreement with these findings, the marked appearance of fibronectin, a marker of the mesenchymal phenotype, was also observed in Tks4-KO cells. Analysis of the expression of well-known EMT transcription factors revealed that Snail2 was strongly overexpressed in cells lacking Tks4. Tks4-KO cells showed increased motility and decreased cell-cell attachment. Collagen matrix invasion assays demonstrated the abundance of invasive solitary cells. Finally, the reintroduction of Tks4 protein in the Tks4-KO cells restored the expression levels of relevant key transcription factors, suggesting that the Tks4 scaffold protein has a specific and novel role in EMT regulation and cancer progression.

Absence of the Tks4 Scaffold Protein Induces Epithelial-Mesenchymal Transition-Like Changes in Human Colon Cancer Cells.

Diverse linkage in polyubiquitin chain structure gives cells an unparalleled complexity to virtually modulate all aspects of cell biology. Substrates can be covalently modified by ubiquitin chains of different topology. Proper DNA damage response takes advantage of this regulatory system and heavily relies on ubiquitin-based signaling. Moreover, increasing evidence suggests that chain specificity dictates DNA repair outcome. In this issue of , Wu and colleagues (pp. 1702-1717) show that Cezanne and Cezanne2, two paralogous deubiquitinating enzymes that are recruited to sites of DNA damage, ensure proper local polyubiquitin chain composition for downstream DNA repair protein assembly. Their study offers a key insight into the mechanism of crosstalk between linkage-specific ubiquitylation at DNA damage sites, while simultaneously raising important questions for future research.

Mixed ubiquitin chains regulate DNA repair.

The SMC5/6 complex, cohesin and condensin are the three mammalian members of the structural maintenance of chromosomes (SMC) family, large ring-like protein complexes that are essential for genome maintenance. The SMC5/6 complex is the least characterized complex in mammals; however, it is known to be involved in homologous recombination repair (HRR) and chromosome segregation.

Interaction between NSMCE4A and GPS1 links the SMC5/6 complex to the COP9 signalosome.

Numerous anti-cancer drugs perturb thymidylate biosynthesis and lead to genomic uracil incorporation contributing to their antiproliferative effect. Still, it is not yet characterized if uracil incorporations have any positional preference. Here, we aimed to uncover genome-wide alterations in uracil pattern upon drug treatments in human cancer cell line models derived from HCT116. We developed a straightforward U-DNA sequencing method (U-DNA-Seq) that was combined with in situ super-resolution imaging. Using a novel robust analysis pipeline, we found broad regions with elevated probability of uracil occurrence both in treated and non-treated cells. Correlation with chromatin markers and other genomic features shows that non-treated cells possess uracil in the late replicating constitutive heterochromatic regions, while drug treatment induced a shift of incorporated uracil towards segments that are normally more active/functional. Data were corroborated by colocalization studies  dSTORM microscopy. This approach can be applied to study the dynamic  nature of genomic uracil.

Genome-wide alterations of uracil distribution patterns in human DNA upon chemotherapeutic treatments.

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway. Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis.

AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity.

D-type cyclins are central regulators of the cell division cycle and are among the most frequently deregulated therapeutic targets in human cancer, but the mechanisms that regulate their turnover are still being debated. Here, by combining biochemical and genetics studies in somatic cells, we identify CRL4 (also known as CRL4) as the ubiquitin ligase that targets all three D-type cyclins for degradation. During development, loss of Ambra1 induces the accumulation of D-type cyclins and retinoblastoma (RB) hyperphosphorylation and hyperproliferation, and results in defects of the nervous system that are reduced by treating pregnant mice with the FDA-approved CDK4 and CDK6 (CDK4/6) inhibitor abemaciclib. Moreover, AMBRA1 acts as a tumour suppressor in mouse models and low AMBRA1 mRNA levels are predictive of poor survival in cancer patients. Cancer hotspot mutations in D-type cyclins abrogate their binding to AMBRA1 and induce their stabilization. Finally, a whole-genome, CRISPR-Cas9 screen identified AMBRA1 as a regulator of the response to CDK4/6 inhibition. Loss of AMBRA1 reduces sensitivity to CDK4/6 inhibitors by promoting the formation of complexes of D-type cyclins with CDK2. Collectively, our results reveal the molecular mechanism that controls the stability of D-type cyclins during cell-cycle progression, in development and in human cancer, and implicate AMBRA1 as a critical regulator of the RB pathway.

