Dataset Information

Identification of DNA methylation reader proteins of Arabidopsis thaliana by DNA-affinity pull-down MS and their characterization by IP-MS.

ABSTRACT: In plants, cytosine DNA methylation (mC) is largely associated with transcriptional repression of transposable elements, but it can also be found in the body of expressed genes, referred to as gene body methylation (GbM). GbM is correlated with ubiquitously expressed genes, however its function, or absence thereof, is highly debated. The different output that mC can have raises questions as to how it is interpreted - or read - differently in these sequence and genomic contexts. To screen for potential mC binding proteins, we performed an unbiased DNA affinity pull-down assay combined with quantitative mass spectrometry using methylated DNA probes for each DNA sequence context. All mC readers known to date were found to preferentially bind to the methylated probes, along with a range of new mC binding protein candidates. Functional characterization of these mC readers, focused on the MBD and SUVH families, was undertaken by ChIP-seq mapping of genome-wide binding sites, their protein interactors, and the impact of high-order mutations on transcriptomic and epigenomic profiles. Together, these results highlight specific context preferences for these proteins, and in particular the ability of MBD2 to bind predominantly to GbM. This comprehensive analysis of Arabidopsis mC readers emphasizes the complexity and interconnectivity between DNA methylation and chromatin remodelling processes in plants.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Whole Body

SUBMITTER: Owen Duncan

LAB HEAD: Ryan Lister

PROVIDER: PXD050777 | Pride | 2025-06-09

REPOSITORIES: Pride

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Characterization of DNA methylation reader proteins in <i>Arabidopsis thaliana</i>.

Cahn Jonathan J Lloyd James P B JPB Karemaker Ino D ID Jansen Pascal W T C PWTC Pflueger Jahnvi J Duncan Owen O Petereit Jakob J Bogdanovic Ozren O Millar A Harvey AH Vermeulen Michiel M Lister Ryan R

Genome research 20241223 12

In plants, cytosine DNA methylation (mC) is largely associated with transcriptional repression of transposable elements, but it can also be found in the body of expressed genes, referred to as gene body methylation (gbM). gbM is correlated with ubiquitously expressed genes; however, its function, or absence thereof, is highly debated. The different outputs that mC can have raise questions as to how it is interpreted-or read-differently in these sequence and genomic contexts. To screen for potent ...[more]

PMID: 39632087

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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.

			Action	DRS
	NBD1.interact.pep.xml	Pepxml
	NBD1.prot.xml	Xml
	NBD1.raw	Raw
	NBD2.interact.pep.xml	Pepxml
	NBD2.prot.xml	Xml

Dataset Information

Identification of DNA methylation reader proteins of Arabidopsis thaliana by DNA-affinity pull-down MS and their characterization by IP-MS.

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Publications

Characterization of DNA methylation reader proteins in <i>Arabidopsis thaliana</i>.

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Dataset Information

Identification of DNA methylation reader proteins of Arabidopsis thaliana by DNA-affinity pull-down MS and their characterization by IP-MS.

Dataset's files

Publications

Characterization of DNA methylation reader proteins in &lt;i&gt;Arabidopsis thaliana&lt;/i&gt;.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Characterization of DNA methylation reader proteins in <i>Arabidopsis thaliana</i>.