Genome-wide mapping of 5-hydroxymethyluracil in the eukaryote parasite Leishmania
© The Author(s). 2017
Received: 25 November 2016
Accepted: 10 January 2017
Published: 30 January 2017
5-Hydroxymethyluracil (5hmU) is a thymine base modification found in the genomes of a diverse range of organisms. To explore the functional importance of 5hmU, we develop a method for the genome-wide mapping of 5hmU-modified loci based on a chemical tagging strategy for the hydroxymethyl group.
We apply the method to generate genome-wide maps of 5hmU in the parasitic protozoan Leishmania sp. In this genus, another thymine modification, 5-(β-glucopyranosyl) hydroxymethyluracil (base J), plays a key role during transcription. To elucidate the relationship between 5hmU and base J, we also map base J loci by introducing a chemical tagging strategy for the glucopyranoside residue.
Observed 5hmU peaks are highly consistent among technical replicates, confirming the robustness of the method. 5hmU is enriched in strand switch regions, telomeric regions, and intergenic regions. Over 90% of 5hmU-enriched loci overlapped with base J-enriched loci, which occurs mostly within strand switch regions. We also identify loci comprising 5hmU but not base J, which are enriched with motifs consisting of a stretch of thymine bases.
By chemically detecting 5hmU we present a method to provide a genome-wide map of this modification, which will help address the emerging interest in the role of 5hmU. This method will also be applicable to other organisms bearing 5hmU.
Keywords5-Hydroxymethyluracil (5hmU) 5-Formyluracil (5fU) Base J Genome-wide mapping Leishmania major Leishmania donovani
Natural chemical modifications to DNA nucleobases have the potential to profoundly influence biology. A number of modified bases have been identified in the genomes of a variety of organisms , such as derivatives of cytosine found in the genomes of mammals . 5-Methylcytosine (5mC) and its oxidised derivatives 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC) have potential roles in gene regulation and epigenetics programming, which are fundamental in biology . Yet, the details of thymine modifications and their roles in nature are still largely elusive. 5-Hydroxymethyluracil (5hmU) is a thymine base modification found in the genomic DNA of diverse organisms ranging from bacteriophages to mammals . While the replicative incorporation of 5hmU is known in bacteriophage genomes [4, 5], the existence of enzyme-mediated pathways to form 5hmU in eukaryotic genomes suggests that this modification has functional importance . The post-replicative formation of 5hmU occurs via hydroxylation of thymine, which can be mediated by the ten-eleven translocation (TET) enzymes and J-binding protein (JBP) family proteins in mammalian and protozoan genomes, respectively [6, 7]. 5hmU has also been reported to affect protein-binding to DNA  and may also be a key intermediate in the generation of site-specific mutations, as it can be excised by DNA glycosylases to create potentially mutagenic abasic sites . As an important step towards understanding the functional importance of 5hmU, we have explored ways to detect and map 5hmU in genomic DNA. A method to map 5hmU:G base mispairs has been reported but will not be applicable to map 5hmU derived from thymine.  Although the single molecule, real-time (SMRT) sequencing approach could be an option , its application to map 5hmU in the actual genomic DNA has not been reported, possibly due to the relatively weak signals derived from 5hmU in SMRT sequencing  compared to the strong signature signals of nucleobases which have been sequenced by the approach successfully (e.g., 4mC, 6mA, and β-glucosylated modified bases) [12–15]. Here we report the first genome-wide map of 5hmU, which we determined for the parasitic protozoan Leishmania sp. using chemical manipulation of 5hmU and next-generation sequencing (NGS) . In Leishmania sp., 5hmU is a key post-replication nucleobase modification that can be further modified enzymatically to form 5-(β-glucopyranosyl) hydroxymethyluracil (base J). mRNA genes in Leishmania sp. are transcribed as large continuous polycistronic gene clusters starting at small numbers of transcription initiation sites, accompanied by simultaneous post-transcriptional processing to produce mature mRNAs [17, 18]. Base J has been reported to play a major role in polycistronic transcription termination and has also been reported to localise at transcription initiation sites marked by histone acetylation, though any individual role of 5hmU has yet to be determined [19–21]. In this study, by individually mapping 5hmU and base J, we demonstrate genomic loci that are enriched exclusively with 5hmU and discuss potential unique consequences of 5hmU at these loci.
