Publications by authors named "Shankar Balasubramanian"

240 Publications

TET2 is a component of the estrogen receptor complex and controls 5mC to 5hmC conversion at estrogen receptor cis-regulatory regions.

Cell Rep 2021 Feb;34(8):108776

Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge CB2 0RE, UK. Electronic address:

Estrogen receptor-α (ER) drives tumor development in ER-positive (ER+) breast cancer. The transcription factor GATA3 has been closely linked to ER function, but its precise role in this setting remains unclear. Quantitative proteomics was used to assess changes to the ER complex in response to GATA3 depletion. Unexpectedly, few proteins were lost from the ER complex in the absence of GATA3, with the only major change being depletion of the dioxygenase TET2. TET2 binding constituted a near-total subset of ER binding in multiple breast cancer models, with loss of TET2 associated with reduced activation of proliferative pathways. TET2 knockdown did not appear to change global methylated cytosine (5mC) levels; however, oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) was significantly reduced, and these events occurred at ER enhancers. These findings implicate TET2 in the maintenance of 5hmC at ER sites, providing a potential mechanism for TET2-mediated regulation of ER target genes.
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http://dx.doi.org/10.1016/j.celrep.2021.108776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921846PMC
February 2021

Propensity matched analysis of short term oncological and perioperative outcomes following robotic and thoracolaparoscopic esophagectomy for carcinoma esophagus- the first Indian experience.

J Robot Surg 2021 Feb 20. Epub 2021 Feb 20.

Department of Surgical Gastroenterology, GEM Hospital and Research Center, Coimbatore, India.

Thoracolaparoscopic esophagectomy (TLE) for carcinoma esophagus has better short-term outcomes compared to open esophagectomy. The precise role of robot-assisted laparoscopic esophagectomy (RALE) is still evolving. Single center retrospective analysis of TLE and RALE performed for carcinoma esophagus between January 2015 and September 2018. Propensity score matching was done between the groups for age, gender, BMI, ASA grade, tumor location, neoadjuvant therapy, the extent of surgical resection (Ivor Lewis or McKeown's), histopathological type (squamous cell carcinoma or adenocarcinoma), clinical T and N stages. The primary outcome parameter was lymph node yield. Secondary outcome parameters were resection margin status, duration of surgery, blood loss, conversion to open procedure, length of hospital stay, length of ICU stay, complications, 90-day mortality and cost. There were 90 patients in TLE and 25 patients in RALE group. After propensity matching, there were 22 patients in each group. The lymph node yield was similar in both the groups (23.95 ± 8.23 vs 22.73 ± 11.63; p = 0.688). There were no conversions or positive resection margins in either group. RALE was associated with longer operating duration (513.18 ± 91.23 min vs 444.77 ± 64.91 min; p = 0.006) and higher cost ($5271.75 ± 456.46 vs $4243.01 ± 474.64; p < 0.001) than TLE. Both were comparable in terms of blood loss (138.86 ± 31.20 ml vs 133.18 ± 34.80 ml; p = 0.572), Clavien-Dindo grade IIIa and above complications (13.64% vs 9.09%; p = 0.634), hospital stay (12.18 ± 6.35 days vs 12.73 ± 7.83 days; p = 0.801), ICU stay (4.91 ± 5.22 days vs 4.77 ± 4.81 days; p = 0.929) and mortality (0 vs 4.55%; p = 0.235). RALE is comparable to TLE in terms of short-term oncological and perioperative outcomes except for longer operating duration when performed for carcinoma esophagus. RALE is costlier than TLE.
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http://dx.doi.org/10.1007/s11701-021-01211-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7896161PMC
February 2021

Selective Chemical Functionalization at N6-Methyladenosine Residues in DNA Enabled by Visible-Light-Mediated Photoredox Catalysis.

J Am Chem Soc 2020 12 11;142(51):21484-21492. Epub 2020 Dec 11.

