Publications by authors named "Giovanni Marsico"

40 Publications

Landscape of G-quadruplex DNA structural regions in breast cancer.

Nat Genet 2020 09 3;52(9):878-883. Epub 2020 Aug 3.

Cancer Research UK Cambridge Institute, Li Ka Shing Centre, University of Cambridge, Cambridge, UK.

Response and resistance to anticancer therapies vary due to intertumor and intratumor heterogeneity. Here, we map differentially enriched G-quadruplex (G4) DNA structure-forming regions (∆G4Rs) in 22 breast cancer patient-derived tumor xenograft (PDTX) models. ∆G4Rs are associated with the promoters of highly amplified genes showing high expression, and with somatic single-nucleotide variants. Differences in ΔG4R landscapes reveal seven transcription factor programs across PDTXs. ∆G4R abundance and locations stratify PDTXs into at least three G4-based subtypes. ∆G4Rs in most PDTXs (14 of 22) were found to associate with more than one breast cancer subtype, which we also call an integrative cluster (IC). This suggests the frequent coexistence of multiple breast cancer states within a PDTX model, the majority of which display aggressive triple-negative IC10 gene activity. Short-term cultures of PDTX models with increased ∆G4R levels are more sensitive to small molecules targeting G4 DNA. Thus, G4 landscapes reveal additional IC-related intratumor heterogeneity in PDTX biopsies, improving breast cancer stratification and potentially identifying new treatment strategies.
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http://dx.doi.org/10.1038/s41588-020-0672-8DOI Listing
September 2020

Correction to: RNA G-quadruplexes at upstream open reading frames cause DHX36- and DHX9-dependent translation of human mRNAs.

Genome Biol 2019 06 18;20(1):124. Epub 2019 Jun 18.

Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.

Following publication of the original article [1], the authors reported the following error in the name of the fourth author.
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http://dx.doi.org/10.1186/s13059-019-1737-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580489PMC
June 2019

Whole genome experimental maps of DNA G-quadruplexes in multiple species.

Nucleic Acids Res 2019 05;47(8):3862-3874

Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Cambridge CB2 0RE, UK.

Genomic maps of DNA G-quadruplexes (G4s) can help elucidate the roles that these secondary structures play in various organisms. Herein, we employ an improved version of a G-quadruplex sequencing method (G4-seq) to generate whole genome G4 maps for 12 species that include widely studied model organisms and also pathogens of clinical relevance. We identify G4 structures that form under physiological K+ conditions and also G4s that are stabilized by the G4-targeting small molecule pyridostatin (PDS). We discuss the various structural features of the experimentally observed G-quadruplexes (OQs), highlighting differences in their prevalence and enrichment across species. Our study describes diversity in sequence composition and genomic location for the OQs in the different species and reveals that the enrichment of OQs in gene promoters is particular to mammals such as mouse and human, among the species studied. The multi-species maps have been made publicly available as a resource to the research community. The maps can serve as blueprints for biological experiments in those model organisms, where G4 structures may play a role.
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http://dx.doi.org/10.1093/nar/gkz179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486626PMC
May 2019

Correction to: RNA G-quadruplexes at upstream open reading frames cause DHX36- and DHX9-dependent translation of human mRNAs.

Genome Biol 2019 01 11;20(1):11. Epub 2019 Jan 11.

Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.

Following publication of the original article [1], the authors reported the following error in the name of the fourth author.
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http://dx.doi.org/10.1186/s13059-019-1623-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6329048PMC
January 2019

RNA G-quadruplexes at upstream open reading frames cause DHX36- and DHX9-dependent translation of human mRNAs.

Genome Biol 2018 12 27;19(1):229. Epub 2018 Dec 27.

Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.

Background: RNA secondary structures in the 5'-untranslated regions (5'-UTR) of mRNAs are key to the post-transcriptional regulation of gene expression. While it is evident that non-canonical Hoogsteen-paired G-quadruplex (rG4) structures somehow contribute to the regulation of translation initiation, the nature and extent of human mRNAs that are regulated by rG4s is not known. Here, we provide new insights into a mechanism by which rG4 formation modulates translation.

Results: Using transcriptome-wide ribosome profiling, we identify rG4-driven mRNAs in HeLa cells and reveal that rG4s in the 5'-UTRs of inefficiently translated mRNAs associate with high ribosome density and the translation of repressive upstream open reading frames (uORF). We demonstrate that depletion of the rG4-unwinding helicases DHX36 and DHX9 promotes translation of rG4-associated uORFs while reducing the translation of coding regions for transcripts that comprise proto-oncogenes, transcription factors and epigenetic regulators. Transcriptome-wide identification of DHX9 binding sites shows that reduced translation is mediated through direct physical interaction between the helicase and its rG4 substrate.

