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HM-DyadCap – Capture and Mapping of Asymmetric 5-Hydroxymethylcytosine/5-Methylcytosine CpG Dyads in Mammalian DNA

ABSTRACT: 5-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.

INSTRUMENT(S): Illumina NovaSeq X

ORGANISM(S): Mus musculus

SUBMITTER: Marlon Zambrano Mila

PROVIDER: E-MTAB-15857 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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HM-DyadCap – Capture and Mapping of Asymmetric 5-Hydroxymethylcytosine/5-Methylcytosine CpG Dyads in Mammalian DNA

Project description:5-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.

2026-04-13 | E-MTAB-15961 | biostudies-arrayexpress

Binding proteins of Hydroxymethylcytosine-modified CpG dyads from mouse brain tissue

Project description:Cytosine (C) modifications within CpG dyads are central regulatory elements of mammalian genomes. In addition to 5-methylcytosine (mC), genomes can also contain high levels of 5-hydroxymethylcytosine (hmC) that is read differently by nuclear proteins than mC. However, hmC can co-exist with C and mC in different combinations in the two strands of CpG dyads, such as hmC/C, hmC/mC, or hmC/hmC, and it is poorly understood, how these combinatorial marks differ in their protein interactomes and regulatory functions. To systematically identify nuclear proteins that bind to distinct hmC-containing CpG dyads, we conducted DNA pulldown experiments using promoter probes harboring defined dyad modifications. In this study, we used nuclear extracts from mouse brain tissue and a DNA probe based on part of the Sp1 promoter sequence. For each dyad configuration, C/C, mC/mC, hmC/mC, hmC/C, and hmC/hmC, five technical replicates were performed.

2025-07-30 | MSV000098682 | MassIVE

Binding proteins of Hydroxymethylcytosine-modified CpG dyads

Project description:Cytosine (C) modifications within CpG dyads are central regulatory elements of mammalian genomes. In addition to 5-methylcytosine (mC), genomes can also contain high levels of 5-hydroxymethylcytosine (hmC) that is read differently by nuclear proteins than mC. However, hmC can co-exist with C and mC in different combinations in the two strands of CpG dyads, and it is poorly understood, how these combinatorial marks differ in their protein interactomes and regulatory functions. To identify nuclear binding proteins, we employed DNA promoter probes with differentially modified CpG dyads in pulldown experiments. The datasets comprise three biological replicates of HEK293T nuclear proteins binding to a VEGFA promoter sequence with differentially modified CpG dyads. Each biological replicate contains five technical replicates per modified CpG dyad, resulting in a total of 15 replicates for C/C, mC/mC and hmC/mC, and 10 replicates for hmC/C and hmC/hmC.

2024-08-15 | MSV000095609 | MassIVE

SCoTCH-seq reveals that 5-hydroxymethylcytosine encodes regulatory information across DNA strands

Project description:In mammalian genomes, cytosine modifications form a layer of regulatory information alongside the genetic code. Decoding this information is crucial to our understanding of biology and disease. Established sequencing methods cannot simultaneously resolve cytosine’s three most common forms—cytosine (C), 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC)—across both strands of the DNA double helix. Thus, how epigenetic information is distributed in DNA remains unclear. We present an accurate and quantitative, base-resolution approach to sequence genomes, together with mC and hmC, in both strands of the same DNA fragment. We show that different forms of cytosine combine across the double helix at CpG sites to form discrete information states in the mouse epigenome. These CpG states have distinct genomic distributions—including at promoters, enhancers, and gene bodies—and have different relationships with transcription. We show that while all possible forms of hydroxymethylation occur, hmC is predominantly asymmetric and that different forms of asymmetric hmC are not equivalent. Our findings demonstrate that 5-hydroxymethylcytosine combines with different cytosine variants across the DNA double helix to form distinct states of regulatory information.

2025-07-16 | GSE263772 | GEO

Differential 5-methylcytosine and 5-hydroxymethylcytosine in Parkinson’s and Alzheimer’s Disease.

