Papers by laura baranello
Control of protein synthesis through mRNA pseudouridylation by dyskerin
Science Advances, Jul 28, 2023
Posttranscriptional modifications of mRNA have emerged as regulators of gene expression. Although... more Posttranscriptional modifications of mRNA have emerged as regulators of gene expression. Although pseudouridylation is the most abundant, its biological role remains poorly understood. Here, we demonstrate that the pseudouridine synthase dyskerin associates with RNA polymerase II, binds to thousands of mRNAs, and is responsible for their pseudouridylation, an action that occurs in chromatin and does not appear to require a guide RNA with full complementarity. In cells lacking dyskerin, mRNA pseudouridylation is reduced, while at the same time, de novo protein synthesis is enhanced, indicating that this modification interferes with translation. Accordingly, mRNAs with fewer pseudouridines due to knockdown of dyskerin are translated more efficiently. Moreover, mRNA pseudouridylation is severely reduced in patients with dyskeratosis congenita caused by inherited mutations in the gene encoding dyskerin (i.e., DKC1 ). Our findings demonstrate that pseudouridylation by dyskerin modulates mRNA translatability, with important implications for both normal development and disease.
Life science alliance, Jan 5, 2021
Cyclin A2 is a key regulator of the cell cycle, implicated both in DNA replication and mitotic en... more Cyclin A2 is a key regulator of the cell cycle, implicated both in DNA replication and mitotic entry. Cyclin A2 participates in feedback loops that activate mitotic kinases in G2 phase, but why active Cyclin A2-CDK2 during the S phase does not trigger mitotic kinase activation remains unclear. Here, we describe a change in localisation of Cyclin A2 from being only nuclear to both nuclear and cytoplasmic at the S/G2 border. We find that Cyclin A2-CDK2 can activate the mitotic kinase PLK1 through phosphorylation of Bora, and that only cytoplasmic Cyclin A2 interacts with Bora and PLK1. Expression of predominately cytoplasmic Cyclin A2 or phospho-mimicking PLK1 T210D can partially rescue a G2 arrest caused by Cyclin A2 depletion. Cytoplasmic presence of Cyclin A2 is restricted by p21, in particular after DNA damage. Cyclin A2 chromatin association during DNA replication and additional mechanisms contribute to Cyclin A2 localisation change in the G2 phase. We find no evidence that such mechanisms involve G2 feedback loops and suggest that cytoplasmic appearance of Cyclin A2 at the S/G2 transition functions as a trigger for mitotic kinase activation.
Supplementary Materials and Methods, Figures 1-6 from MYCMI-7: A Small MYC-Binding Compound that Inhibits MYC: MAX Interaction and Tumor Growth in a MYC-Dependent Manner
Supplementary Figure 1. MYCMI-7 selectively inhibits the MYC:MAX interaction in cells and blocks ... more Supplementary Figure 1. MYCMI-7 selectively inhibits the MYC:MAX interaction in cells and blocks MYC's association with chromatin. Supplementary Figure 2. MYCMI-7 downregulates MYC and MYCN protein expression. Supplementary Figure 3. MYCMI-7 reduces tumor cell growth/viability of MYC-driven tumor cells and inhibits oncogenic transformation in 2D and 3D cultures. Supplementary Figure 4. Effects of MYCMI-7 is relation to TOP2A activity in vitro and in cells. Supplementary Figure 5. Treatment of MYC/BCL-XL-driven acute myeloid leukemia with MYCMI-7. Supplementary Figure 6. MYCMI-7 inhibits growth of MDA-MB-231 breast cancer cell and SK-N-DZ MYCN-amplified neuroblastoma cell xenografts in vivo.
NEDDylated Cullin 3 mediates the adaptive response to topoisomerase 1 inhibitors
Science Advances, Dec 9, 2022
DNA topoisomerase 1 (TOP1) inhibitors are mainstays of anticancer therapy. These drugs trap TOP1 ... more DNA topoisomerase 1 (TOP1) inhibitors are mainstays of anticancer therapy. These drugs trap TOP1 on DNA, stabilizing the TOP1-cleavage complex (TOP1-cc). The accumulation of TOP1-ccs perturbs DNA replication fork progression, leading to DNA breaks and cell death. By analyzing the genomic occupancy and activity of TOP1, we show that cells adapt to treatment with multiple doses of TOP1 inhibitor by promoting the degradation of TOP1-ccs, allowing cells to better tolerate subsequent doses of TOP1 inhibitor. The E3-RING Cullin 3 ligase in complex with the BTBD1 and BTBD2 adaptor proteins promotes TOP1-cc ubiquitination and subsequent proteasomal degradation. NEDDylation of Cullin 3 activates this pathway, and inhibition of protein NEDDylation or depletion of Cullin 3 sensitizes cancer cells to TOP1 inhibitors. Collectively, our data uncover a previously unidentified NEDD8–Cullin 3 pathway involved in the adaptive response to TOP1 inhibitors, which can be targeted to improve the efficacy of TOP1 drugs in cancer therapy.
