biostudies-arrayexpressMarlon ZambranoMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-159615-Methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) are the two main epigenetic modifications of mammalian DNA, and play crucial roles in cell differentiation, development, and tumorigenesis. Both modifications co-exist with unmodified cytosine in palindromic CpG dyads in different strand-symmetric and -asymmetric combinations, each having unique regulatory potential. To facilitate investigating the individual functions of such dyad modifications, we here report HM-DyadCap. This method employs MECP2 HM – an evolved methyl-CpG-binding domain of the mC reader MECP2 – for the simple capture and sequencing of DNA fragments containing the CpG dyad hmC/mC that frequently occurs in mouse embryonic stem cell and brain genomes. In vitro binding studies reveal a high discrimination of MECP2 HM against diverse off-target dinucleotides in vitro. We conduct comparative mapping experiments for mESC genomes with HM-DyadCap, standard MethylCap based on wild type MECP2, as well as the antibody-based MeDIP and hMeDIP protocols. We find that MECP2 HM is blocked by hmC glucosylation, and conduct control enrichments with glucosylated genomes that indicate highly selective enrichment of hmC/mC dyads by MECP2 HM. Metagene profiles correlate hmC/mC with actively transcribed genes, and reveal global enrichment in gene bodies as well as depletion at transcription start sites. We anticipate that HM-DyadCap will enable effective mapping of hmC/mC with implications for biomarker discovery and unravelling the function of this dyad in diverse aspects of chromatin biology.biostudies-arrayexpressGrowth Protocol - E14tg2a mouse embryonic stem cells were grown in dishes pre-coated with 0.1 % gelatin (w/vol, Sigma) in GMEM supplemented with 10% fetal bovine serum, sodium pyruvate, 50 μM β-mercaptoethanol, glutamax, non-essential amino acids (all from Gibco/ThermoFisher) and 10 ng/ml murine leukemia inhibitory factor (LIF, Protein Expression Facility, MPI Dortmund).Sequencing - Illumina NovaSeq X - IlluminaLibrary Construction - For each immunoprecipitation, 1 µg of sheared gDNA was used as input. DIPs were performed according to the MagMeDIP-seq Package V2 protocol (Diagenode, C02010041), using either the provided anti-5mC antibody for MeDIP or 0.6 µg per IP of the mouse monoclonal anti-hmC antibody (Diagenode, C15200200) for hMeDIP. Additionally, a control IP with 0.6 µg of mouse IgG (Diagenode, C15400001) was included. Illumina libraries were prepared according to the Diagenode MeDIP-seq library preparation protocol. Final library concentrations were quantified using the Quantus Fluorometer (Promega) and fragment size distributions were assessed with a TapeStation (Agilent Technologies) using a D1000 ScreenTape. Libraries were sequenced on an Illumina NovaSeq X-25B instrument using paired-end 2x 150 bp reads with a target depth of 50 million reads per sample.Sample Collection - Cells were passaged every 2-3 days to maintain cultures between 10% and 90% confluency. For analysis, near-confluent cultures were released from culture vessels with trypsin, spun down, and snap-frozen in liquid nitrogen before further processing.Nucleic Acid Extraction - gDNA was isolated from E12tg2a mESCs using the Monarch Genomic DNA Purification Kit (NEB, T3010) according to the manufacturer’s instructions. gDNA was sheared to an average fragment size of 200 bp using a Bioruptor Pico sonication device (Diagenode)OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Counts Per Million (CPM) normalizationSequence Alignment - Initial quality assessment of raw reads was carried out using FastQC, followed by adapter trimming and removal of low-quality bases using Trim Galore. The cleaned reads were then aligned to the Mus musculus reference genome mm10 using Bowtie2. Retaining only properly paired reads and removing PCR duplicates was done using Samtools. In the case of DIP-seq, enriched regions were obtained using MACS2, with either IgG or input from mESCs serving as controls. True positive regions were defined as those enriched regions identified for both IgG and input controls, as described by Lentini et al., 2018UnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq XMeDIP-seqMus musculusDaniel SummererMarlon ZambranoLena EngelhardSidney BeckerfalseHM-DyadCap – Capture and Mapping of Asymmetric 5-Hydroxymethylcytosine/5-Methylcytosine CpG Dyads in Mammalian DNA5-Methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) are the two main epigenetic modifications of mammalian DNA, and play crucial roles in cell differentiation, development, and tumorigenesis. Both modifications co-exist with unmodified cytosine in palindromic CpG dyads in different strand-symmetric and -asymmetric combinations, each having unique regulatory potential. To facilitate investigating the individual functions of such dyad modifications, we here report HM-DyadCap. This method employs MECP2 HM – an evolved methyl-CpG-binding domain of the mC reader MECP2 – for the simple capture and sequencing of DNA fragments containing the CpG dyad hmC/mC that frequently occurs in mouse embryonic stem cell and brain genomes. In vitro binding studies reveal a high discrimination of MECP2 HM against diverse off-target dinucleotides in vitro. We conduct comparative mapping experiments for mESC genomes with HM-DyadCap, standard MethylCap based on wild type MECP2, as well as the antibody-based MeDIP and hMeDIP protocols. We find that MECP2 HM is blocked by hmC glucosylation, and conduct control enrichments with glucosylated genomes that indicate highly selective enrichment of hmC/mC dyads by MECP2 HM. Metagene profiles correlate hmC/mC with actively transcribed genes, and reveal global enrichment in gene bodies as well as depletion at transcription start sites. We anticipate that HM-DyadCap will enable effective mapping of hmC/mC with implications for biomarker discovery and unravelling the function of this dyad in diverse aspects of chromatin biology.2026-04-13T00:00:00Z2026-04-13T08:17:17.988Z2025-11-01T19:02:05.298ZE-MTAB-15961ERP183509E-MTAB-15857EFO_0002944EFO_0004170EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0003749EFO_0004184