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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE263772 | GEO | 2025/07/16

REPOSITORIES: GEO

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Project description:Tet proteins oxidize 5-methylcytosine (mC) to generate 5-hydroxymethyl (hmC), 5-formyl (fC) and 5-carboxylcytosine (caC). The exact function of these oxidative cytosine bases remains elusive. We applied quantitative mass spectrometry-based proteomics to identify readers for mC and hmC in mouse embryonic stem cells (mESC), neuronal progenitor cells (NPC) and adult mouse brain tissue. Readers for these modifications are only partially overlapping and some readers, such as Rfx proteins, display strong specificity. Interactions are dynamic during differentiation, as for example evidenced by the mESC-specific binding of Klf4 to mC and the NPC-specific binding of Uhrf2 to hmC, suggesting specific biological roles for mC and hmC. Oxidized derivatives of mC recruit distinct transcription regulators as well as a large number of DNA repair proteins in mouse ES cells, implicating the DNA damage response as a major player in active DNA demethylation. Peptides were separated on an EASY-nLC (Proxeon) connected online to an LTQ-Orbitrap-Velos mass spectrometer. Spectra were recorded in CID mode. A gradient of organic solvent (5-30% acetonitrile) was applied (120 minutes) and the top 15 most abundant peptides were fragmented for MS/MS, using an exclusion list of 500 proteins for 45 seconds. Raw data were analyzed using Maxquant version 1.2.2.5 and the integrated Andromeda search engine against protein database ipi.MOUSE.v3.68. Using Perseus, data was filtered for contaminants, reverse hits, number of peptides (>1) and unique peptides (>0). Ratios were logarithmitized (log2) and groups (consisting of forward and reverse) were defined. Proteins were filtered to have at least 2 valid values in one of the groups and missing values were imputed based on a normal distribution (width=0.2 and shift=0), after which Significance B was calculated (Benj.Hoch.FDR=0.05). Scatterplots were made using R. Proteins were defined to be significant when both forward and reverse significance p<0.05 and minimal ratios were >2 in both experiments. The H/L ratios shown in Figure2A-C were calculated using the formula (log(forward ratio) - log(reverse ratio))/2.

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

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