CRL4 is a master regulator of D-type cyclins.

The risk of zoonotic coronavirus spillover into the human population, as highlighted by the SARS-CoV-2 pandemic, demands the development of pan-coronavirus antivirals. The efficacy of existing antiviral ribonucleoside/ribonucleotide analogs, such as remdesivir, is decreased by the viral proofreading exonuclease NSP14-NSP10 complex. Here, using a novel assay and in silico modeling and screening, we identified NSP14-NSP10 inhibitors that increase remdesivir's potency. A model compound, sofalcone, both inhibits the exonuclease activity of SARS-CoV-2, SARS-CoV, and MERS-CoV in vitro, and synergistically enhances the antiviral effect of remdesivir, suppressing the replication of SARS-CoV-2 and the related human coronavirus OC43. The validation of top hits from our primary screenings using cellular systems provides proof-of-concept for the NSP14 complex as a therapeutic target.

The NSP14/NSP10 RNA repair complex as a Pan-coronavirus therapeutic target.

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine lung cancer. Oncogenic MYC amplifications drive SCLC heterogeneity, but the genetic mechanisms of MYC amplification and phenotypic plasticity, characterized by neuroendocrine and nonneuroendocrine cell states, are not known. Here, we integrate whole-genome sequencing, long-range optical mapping, single-cell DNA sequencing, and fluorescence in situ hybridization to find extrachromosomal DNA (ecDNA) as a primary source of SCLC oncogene amplifications and driver fusions. ecDNAs bring to proximity enhancer elements and oncogenes, creating SCLC transcription-amplifying units, driving exceptionally high MYC gene dosage. We demonstrate that cell-free nucleosome profiling can noninvasively detect ecDNA amplifications in plasma, facilitating its genome-wide interrogation in SCLC and other cancers. Altogether, our work provides the first comprehensive map of SCLC ecDNA and describes a new mechanism that governs MYC-driven SCLC heterogeneity. ecDNA-enabled transcriptional flexibility may explain the significantly worse survival outcomes of SCLC harboring complex ecDNA amplifications.

ArticleTotal : 2	Year
Publication title Cited by 6	2021
Publication title Cited by 2	2023

Article	Year
Cyclin F-Mediated Degradation of SLBP Limits H2A.X Accumulation and Apoptosis upon Genotoxic Stress in G2. Molecular cell\| PubMed ID: 27773672	2016
Identification of Extracellular Segments by Mass Spectrometry Improves Topology Prediction of Transmembrane Proteins. Scientific reports\| PubMed ID: 28211907	2017
PARP1-dependent recruitment of the FBXL10-RNF68-RNF2 ubiquitin ligase to sites of DNA damage controls H2A.Z loading. eLife\| PubMed ID: 29985131	2018
CRISPR/Cas9-Mediated Knock-Out of dUTPase in Mice Leads to Early Embryonic Lethality. Biomolecules\| PubMed ID: 30987342	2019
Absence of the Tks4 Scaffold Protein Induces Epithelial-Mesenchymal Transition-Like Changes in Human Colon Cancer Cells. Cells\| PubMed ID: 31671862	2019
Mixed ubiquitin chains regulate DNA repair. Genes & development\| PubMed ID: 31792015	2019
Interaction between NSMCE4A and GPS1 links the SMC5/6 complex to the COP9 signalosome. BMC molecular and cell biology\| PubMed ID: 32384871	2020
Genome-wide alterations of uracil distribution patterns in human DNA upon chemotherapeutic treatments. eLife\| PubMed ID: 32956035	2020
AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity. Nature\| PubMed ID: 33854232	2021
CRL4 is a master regulator of D-type cyclins. Nature\| PubMed ID: 33854235	2021
The NSP14/NSP10 RNA repair complex as a Pan-coronavirus therapeutic target. Cell death and differentiation\| PubMed ID: 34862481	2022
Extrachromosomal DNA Amplification Contributes to Small Cell Lung Cancer Heterogeneity and Is Associated with Worse Outcomes. Cancer discovery\| PubMed ID: 36715552	2023

Gergely Rona

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