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Protein Interactions at Oxidized 5-Methylcytosine Bases.

ABSTRACT: 5-Methylcytosine (5mC), the major modified DNA base in mammalian cells, can be oxidized enzymatically to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by the Ten-Eleven-Translocation (TET) family of proteins. Whereas 5fC and 5caC are recognized and removed by base excision repair proteins, the 5hmC base accumulates to substantial levels in certain cell types such as brain-derived neurons and is viewed as a relatively stable DNA base. As such, the existence of "reader" proteins that recognize 5hmC would be a logical assumption, and various searches have been undertaken to identify proteins that specifically bind to 5hmC and the other oxidized 5mC bases. However, the existence of definitive 5hmC "readers" has remained unclear and proteins interacting specifically with 5fC or 5caC are also very few. On the other hand, 5hmC is incapable of interacting with a number of proteins that recognize 5mC at CpG sequences, suggesting that 5hmC is an anti-reader modification that may serve to displace 5mC readers from DNA. In this review article, we discuss candidate proteins that may interact with oxidized 5mC bases.

SUBMITTER: Pfeifer GP

PROVIDER: S-EPMC7007358 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Protein Interactions at Oxidized 5-Methylcytosine Bases.

Pfeifer Gerd P GP Szabó Piroska E PE Song Jikui J

Journal of molecular biology 20190808 6

5-Methylcytosine (5mC), the major modified DNA base in mammalian cells, can be oxidized enzymatically to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by the Ten-Eleven-Translocation (TET) family of proteins. Whereas 5fC and 5caC are recognized and removed by base excision repair proteins, the 5hmC base accumulates to substantial levels in certain cell types such as brain-derived neurons and is viewed as a relatively stable DNA base. As such, the existence ...[more]

PMID: 31401118

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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.

Dataset Information

Protein Interactions at Oxidized 5-Methylcytosine Bases.

Publications

Protein Interactions at Oxidized 5-Methylcytosine Bases.

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