Shankar Balasubramanian

Cancer Research UK Cambridge Institute, Cambridge, England, United Kingdom

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Publications (173)1440.67 Total impact

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    ABSTRACT: The modified base 5-formylcytosine (5fC) was recently identified in mammalian DNA and might be considered to be the 'seventh' base of the genome. This nucleotide has been implicated in active demethylation mediated by the base excision repair enzyme thymine DNA glycosylase. Genomics and proteomics studies have suggested an additional role for 5fC in transcription regulation through chromatin remodeling. Here we propose that 5fC might affect these processes through its effect on DNA conformation. Biophysical and structural analysis revealed that 5fC alters the structure of the DNA double helix and leads to a conformation unique among known DNA structures including those comprising other cytosine modifications. The 1.4-Å-resolution X-ray crystal structure of a DNA dodecamer comprising three 5fCpG sites shows how 5fC changes the geometry of the grooves and base pairs associated with the modified base, leading to helical underwinding.
    Nature Structural & Molecular Biology 12/2014; · 11.63 Impact Factor
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    ABSTRACT: In investigating the binding interactions between the human telomeric RNA (TERRA) G-quadruplex (GQ) and its ligands, it was found that the small molecule carboxypyridostatin (cPDS) and the GQ-selective antibody BG4 simultaneously bind the TERRA GQ. We previously showed that the overall binding affinity of BG4 for RNA GQs is not significantly affected in the presence of cPDS. However, single-molecule mechanical unfolding experiments revealed a population (48 %) with substantially increased mechanical and thermodynamic stability. Force-jump kinetic investigations suggested competitive binding of cPDS and BG4 to the TERRA GQ. Following this, the two bound ligands slowly rearrange, thereby leading to the minor population with increased stability. Given the relevance of G-quadruplexes in the regulation of biological processes, we anticipate that the unprecedented conformational rearrangement observed in the TERRA-GQ–ligand complex may inspire new strategies for the selective stabilization of G-quadruplexes in cells.
    Angewandte Chemie 11/2014;
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    ABSTRACT: In investigating the binding interactions between the human telomeric RNA (TERRA) G-quadruplex (GQ) and its ligands, it was found that the small molecule carboxypyridostatin (cPDS) and the GQ-selective antibody BG4 simultaneously bind the TERRA GQ. We previously showed that the overall binding affinity of BG4 for RNA GQs is not significantly affected in the presence of cPDS. However, single-molecule mechanical unfolding experiments revealed a population (48 %) with substantially increased mechanical and thermodynamic stability. Force-jump kinetic investigations suggested competitive binding of cPDS and BG4 to the TERRA GQ. Following this, the two bound ligands slowly rearrange, thereby leading to the minor population with increased stability. Given the relevance of G-quadruplexes in the regulation of biological processes, we anticipate that the unprecedented conformational rearrangement observed in the TERRA-GQ–ligand complex may inspire new strategies for the selective stabilization of G-quadruplexes in cells.
    Angewandte Chemie International Edition 11/2014; · 11.34 Impact Factor
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    ABSTRACT: 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.
    Nature Communications 11/2014; 5:5165. · 10.74 Impact Factor
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    ABSTRACT: 5-Hydroxymethylcytosine ( hmC) is an oxidation product of 5-methylcytosine which is present in the deoxyribonucleic acid (DNA) of most mammalian cells. Reduction of hmC levels in DNA is a hallmark of cancers. Elucidating the dynamics of this oxidation reaction and the lifetime of hmC in DNA is fundamental to understanding hmC function. Using stable isotope labelling of cytosine derivatives in the DNA of mammalian cells and ultrasensitive tandem liquid–chromatography mass spectrometry, we show that the majority of hmC is a stable modification, as opposed to a transient intermediate. In contrast with DNA methylation, which occurs immediately during replication, hmC forms slowly during the first 30 hours following DNA synthesis. Isotopic labelling of DNA in mouse tissues confirmed the stability of hmC in vivo and demonstrated a relationship between global levels of hmC and cell proliferation. These insights have important implications for understanding the states of chemically modified DNA bases in health and disease.
    Nature Chemistry 09/2014; · 21.76 Impact Factor