We also assessed the reactivity of other aldehyde-bearing components that could potentially exist in genomic DNA, in particular abasic sites (APs) and 5fC, the latter of which can also derive from 5hmC during KRuO4-mediated oxidation. In the biotinylation condition we employed, only trace biotinylation of an AP-bearing ODN occurs whereas complete biotinylation of ODN-5fU was observed. Furthermore, the ODN was fragmented at the abasic site in the basic DNA denaturing condition followed by oxidization of 5hmU (Additional file 1: Figure S1b), so DNA fragments with abasic sites will not be subjected to sequencing since adapter sequences at both ends of the fragment are absolutely required. Taken together with the observation that AP-bearing DNA is also subjected to polymerase stalling during PCR (i.e., no amplification),  the effect of abasic sites in 5hmU enrichment would be negligible in the final sequenced data. Our DNA elution method would also reduce any background due to 5fC (which was absent in the Leishmania DNA we used in this study but present in mammalian genomes), as the biotin tag coupled to 5fU was removed faster and more selectively compared to the corresponding 5fC derivative in the optimum conditions (Additional file 1: Figure S1c). We then applied the enrichment chemistry to NGS library preparation (Step 3 in Fig. 1b). We also carried out the following controls in parallel (Additional file 1: Table S3): i) libraries prepared without the biotinylation and enrichment processes (i.e., “input” controls); ii) libraries prepared with the chemical pull-down without prior oxidation to control for peaks derived from any pre-existing modifications that react with the biotinylation probe as well as any biases caused by the enrichment process (i.e., “no-oxidation” controls).
In the currently suggested mechanism of thymine modifications in trypanosomatids, the initial step of 5hmU formation is binding of the JBP family of proteins to base J, followed by hydroxymethylation of proximal thymine [25, 29]. The presence of base J-specific loci may suggest that any 5hmU formed is quickly enzymatically glycosylated to form base J in the loci. Although 5hmU-specific loci may have arisen from DNA oxidative damage regularly occurring in the genome, it is also possible that these loci form naturally via a JBP-independent mechanism. While previous studies have proposed that base J-associated glucosyltransferase in trypanosomatids readily converts 5hmU to base J promiscuously without sequence biases [15, 25, 30], the presence of 5hmU-specific loci suggests that some 5hmUs may exist in sequence contexts that are less preferred by this glucosyltransferase and are only slowly converted to base J. To assess the potential artifacts and biases caused by 5hmU chemical enrichment, especially due to spontaneous oxidation of T during library preparation, we carried out mock chemical 5hmU enrichment using a randomly sequenced ODN bearing no nucleoside modification, which will mimic the background signal. We did not observe any significant change in the nucleoside composition and enrichment of specific motifs, suggesting chemical 5hmU enrichment does not cause artifacts and biases (Additional file 1: Figure S5), which supports our interpretation of 5hmU-specific loci.
We provide the first genome-wide map of 5hmU in Leishmania sp. genomes and investigated the relationship of 5hmU with the hypermodified base J. To achieve this goal, we established methods to selectively tag each modified derivative of the thymine base. The observed accumulation of 5hmU in T(A) rich sequence contexts, distinct from the sequence context of base J loci, suggests 5hmU remains intact or is less reactive to glucosylation in these sequence contexts. The methods presented herein provide powerful tools to help address the roles of modified T nucleobases, which are of emerging interest due to their relevance in biological function and disease [31–33].
