Identification and annotation of conserved promoters and macrophage-expressed genes in the pig genome.

BACKGROUND: The FANTOM5 consortium used Cap Analysis of Gene Expression (CAGE) tag sequencing to produce a comprehensive atlas of promoters and enhancers within the human and mouse genomes. We reasoned that the mapping of these regulatory elements to the pig genome could provide useful annotation and evidence to support assignment of orthology. RESULTS: For human transcription start sites (TSS) associated with annotated human-mouse orthologs, 17% mapped to the pig genome but not to the mouse, 10% mapped only to the mouse, and 55% mapped to both pig and mouse. Around 17% did not map to either species. The mapping percentages were lower where there was not clear orthology relationship, but in every case, mapping to pig was greater than to mouse, and the degree of homology was also greater. Combined mapping of mouse and human CAGE-defined promoters identified at least one putative conserved TSS for >16,000 protein-coding genes. About 54% of the predicted locations of regulatory elements in the pig genome were supported by CAGE and/or RNA-Seq analysis from pig macrophages. CONCLUSIONS: Comparative mapping of promoters and enhancers from humans and mice can provide useful preliminary annotation of other animal genomes. The data also confirm extensive gain and loss of regulatory elements between species, and the likelihood that pigs provide a better model than mice for human gene regulation and function

Accurate measurement of 5-methylcytosine and 5-hydroxymethylcytosine in human cerebellum DNA by oxidative bisulfite on an array (OxBS-array).

The Infinium 450K Methylation array is an established tool for measuring methylation. However, the bisulfite (BS) reaction commonly used with the 450K array cannot distinguish between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). The oxidative-bisulfite assay disambiguates 5mC and 5hmC. We describe the use of oxBS in conjunction with the 450K array (oxBS-array) to analyse 5hmC/5mC in cerebellum DNA. The "methylation" level derived by the BS reaction is the combined level of 5mC and 5hmC at a given base, while the oxBS reaction gives the level of 5mC alone. The level of 5hmC is derived by subtracting the oxBS level from the BS level. Here we present an analysis method that distinguishes genuine positive levels of 5hmC at levels as low as 3%. We performed four replicates of the same sample of cerebellum and found a high level of reproducibility (average r for BS = 98.3, and average r for oxBS = 96.8). In total, 114,734 probes showed a significant positive measurement for 5hmC. The range at which we were able to distinguish 5hmC occupancy was between 3% and 42%. In order to investigate the effects of multiple replicates on 5hmC detection we also simulated fewer replicates and found that decreasing the number of replicates to two reduced the number of positive probes identified by > 50%. We validated our results using qPCR in conjunction with glucosylation of 5hmC sites followed by MspI digestion and we found good concordance with the array estimates (r = 0.94). This experiment provides a map of 5hmC in the cerebellum and a robust dataset for use as a standard in future 5hmC analyses. We also provide a novel method for validating the presence of 5hmC at low levels, and highlight some of the pitfalls associated with measuring 5hmC and 5mC.S. Balasubramanian is a Senior Investigator of The Wellcome Trust and the Balasubramanian group is core-funded by Cancer Research UK. We would like to thank Tobias Ost and Christine Clark of Cambridge Epigenetix Ltd. for valuable discussions and development of the method.This article was originally published in PLOS ONE (Field SF, Beraldi D, Bachman M, Stewart SK, Beck S, Balasubramanian S, PLoS ONE 2015, 10(2): e0118202. doi:10.1371/journal.pone.0118202

Genome-wide mapping of 5-hydroxymethyluracil in the eukaryote parasite Leishmania.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.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)

Enhanced Methylation Analysis by Recovery of Unsequenceable Fragments.