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

Selective chemistry that modifies the structure of DNA and RNA is essential to understanding the role of epigenetic modifications. We report a visible-light-activated photocatalytic process that introduces a covalent modification at a C(sp)-H bond in the methyl group of N6-methyl deoxyadenosine and N6-methyl adenosine, epigenetic modifications of emerging importance. A carefully orchestrated reaction combines reduction of a nitropyridine to form a nitrosopyridine spin-trapping reagent and an exquisitely selective tertiary amine-mediated hydrogen-atom abstraction at the N6-methyl group to form an α-amino radical. Cross-coupling of the putative α-amino radical with nitrosopyridine leads to a stable conjugate, installing a label at N6-methyl-adenosine. We show that N6-methyl deoxyadenosine-containing oligonucleotides can be enriched from complex mixtures, paving the way for applications to identify this modification in genomic DNA and RNA.
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http://dx.doi.org/10.1021/jacs.0c10616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760100PMC
December 2020

Activation-induced cytidine deaminase localizes to G-quadruplex motifs at mutation hotspots in lymphoma.

NAR Cancer 2020 Dec 13;2(4):zcaa029. Epub 2020 Oct 13.

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.

Diffuse large B-cell lymphoma (DLBCL) is a molecularly heterogeneous group of malignancies with frequent genetic abnormalities. G-quadruplex (G4) DNA structures may facilitate this genomic instability through association with activation-induced cytidine deaminase (AID), an antibody diversification enzyme implicated in mutation of oncogenes in B-cell lymphomas. Chromatin immunoprecipitation sequencing analyses in this study revealed that AID hotspots in both activated B cells and lymphoma cells were highly enriched for G4 elements. A representative set of these targeted sequences was validated for characteristic, stable G4 structure formation including previously unknown G4s in lymphoma-associated genes, , , ,  and , along with the established and structures. Frequent genome-wide G4 formation was also detected for the first time in DLBCL patient-derived tissues using BG4, a structure-specific G4 antibody. Tumors with greater staining were more likely to have concurrent and oncogene amplification and mutations. Ninety-seven percent of the mutations occurred within G4 sites that overlapped with AID binding. G4 localization at sites of mutation, and within aggressive DLBCL tumors harboring amplified and , supports a role for G4 structures in events that lead to a loss of genomic integrity, a critical step in B-cell lymphomagenesis.
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http://dx.doi.org/10.1093/narcan/zcaa029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556405PMC
December 2020

The Structure and Function of DNA G-Quadruplexes.

Trends Chem 2020 Feb;2(2):123-136

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

Guanine-rich DNA sequences can fold into four-stranded, noncanonical secondary structures called G-quadruplexes (G4s). G4s were initially considered a structural curiosity, but recent evidence suggests their involvement in key genome functions such as transcription, replication, genome stability, and epigenetic regulation, together with numerous connections to cancer biology. Collectively, these advances have stimulated research probing G4 mechanisms and consequent opportunities for therapeutic intervention. Here, we provide a perspective on the structure and function of G4s with an emphasis on key molecules and methodological advances that enable the study of G4 structures in human cells. We also critically examine recent mechanistic insights into G4 biology and protein interaction partners and highlight opportunities for drug discovery.
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http://dx.doi.org/10.1016/j.trechm.2019.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472594PMC
February 2020

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

Single-molecule visualization of DNA G-quadruplex formation in live cells.

Nat Chem 2020 09 20;12(9):832-837. Epub 2020 Jul 20.

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

Substantial evidence now exists to support that formation of DNA G-quadruplexes (G4s) is coupled to altered gene expression. However, approaches that allow us to probe G4s in living cells without perturbing their folding dynamics are required to understand their biological roles in greater detail. Herein, we report a G4-specific fluorescent probe (SiR-PyPDS) that enables single-molecule and real-time detection of individual G4 structures in living cells. Live-cell single-molecule fluorescence imaging of G4s was carried out under conditions that use low concentrations of SiR-PyPDS (20 nM) to provide informative measurements representative of the population of G4s in living cells, without globally perturbing G4 formation and dynamics. Single-molecule fluorescence imaging and time-dependent chemical trapping of unfolded G4s in living cells reveal that G4s fluctuate between folded and unfolded states. We also demonstrate that G4 formation in live cells is cell-cycle-dependent and disrupted by chemical inhibition of transcription and replication. Our observations provide robust evidence in support of dynamic G4 formation in living cells.
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http://dx.doi.org/10.1038/s41557-020-0506-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610488PMC
September 2020

Genome-wide DNA Methylation Signatures Are Determined by DNMT3A/B Sequence Preferences.