Project description:Background: 5-methylcytosine (5-mC) and its oxidized form, 5-hydroxymethylcytosine (5-hmC), are distinct epigenetic marks that help regulate gene expression in the mammalian brain. Existing studies have identified associations between 5-mC and neurodegenerative disease, but little work has examined the potential role of 5-hmC in Parkinson’s disease (PD) or Alzheimer’s disease (AD). Here, we utilized PD postmortem brain tissue and a public dataset from postmortem AD brains to analyze the effect of neurodegenerative disease on paired 5-mC and 5-hmC levels. Results: In PD samples, we measured genome-wide 5-mC and 5-hmC from control (n=3) and PD (n=6) brains using the Illumina EPIC array combined with bisulfite and oxidative bisulfite treatments (BS/oxBS-EPIC). In publicly sourced data, genome-wide 5-mC and 5-hmC were measured from control (n=25) and AD (n=62) human entorhinal cortex tissue using the Illumina 450K array combined with bisulfite and oxidative bisulfite treatment (BS/oxBS-450K). Paired 5-mC and 5-hmC beta values were generated using a custom pipeline of bioinformatics tools, and we modeled the effect of disease on paired 5-mC and 5-hmC data using a mixed effects beta regression model with a random effect for ID and an interaction term between disease category and DNA modification category (“5-mC” or “5-hmC”). We identified a number of CpG probes (AD: n=699, PD: total n = 80) that showed a significant interaction between disease status and DNA modification category (p-value < 2.4x10-7). Conclusions: While our PD data requires additional validation, our analyses suggest that there are widespread shifts in the balance between 5-mC and 5-hmC in Parkinson’s and Alzheimer’s disease.

2019-10-09 | GSE138597 | GEO

5-hmC in the brain: abundance in synaptic genes and differences at the exon-intron boundary (tiling array)

Project description:5-hydroxymethylcytosine (5-hmC), a derivative of 5-methylcytosine (5-mC), is abundant in the brain for unknown reasons. We mapped the genomic distribution of 5-hmC and 5-mC in human and mouse tissues using glucosylation of 5-hmC coupled with restriction enzyme digestion, and interrogation on microarrays. We detected 5-hmC enrichment in genes with synapse-related functions in the brain. We also identified significant, tissue-specific differential distributions of these DNA modifications at the exon-intron boundary, in both human and mouse. This boundary change was mainly due to 5-hmC in the brain, but due to 5-mC in non-neural contexts. This pattern was replicated in multiple independent datasets, and the brain-specific change in 5-hmC was validated using single-molecule sequencing. Moreover, in the brain, constitutive exons contained higher levels of 5-hmC, relative to alternatively-spliced exons. Our study suggests a novel role for 5-hmC in RNA splicing and synaptic function in the brain

2012-08-24 | GSE40166 | GEO

5-hmC in the brain: abundance in synaptic genes and differences at the exon-intron boundary (MRE-Seq)

2012-08-24 | GSE40158 | GEO

In vivo BioID Mouse heart masspec data

Project description:Cardiac excitation-contraction (E-C) coupling requires dyads, the nanoscopic microdomains formed adjacent to Z-lines by apposition of transverse (T)-tubules and junctional sarcoplasmic reticulum (jSR). Disruption of dyad architecture and function are common features of diseased cardiomyocytes. However, little is known about the mechanisms that modulate dyad organization during cardiac homeostasis and disease. Here, we used proximity proteomics in intact, living hearts to identify proteins enriched near dyads.

2022-02-21 | PXD028960 | Pride

WDR76 promotes MLL-rearranged leukemia via highly-selective binding of 5-hydroxymethylcytosine in DNA

Project description:Although rare, the distribution of the 5-hydroxymethylcytosine (hmC) modification in mammalian DNA is tissue- and gene-specific, yet distinct from its repressive methylcytosine (mC) precursor, suggesting unique functions. To examine this possibility, we fractionated mammalian brain extracts to discover binding partners specific for oxidized states of mC. We demonstrate that one such factor, WDR76, is a highly hmC-specific binding protein that modulates gene expression within chromosomal regions enriched in hmC where it binds. In human cell lines and mouse models, WDR76 recruitment by hmC is critical for the initiation and maintenance of MLL-rearranged leukemias. Beyond its canonical role as an intermediate in mC remediation, we show that hmC can be an epigenetic mark whose recognition drives leukemogenesis, portending analogous signaling pathways for other rare DNA modifications.

2021-01-06 | GSE108832 | GEO

Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia

Project description:We studied global distribution of hydroxymethylation (5hmC) and DNA methylation (5mC) in during normal B-cell development and CLL maturation. To derive disease-specific epigenetic changes we have compared CLL subgroups to their corresponding cell of origin. Our data showed that naïve B-cells, compared to non-class switched and class switched memory B-cells, had higher levels of 5-hmC and 5-mC. By comparing with ChIP sequencing data of H3K27Ac and H3K4me1 modifications of CLL samples with 5hmC and 5mC levels, we observed an increase of 5-hmC at overall gene body regions, CpG island shores and regulatory elements. Comparing 5-hmC distribution with RNA sequencing we showed that genes with higher transcriptional activity had higher 5-hmC and vice versa. Thus, our study suggests that the global loss of 5-hmC levels, with significant increase at gene regulatory regions, constitute a novel hallmark of CLL pathogenesis.

2019-01-23 | GSE113386 | GEO

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