bioRxiv (Cold Spring Harbor Laboratory), Feb 12, 2023
Pancreatic carcinoma is one of the most lethal cancers and the absence of efficient therapeutic s... more Pancreatic carcinoma is one of the most lethal cancers and the absence of efficient therapeutic strategies results in poor prognosis. Transcriptional dysregulation due to alterations in KRAS and MYC impacts initiation, development, and survival of this tumor type. Using patient-derived xenografts of pancreatic carcinoma driven by KRAS and MYC oncogenic transcription, we show that co-inhibition of Topoisomerase 1 (TOP1) and bromodomain containing protein 4 (BRD4) synergistically induce tumor regression through targeting promoter pause-release, a rate-limiting step in transcription elongation. By comparing the nascent transcriptome with the recruitment of elongation and termination factors along genes, we found that co-inhibition of TOP1 and BRD4, while globally impairing RNA production, disturbs recruitment of proteins involved in termination. Thus, RNA polymerases continue transcribing downstream of genes for hundreds of kilobases leading to readthrough transcription. This pervasive transcription also occurs during replication, perturbing replisome preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
TOP1 CAD-seq: A protocol to map catalytically engaged topoisomerase 1 in human cells
STAR protocols, Sep 1, 2022
Deregulated expression of MYC family oncogenes occurs frequently in human cancer and is often ass... more Deregulated expression of MYC family oncogenes occurs frequently in human cancer and is often associated with aggressive disease and poor prognosis. While MYC is a highly warranted target, it has been considered “undruggable,” and no specific anti-MYC drugs are available in the clinic. We recently identified molecules named MYCMIs that inhibit the interaction between MYC and its essential partner MAX. Here we show that one of these molecules, MYCMI-7, efficiently and selectively inhibits MYC:MAX and MYCN:MAX interactions in cells, binds directly to recombinant MYC, and reduces MYC-driven transcription. In addition, MYCMI-7 induces degradation of MYC and MYCN proteins. MYCMI-7 potently induces growth arrest/apoptosis in tumor cells in a MYC/MYCN-dependent manner and downregulates the MYC pathway on a global level as determined by RNA sequencing. Sensitivity to MYCMI-7 correlates with MYC expression in a panel of 60 tumor cell lines and MYCMI-7 shows high efficacy toward a collection ...
Supplementary Materials and Methods, Figures 1-6 from MYCMI-7: A Small MYC-Binding Compound that Inhibits MYC: MAX Interaction and Tumor Growth in a MYC-Dependent Manner
Supplementary Figure 1. MYCMI-7 selectively inhibits the MYC:MAX interaction in cells and blocks ... more Supplementary Figure 1. MYCMI-7 selectively inhibits the MYC:MAX interaction in cells and blocks MYC's association with chromatin. Supplementary Figure 2. MYCMI-7 downregulates MYC and MYCN protein expression. Supplementary Figure 3. MYCMI-7 reduces tumor cell growth/viability of MYC-driven tumor cells and inhibits oncogenic transformation in 2D and 3D cultures. Supplementary Figure 4. Effects of MYCMI-7 is relation to TOP2A activity in vitro and in cells. Supplementary Figure 5. Treatment of MYC/BCL-XL-driven acute myeloid leukemia with MYCMI-7. Supplementary Figure 6. MYCMI-7 inhibits growth of MDA-MB-231 breast cancer cell and SK-N-DZ MYCN-amplified neuroblastoma cell xenografts in vivo.