Biochemistry 2020 07 28;59(27):2541-2550. Epub 2020 Jun 28.

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

Cytosine methylation is an important epigenetic mark, but how the distinctive patterns of DNA methylation arise remains elusive. For the first time, we systematically investigated how these patterns can be imparted by the inherent enzymatic preferences of mammalian DNA methyltransferases and the extent to which this applies in cells. In a biochemical experiment, we subjected a wide variety of DNA sequences to methylation by DNMT3A or DNMT3B and then applied deep bisulfite sequencing to quantitatively determine the sequence preferences for methylation. The data show that DNMT3A prefers CpG and non-CpG sites followed by a 3'-pyrimidine, whereas DNMT3B favors a 3'-purine. Overall, we show that DNMT3A has a sequence preference for a TNC[G/A]CC context, while DNMT3B prefers TAC[G/A]GC. We extended our finding using publicly available data from mouse Dnmt1/3a/3b triple-knockout cells in which reintroduction of either DNMT3A or DNMT3B expression results in the acquisition of the same enzyme specific signature sequences observed . Furthermore, loss of DNMT3A or DNMT3B in human embryonic stem cells leads to a loss of methylation at the corresponding enzyme specific signatures. Therefore, the global DNA methylation landscape of the mammalian genome can be fundamentally determined by the inherent sequence preference of methyltransferases.
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http://dx.doi.org/10.1021/acs.biochem.0c00339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7364778PMC
July 2020

Is the Current Micronutrient Supplementation Adequate in Preventing Deficiencies in Indian Patients? Short- and Mid-Term Comparison of Sleeve Gastrectomy and Roux-en-Y Gastric Bypass.

Obes Surg 2020 Sep;30(9):3480-3488

Department of bariatric and metabolic surgery, GEM hospital & research center, Coimbatore, Tamil Nadu, 641045, India.

Purpose: Bariatric procedures reduce the capacity of the gut and alter the gastrointestinal transit time predisposing to micro-nutritional deficiencies. This study analyzed and compared the micro-nutritional parameters following laparoscopic sleeve gastrectomy (LSG) and Roux-en-Y gastric bypass (RYGB) in the Indian population.

Materials And Methods: This is a retrospective study of patients who underwent LSG or RYGB for morbid obesity at a tertiary care center between January 2015 and December 2016. The micronutrient parameters, namely, serum ferritin, vitamin B12, ionized calcium, vitamin D3, and parathormone (PTH) in the preoperative settings and subsequently at 1, 2, and 3 years were analyzed.

Results: A total of 390 patients were studied, of which 258 (66.15%) underwent LSG while 132 (33.85%) underwent RYGB. Baseline micronutrient parameters were comparable in the two groups. Anemia (58.1% vs. 59.1%), deficiencies of ferritin (31.7% vs. 34.3%), vitamin B12 (18.8% vs. 36.4%), ionized calcium (65.1% vs. 72.7%), vitamin D3 (95.3% vs. 90.9%), and secondary hyperparathyroidism (45.5% vs. 58.1%) were seen following LSG and RYGB at the end of 3 years, respectively. There was no significant difference found between LSG and RYGB in terms of micronutrient deficiencies studied, including rising in PTH at 1, 2, and 3 years. Vitamin D3 levels were significantly lower at 2 and 3 years following RYGB (p = 0.035 and p = 0.032, respectively).