Conclusion: This study identifies human mRNAs whose translation efficiency is modulated by the DHX36- and DHX9-dependent folding/unfolding of rG4s within their 5'-UTRs. We reveal a previously unknown mechanism for translation regulation in which unresolved rG4s within 5'-UTRs promote 80S ribosome formation on upstream start codons, causing inhibition of translation of the downstream main open reading frames. Our findings suggest that the interaction of helicases with rG4s could be targeted for future therapeutic intervention.
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http://dx.doi.org/10.1186/s13059-018-1602-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307142PMC
December 2018

DNA G-quadruplex structures mold the DNA methylome.

Nat Struct Mol Biol 2018 10 1;25(10):951-957. Epub 2018 Oct 1.

Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Cambridge, UK.

Control of DNA methylation level is critical for gene regulation, and the factors that govern hypomethylation at CpG islands (CGIs) are still being uncovered. Here, we provide evidence that G-quadruplex (G4) DNA secondary structures are genomic features that influence methylation at CGIs. We show that the presence of G4 structure is tightly associated with CGI hypomethylation in the human genome. Surprisingly, we find that these G4 sites are enriched for DNA methyltransferase 1 (DNMT1) occupancy, which is consistent with our biophysical observations that DNMT1 exhibits higher binding affinity for G4s as compared to duplex, hemi-methylated, or single-stranded DNA. The biochemical assays also show that the G4 structure itself, rather than sequence, inhibits DNMT1 enzymatic activity. Based on these data, we propose that G4 formation sequesters DNMT1 thereby protecting certain CGIs from methylation and inhibiting local methylation.
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http://dx.doi.org/10.1038/s41594-018-0131-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173298PMC
October 2018

Analysis of NRAS RNA G-quadruplex binding proteins reveals DDX3X as a novel interactor of cellular G-quadruplex containing transcripts.

Nucleic Acids Res 2018 11;46(21):11592-11604

Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK.

RNA G-quadruplexes (rG4s) are secondary structures in mRNAs known to influence RNA post-transcriptional mechanisms thereby impacting neurodegenerative disease and cancer. A detailed knowledge of rG4-protein interactions is vital to understand rG4 function. Herein, we describe a systematic affinity proteomics approach that identified 80 high-confidence interactors that assemble on the rG4 located in the 5'-untranslated region (UTR) of the NRAS oncogene. Novel rG4 interactors included DDX3X, DDX5, DDX17, GRSF1 and NSUN5. The majority of identified proteins contained a glycine-arginine (GAR) domain and notably GAR-domain mutation in DDX3X and DDX17 abrogated rG4 binding. Identification of DDX3X targets by transcriptome-wide individual-nucleotide resolution UV-crosslinking and affinity enrichment (iCLAE) revealed a striking association with 5'-UTR rG4-containing transcripts which was reduced upon GAR-domain mutation. Our work highlights hitherto unrecognized features of rG4 structure-protein interactions that highlight new roles of rG4 structures in mRNA post-transcriptional control.
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http://dx.doi.org/10.1093/nar/gky861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265444PMC
November 2018

Detecting RNA G-Quadruplexes (rG4s) in the Transcriptome.

Cold Spring Harb Perspect Biol 2018 07 2;10(7). Epub 2018 Jul 2.

Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom.

RNA G-quadruplex (rG4) secondary structures are proposed to play key roles in fundamental biological processes that include the modulation of transcriptional, co-transcriptional, and posttranscriptional events. Recent methodological developments that include predictive algorithms and structure-based sequencing have enabled the detection and mapping of rG4 structures on a transcriptome-wide scale at high sensitivity and resolution. The data generated by these studies provide valuable insights into the potentially diverse roles of rG4s in biology and open up a number of mechanistic hypotheses. Herein we highlight these methodologies and discuss the associated findings in relation to rG4-related biological mechanisms.
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http://dx.doi.org/10.1101/cshperspect.a032284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028067PMC
July 2018

Genome-wide mapping of endogenous G-quadruplex DNA structures by chromatin immunoprecipitation and high-throughput sequencing.

Nat Protoc 2018 03 22;13(3):551-564. Epub 2018 Feb 22.

Cancer Research UK, Cambridge Institute, Li Ka Shing Centre, Cambridge, UK.

G-rich DNA sequences can form four-stranded G-quadruplex (G4) secondary structures and are linked to fundamental biological processes such as transcription, replication and telomere maintenance. G4s are also implicated in promoting genome instability, cancer and other diseases. Here, we describe a detailed G4 ChIP-seq method that robustly enables the determination of G4 structure formation genome-wide in chromatin. This protocol adapts traditional ChIP-seq for the detection of DNA secondary structures through the use of a G4-structure-specific single-chain antibody with refinements in chromatin immunoprecipitation followed by high-throughput sequencing. This technology does not require expression of the G4 antibody in situ, enabling broad applicability to theoretically all chromatin sources. Beginning with chromatin isolation and antibody preparation, the entire protocol can be completed in <1 week, including basic computational analysis.
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http://dx.doi.org/10.1038/nprot.2017.150DOI Listing
March 2018

Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule.

J Med Chem 2018 03 7;61(6):2500-2517. Epub 2018 Feb 7.

UCL School of Pharmacy , University College London , 29-39 Brunswick Square , London WC1N 1AX , U.K.

Human pancreatic ductal adenocarcinoma (PDAC) involves the dysregulation of multiple signaling pathways. A novel approach to the treatment of PDAC is described, involving the targeting of cancer genes in PDAC pathways having over-representation of G-quadruplexes, using the trisubstituted naphthalene diimide quadruplex-binding compound 2,7-bis(3-morpholinopropyl)-4-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[ lmn][3,8]phenanthroline-1,3,6,8(2 H,7 H)-tetraone (CM03). This compound has been designed by computer modeling, is a potent inhibitor of cell growth in PDAC cell lines, and has anticancer activity in PDAC models, with a superior profile compared to gemcitabine, a commonly used therapy. Whole-transcriptome RNA-seq methodology has been used to analyze the effects of this quadruplex-binding small molecule on global gene expression. This has revealed the down-regulation of a large number of genes, rich in putative quadruplex elements and involved in essential pathways of PDAC survival, metastasis, and drug resistance. The changes produced by CM03 represent a global response to the complexity of human PDAC and may be applicable to other currently hard-to-treat cancers.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5867665PMC
March 2018

Machine learning model for sequence-driven DNA G-quadruplex formation.

Sci Rep 2017 11 6;7(1):14535. Epub 2017 Nov 6.

Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.

We describe a sequence-based computational model to predict DNA G-quadruplex (G4) formation. The model was developed using large-scale machine learning from an extensive experimental G4-formation dataset, recently obtained for the human genome via G4-seq methodology. Our model differentiates many widely accepted putative quadruplex sequences that do not actually form stable genomic G4 structures, correctly assessing the G4 folding potential of over 700,000 such sequences in the human genome. Moreover, our approach reveals the relative importance of sequence-based features coming from both within the G4 motifs and their flanking regions. The developed model can be applied to any DNA sequence or genome to characterise sequence-driven intramolecular G4 formation propensities.
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http://dx.doi.org/10.1038/s41598-017-14017-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5673958PMC
November 2017

Functional properties of hepatocytes in vitro are correlated with cell polarity maintenance.

Exp Cell Res 2017 Jan 1;350(1):242-252. Epub 2016 Dec 1.

Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany. Electronic address:

Exploring the cell biology of hepatocytes in vitro could be a powerful strategy to dissect the molecular mechanisms underlying the structure and function of the liver in vivo. However, this approach relies on appropriate in vitro cell culture systems that can recapitulate the cell biological and metabolic features of the hepatocytes in the liver whilst being accessible to experimental manipulations. Here, we adapted protocols for high-resolution fluorescence microscopy and quantitative image analysis to compare two primary hepatocyte culture systems, monolayer and collagen sandwich, with respect to the distribution of two distinct populations of early endosomes (APPL1 and EEA1-positive), endocytic capacity, metabolic and signaling activities. In addition to the re-acquisition of hepatocellular polarity, primary hepatocytes grown in collagen sandwich but not in monolayer culture recapitulated the apico-basal distribution of EEA1 endosomes observed in liver tissue. We found that such distribution correlated with the organization of the actin cytoskeleton in vitro and, surprisingly, was dependent on the nutritional state in vivo. Hepatocytes in collagen sandwich also exhibited faster kinetics of low-density lipoprotein (LDL) and epidermal growth factor (EGF) internalization, showed improved insulin sensitivity and preserved their ability for glucose production, compared to hepatocytes in monolayer cultures. Although no in vitro culture system can reproduce the exquisite structural features of liver tissue, our data nevertheless highlight the ability of the collagen sandwich system to recapitulate key structural and functional properties of the hepatocytes in the liver and, therefore, support the usage of this system to study aspects of hepatocellular biology in vitro.
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http://dx.doi.org/10.1016/j.yexcr.2016.11.027DOI Listing
January 2017

Clinical evaluation and molecular screening of a large consecutive series of albino patients.

