In vivo genome-wide profiling reveals a tissue-specific role for 5-formylcytosine
- Mario Iurlaro†1View ORCID ID profile,
- Gordon R. McInroy†2,
- Heather E. Burgess1,
- Wendy Dean1,
- Eun-Ang Raiber3,
- Martin Bachman2, 3, 4,
- Dario Beraldi2,
- Shankar Balasubramanian2, 3, 5Email author and
- Wolf Reik1, 6, 7Email author
© The Author(s). 2016
Received: 4 March 2016
Accepted: 6 June 2016
Published: 29 June 2016
Genome-wide methylation of cytosine can be modulated in the presence of TET and thymine DNA glycosylase (TDG) enzymes. TET is able to oxidise 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). TDG can excise the oxidative products 5fC and 5caC, initiating base excision repair. These modified bases are stable and detectable in the genome, suggesting that they could have epigenetic functions in their own right. However, functional investigation of the genome-wide distribution of 5fC has been restricted to cell culture-based systems, while its in vivo profile remains unknown.
Here, we describe the first analysis of the in vivo genome-wide profile of 5fC across a range of tissues from both wild-type and Tdg-deficient E11.5 mouse embryos. Changes in the formylation profile of cytosine upon depletion of TDG suggest TET/TDG-mediated active demethylation occurs preferentially at intron-exon boundaries and reveals a major role for TDG in shaping 5fC distribution at CpG islands. Moreover, we find that active enhancer regions specifically exhibit high levels of 5fC, resulting in characteristic tissue-diagnostic patterns, which suggest a role in embryonic development.
The tissue-specific distribution of 5fC can be regulated by the collective contribution of TET-mediated oxidation and excision by TDG. The in vivo profile of 5fC during embryonic development resembles that of embryonic stem cells, sharing key features including enrichment of 5fC in enhancer and intragenic regions. Additionally, by investigating mouse embryo 5fC profiles in a tissue-specific manner, we identify targeted enrichment at active enhancers involved in tissue development.
Active DNA demethylation in mammals can be achieved via iterative oxidation of 5-methylcytosine (5mC) by one or more of the TET enzymes to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) [1–5]. 5fC and 5caC are recognised and excised by the thymine DNA glycosylase (TDG) enzyme, initiating base excision repair (BER). Alternatively, 5fC may be lost passively during DNA replication [5, 6]. Following the initial identification of 5fC as an oxidative product of 5mC, 5fC has been detected in a variety of tissues and cell systems, albeit at relatively low levels compared to 5mC and 5hmC [4, 7]. Recently we demonstrated that 5fC is not only detectable but also stable in vivo , lending credence to a biological role for 5fC beyond that of a demethylation intermediate. Indeed, several proteins have been found to specifically bind 5fC in mouse embryonic stem cells (mESCs) and neural precursor cells (NPCs) with putative roles in transcription, chromatin regulation and DNA repair [9, 10]. The mechanisms by which 5fC affects these processes are not known, but it has recently been proposed that changes in the physical properties of the DNA double-stranded helix may be involved .
In the past three years, a number of different methods have been described for the mapping of 5fC in the genome of mESCs. These methods fall broadly into two categories: (1) affinity enrichment methods relying on antibody or chemical pull-downs [12–14]; and (2) base-resolution methods based on modifications to the classical bisulphite sequencing (BS-seq) technique [13, 15–17]. Recently two bisulphite-free base-resolution methods have been described that employ either the change in base-pairing properties (C to T) upon chemical modification  or the use of a specific restriction enzyme followed by an enrichment step . Despite the methodological differences between these techniques, a consistent picture of the distribution of 5fC in mESCs is emerging. 5fC is enriched at enhancers, repetitive elements and exons, but how this relates to its distribution in tissues of a whole organism is currently unexplored. Considering the dynamic nature of the modification and its enrichment at regulatory regions, this becomes extremely relevant especially in the context of development, where epigenetic modifications are subject to changes.
Here we present the first in vivo genome-wide profile of 5fC, in tissues of the early mouse embryo. In particular, we describe the distribution of 5fC in the hindbrain, heart, liver and carcass of embryonic day (E) 11.5 wild-type (WT) and Tdg null embryos. The use of Tdg null embryos enables the identification of sites marked by active demethylation driven by TET/TDG activity in these tissues, highlighting a potential role for 5fC in tissue development and differentiation.