Conclusion: LSG and RYGB have comparable micronutrient deficiencies in the short- and mid-term except for vitamin D3, which is higher following RYGB. Long-term studies are needed to define optimum micronutrient supplement dosages for the Indian population.
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http://dx.doi.org/10.1007/s11695-020-04674-2DOI Listing
September 2020

The regulation and functions of DNA and RNA G-quadruplexes.

Nat Rev Mol Cell Biol 2020 08 20;21(8):459-474. Epub 2020 Apr 20.

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

DNA and RNA can adopt various secondary structures. Four-stranded G-quadruplex (G4) structures form through self-recognition of guanines into stacked tetrads, and considerable biophysical and structural evidence exists for G4 formation in vitro. Computational studies and sequencing methods have revealed the prevalence of G4 sequence motifs at gene regulatory regions in various genomes, including in humans. Experiments using chemical, molecular and cell biology methods have demonstrated that G4s exist in chromatin DNA and in RNA, and have linked G4 formation with key biological processes ranging from transcription and translation to genome instability and cancer. In this Review, we first discuss the identification of G4s and evidence for their formation in cells using chemical biology, imaging and genomic technologies. We then discuss possible functions of DNA G4s and their interacting proteins, particularly in transcription, telomere biology and genome instability. Roles of RNA G4s in RNA biology, especially in translation, are also discussed. Furthermore, we consider the emerging relationships of G4s with chromatin and with RNA modifications. Finally, we discuss the connection between G4 formation and synthetic lethality in cancer cells, and recent progress towards considering G4s as therapeutic targets in human diseases.
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http://dx.doi.org/10.1038/s41580-020-0236-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115845PMC
August 2020

Affinity-Selected Bicyclic Peptide G-Quadruplex Ligands Mimic a Protein-like Binding Mechanism.

J Am Chem Soc 2020 05 21;142(18):8367-8373. Epub 2020 Apr 21.

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

The study of G-quadruplexes (G4s) in a cellular context has demonstrated links between these nucleic acid secondary structures, gene expression, and DNA replication. Ligands that bind to the G4 structure therefore present an excellent opportunity for influencing gene expression through the targeting of a nucleic acid structure rather than sequence. Here, we explore cyclic peptides as an alternative class of G4 ligands. Specifically, we describe the development of G4-binding bicyclic peptides selected by phage display. Selected bicyclic peptides display submicromolar affinity to G4 structures and high selectivity over double helix DNA. Molecular simulations of the bicyclic peptide-G4 complexes corroborate the experimental binding strengths and reveal molecular insights into G4 recognition by bicyclic peptides via the precise positioning of amino acid side chains, a binding mechanism reminiscent of endogenous G4-binding proteins. Overall, our results demonstrate that selection of (bi)cyclic peptides unlocks a valuable chemical space for targeting nucleic acid structures.
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http://dx.doi.org/10.1021/jacs.0c01879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212521PMC
May 2020

Natural, modified DNA bases.

Curr Opin Chem Biol 2020 08 4;57:1-7. Epub 2020 Mar 4.

Department of Chemistry, University of Cambridge, Cambridge, CB2 1EW, United Kingdom; Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, CB2 0RE, United Kingdom; School of Clinical Medicine, University of Cambridge, Cambridge, CB2 0SP, United Kingdom. Electronic address:

The four canonical bases that make up genomic DNA are subject to a variety of chemical modifications in living systems. Recent years have witnessed the discovery of various new modified bases and of the enzymes responsible for their processing. Here, we review the range of DNA base modifications currently known and recent advances in chemical methodology that have driven progress in this field, in particular regarding their detection and sequencing. Elucidating the cellular functions of modifications remains an ongoing challenge; we discuss recent contributions to this area before exploring their relevance in medicine.
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http://dx.doi.org/10.1016/j.cbpa.2020.01.014DOI Listing
August 2020

An Activatable Cancer-Targeted Hydrogen Peroxide Probe for Photoacoustic and Fluorescence Imaging.

Cancer Res 2019 10 27;79(20):5407-5417. Epub 2019 Aug 27.