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    ABSTRACT: REV1-deficient chicken DT40 cells are compromised in replicating G quadruplex (G4)-forming DNA. This results in localised, stochastic loss of parental chromatin marks and changes in gene expression. We previously proposed that this epigenetic instability arises from G4-induced replication fork stalls disrupting the accurate propagation of chromatin structure through replication. Here, we test this model by showing that a single G4 motif is responsible for the epigenetic instability of the BU-1 locus in REV1-deficient cells, despite its location 3.5 kb from the transcription start site (TSS). The effect of the G4 is dependent on it residing on the leading strand template, but is independent of its in vitro thermal stability. Moving the motif to more than 4 kb from the TSS stabilises expression of the gene. However, loss of histone modifications (H3K4me3 and H3K9/14ac) around the transcription start site correlates with the position of the G4 motif, expression being lost only when the promoter is affected. This supports the idea that processive replication is required to maintain the histone modification pattern and full transcription of this model locus.
    The EMBO Journal 09/2014; · 10.75 Impact Factor
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    ABSTRACT: DNA methylation analysis has become an integral part of biomedical research. For high-throughput applications such as epigenome-wide association studies, the Infinium HumanMethylation450 (450K) BeadChip is currently the platform of choice. However, BeadChip processing relies on traditional bisulfite (BS) based protocols which cannot discriminate between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Here, we report the adaptation of the recently developed oxidative bisulfite (oxBS) chemistry to specifically detect both 5mC and 5hmC in a single workflow using 450K BeadChips, termed oxBS-450K. Supported by validation using mass spectrometry and pyrosequencing, we demonstrate reproducible (R(2) > 0.99) detection of 5hmC in human brain tissue using the optimised oxBS-450K protocol described here.
    Methods 08/2014; · 3.22 Impact Factor
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    ABSTRACT: Non-coding Y RNAs are small stem-loop RNAs that are involved in different cellular processes, including the regulation of DNA replication. An evolutionarily conserved small domain in the upper stem of vertebrate Y RNAs has an essential function for the initiation of chromosomal DNA replication. Here we provide a structure-function analysis of this essential RNA domain under physiological conditions. Solution state NMR and far-UV CD spectroscopy show that the upper stem domain of human Y1 RNA adopts a locally destabilised A-form helical structure involving eight Watson-Crick base pairings. Within this helix, two G:C base pairs are highly stable even at elevated temperatures, and therefore may serve as clamps to maintain the local structure of the helix. These two stable G:C base pairs frame three unstable base pairs, which are located centrally between them. Systematic substitution mutagenesis results in a disruption of the ordered A-form helical structure and in the loss of DNA replication initiation activity, establishing a positive correlation between folding stability and function. Our data thus provide a structural basis for the evolutionary conservation of key nucleotides in this RNA domain that are essential for the functionality of non-coding Y RNAs during the initiation of DNA replication.
    Biochemistry 08/2014; · 3.38 Impact Factor
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    ABSTRACT: G-quadruplexes are functional DNA elements and small molecules are formidable to study their biological roles and may have potential for therapeutic development. We previously identified a trimeric quinoline-based oligoamide macrocycle and helical oligoamide foldamers as selective ligands. Their helical structure may permit the targeting of the backbone loops and grooves of G-quadruplexes instead of the G-tetrads. Given the vast array of morphologies G-quadruplex structures can adopt, this may be a way to elicit sequence selective binding. Herein, we describe the design and synthesis of molecules based on macrocyclic and helically folded oligoamides. We tested them for the ability to interact with the human telomeric G-quadruplex and an array of promoter G-quadruplexes using a well established Förster Resonance Energy Transfer (FRET) melting assay and single molecule FRET. Our results show that they constitute very potent ligands, comparable to the best reported in the literature. Their mode of interaction differs from that of traditional tetrad binders, opening avenues for the development of molecules specific for certain G-quadruplex conformations.
    ChemBioChem 08/2014; · 3.06 Impact Factor
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    Gordon Ross McInroy, Eun-Ang Raiber, Shankar Balasubramanian
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    ABSTRACT: The exquisite selectivity of chemical reactions enables the study of rare DNA bases. However, chemical modification of the genome can affect downstream analysis. We report a PCR bias caused by such modification, and exemplify a solution with the synthesis and characterization of a cleavable aldehyde-reactive biotinylation probe.
    Chemical Communications 08/2014; · 6.38 Impact Factor
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    Michael J Booth, Eun-Ang Raiber, Shankar Balasubramanian
    Chemical Reviews 08/2014; · 45.66 Impact Factor