J Hum Genet 2017 Feb 13;62(2):277-290. Epub 2016 Oct 13.

Pediatric Ophthalmology Unit, ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy.

Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. In this study we recruited 321 albino patients and screened them for the genes known to cause oculocutaneous albinism (OCA1-4 and OCA6) and ocular albinism (OA1). Our purpose was to detect mutations and genetic frequencies of the main causative genes, offering to albino patients an exhaustive diagnostic assessment within a multidisciplinary approach including ophthalmological, dermatological, audiological and genetic evaluations. We report 70 novel mutations and the frequencies of the major causative OCA genes that are as follows: TYR (44%), OCA2 (17%), TYRP1 (1%), SLC45A2 (7%) and SLC24A5 (<0.5%). An additional 5% of patients had GPR143 mutations. In 19% of cases, a second reliable mutation was not detected, whereas 7% of our patients remain still molecularly undiagnosed. This comprehensive study of a consecutive series of OCA/OA1 patients allowed us to perform a clinical evaluation of the different OCA forms.
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http://dx.doi.org/10.1038/jhg.2016.123DOI Listing
February 2017

G-quadruplex structures mark human regulatory chromatin.

Nat Genet 2016 10 12;48(10):1267-72. Epub 2016 Sep 12.

Cancer Research UK, Cambridge Institute, Li Ka Shing Centre, Cambridge, UK.

G-quadruplex (G4) structural motifs have been linked to transcription, replication and genome instability and are implicated in cancer and other diseases. However, it is crucial to demonstrate the bona fide formation of G4 structures within an endogenous chromatin context. Herein we address this through the development of G4 ChIP-seq, an antibody-based G4 chromatin immunoprecipitation and high-throughput sequencing approach. We find ∼10,000 G4 structures in human chromatin, predominantly in regulatory, nucleosome-depleted regions. G4 structures are enriched in the promoters and 5' UTRs of highly transcribed genes, particularly in genes related to cancer and in somatic copy number amplifications, such as MYC. Strikingly, de novo and enhanced G4 formation are associated with increased transcriptional activity, as shown by HDAC inhibitor-induced chromatin relaxation and observed in immortalized as compared to normal cellular states. Our findings show that regulatory, nucleosome-depleted chromatin and elevated transcription shape the endogenous human G4 DNA landscape.
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http://dx.doi.org/10.1038/ng.3662DOI Listing
October 2016

rG4-seq reveals widespread formation of G-quadruplex structures in the human transcriptome.

Nat Methods 2016 10 29;13(10):841-4. Epub 2016 Aug 29.

Department of Chemistry, University of Cambridge, Cambridge, UK.

We introduce RNA G-quadruplex sequencing (rG4-seq), a transcriptome-wide RNA G-quadruplex (rG4) profiling method that couples rG4-mediated reverse transcriptase stalling with next-generation sequencing. Using rG4-seq on polyadenylated-enriched HeLa RNA, we generated a global in vitro map of thousands of canonical and noncanonical rG4 structures. We characterize rG4 formation relative to cytosine content and alternative RNA structure stability, uncover rG4-dependent differences in RNA folding and show evolutionarily conserved enrichment in transcripts mediating RNA processing and stability.
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http://dx.doi.org/10.1038/nmeth.3965DOI Listing
October 2016

DSBCapture: in situ capture and sequencing of DNA breaks.

Nat Methods 2016 10 15;13(10):855-7. Epub 2016 Aug 15.

Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK.

Double-strand DNA breaks (DSBs) continuously arise and cause mutations and chromosomal rearrangements. Here, we present DSBCapture, a sequencing-based method that captures DSBs in situ and directly maps these at single-nucleotide resolution, enabling the study of DSB origin. DSBCapture shows substantially increased sensitivity and data yield compared with other methods. Using DSBCapture, we uncovered a striking relationship between DSBs and elevated transcription within nucleosome-depleted chromatin.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045719PMC
http://dx.doi.org/10.1038/nmeth.3960DOI Listing
October 2016

A versatile pipeline for the multi-scale digital reconstruction and quantitative analysis of 3D tissue architecture.

Elife 2015 Dec 27;4. Epub 2015 Dec 27.

Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

A prerequisite for the systems biology analysis of tissues is an accurate digital three-dimensional reconstruction of tissue structure based on images of markers covering multiple scales. Here, we designed a flexible pipeline for the multi-scale reconstruction and quantitative morphological analysis of tissue architecture from microscopy images. Our pipeline includes newly developed algorithms that address specific challenges of thick dense tissue reconstruction. Our implementation allows for a flexible workflow, scalable to high-throughput analysis and applicable to various mammalian tissues. We applied it to the analysis of liver tissue and extracted quantitative parameters of sinusoids, bile canaliculi and cell shapes, recognizing different liver cell types with high accuracy. Using our platform, we uncovered an unexpected zonation pattern of hepatocytes with different size, nuclei and DNA content, thus revealing new features of liver tissue organization. The pipeline also proved effective to analyse lung and kidney tissue, demonstrating its generality and robustness.
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http://dx.doi.org/10.7554/eLife.11214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4764584PMC
December 2015

Small molecule inhibition of FOXM1: How to bring a novel compound into genomic context.

Genom Data 2015 Mar 22;3:19-23. Epub 2014 Oct 22.

Cancer Research UK, Cambridge Institute, Li Ka Shing Centre, Cambridge CB2 0RE, UK.

Deregulation of transcription factor (TF) networks is emerging as a major pathogenic event in many human cancers (Darnell, 2002 [1]; Libermann and Zerbini, 2006 [2]; Laoukili et al., 2007 [3]). Small molecule intervention is an attractive avenue to understand TF regulatory mechanisms in healthy and disease state, as well as for exploiting these targets therapeutically (Koehler et al., 2003 [4]; Berg, 2008 [5]; Koehler, 2010 [6]). However, because of their physico-chemical properties, TF targeting has been proven to be difficult (Verdine and Walensky, 2007 [7]). The TF FOXM1 is an important mitotic player (Wonsey and Follettie, 2005 [8]; Laoukili et al., 2005 [9]; McDonald, 2005 [10]) also implicated in cancer progression (Laoukili et al., 2007 [3]; Teh, 2011 [11]; Koo, 2012 [12]) and drug resistance development (Kwok et al., 2010 [13]; Carr et al., [14]). Therefore, its inhibition is an attractive goal for cancer therapy. Here, we describe a computational biology approach, by giving detailed insights into methodologies and technical results, which was used to analyze the transcriptional RNA-Seq data presented in our previous work (Gormally et al., 2014 [20]). Our Bioinformatics analysis shed light on the cellular effect of a novel FOXM1 inhibitor (FDI-6) newly identified through a biophysical screen. The data for this report is available at the public GEO repository (accession number http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58626).
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http://dx.doi.org/10.1016/j.gdata.2014.10.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4535965PMC
March 2015

Identification of siRNA delivery enhancers by a chemical library screen.

Nucleic Acids Res 2015 Sep 28;43(16):7984-8001. Epub 2015 Jul 28.

Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108 01307, Dresden, Germany

Most delivery systems for small interfering RNA therapeutics depend on endocytosis and release from endo-lysosomal compartments. One approach to improve delivery is to identify small molecules enhancing these steps. It is unclear to what extent such enhancers can be universally applied to different delivery systems and cell types. Here, we performed a compound library screen on two well-established siRNA delivery systems, lipid nanoparticles and cholesterol conjugated-siRNAs. We identified fifty-one enhancers improving gene silencing 2-5 fold. Strikingly, most enhancers displayed specificity for one delivery system only. By a combination of quantitative fluorescence and electron microscopy we found that the enhancers substantially differed in their mechanism of action, increasing either endocytic uptake or release of siRNAs from endosomes. Furthermore, they acted either on the delivery system itself or the cell, by modulating the endocytic system via distinct mechanisms. Interestingly, several compounds displayed activity on different cell types. As proof of principle, we showed that one compound enhanced siRNA delivery in primary endothelial cells in vitro and in the endocardium in the mouse heart. This study suggests that a pharmacological approach can improve the delivery of siRNAs in a system-specific fashion, by exploiting distinct mechanisms and acting upon multiple cell types.
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http://dx.doi.org/10.1093/nar/gkv762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4652771PMC
September 2015

High-throughput sequencing of DNA G-quadruplex structures in the human genome.

Nat Biotechnol 2015 Aug 20;33(8):877-81. Epub 2015 Jul 20.

1] Department of Chemistry, University of Cambridge, Cambridge, UK. [2] Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Cambridge, UK. [3] School of Clinical Medicine, University of Cambridge, Cambridge, UK.