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

Reactive oxygen species play an important role in cancer, however, their promiscuous reactivity, low abundance, and short-lived nature limit our ability to study them in real time in living subjects with conventional noninvasive imaging methods. Photoacoustic imaging is an emerging modality for visualization of molecular processes with deep tissue penetration and high spatiotemporal resolution. Here, we describe the design and synthesis of a targeted, activatable probe for photoacoustic imaging, which is responsive to one of the major and abundant reactive oxygen species, hydrogen peroxide (HO). This bifunctional probe, which is also detectable with fluorescence imaging, is composed of a heptamethine carbocyanine dye scaffold for signal generation, a 2-deoxyglucose cancer localization moiety, and a boronic ester functionality that specifically detects and reacts to HO. The optical properties of the probe were characterized using absorption, fluorescence, and photoacoustic measurements; upon addition of pathophysiologic HO concentrations, a clear increase in fluorescence and red-shift of the absorption and photoacoustic spectra were observed. Studies performed showed no significant toxicity and specific uptake of the probe into the cytosol in breast cancer cell lines. Importantly, intravenous injection of the probe led to targeted uptake and accumulation in solid tumors, which enabled noninvasive photoacoustic and fluorescence imaging of HO. In conclusion, the reported probe shows promise for the visualization of hydrogen peroxide. SIGNIFICANCE: This study presents the first activatable and cancer-targeted hydrogen peroxide probe for photoacoustic molecular imaging, paving the way for visualization of hydrogen peroxide at high spatiotemporal resolution in living subjects. http://cancerres.aacrjournals.org/content/canres/79/20/5407/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-0691DOI Listing
October 2019

Unusual Activity of a TET/JBP Family Enzyme.

Biochemistry 2019 09 22;58(35):3627-3629. Epub 2019 Aug 22.

La Jolla Institute for Immunology and Sanford Consortium for Regenerative Medicine , La Jolla , California 92037 , United States.

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http://dx.doi.org/10.1021/acs.biochem.9b00609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416651PMC
September 2019

A Spontaneous Ring-Opening Reaction Leads to a Repair-Resistant Thymine Oxidation Product in Genomic DNA.

Chembiochem 2020 02 23;21(3):320-323. Epub 2019 Oct 23.

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

The alphabet of modified DNA bases goes beyond the conventional four letters, with biological roles being found for many such modifications. Herein, we describe the observation of a modified thymine base that arises from spontaneous N -C ring opening of the oxidation product 5-formyl uracil, after N deprotonation. We first observed this phenomenon in silico through ab initio calculations, followed by in vitro experiments to verify its formation at a mononucleoside level and in a synthetic DNA oligonucleotide context. We show that the new base modification (T , thymine ring expunged) can form under physiological conditions, and is resistant to the action of common repair machineries. Furthermore, we found cases of the natural existence of T while screening a number of human cell types and mESC (E14), thus suggesting potential biological relevance of this modification.
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http://dx.doi.org/10.1002/cbic.201900484DOI Listing
February 2020

Genetic interactions of G-quadruplexes in humans.

Elife 2019 07 9;8. Epub 2019 Jul 9.

Cancer Research United Kingdom Cambridge Institute, Cambridge, United Kingdom.

G-quadruplexes (G4) are alternative nucleic acid structures involved in transcription, translation and replication. Aberrant G4 formation and stabilisation is linked to genome instability and cancer. G4 ligand treatment disrupts key biological processes leading to cell death. To discover genes and pathways involved with G4s and gain mechanistic insights into G4 biology, we present the first unbiased genome-wide study to systematically identify human genes that promote cell death when silenced by shRNA in the presence of G4-stabilising small molecules. Many novel genetic vulnerabilities were revealed opening up new therapeutic possibilities in cancer, which we exemplified by an orthogonal pharmacological inhibition approach that phenocopies gene silencing. We find that targeting the WEE1 cell cycle kinase or USP1 deubiquitinase in combination with G4 ligand treatment enhances cell killing. We also identify new genes and pathways regulating or interacting with G4s and demonstrate that the DDX42 DEAD-box helicase is a newly discovered G4-binding protein.
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http://dx.doi.org/10.7554/eLife.46793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6615864PMC
July 2019

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

Sequencing abasic sites in DNA at single-nucleotide resolution.