Results and discussion
Genomic levels of 5fC are heterogeneous in mid-gestation staged embryos
Genome-wide profiling of 5fC with a traceless chemical probe
In order to better understand the biological role of 5fC during embryonic development we generated genome-wide profiles for 5fC in dissected tissues from E11.5 WT and Tdg null embryos. To this end, we developed an optimised protocol based on a previously reported 5fC-pull-down method , additionally employing a cleavable aldehyde-reactive biotinylation probe as previously described  (Additional file 1: Figure S2A). The hydroxylamine moiety exploits the unique reactivity of 5fC among the other DNA bases to install a biotin tag, enabling subsequent enrichment with streptavidin coated magnetic beads. The presence of a chemical cleavage site (Additional file 1: Figure S2B) enables removal of the biotin tag following enrichment, which alleviates PCR biases introduced by polymerase stalling at modified sites . This new probe and methodology resulted in substantially increased enrichment efficiencies over 30-fold higher than the previously reported method. Through simultaneously obtaining an enhanced signal/noise ratio and increasing resolution, we were able to perform sensitive and robust profiling of 5fC from relatively small amounts of embryonic-tissue-derived genomic DNA (Additional file 1: Figure S2C). We used this capture method to systematically perform genome-wide profiling of 5fC in the hindbrain, heart, liver and carcass of E11.5 embryos. At least two biological replicates were performed for each tissue and WT and Tdg null samples were paired from the same litter to account for litter-induced variability. A full list of replicates is available in Additional file 1: Figure S3A. In accordance with the data obtained by LC/MS, Tdg null embryos show substantially higher levels of 5fC in all tissues analysed, reflected in the higher signal and increased number of 5fC peaks in all Tdg null samples compared to their WT counterparts. Biological replicates for the same tissues from different embryos showed an extremely high degree of similarity, suggesting a tight regulation of TET-driven formation and TDG-mediated excision of 5fC during development. Unbiased hierarchical clustering of all individual replicates accurately separates all samples derived from WT and Tdg null embryos, while simultaneously pairing replicates from the same tissues (Additional file 1: Figure S3B). One replicate for the Tdg null liver was of poor quality and therefore was excluded from the following analyses.
Finally, we observed a substantial enrichment for 5fC over exon/intron boundaries in both WT and knockout tissues (Fig. 2e, f and Additional file 1: Figure S6). This has also been observed for 5hmC and 5fC in embryonic stem cells and suggests a role for oxidised cytosine derivatives in regulating splicing [14, 25]. The presence of 5fC at these sites is particularly interesting, as 5fC has been found to reduce the rate and the fidelity of transcription by RNA polymerase II .
5fC is enriched at active enhancers in the mouse embryo
We next asked whether 5fC enrichment was specific for regions marked by activating histone modifications. To this end, we analysed the distribution of 5fC peaks in the hindbrain at regions marked by tri-methylation of histone H3 lysine 36 (H3K36me3), which normally labels transcriptional elongation and hence gene bodies, or by tri-methylation of lysine 27 (H3K27me3), a repressive mark deposited by the PRC2 complex. Notably, 5fC shows very little enrichment over these regions (Fig. 3c). Taken together, these data show a strong and specific enrichment of 5fC at active enhancer regions during embryonic development. This constitutes a significant difference in the profile of 5fC between mESCs and embryonic tissues and could reflect a different need for dynamic regulation in developing embryos compared to the stem cell state.
Tissue-specific cytosine formylation at developmental enhancers
Finally, we investigated the relationship between tissue-specific 5fC formation and gene function. To this end, we subdivided 5fC peaks in the individual tissues from Tdg null embryos into shared and tissue-specific peaks. Notably, heart-specific 5fC peaks were enriched in the proximity of genes involved in heart development, whereas hindbrain-specific peaks were enriched proximal to genes involved in neuron development (Fig. 4b and example in Additional file 1: Figure S8). Interestingly we could not find a similar enrichment for liver-specific 5fC peaks, which may be due to the mixed endoderm/mesoderm lineage of this tissue . Taken together, these results clearly indicate the presence of a tissue-specific role for 5fC formation at the level of genes involved in the development of the cognate tissues.