G-quadruplexes (G4s) are nucleic acid secondary structures that form within guanine-rich DNA or RNA sequences. G4 formation can affect chromatin architecture and gene regulation and has been associated with genomic instability, genetic diseases and cancer progression. Here we present a high-resolution sequencing-based method to detect G4s in the human genome. We identified 716,310 distinct G4 structures, 451,646 of which were not predicted by computational methods. These included previously uncharacterized noncanonical long loop and bulged structures. We observed a high G4 density in functional regions, such as 5' untranslated regions and splicing sites, as well as in genes previously not predicted to contain these structures (such as BRCA2). G4 formation was significantly associated with oncogenes, tumor suppressors and somatic copy number alterations related to cancer development. The G4s identified in this study may therefore represent promising targets for cancer intervention.
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http://dx.doi.org/10.1038/nbt.3295DOI Listing
August 2015

FOXM1 binds directly to non-consensus sequences in the human genome.

Genome Biol 2015 Jun 23;16:130. Epub 2015 Jun 23.

Cancer Research UK, Cambridge Research Institute, Li Ka Shing Center, Robinson Way, Cambridge, CB2 0RE, UK.

Background: The Forkhead (FKH) transcription factor FOXM1 is a key regulator of the cell cycle and is overexpressed in most types of cancer. FOXM1, similar to other FKH factors, binds to a canonical FKH motif in vitro. However, genome-wide mapping studies in different cell lines have shown a lack of enrichment of the FKH motif, suggesting an alternative mode of chromatin recruitment. We have investigated the role of direct versus indirect DNA binding in FOXM1 recruitment by performing ChIP-seq with wild-type and DNA binding deficient FOXM1.

Results: An in vitro fluorescence polarization assay identified point mutations in the DNA binding domain of FOXM1 that inhibit binding to a FKH consensus sequence. Cell lines expressing either wild-type or DNA binding deficient GFP-tagged FOXM1 were used for genome-wide mapping studies comparing the distribution of the DNA binding deficient protein to the wild-type. This shows that interaction of the FOXM1 DNA binding domain with target DNA is essential for recruitment. Moreover, analysis of the protein interactome of wild-type versus DNA binding deficient FOXM1 shows that the reduced recruitment is not due to inhibition of protein-protein interactions.

Conclusions: A functional DNA binding domain is essential for FOXM1 chromatin recruitment. Even in FOXM1 mutants with almost complete loss of binding, the protein-protein interactions and pattern of phosphorylation are largely unaffected. These results strongly support a model whereby FOXM1 is specifically recruited to chromatin through co-factor interactions by binding directly to non-canonical DNA sequences.
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http://dx.doi.org/10.1186/s13059-015-0696-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4492089PMC
June 2015

Regulation of liver metabolism by the endosomal GTPase Rab5.

Cell Rep 2015 May 30;11(6):884-892. Epub 2015 Apr 30.

Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany. Electronic address:

The liver maintains glucose and lipid homeostasis by adapting its metabolic activity to the energy needs of the organism. Communication between hepatocytes and extracellular environment via endocytosis is key to such homeostasis. Here, we addressed the question of whether endosomes are required for gluconeogenic gene expression. We took advantage of the loss of endosomes in the mouse liver upon Rab5 silencing. Strikingly, we found hepatomegaly and severe metabolic defects such as hypoglycemia, hypercholesterolemia, hyperlipidemia, and glycogen accumulation that phenocopied those found in von Gierke's disease, a glucose-6-phosphatase (G6Pase) deficiency. G6Pase deficiency alone can account for the reduction in hepatic glucose output and glycogen accumulation as determined by mathematical modeling. Interestingly, we uncovered functional alterations in the transcription factors, which regulate G6Pase expression. Our data highlight a requirement of Rab5 and the endosomal system for the regulation of gluconeogenic gene expression that has important implications for metabolic diseases.
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http://dx.doi.org/10.1016/j.celrep.2015.04.018DOI Listing
May 2015

Complex pulmonary aspergilloma treated by cavernostomy.

Rev Col Bras Cir 2014 Nov-Dec;41(6):406-11

Andaraí Federal Hospital, Ministry of Health, Rio de Janeiro, RJ, Brazil.

Objective: To evaluate the effectiveness of cavernostomy in patients with complex fungal balls.

Methods: We analyzed the medical records of patients undergoing cavernostomy between January 2005 and May 2013, evaluating: age, gender, preoperative signs and symptoms, predisposing disease, preoperative tests, location of the aspergilloma, etiologic agent, cavernostomy indication, postoperative outcome.