Nat Chem 2019 07 17;11(7):629-637. Epub 2019 Jun 17.

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

In DNA, the loss of a nucleobase by hydrolysis generates an abasic site. Formed as a result of DNA damage, as well as a key intermediate during the base excision repair pathway, abasic sites are frequent DNA lesions that can lead to mutations and strand breaks. Here we present snAP-seq, a chemical approach that selectively exploits the reactive aldehyde moiety at abasic sites to reveal their location within DNA at single-nucleotide resolution. Importantly, the approach resolves abasic sites from other aldehyde functionalities known to exist in genomic DNA. snAP-seq was validated on synthetic DNA and then applied to two separate genomes. We studied the distribution of thymine modifications in the Leishmania major genome by enzymatically converting these modifications into abasic sites followed by abasic site mapping. We also applied snAP-seq directly to HeLa DNA to provide a map of endogenous abasic sites in the human genome.
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http://dx.doi.org/10.1038/s41557-019-0279-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589398PMC
July 2019

METTL1 Promotes let-7 MicroRNA Processing via m7G Methylation.

Mol Cell 2019 06 25;74(6):1278-1290.e9. Epub 2019 Apr 25.

The Gurdon Institute and Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK. Electronic address:

7-methylguanosine (m7G) is present at mRNA caps and at defined internal positions within tRNAs and rRNAs. However, its detection within low-abundance mRNAs and microRNAs (miRNAs) has been hampered by a lack of sensitive detection strategies. Here, we adapt a chemical reactivity assay to detect internal m7G in miRNAs. Using this technique (Borohydride Reduction sequencing [BoRed-seq]) alongside RNA immunoprecipitation, we identify m7G within a subset of miRNAs that inhibit cell migration. We show that the METTL1 methyltransferase mediates m7G methylation within miRNAs and that this enzyme regulates cell migration via its catalytic activity. Using refined mass spectrometry methods, we map m7G to a single guanosine within the let-7e-5p miRNA. We show that METTL1-mediated methylation augments let-7 miRNA processing by disrupting an inhibitory secondary structure within the primary miRNA transcript (pri-miRNA). These results identify METTL1-dependent N7-methylation of guanosine as a new RNA modification pathway that regulates miRNA structure, biogenesis, and cell migration.
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http://dx.doi.org/10.1016/j.molcel.2019.03.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6591002PMC
June 2019

Publisher Correction: DNA sequencing at 40: past, present and future.

Nature 2019 Apr;568(7752):E11

Department of Genome Sciences, University of Washington, Seattle, Washington, USA.

In this Review, the year of publication of reference 54 should be 2005, not 2015. In Box 2, "1982: GenBank ( https://www.ncbi.nlm.nih.gov/genbank/statistics/ )" should read "1982: Genbank/ENA/DDBJ" and "2007: NCBI Short Read Archive" should read "2007: NCBI and ENA Short Read Archives"; this is because the launches of these American, European and Japanese databases were coordinated. These errors have not been corrected.
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http://dx.doi.org/10.1038/s41586-019-1120-8DOI Listing
April 2019

Detection, Structure and Function of Modified DNA Bases.

J Am Chem Soc 2019 04 28;141(16):6420-6429. Epub 2019 Mar 28.

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

While some DNA base modifications such as 5-methylcytosine have been known and studied for decades, recent discoveries of a number of other modified bases have stimulated research to understand their origin and function. Chemistry-based methods for their detection and analysis have proven to be important for advancing the field. Here, we feature a selection of methods that have helped advance the field, along with some key advances in the understanding of how the chemistry of modified bases affects biological functions. We also discuss fundamental questions in the field that remain unanswered.
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http://dx.doi.org/10.1021/jacs.9b01915DOI Listing
April 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

A Photo-responsive Small-Molecule Approach for the Opto-epigenetic Modulation of DNA Methylation.

Angew Chem Int Ed Engl 2019 05 12;58(20):6620-6624. Epub 2019 Apr 12.

Department of Chemistry, Institute for Structural and Molecular Biology, University College London, 20 Gordon Street, London, WC1H 0AJ, UK.

Controlling the functional dynamics of DNA within living cells is essential in biomedical research. Epigenetic modifications such as DNA methylation play a key role in this endeavour. DNA methylation can be controlled by genetic means. Yet there are few chemical tools available for the spatial and temporal modulation of this modification. Herein, we present a small-molecule approach to modulate DNA methylation with light. The strategy uses a photo-tuneable version of a clinically used drug (5-aza-2'-deoxycytidine) to alter the catalytic activity of DNA methyltransferases, the enzymes that methylate DNA. After uptake by cells, the photo-regulated molecule can be light-controlled to reduce genome-wide DNA methylation levels in proliferating cells. The chemical tool complements genetic, biochemical, and pharmacological approaches to study the role of DNA methylation in biology and medicine.
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http://dx.doi.org/10.1002/anie.201901139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7027477PMC
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

Structure of a (3+1) hybrid G-quadruplex in the PARP1 promoter.

Nucleic Acids Res 2019 02;47(3):1564-1572

School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore 637371, Singapore.

Poly (ADP-ribose) polymerase 1 (PARP1) has emerged as an attractive target for cancer therapy due to its key role in DNA repair processes. Inhibition of PARP1 in BRCA-mutated cancers has been observed to be clinically beneficial. Recent genome-mapping experiments have identified a non-canonical G-quadruplex-forming sequence containing bulges within the PARP1 promoter. Structural features, like bulges, provide opportunities for selective chemical targeting of the non-canonical G-quadruplex structure within the PARP1 promoter, which could serve as an alternative therapeutic approach for the regulation of PARP1 expression. Here we report the G-quadruplex structure formed by a 23-nucleotide G-rich sequence in the PARP1 promoter. Our study revealed a three-layered intramolecular (3+1) hybrid G-quadruplex scaffold, in which three strands are oriented in one direction and the fourth in the opposite direction. This structure exhibits unique structural features such as an adenine bulge and a G·G·T base triple capping structure formed between the central edgewise loop, propeller loop and 5' flanking terminal. Given the highly important role of PARP1 in DNA repair and cancer intervention, this structure presents an attractive opportunity to explore the therapeutic potential of PARP1 inhibition via G-quadruplex DNA targeting.
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http://dx.doi.org/10.1093/nar/gky1179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379715PMC
February 2019

5-Formylcytosine organizes nucleosomes and forms Schiff base interactions with histones in mouse embryonic stem cells.

Nat Chem 2018 12 22;10(12):1258-1266. Epub 2018 Oct 22.

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

Nucleosomes are the basic unit of chromatin that help the packaging of genetic material while controlling access to the genetic information. The underlying DNA sequence, together with transcription-associated proteins and chromatin remodelling complexes, are important factors that influence the organization of nucleosomes. Here, we show that the naturally occurring DNA modification, 5-formylcytosine (5fC) is linked to tissue-specific nucleosome organization. Our study reveals that 5fC is associated with increased nucleosome occupancy in vitro and in vivo. We demonstrate that 5fC-associated nucleosomes at enhancers in the mammalian hindbrain and heart are linked to elevated gene expression. Our study also reveals the formation of a reversible-covalent Schiff base linkage between lysines of histone proteins and 5fC within nucleosomes in a cellular environment. We define their specific genomic loci in mouse embryonic stem cells and look into the biological consequences of these DNA-histone Schiff base sites. Collectively, our findings show that 5fC is a determinant of nucleosome organization and plays a role in establishing distinct regulatory regions that control transcription.
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http://dx.doi.org/10.1038/s41557-018-0149-xDOI Listing
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