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    ABSTRACT: Four-stranded G-quadruplex DNA secondary structures have recently been visualized in the nuclei of human cultured cells. Here, we show that BG4, a G-quadruplex-specific antibody, can be used to stain DNA G-quadruplex structures in patient-derived tissues using immunohistochemistry. We observe a significantly elevated number of G-quadruplex-positive nuclei in human cancers of the liver and stomach as compared to background non-neoplastic tissue. Our results suggest that G-quadruplex formation can be detected and measured in patient-derived material and that elevated G-quadruplex formation may be a characteristic of some cancers.
    PLoS ONE 07/2014; 9(7):e102711. · 3.53 Impact Factor
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    ABSTRACT: 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.
    Nature Chemistry 05/2014; 6(5):435-40. · 21.76 Impact Factor
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    Marco Di Antonio, Keith I E McLuckie, Shankar Balasubramanian
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    ABSTRACT: The nitrogen mustard Chlorambucil (Chl) generates covalent adducts with double-helical DNA and inhibits cell proliferation. Among these adducts inter-strand crosslinks (ICLs) are the most toxic, as they stall replication by generating DNA double strand breaks (DSBs). Intra-strand crosslinks generated by Chl are repaired by a dedicated Nucleotide Excision Repair (NER) enzyme. We synthesized a novel crosslinking agent that combines Chl with the G-quadruplex (G4) ligand PDS (PDS-Chl). We demonstrated that PDS-Chl alkylates G4 structures at low μM doses, without reactivity towards double- or single-stranded DNA. Since intra-molecular G4s arise from a single DNA strand, we reasoned that preferential alkylation of such structures might prevent the generation of ICLs, whilst favoring intra-strand crosslinks. We observed that PDS-Chl selectively impairs growth in cells genetically deficient in NER, but did not show any sensitivity to the repair gene BRCA2, involved in double-stranded break repair. Our findings suggest that G4 targeting of this clinically important alkylating agent alters the overall mechanism of action. These insights may inspire new opportunities for intervention in diseases specifically characterised by genetic impairment of NER, such as skin and testicular cancers.
    Journal of the American Chemical Society 04/2014; · 11.44 Impact Factor
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    ABSTRACT: Viruses that establish latent infections have evolved unique mechanisms to avoid host immune recognition. Maintenance proteins of these viruses regulate their synthesis to levels sufficient for maintaining persistent infection but below threshold levels for host immune detection. The mechanisms governing this finely tuned regulation of viral latency are unknown. Here we show that mRNAs encoding gammaherpesviral maintenance proteins contain within their open reading frames clusters of unusual structural elements, G-quadruplexes, which are responsible for the cis-acting regulation of viral mRNA translation. By studying the Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1) mRNA, we demonstrate that destabilization of G-quadruplexes using antisense oligonucleotides increases EBNA1 mRNA translation. In contrast, pretreatment with a G-quadruplex-stabilizing small molecule, pyridostatin, decreases EBNA1 synthesis, highlighting the importance of G-quadruplexes within virally encoded transcripts as unique regulatory signals for translational control and immune evasion. Furthermore, these findings suggest alternative therapeutic strategies focused on targeting RNA structure within viral ORFs.
    Nature Chemical Biology 03/2014; · 12.95 Impact Factor
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    Giulia Biffi, Marco Di Antonio, David Tannahill, Shankar Balasubramanian
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    ABSTRACT: Following extensive evidence for the formation of four-stranded DNA G-quadruplex structures in vitro, DNA G-quadruplexes have been observed within human cells. Although chemically distinct, RNA can also fold in vitro into G-quadruplex structures that are highly stable because of the 2'-hydroxyl group. However, RNA G-quadruplexes have not yet been reported in cells. Here, we demonstrate the visualization of RNA G-quadruplex structures within the cytoplasm of human cells using a G-quadruplex structure-specific antibody. We also demonstrate that small molecules that bind to G-quadruplexes in vitro can trap endogenous RNA G-quadruplexes when applied to cells. Furthermore, a small molecule that exhibits a preference for RNA G-quadruplexes rather than DNA G-quadruplexes in biophysical experiments also shows the same selectivity within a cellular context. Our findings provide substantive evidence for RNA G-quadruplex formation in the human transcriptome, and corroborate the selectivity and application of stabilizing ligands that target G-quadruplexes within a cellular context.
    Nature Chemistry 01/2014; 6(1):75-80. · 21.76 Impact Factor
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    Pierre Murat, Shankar Balasubramanian
