Reversible chromatin condensation by the DNA repair and demethylation factor thymine DNA glycosylase

Abstract

Chromatin structures (and modulators thereof) play a central role in genome organization and function. Herein, we report that thymine DNA glycosylase (TDG), an essential enzyme involved in DNA repair and demethylation, has the capacity to alter chromatin structure directly through its physical interactions with DNA. Using chemically defined nucleosome arrays, we demonstrate that TDG induces decompaction of individual chromatin fibers upon binding and promotes self-association of nucleosome arrays into higher-order oligomeric structures (i.e. condensation). Chromatin condensation is mediated by TDG's disordered polycationic N-terminal domain, whereas its C-terminal domain antagonizes this process. Furthermore, we demonstrate that TDG-mediated chromatin condensation is reversible by growth arrest and DNA damage 45 alpha (GADD45a), implying that TDG cooperates with its binding partners to dynamically control chromatin architecture. Finally, we show that chromatin condensation by TDG is sensitive to the methylation status of the underlying DNA. This new paradigm for TDG has specific implications for associated processes, such as DNA repair, DNA demethylation, and transcription, and general implications for the role of DNA modification 'readers' in controlling chromatin organization.

Figure 1.

TDG binds and locally opens chromatin fibers. (A) Intra-array FRET-based assay to measure the extent of chromatin fiber compaction (16). (B) FRET analysis of compact 12-mer arrays (2 mM Mg²⁺) in the presence of TDG (200 nM) or FOXA1 (1 μM). FRET efficiency was normalized to the compact array sample. The extended array sample does not contain Mg²⁺ in the buffer. Raw FRET efficiency is provided in Supplementary Figure S1h. ****P < 0.0001. (C) Saturation plots for binding of TDG to naked 601 DNA or mononucleosomes having different arrangements of linker DNA. The K_d is listed below each substrate. Error bars represent standard deviation from at least three independent experiments. (D) MNase digestion of nucleosome arrays in the presence of TDG. The concentration of TDG (nM) used in each experiment is listed to the right.

Figure 2.

TDG promotes chromatin condensation. (A) Precipitation assay to monitor nucleosome array oligomerization. Nucleosome arrays were incubated with the indicated protein, oligomers were removed by centrifugation, and the percentage of arrays remaining in solution was determined by gel electrophoresis. (B) FRET-based assay to monitor inter-fiber oligomerization. (C) Mg²⁺-induced oligomerization of nucleosome arrays. Precipitation data (black) is shown on the left Y-axis, and inter-fiber FRET efficiency (red) is shown on the right Y-axis. (D) Comparison of the inter-fiber FRET efficiency for arrays treated with Mg²⁺ or TDG. