Dataset Information

Evolved DNA Duplex Readers for Strand-Asymmetrically Modified 5-Hydroxymethylcytosine/5-Methylcytosine CpG Dyads.

ABSTRACT: 5-Methylcytosine (mC) and 5-hydroxymethylcytosine (hmC), the two main epigenetic modifications of mammalian DNA, exist in symmetric and asymmetric combinations in the two strands of CpG dyads. However, revealing such combinations in single DNA duplexes is a significant challenge. Here, we evolve methyl-CpG-binding domains (MBDs) derived from MeCP2 by bacterial cell surface display, resulting in the first affinity probes for hmC/mC CpGs. One mutant has low nanomolar affinity for a single hmC/mC CpG, discriminates against all 14 other modified CpG dyads, and rivals the selectivity of wild-type MeCP2. Structural studies indicate that this protein has a conserved scaffold and recognizes hmC and mC with two dedicated sets of residues. The mutant allows us to selectively address and enrich hmC/mC-containing DNA fragments from genomic DNA backgrounds. We anticipate that this novel probe will be a versatile tool to unravel the function of hmC/mC marks in diverse aspects of chromatin biology.

SUBMITTER: Buchmuller BC

PROVIDER: S-EPMC8874921 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Evolved DNA Duplex Readers for Strand-Asymmetrically Modified 5-Hydroxymethylcytosine/5-Methylcytosine CpG Dyads.

Buchmuller Benjamin C BC Dröden Jessica J Singh Himanshu H Palei Shubhendu S Drescher Malte M Linser Rasmus R Summerer Daniel D

Journal of the American Chemical Society 20220214 7

5-Methylcytosine (mC) and 5-hydroxymethylcytosine (hmC), the two main epigenetic modifications of mammalian DNA, exist in symmetric and asymmetric combinations in the two strands of CpG dyads. However, revealing such combinations in single DNA duplexes is a significant challenge. Here, we evolve methyl-CpG-binding domains (MBDs) derived from MeCP2 by bacterial cell surface display, resulting in the first affinity probes for hmC/mC CpGs. One mutant has low nanomolar affinity for a single hmC/mC C ...[more]

PMID: 35157801

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Project description:BACKGROUND:In mammals, the regulation of imprinted genes is controlled by differential methylation at imprinting control regions which acquire parent of origin-specific methylation patterns during gametogenesis and retain differences in allelic methylation status throughout fertilization and subsequent somatic cell divisions. In addition, many imprinted genes acquire differential methylation during post-implantation development; these secondary differentially methylated regions appear necessary to maintain the imprinted expression state of individual genes. Despite the requirement for both types of differentially methylated sequence elements to achieve proper expression across imprinting clusters, methylation patterns are more labile at secondary differentially methylated regions. To understand the nature of this variability, we analyzed CpG dyad methylation patterns at both paternally and maternally methylated imprinted loci within multiple imprinting clusters. RESULTS:We determined that both paternally and maternally methylated secondary differentially methylated regions associated with imprinted genes display high levels of hemimethylation, 29-49%, in comparison to imprinting control regions which exhibited 8-12% hemimethylation. To explore how hemimethylation could arise, we assessed the differentially methylated regions for the presence of 5-hydroxymethylcytosine which could cause methylation to be lost via either passive and/or active demethylation mechanisms. We found enrichment of 5-hydroxymethylcytosine at paternally methylated secondary differentially methylated regions, but not at the maternally methylated sites we analyzed in this study. CONCLUSIONS:We found high levels of hemimethylation to be a generalizable characteristic of secondary differentially methylated regions associated with imprinted genes. We propose that 5-hydroxymethylcytosine enrichment may be responsible for the variability in methylation status at paternally methylated secondary differentially methylated regions associated with imprinted genes. We further suggest that the high incidence of hemimethylation at secondary differentially methylated regions must be counteracted by continuous methylation acquisition at these loci.

Dataset Information

Evolved DNA Duplex Readers for Strand-Asymmetrically Modified 5-Hydroxymethylcytosine/5-Methylcytosine CpG Dyads.

Publications

Evolved DNA Duplex Readers for Strand-Asymmetrically Modified 5-Hydroxymethylcytosine/5-Methylcytosine CpG Dyads.

Similar Datasets

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