The genomic DNA samples for L. major ATCC 30012D (lot 60685413, lot 63717803) as well as a set of genomic DNA and RNA from the same culture (lot ATCC-CUST-30012D/R) were obtained from ATCC. The genomic DNA sample for L. donovani GR383WT1 (single batch) was provided courtesy of Prof. Charles L. Jaffe (National Center for Leishmaniasis, Kuvin Centre for Study of Tropical & Infectious Diseases, Jerusalem). Detailed information of samples used to generate each sequencing result is available in Additional file 1: Table S3.
The modified adapter solution
A 5′-O-methylated ODN with a sequence of 5′-MeO-GAATGATACGGCGACCACCGAGATCTA CACTCTTTCCCTACACGACGCTCTTCCGATCT-3′ (Eurogentec) and a 3′-O-phosphorylated ODN with a sequence of 5′-GATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCT TCTGCTTG-O-phosphate-3′ (Sigma Aldrich), where NNNNNN corresponds to TruSeq (Illumina) barcoding, were made up to a solution (25 μM each) in 50 mM NaCl and 20 mM Tris-HCl (pH 7) and annealed prior to use.
Adapter ligation for chemical 5hmU enrichment
Fragmented DNA samples (200 ng) with an average size of 200 bp and spike-in control ODNs (100 pg each; see Additional file 1: Table S2) were mixed and subjected to adapter ligation using the standard protocol using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (New England Biolabs), except the use of the modified adapter solution (2.5 μL) instead of a standard adapter solution. Adapter-ligated DNA samples were bound to AMPure® XP Beads (Beckman Coulter), washed two times with 80% aqueous acetonitrile (1.0 mL), eluted with 25 μL of water, then passed through Micro Bio-Spin™ P-6 Gel Columns in SSC Buffer (Bio-Rad) pre-washed five times with ultrapure water (0.5 mL). It was essential to avoid use of ethanol during the DNA sample purification.
Chemical modifications of 5hmU
The adapter ligated DNA sample in 50 mM NaOH (24 μL) was denatured by incubating at 37 °C for 30 min. A ten-fold diluted oxidant solution (1 μL) provided in the TrueMethyl™ kit (Cambridge Epigenetics) was added to the DNA solution and the resultant mixture was incubated at 40 °C for 30 min, followed by immediate purification by Micro Bio-Spin™ P-6 Gel Columns (Bio-Rad). The resultant DNA sample (in ca. 25 μL solution), 100 mM phosphate buffer (pH 6, 10 μL), and 17 mM (+)-biotinamidohexanoic acid hydrazide (15 μL) were mixed and incubated at 40 °C for 3–12 h. The reaction mixture was purified by Micro Bio-Spin™ P-6 Gel Columns (Bio-Rad) and used immediately for enrichment.
Chemical 5hmU enrichment sequencing
The biotinylated DNA sample (in ca. 50 μL solution) and the binding buffer 1 (2×, 50 μL; see Additional file 1 for the buffer composition) were mixed and added to Dynabeads® MyOne™ Streptavidin C1 (Life Technologies) which had been pre-washed two times with the binding buffer 1 (200 μL). The mixture was gently mixed by rotation at 4 °C for 30 min, placed on a magnetic rack, and the supernatant was removed. The beads were washed five times with the binding buffer 1 (1×, 100 μL). The DNA was eluted by incubating the beads in elution buffer (1×, 50 μL; see Additional file 1 for the buffer composition) at 40 °C with vigorous shaking (1400 rpm) for 30 min. The mixture was then placed on a magnetic rack and the supernatant containing the eluted DNA was collected. The elution process was repeated once more. The combined eluted DNA solutions were purified using the GeneJET PCR purification kit (Thermo Scientific). The obtained 5hmU enriched libraries were amplified by PCR Master Mix and PCR Primer Cocktail provided in the TruSeq DNA Sample Preparation Kit v3 (Illumina) and quantified using a KAPA Library Quantification Kit (KAPA Biosystems). Experiments were carried out in technical triplicates for L. major and technical duplicates for L. donovani. The efficacy of 5hmU enrichment was confirmed by read counts for 5hmU-modified spike-in controls over non-modified controls (Additional file 1: Table S3).