NEDDylated Cullin 3 mediates the adaptive response to topoisomerase 1 inhibitors
Science Advances
DNA topoisomerase 1 (TOP1) inhibitors are mainstays of anticancer therapy. These drugs trap TOP1 ... more DNA topoisomerase 1 (TOP1) inhibitors are mainstays of anticancer therapy. These drugs trap TOP1 on DNA, stabilizing the TOP1-cleavage complex (TOP1-cc). The accumulation of TOP1-ccs perturbs DNA replication fork progression, leading to DNA breaks and cell death. By analyzing the genomic occupancy and activity of TOP1, we show that cells adapt to treatment with multiple doses of TOP1 inhibitor by promoting the degradation of TOP1-ccs, allowing cells to better tolerate subsequent doses of TOP1 inhibitor. The E3-RING Cullin 3 ligase in complex with the BTBD1 and BTBD2 adaptor proteins promotes TOP1-cc ubiquitination and subsequent proteasomal degradation. NEDDylation of Cullin 3 activates this pathway, and inhibition of protein NEDDylation or depletion of Cullin 3 sensitizes cancer cells to TOP1 inhibitors. Collectively, our data uncover a previously unidentified NEDD8–Cullin 3 pathway involved in the adaptive response to TOP1 inhibitors, which can be targeted to improve the efficacy ...
Cancer Research Communications, 2022
Deregulated expression of MYC family oncogenes occurs frequently in human cancer and is often ass... more Deregulated expression of MYC family oncogenes occurs frequently in human cancer and is often associated with aggressive disease and poor prognosis. While MYC is a highly warranted target, it has been considered “undruggable,” and no specific anti-MYC drugs are available in the clinic. We recently identified molecules named MYCMIs that inhibit the interaction between MYC and its essential partner MAX. Here we show that one of these molecules, MYCMI-7, efficiently and selectively inhibits MYC:MAX and MYCN:MAX interactions in cells, binds directly to recombinant MYC, and reduces MYC-driven transcription. In addition, MYCMI-7 induces degradation of MYC and MYCN proteins. MYCMI-7 potently induces growth arrest/apoptosis in tumor cells in a MYC/MYCN-dependent manner and downregulates the MYC pathway on a global level as determined by RNA sequencing. Sensitivity to MYCMI-7 correlates with MYC expression in a panel of 60 tumor cell lines and MYCMI-7 shows high efficacy toward a collection ...
2021 Wu et al PLoS Genetics Uncropped Images for SFig_7D
Immunoflourescence staining of Rpb1 in G2/M<i> </i>arrested<i> Saccharomyces ce... more Immunoflourescence staining of Rpb1 in G2/M<i> </i>arrested<i> Saccharomyces cereivisae, </i>with and without rapamycin treatment for anchoring away of Rpb1. Images show loss of nuclear accumulation of Rpb1 after rapamycin incubation. This is a control experiment for the investigation of the effect of inhibiting transcription on damage-induced cohesion.<br>
Communication DNA Break Mapping Reveals Topoisomerase II Activity Genome-Wide
Abstract: Genomic DNA is under constant assault by endogenous and exogenous DNA damaging agents. ... more Abstract: Genomic DNA is under constant assault by endogenous and exogenous DNA damaging agents. DNA breakage can represent a major threat to genome integrity but can also be necessary for genome function. Here we present approaches to map DNA double-strand breaks (DSBs) and single-strand breaks (SSBs) at the genome-wide scale by two methods called DSB- and SSB-Seq, respectively. We tested these methods in human colon cancer cells and validated the results using the Topoisomerase II (Top2)-poisoning agent etoposide (ETO). Our results show that the combination of ETO treatment with break-mapping techniques is a powerful method to elaborate the pattern of Top2 enzymatic activity across the genome.
Pancreatic carcinoma is one of the most lethal cancers and the absence of efficient therapeutic s... more Pancreatic carcinoma is one of the most lethal cancers and the absence of efficient therapeutic strategies results in poor prognosis. Transcriptional dysregulation due to alterations in KRAS and MYC impacts initiation, development, and survival of this tumor type. Using patient-derived xenografts of pancreatic carcinoma driven by KRAS and MYC oncogenic transcription, we show that co-inhibition of Topoisomerase 1 (TOP1) and bromodomain containing protein 4 (BRD4) synergistically induce tumor regression through targeting promoter pause-release, a rate-limiting step in transcription elongation. By comparing the nascent transcriptome with the recruitment of elongation and termination factors along genes, we found that co-inhibition of TOP1 and BRD4, while globally impairing RNA production, disturbs recruitment of proteins involved in termination. Thus, RNA polymerases continue transcribing downstream of genes for hundreds of kilobases leading to readthrough transcription. This pervasive...
