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. 2017 Nov 6;56(45):14130-14134.
doi: 10.1002/anie.201708286. Epub 2017 Oct 6.

Reversible DNA-Protein Cross-Linking at Epigenetic DNA Marks

Shaofei Ji  1 , Hongzhao Shao  1 , Qiyuan Han  2 , Christopher L Seiler  3   4 , Natalia Y Tretyakova  3   4
Affiliations

Reversible DNA-Protein Cross-Linking at Epigenetic DNA Marks

Shaofei Ji et al. Angew Chem Int Ed Engl. .

Abstract

5-Formylcytosine (5fC) is an endogenous DNA modification frequently found within regulatory elements of mammalian genes. Although 5fC is an oxidation product of 5-methylcytosine (5mC), the two epigenetic marks show distinct genome-wide distributions and protein affinities, suggesting that they perform different functions in epigenetic signaling. A unique feature of 5fC is the presence of a potentially reactive aldehyde group in its structure. Herein, we show that 5fC bases in DNA readily form Schiff-base conjugates with Lys side chains of nuclear proteins in vitro and in vivo. These covalent protein-DNA complexes are reversible (t1/2 =1.8 h), suggesting that they contribute to transcriptional regulation and chromatin remodeling. On the other hand, 5fC-mediated DNA-protein cross-links, if present at replication forks or actively transcribed regions, may interfere with DNA replication and transcription.

Keywords: DNA methylation; DNA-protein cross-linking; aldehydes; epigenetics; histones.

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Figures

Figure 1
Figure 1
Denaturing SDS-PAGE analysis of DNA-protein cross-links using (A) 32P-labeled 5fC DNA and (B) SimplyBlue Protein staining (left panel) and SYBR Green DNA staining (right panel).
Figure 2
Figure 2
ESI+ mass spectrum of histone H4 (M = 11,241) cross-linked to 5fC-containing 12-mer oligonucleotide (M = 3704.4). The cross-links were stabilized by NaCNBH3 treatment. The signal at 14,928 corresponds to DPC structure shown on top.
Figure 3
Figure 3
Effects of pH on DNA-protein and DNA-peptide cross-links formation.
Figure 4
Figure 4
Time-dependent DPC formation between 5-formly-dC containing DNA and Histone H2A. The time course of DPC formation fits pseudo first-order reversible kinetics in the presence of 35 molar excess of histone H2A.
Figure 5
Figure 5
MS (A) and MS2 (B) spectrum of 11-mer peptide cross-linked to 5-formyl-dC.
Figure 6
Figure 6
Representative NanoLC-ESI+-MS/MS chromatograms of AccQ-Tag derivatized 5fC-lys conjugates from NaCNBH3 treated nuclei from HEK293T cells.
Figure 7
Figure 7
Crystal structure of (A) Histone H4 and (B) Histone H2A (PDB IKX5) showing Lys and Arg residues participating in DPC formation.
Scheme 1
Scheme 1
Schiff base formation between 5-formyl-dC in DNA and basic side chains of proteins and its chemical stabilization via NaCNBH3 reduction.
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