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    ABSTRACT: While the discovery of B-form DNA 60 years ago has defined our molecular view of the genetic code, other postulated DNA secondary structures, such as A-DNA, Z-DNA, H-DNA, cruciform and slipped structures have provoked consideration of DNA as a more dynamic structure. Four-stranded G-quadruplex DNA does not use Watson-Crick base pairing and has been subject of considerable speculation and investigation during the past decade, particularly with regard to its potential relevance to genome integrity and gene expression. Here, we discuss recent data that collectively support the formation of G-quadruplexes in genomic DNA and the consequences of formation of this structural motif in biological processes.
    Current opinion in genetics & development 12/2013; 25C:22-29. · 8.99 Impact Factor
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    ABSTRACT: RNA-protein interactions are vital throughout the HIV-1 life cycle for the successful production of infectious virus particles. One such essential RNA-protein interaction occurs between the full-length genomic viral RNA and the major structural protein of the virus. The initial interaction is between the Gag polyprotein and the viral RNA packaging signal (psi or Ψ), a highly conserved RNA structural element within the 5'-UTR of the HIV-1 genome, which has gained attention as a potential therapeutic target. Here, we report the application of a target-based assay to identify small molecules, which modulate the interaction between Gag and Ψ. We then demonstrate that one such molecule exhibits potent inhibitory activity in a viral replication assay. The mode of binding of the lead molecules to the RNA target was characterized by (1)H NMR spectroscopy.
    Biochemistry 12/2013; 52(51):9269-74. · 3.38 Impact Factor
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    ABSTRACT: Long non--coding RNAs (lncRNAs) play a key role in the epigenetic regulation of cells. Many of these lncRNAs function by interacting with histone repressive proteins of the Polycomb group (PcG) family, recruiting them to gene loci to facilitate silencing. Although there are now many RNAs known to interact with the PRC2 complex, little is known about the details of the molecular interactions. Here we show that the PcG protein heterodimer EZH2--EED is necessary and sufficient for binding to the lncRNA HOTAIR. We also show that protein recognition occurs within a folded 89-mer domain of HOTAIR. This 89--mer represents a minimal binding motif, as further deletion of nucleotides results in substantial loss of affinity for PRC2. These findings provide molecular insights into an important system involved in epigenetic regulation.
    Biochemistry 12/2013; · 3.38 Impact Factor
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    ABSTRACT: DNA methylation (5mC) plays important roles in epigenetic regulation of genome function. Recently, TET hydroxylases have been found to oxidize 5mC to hydroxymethylcytosine (5hmC), formylcytosine (5fC) and carboxylcytosine (5caC) in DNA. These derivatives have a role in demethylation of DNA but in addition may have epigenetic signaling functions in their own right. A recent study identified proteins which showed preferential binding to 5-methylcytosine (5mC) and its oxidized forms, where readers for 5mC and 5hmC showed little overlap, and proteins bound to further oxidation forms were enriched for repair proteins and transcription regulators. We extend this study by using promoter sequences as baits and compare protein binding patterns to unmodified or modified cytosine using DNA from mouse embryonic stem cell extracts. We compared protein enrichments from two DNA probes with different CpG composition and show that, whereas some of the enriched proteins show specificity to cytosine modifications, others are selective for both modification and target sequences. Only a few proteins were identified with a preference for 5hmC (such as RPL26, PRP8 and the DNA mismatch repair protein MHS6), but proteins with a strong preference for 5fC were more numerous, including transcriptional regulators (FOXK1, FOXK2, FOXP1, FOXP4 and FOXI3), DNA repair factors (TDG and MPG) and chromatin regulators (EHMT1, L3MBTL2 and all components of the NuRD complex). Our screen has identified novel proteins that bind to 5fC in genomic sequences with different CpG composition and suggests they regulate transcription and chromatin, hence opening up functional investigations of 5fC readers.
    Genome biology 10/2013; 14(10):R119. · 10.47 Impact Factor

Publication Stats

6k Citations
1,440.67 Total Impact Points

Institutions

  • 2010–2014
    • Cancer Research UK Cambridge Institute
      Cambridge, England, United Kingdom
  • 1991–2014
    • University of Cambridge
      • Department of Chemistry
      Cambridge, England, United Kingdom
  • 2011–2013
    • Kent State University
      • • Department of Chemistry and Biochemistry
      • • School of Biomedical Sciences
      Kent, OH, United States
  • 2010–2013
    • Cambridge Institute for Medical Research
      Cambridge, England, United Kingdom
  • 2005
    • Tata Institute of Fundamental Research
      • National Centre for Biological Sciences
      Mumbai, State of Maharashtra, India
    • The School of Pharmacy
      • Cancer Research UK Biomolecular Structure Group
      Londinium, England, United Kingdom