bioRxiv, 2021
The structural maintenance of chromosome (SMC) complex cohesin mediates sister chromatid cohesion... more The structural maintenance of chromosome (SMC) complex cohesin mediates sister chromatid cohesion established during replication, and damage-induced cohesion formed in response to DSBs post-replication. The translesion synthesis polymerase Polη is required for damage-induced cohesion through a hitherto unknown mechanism. Since Polη is functionally associated with transcription, and transcription triggers de novo cohesion in Schizosaccharomyces pombe, we hypothesized that transcription facilitates damage-induced cohesion in Saccharomyces cerevisiae. Here, we show dysregulated transcriptional profiles in Polη-depleted cells (rad30Δ), where genes involved in chromatin assembly and positive transcription regulation were downregulated. In addition, chromatin association of RNA polymerase II was reduced at promoters and coding regions in rad30Δ compared to WT cells, while occupancy of the H2A.Z variant (Htz1) at promoters was increased in rad30Δ cells. Perturbing histone exchange at promo...
Analysis of Myc Chromatin Binding by Calibrated ChIP-Seq Approach
Methods in molecular biology, 2021
Here, we present a strategy to map and quantify the interactions between Myc and chromatin using ... more Here, we present a strategy to map and quantify the interactions between Myc and chromatin using a calibrated Myc ChIP-seq approach. We recommend the use of an internal spike-in control for post-sequencing normalization to enable detection of broad changes in Myc binding as can occur under conditions with varied Myc abundance. We also highlight a range of bioinformatic analyses that can dissect the downstream effects of Myc binding. These methods include peak calling, mapping Myc onto an integrated metagenome, juxtaposing ChIP-seq data with matching RNA-seq data, and identifying gene ontologies enriched for genes with high Myc binding. Our aim is to provide a guided strategy, from cell harvest through to bioinformatic analysis, to elucidate the global effects of Myc on transcription.
DNA Topoisomerases and Cancer, 2011
The effects of camptothecin on RNA polymerase II transcription: Roles of DNA topoisomerase I
Biochimie, 2007
Eukaryotic DNA topoisomerase I is active in transcribed chromatin domains to modulate transcripti... more Eukaryotic DNA topoisomerase I is active in transcribed chromatin domains to modulate transcription-generated DNA torsional tension. Camptothecin and other agents targeting DNA topoisomerase I are used in the treatment of human solid cancers with significant clinical efficacy. Major progress has been achieved in recent years in the understanding of enzyme structures and basic cellular functions of DNA topoisomerase I. Nevertheless, the precise enzyme functions and mechanisms during transcription-related processes remain unclear. The current understanding of the molecular action of camptothecin emphasizes the drug action against the enzyme and the production of irreversible breaks in the cellular DNA. However, the high drug potency is hardly fully explained by the DNA damage outcome only. In the recent past, several unexpected findings have been reported in relation to the role of eukaryotic topoisomerase I during transcription. In particular, the function of DNA topoisomerase I and the molecular effects of its inhibition on transcription-coupled processes constitute a very active research area. Here, we will briefly review relevant investigations on topoisomerase I involvement in different stages of transcription, discussing both enzyme functions and drug effects on molecular processes.
Genomic
dependent dynamic supercoiling is a short-range
The Epstein-Barr virus ubiquitin deconjugase BPLF1 regulates the activity of Topoisomerase II during virus replication
Topoisomerases are essential for the replication of herpesviruses but the mechanisms by which the... more Topoisomerases are essential for the replication of herpesviruses but the mechanisms by which the viruses hijack the cellular enzymes are largely unknown. We found that topoisomerase-II (TOP2) is a substrate of the Epstein-Barr virus (EBV) ubiquitin deconjugase BPLF1. BPLF1 selectively inhibited the ubiquitination of TOP2 following treatment with topoisomerase poisons, interacted with TOP2α and TOP2β in co-immunoprecipitation and in vitro pull-down, stabilized Etoposide-trapped TOP2 cleavage complexes (TOP2cc) and promoted TOP2 SUMOylation, which halted the DNA-damage response and reduced Etoposide toxicity. Induction of the productive virus cycle promoted the accumulation of TOP2βcc, enhanced TOP2β SUMOylation, and reduced Etoposide toxicity in lymphoblastoid cell lines carrying recombinant EBV encoding the active enzyme. Attenuation of this phenotype upon expression of a catalytic mutant BPLF1-C61A impaired viral DNA synthesis and virus release. These findings highlight a previous...
MYC assembles and stimulates topoisomerases 1 and 2 in a “topoisome”
Molecular Cell
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Papers by laura baranello