Wilms tumor protein recognizes 5-carboxylcytosine within a specific DNA sequence

Abstract

In mammalian DNA, cytosine occurs in several chemical forms, including unmodified cytosine (C), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5mC is a major epigenetic signal that acts to regulate gene expression. 5hmC, 5fC, and 5caC are oxidized derivatives that might also act as distinct epigenetic signals. We investigated the response of the zinc finger DNAbinding domains of transcription factors early growth response protein 1 (Egr1) and Wilms tumor protein 1 (WT1) to different forms of modified cytosine within their recognition sequence, 59-GCG(T/G)GGGCG-39. Both displayed high affinity for the sequence when C or 5mC was present and much reduced affinity when 5hmC or 5fC was present, indicating that they differentiate primarily oxidized C from unoxidized C, rather than methylated C from unmethylated C. 5caC affected the two proteins differently, abolishing binding by Egr1 but not by WT1. We ascribe this difference to electrostatic interactions in the binding sites. In Egr1, a negatively charged glutamate conflicts with the negatively charged carboxylate of 5caC, whereas the corresponding glutamine of WT1 interacts with this group favorably. Our analyses shows that zinc finger proteins (and their splice variants) can respond in modulated ways to alternative modifications within their binding sequence.

5-Hydroxymethylcytosine (5hmC), or How to Identify Your Favorite Cell

“…Interestingly, 5fC and 5caC seem to be associated with numerous molecular readers [28,[33][34][35]. Of note, these methylated variations have been shown to cause pause at the transcription rate generated by RNA Polymerase II (Pol II) binding.…”

Tet proteins influence the balance between neuroectodermal and mesodermal fate choice by inhibiting Wnt signaling

“…The three mammalian Tet proteins, Tet1, Tet2, and Tet3, possess homologous C-terminal catalytic domains as well as CXXC domains that bind unmodified CpGs (4), except that the CXXC domain of TET2 became separated from the catalytic domain during evolution and is now a separate protein known as IDAX/CXXC4 (5). The oxidized methylcytosine (oxi-mC) species generated by Tet enzymes facilitate DNA demethylation through both passive (replication-dependent) and active (replicationindependent) mechanisms (4); they also function as epigenetic marks that bind transcription factors and chromatin-associated proteins, thereby influencing chromatin structure and gene expression (6)(7)(8).…”

The Mechanisms of Generation, Recognition, and Erasure of DNA 5-Methylcytosine and Thymine Oxidations

“…Furthermore, the structurally related E. coli mismatch uracil glycosylase (eMUG) can excise 5caC and 5fC as well (74,82). Both 5fC and 5caC exhibit an intrabase hydrogen bond between their formyl or carboxyl oxygen atoms, respectively, and the adjacent cytosine N 4 exocyclic amine nitrogen atom, both in the free form (83) and in the protein-bound form (58) (Fig. 3, h and i, top panels).…”

“…In contrast, certain Cys2-His2 (C2H2) ZnF proteins bind preferentially to DNA when CpG sites embedded within their recognition sequences are methylated (54). The structures of five ZnF domains bound to 5mC-containing DNA have been solved, including the transcription factors Kaiso, Zfp57, Krüp-pel-like factor 4 (Klf4), growth response protein 1 (Egr1), and Wilms tumor protein 1 (WT1) (55)(56)(57)(58). Kaiso recognizes either methylated CpG dinucleotides (59) or an unmodified sequence with a TpG in the place of 5mCpG (60).…”