Results: Ten patients were male. The mean age was 42.9 years (34-56). The most frequent symptom was repeated pulmonary bleeding. Cavernostomy was proposed for patients at high risk for lung resection. It was performed in 17 patients and all of them had pulmonary tuberculosis sequelae, with cavitations. The indication in all cases was hemoptysis and elimination of phlegm. The cavernostomies were performed in a single surgical procedure. In all 17 patients the cavity was left open after the withdrawal of the mycetoma. In all patients hemoptysis ceased immediately. Operative mortality was 9.5% (1).

Conclusion: cavernostomy is an effective treatment alternative in patients at high risk. It may be useful in some patients with complex aspergilloma, irrespective of lung function or bilateral disease. It is technically easy, has low-risk, saves parenchyma, and may be performed in a single operative time.
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http://dx.doi.org/10.1590/0100-69912014006005DOI Listing
December 2016

Suppression of the FOXM1 transcriptional programme via novel small molecule inhibition.

Nat Commun 2014 Nov 12;5:5165. Epub 2014 Nov 12.

1] University Chemical Laboratory, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK [2] Cancer Research UK, Li Ka Shing Centre, Cambridge Institute, Cambridge CB2 0RE, UK.

The transcription factor FOXM1 binds to sequence-specific motifs on DNA (C/TAAACA) through its DNA-binding domain (DBD) and activates proliferation- and differentiation-associated genes. Aberrant overexpression of FOXM1 is a key feature in oncogenesis and progression of many human cancers. Here--from a high-throughput screen applied to a library of 54,211 small molecules--we identify novel small molecule inhibitors of FOXM1 that block DNA binding. One of the identified compounds, FDI-6 (NCGC00099374), is characterized in depth and is shown to bind directly to FOXM1 protein, to displace FOXM1 from genomic targets in MCF-7 breast cancer cells, and induce concomitant transcriptional downregulation. Global transcript profiling of MCF-7 cells by RNA-seq shows that FDI-6 specifically downregulates FOXM1-activated genes with FOXM1 occupancy confirmed by ChIP-PCR. This small molecule-mediated effect is selective for FOXM1-controlled genes with no effect on genes regulated by homologous forkhead family factors.
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http://dx.doi.org/10.1038/ncomms6165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4258842PMC
November 2014

Revealing molecular mechanisms by integrating high-dimensional functional screens with protein interaction data.

PLoS Comput Biol 2014 Sep 4;10(9):e1003801. Epub 2014 Sep 4.

Biotechnology Center, TU Dresden, Dresden, Germany; Center for Regenerative Therapy, Dresden, Germany; University of Cologne, Cologne, Germany.

Functional genomics screens using multi-parametric assays are powerful approaches for identifying genes involved in particular cellular processes. However, they suffer from problems like noise, and often provide little insight into molecular mechanisms. A bottleneck for addressing these issues is the lack of computational methods for the systematic integration of multi-parametric phenotypic datasets with molecular interactions. Here, we present Integrative Multi Profile Analysis of Cellular Traits (IMPACT). The main goal of IMPACT is to identify the most consistent phenotypic profile among interacting genes. This approach utilizes two types of external information: sets of related genes (IMPACT-sets) and network information (IMPACT-modules). Based on the notion that interacting genes are more likely to be involved in similar functions than non-interacting genes, this data is used as a prior to inform the filtering of phenotypic profiles that are similar among interacting genes. IMPACT-sets selects the most frequent profile among a set of related genes. IMPACT-modules identifies sub-networks containing genes with similar phenotype profiles. The statistical significance of these selections is subsequently quantified via permutations of the data. IMPACT (1) handles multiple profiles per gene, (2) rescues genes with weak phenotypes and (3) accounts for multiple biases e.g. caused by the network topology. Application to a genome-wide RNAi screen on endocytosis showed that IMPACT improved the recovery of known endocytosis-related genes, decreased off-target effects, and detected consistent phenotypes. Those findings were confirmed by rescreening 468 genes. Additionally we validated an unexpected influence of the IGF-receptor on EGF-endocytosis. IMPACT facilitates the selection of high-quality phenotypic profiles using different types of independent information, thereby supporting the molecular interpretation of functional screens.
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http://dx.doi.org/10.1371/journal.pcbi.1003801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4154648PMC
September 2014

Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution.

Nat Chem 2014 May 23;6(5):435-40. Epub 2014 Mar 23.

1] Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK [2] Cancer Research UK, Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK.