We have generated the first in vivo genome-wide profile for 5fC in tissues obtained from WT and Tdg null E11.5 mouse embryos; namely from dissected heart, hindbrain and liver tissues and the carcass. While showing an overall similar profile to the ones reported in mESCs, with enrichment at enhancers and exons, we also find that 5fC is specifically enriched at active enhancers in the developing embryo. Moreover, we report clear tissue specificity behind the distribution of 5fC in developmentally related enhancers. This highlights a complex regulation of 5fC, balancing of TET oxidation and TDG excision, and thus suggesting a key role for dynamic demethylation at those sites. The relationship between this process and the striking phenotype displayed by Tdg-deficient embryos warrants future investigation.
Knockout mouse and tissue dissection
TDG KO/WT forward: 5′-GGGCAGCAAGGATCTGTCTA-3′
TDG KO/WT reverse: 5′-GCCAGTTCCTCTGACACTTAGC-3′
Mice were maintained as heterozygotes and intercrossed to generate homozygous knockout embryos for analysis. Tissues were collected from timed matings with the day of the copulation plug as E0.5. Embryos were dissected into hindbrain, heart, liver and carcass.
DNA and RNA extraction
Embryonic tissues were lysed in RLT plus buffer (Qiagen), homogenised via centrifugation through a QIAshreddred column (Qiagen) at full speed for 2 min in a microcentrifuge. DNA and RNA were isolated using AllPrep DNA/RNA kit (Qiagen) following the manufacturer’s instructions.
Mass spectrometry of DNA
A total of 100–300 ng of genomic DNA was treated with 5 U of DNA Degradase Plus (Zymo Research) for 3 h and analysed on a nanoHPLC combined with a Q Exactive mass spectrometer, as previously described .
Genomic DNA (500 ng in 10 mM Tris–HCl pH 8, 1 mM EDTA) was sonicated to an average of 250 bp with the Covaris M220 Focused-ultrasonicator. Synthetic spike-in controls were added after sonication to monitor the enrichment efficiency (full details on the spike-in oligos and conditions used are in Additional file 3). The fragmented DNA was incubated with a cleavable aldehyde reactive biotinylation probe  (400 μM), p-anisidine (100 mM) and NH4OAc (40 mM, adjusted to pH 5 with AcOH) in a final volume of 100 μL at 37 °C with a 65 °C heated lid for 24 h. The DNA was purified on silica-based spin columns (GeneJET PCR Purification Kit), with the inclusion of an additional wash step, before standard library preparation.
Streptavidin paramagnetic particles (50 μL, Promega Magnesphere PMPs) were washed with 1× binding buffer (5 mM Tris-HCl pH 7.2, 0.5 mM EDTA, 1 M NaCl, 0.05 % Tween-20) (four times 500 μL) and resuspended in 2× binding buffer (50 μL). The washed beads (50 μL), dIdC (2 μL of 1 μg/μL), spike-in control sequences (1 pg positive control; 10 pg negative control), DNA library and water were combined in a 1.5 mL low binding tube (Eppendorf DNA LoBind) to give 100 μL. The mixture was incubated at room temperature for 20 min, before the tube was placed on a magnetic rack and left for 5 min while the magnetic beads formed a pellet. Once the solution was clear the supernatant was discarded, and 400 μL 1× binding buffer was added to the tube. The beads were washed by rapidly rotating the tube 180° and allowing the beads to recollect against the magnet; this washing procedure was repeated four times. The beads were resuspended in 400 μL 1× binding buffer with vortexing and transferred to a new low binding tube before the washing procedure was repeated another four times. A final tube transfer and washing cycle was performed, giving a total of two tube changes and 12 washes. A final wash with Tris–HCl (10 mM) was performed, with the beads remaining on the magnet at all times due to poor pellet formation in this buffer.