Bisulfite sequencing is a valuable tool for mapping the position of 5-methylcytosine in the genome at single base resolution. However, the associated chemical treatment causes strand scission, which depletes the number of sequenceable DNA fragments in a library and thus necessitates PCR amplification. The AT-rich nature of the library generated from bisulfite treatment adversely affects this amplification, resulting in the introduction of major biases that can confound methylation analysis. Here, we report a method that enables more accurate methylation analysis, by rebuilding bisulfite-damaged components of a DNA library. This recovery after bisulfite treatment (ReBuilT) approach enables PCR-free bisulfite sequencing from low nanogram quantities of genomic DNA. We apply the ReBuilT method for the first whole methylome analysis of the highly AT-rich genome of Plasmodium berghei. Side-by-side comparison to a commercial protocol involving amplification demonstrates a substantial improvement in uniformity of coverage and reduction of sequence context bias. Our method will be widely applicable for quantitative methylation analysis, even for technically challenging genomes, and where limited sample DNA is available.GRM is supported by funding from Trinity College Cambridge and Herchel Smith. DB is supported by funding from the Wellcome Trust and Herchel Smith. EAR is a Herchel Smith Fellow. PVD is a Marie Curie Fellow of the European Union (FP7-PEOPLE-2013-IEF/624885). The Balasubramanian lab is supported by a Senior Investigator Award from the Wellcome Trust (099232/Z/12/Z to SB) and by core funding from Cancer Research UK.This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pone.015232

Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication

Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genome-wide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal but in each case growth slows with time and leads to DNA damage and accumulation of cells with aberrant DNA content. Despite these similarities, we show that only loss of RAD51 or RAD51-3 impairs DNA synthesis and causes elevated levels of genome-wide mutation. Furthermore, we show that these two HR factors act in distinct ways, since ablation of RAD51, but not RAD51-3, has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote

Genome duplication in Leishmania major relies on persistent subtelomeric DNA replication

DNA replication is needed to duplicate a cell’s genome in S phase and segregate it during cell division. Previous work in Leishmania detected DNA replication initiation at just a single region in each chromosome, an organisation predicted to be insufficient for complete genome duplication within S phase. Here, we show that acetylated histone H3 (AcH3), base J and a kinetochore factor co-localise in each chromosome at only a single locus, which corresponds with previously mapped DNA replication initiation regions and is demarcated by localised G/T skew and G4 patterns. In addition, we describe previously undetected subtelomeric DNA replication in G2/M and G1-phase-enriched cells. Finally, we show that subtelomeric DNA replication, unlike chromosome-internal DNA replication, is sensitive to hydroxyurea and dependent on 9-1-1 activity. These findings indicate that Leishmania’s genome duplication programme employs subtelomeric DNA replication initiation, possibly extending beyond S phase, to support predominantly chromosome-internal DNA replication initiation within S phase

In vivo genome-wide profiling reveals a tissue-specific role for 5-formylcytosine.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.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.This is the final version of the article. It first appeared from BioMed Central via http://dx.doi.org/10.1186/s13059-016-1001-5

DNA G-quadruplex structures mould the DNA methylome

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.This work is supported by a core CRUK award (C14303/A17197). S.B. is a Senior Investigator of the Wellcome Trust (grant no. 099232/z/12/z). JS is a Marie Curie Fellow of the European Union (747297-QAPs-H2020-MSCA-IF-2016)

Genome-wide mapping of FOXM1 binding reveals co-binding with estrogen receptor alpha in breast cancer cells.

BACKGROUND: The forkhead transcription factor FOXM1 is a key regulator of the cell cycle. It is frequently over-expressed in cancer and is emerging as an important therapeutic target. In breast cancer FOXM1 expression is linked with estrogen receptor (ERα) activity and resistance to endocrine therapies, with high levels correlated with poor prognosis. However, the precise role of FOXM1 in ER positive breast cancer is not yet fully understood. RESULTS: The study utilizes chromatin immunoprecipitation followed by high-throughput sequencing to map FOXM1 binding in both ERα-positive and -negative breast cancer cell lines. The comparison between binding site distributions in the two cell lines uncovered a previously undescribed relationship between binding of FOXM1 and ERα. Further molecular analyses demonstrated that these two factors can bind simultaneously at genomic sites and furthermore that FOXM1 regulates the transcriptional activity of ERα via interaction with the coactivator CARM1. Inhibition of FOXM1 activity using the natural product thiostrepton revealed down-regulation of a set of FOXM1-regulated genes that are correlated with patient outcome in clinical breast cancer samples. CONCLUSIONS: These findings reveal a novel role for FOXM1 in ERα transcriptional activity in breast cancer and uncover a FOXM1-regulated gene signature associated with ER-positive breast cancer patient prognosis