Abstract
Methylcytosine derivatives dynamically bind distinct readers, including DNA repair proteins.
Major finding: Methylcytosine derivatives dynamically bind distinct readers, including DNA repair proteins.
Approach: Quantitative MS identified readers in ESCs, neuronal progenitors, and adult brain tissue.
Impact: Regulation of methyl–CpG-specific readers is critical for normal development and homeostasis.
DNA methylation at cytosine residues is associated with transcriptional repression, and mutations that deregulate this process have been implicated in tumorigenesis. Oxidation of 5-methylcytosine (mC) by tet methylcytosine dioxygenase (TET) enzymes, particularly in embryonic stem cells (ESC) and the brain, generates 5-hydroxymethylcytosine (hmC) and subsequently 5-formylcytosine (fC) and 5-carboxylcytosine (caC). However, the function of these oxidized derivatives and the identity of the specific binding proteins, or readers, that are recruited to these modified bases are unknown. To address these questions, Spruijt and colleagues performed a quantitative mass spectrometry (MS)-based proteomic analysis of mouse ESCs, neuronal progenitor cells (NPC), and adult brain tissue and identified readers that interact with each type of modified cytosine residue. Proteins recruited to individual modifications showed little overlap and were distinct from the large cluster of proteins that preferentially bound nonmethylated DNA, suggestive of unique functions for each modification. In contrast to mC, oxidized cytosine derivatives were enriched for interactions with transcriptional regulators, DNA glycosylases, and DNA damage response and repair proteins, suggesting that these modifications trigger active DNA demethylation via base excision repair. Furthermore, different sets of readers were detected in each cell type, including binding of the stem-cell reprogramming factor Kruppel-like factor 4 to mC in ESCs and ubiquitin-like with PHD and ring finger domains 2, E3 ubiquitin protein ligase (UHRF2) to hmC in NPCs, which promoted repetitive TET1-mediated oxidation of mC. Additionally, more hmC-specific readers were found in adult brain extracts compared with ESCs and NPCs, indicative of dynamic changes in methyl–CpG-specific reader recruitment during cell differentiation. This differential recruitment resulted largely from variability in the abundance of reader proteins or their cofactors between cell types. These results identify dynamic recruitment of readers as a critical regulator of normal cell homeostasis.
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