The DNA was eluted from the beads by chemical cleavage of the probe with 100 μL of tris(2-carboxyethyl)phosphine (100 mM) and Tris–HCl (200 mM) shaken at 65 °C for 15 min. NaOAc (40 μL of 5 M, pH 5), ethanol (1 mL) and water (100 μL) were added to the isolated supernatant, and the mixture held at −80 °C for 45 min. The precipitated DNA was pelleted by centrifugation at 16,000 rcf for 45 min, washed with 70 % EtOH and centrifuged again at 16,000 rcf for 30 min. The pellet was air dried and dissolved in water (20 μL). At this point the enrichment efficiency was examined by qPCR of the control sequences spiked in before pull-down. One microliter of enriched DNA was diluted tenfold, from which 1 μL aliquots were amplified with primer sets for the positive and negative controls. Comparison to a non-enriched input gave the enrichment factor (Additional file 1: Figure S9 and Additional file 3). Although the enrichment was extremely high (~54,000-fold), there will be a proportion of false-positive regions arising from the excess of non-formylated DNA in genomic context.
Library preparation and sequencing
Sequencing libraries were generated using the NEBNext library preparation modules. Fourteen cycles of PCR amplification were performed with the Illumina PCR Master Mix and PCR Primer Cocktail. Prior to sequencing, libraries were quantified by qPCR (KAPA Biosystems) and their fragment size profile analysed by TapeStation (Agilent). Sequencing was performed on an Illumina Nextseq 500, run in paired-end mode with 100 read cycles.
Raw sequencing files and peak files can be found in the GEO under accession number GSE77447. Data were adapter and quality trimmed using trim_galore v0.4.1 (bioinformatics.babraham.ac.uk/projects/trim_galore/) using default settings. Trimmed sequencing reads were aligned to mouse genome assembly NCBIM37 (mm9) using Bowtie2 v2.2.6 using default parameters  and was converted to BAM format using samtools v0.1.18 . Peaks were called in the SeqMonk software (bioinformatics.babraham.ac.uk/projects/seqmonk/) using the MACS peak caller , with a p value cutoff of 1 × 10−5 and a fragment size of 200 bp. Data manipulations were facilitated by BEDTools  and data analysis was performed in Seqmonk or in R version 3.1.2 .
Functional annotation enrichment analyses were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 .
5caC, 5-carboxylcytosine; 5fC, 5-formylcytosine; 5hmC, 5-hydroxymethylcytosine; 5mC, 5-methylcytosine; BER, base excision repair; BS-seq, bisulphite sequencing; CGI, CpG island; GAT, genome association tester; LC/MS, liquid chromatography/mass spectrometry; mESC, mouse embryonic stem cells; NPC, neural precursor cells; TDG, thymine DNA glycosylase; WT, wild type.
We would like to thank Simon Andrews for his bioinformatics support, Primo Schär for the Tdg KO mice and David Oxley and Judith Webster for their help with mass spectrometry.
MI is supported by the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007-2013/under REA grant agreement no. 290123. GRM was supported by Trinity College and Herchel Smith studentships. MB was supported by the CRUK PhD Training Programme in Chemical Biology and Molecular Medicine. DB is supported by funding from the Wellcome Trust and Herchel Smith. The WR lab is supported by BBSRC, MRC, the Wellcome Trust, EU EpiGeneSys and BLUEPRINT. The SB lab is supported by core funding from Cancer Research UK and a Wellcome Trust Senior Investigator Award.
Availability of data and materials
The data discussed in this publication are accessible through GEO Series accession number GSE77447 (https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/geo/query/acc.cgi?acc=GSE77447).
MI and GRM conceived the study, performed the experiments, analysed the data and wrote the manuscript. HB and MB generated mass spectrometry data. WD performed tissue dissections. EAR and HB conceived the study and analysed the data. DB performed bioinformatics analysis. WD, SB and WR conceived the study and provided supervision. All authors have interpreted the data, read and approved the manuscript.
SB and WR are advisors and shareholders of Cambridge Epigenetix, Ltd.
Ethics approval and consent to participate
All experimental procedures were approved by the Animal Welfare, Experimentation and Ethics Committee at the Babraham Institute and were performed under licenses by the Home Office (UK) in accordance with the Animals (Scientific Procedures) Act 1986.
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