Chemical base J enrichment sequencing
A solution of the fragmented DNA samples (250 ng) with an average length of 200 bp, 50 mM NaIO4 in 50 mM sodium acetate buffer (pH 5.5) was incubated at 40 °C for 3 h followed by work-up with Micro Bio-Spin™ P-6 Gel Columns (Bio Rad) or a GeneJET PCR purification kit (Thermo Scientific). The resultant DNA solution (25 μL), 100 mM phosphate buffer (pH 6, 20 μL), 100 mM (+)-biotinamidohexanoic acid hydrazide (4 μL), and 100 mM p-anisidine (1 μL) were mixed and incubated at 40 °C for 4–12 h. The reaction mixture was purified by Micro Bio-Spin™ P-6 Gel Columns (Bio Rad) or a GeneJET PCR purification kit (Thermo Scientific). The samples were then subjected to NGS adapter ligation using reagents provided in the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (New England Biolabs) and adapter solutions provided in the TruSeq DNA Sample Preparation Kit v3 (Illumina). Downstream procedures for base J enrichment were carried out as described in the chemical 5hmU pull-down sequencing procedure except for the use of KAPA HiFi HotStart Uracil + ReadyMix (KAPA Biosystems) as a PCR master mix.
Sequence alignment and peak calling
5hmU and base J enriched libraries as well as control libraries were sequenced on an Illumina MiSeq with a read length of 151 bp. Reads were trimmed to remove adapters and low quality 3′ ends using cutadapt version 1.7 . Trimmed reads were then aligned to the reference genome using bwa mem version 0.7.10 . Only primary alignments with a mapping quality of 10 or more were retained for further analyses. Alignment filtering and manipulations were performed with samtools version 1.1 . See Additional file 1: Table S3 for a summary of the mapping step. Peaks of read enrichment were detected using macs  version 220.127.116.1140616. Further analyses were performed by means of bedtools , deepTools , DREME , and custom bash, R, and python scripts.
- base J:
Liquid chromatography–mass spectrometry
Strand switch region
The genomic DNA sample for Leishmania donovani GR383WT1 was provided courtesy of Prof. Charles L. Jaffe (National Center for Leishmaniasis, Kuvin Centre for Study of Tropical & Infectious Diseases, Jerusalem). We thank Prof. Matt Berriman (Wellcome Trust Sanger Institute, Cambridge) for useful discussions.
FK is supported by the Wellcome Trust, DB is supported by the Herchel Smith Fund, REH is supported by the University of Cambridge and the Herchel Smith Fund, GRM was supported by Trinity College and the Herchel Smith fund, PVD is supported by Marie Curie fellowship and the Wellcome Trust. The Balasubramanian group is core-funded by a Wellcome Trust Senior Investigator Award (099232/Z/12/Z) and Cancer Research UK(C14303/A17197).
Availability of data and materials
The datasets (fastq files and peak files for each library) supporting the conclusions of this article are available in NCBI’s Gene Expression Omnibus, using GEO series accession GSE83384 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE83384). Detailed protocols for bioinformatics analysis, including statistical analysis, are available from the GitHub repository (https://github.com/sblab-bioinformatics/mapping-5hmU-in-Leishmania).
FK designed and performed the study; DB performed data analysis; GRM assisted the sequencing study; REH assisted the model study and synthesized stable isotope labelled mononucleoside standards; PVD and REH performed the LC-MS/MS2 study. SB had input in study design, data interpretation, and writing the paper. All authors verified the data and contributed to writing the final manuscript. All authors read and approved the final manuscript.
SB is a founder, shareholder, and director of Cambridge Epigenetix Ltd.
Ethics approval and consent to participate
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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