Recently, the cytosine modifications 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) were found to exist in the genomic deoxyribonucleic acid (DNA) of a wide range of mammalian cell types. It is now important to understand their role in normal biological function and disease. Here we introduce reduced bisulfite sequencing (redBS-Seq), a quantitative method to decode 5fC in DNA at single-base resolution, based on a selective chemical reduction of 5fC to 5hmC followed by bisulfite treatment. After extensive validation on synthetic and genomic DNA, we combined redBS-Seq and oxidative bisulfite sequencing (oxBS-Seq) to generate the first combined genomic map of 5-methylcytosine, 5hmC and 5fC in mouse embryonic stem cells. Our experiments revealed that in certain genomic locations 5fC is present at comparable levels to 5hmC and 5mC. The combination of these chemical methods can quantify and precisely map these three cytosine derivatives in the genome and will help provide insights into their function.
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http://dx.doi.org/10.1038/nchem.1893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4188980PMC
May 2014

Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape.

Nat Biotechnol 2013 Jul 23;31(7):638-46. Epub 2013 Jun 23.

Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

Delivery of short interfering RNAs (siRNAs) remains a key challenge in the development of RNA interference (RNAi) therapeutics. A better understanding of the mechanisms of siRNA cellular uptake, intracellular transport and endosomal release could critically contribute to the improvement of delivery methods. Here we monitored the uptake of lipid nanoparticles (LNPs) loaded with traceable siRNAs in different cell types in vitro and in mouse liver by quantitative fluorescence imaging and electron microscopy. We found that LNPs enter cells by both constitutive and inducible pathways in a cell type-specific manner using clathrin-mediated endocytosis as well as macropinocytosis. By directly detecting colloidal-gold particles conjugated to siRNAs, we estimated that escape of siRNAs from endosomes into the cytosol occurs at low efficiency (1-2%) and only during a limited window of time when the LNPs reside in a specific compartment sharing early and late endosomal characteristics. Our results provide insights into LNP-mediated siRNA delivery that can guide development of the next generation of delivery systems for RNAi therapeutics.
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http://dx.doi.org/10.1038/nbt.2612DOI Listing
July 2013

Integration of chemical and RNAi multiparametric profiles identifies triggers of intracellular mycobacterial killing.

Cell Host Microbe 2013 Feb;13(2):129-42

Max-Planck Institute of Molecular Cell Biology and Genetics, 108 Pfotenhauerstrasse, 01307 Dresden, Germany.

Pharmacological modulators of host-microbial interactions can in principle be identified using high-content screens. However, a severe limitation of this approach is the lack of insights into the mode of action of compounds selected during the primary screen. To overcome this problem, we developed a combined experimental and computational approach. We designed a quantitative multiparametric image-based assay to measure intracellular mycobacteria in primary human macrophages, screened a chemical library containing FDA-approved drugs, and validated three compounds for intracellular killing of M. tuberculosis. By integrating the multiparametric profiles of the chemicals with those of siRNAs from a genome-wide survey on endocytosis, we predicted and experimentally verified that two compounds modulate autophagy, whereas the third accelerates endosomal progression. Our findings demonstrate the value of integrating small molecules and genetic screens for identifying cellular mechanisms modulated by chemicals. Furthermore, selective pharmacological modulation of host trafficking pathways can be applied to intracellular pathogens beyond mycobacteria.
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http://dx.doi.org/10.1016/j.chom.2013.01.008DOI Listing
February 2013

Environmental toxins trigger PD-like progression via increased alpha-synuclein release from enteric neurons in mice.

Sci Rep 2012 30;2:898. Epub 2012 Nov 30.

Institute for Anatomy, TU-Dresden , Fetscherstr. 74, 01307, Dresden, Germany.

Pathological studies on Parkinson's disease (PD) patients suggest that PD pathology progresses from the enteric nervous system (ENS) and the olfactory bulb into the central nervous system. We have previously shown that environmental toxins acting locally on the ENS mimic this PD-like pathology progression pattern in mice. Here, we show for the first time that the resection of the autonomic nerves stops this progression. Moreover, our results show that an environmental toxin (i.e. rotenone) promotes the release of alpha-synuclein by enteric neurons and that released enteric alpha-synuclein is up-taken by presynaptic sympathetic neurites and retrogradely transported to the soma, where it accumulates. These results strongly suggest that pesticides can initiate the progression of PD pathology and that this progression is based on the transneuronal and retrograde axonal transport of alpha-synuclein. If confirmed in patients, this study would have crucial implications in the strategies used to prevent and treat PD.
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http://dx.doi.org/10.1038/srep00898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3510466PMC
May 2013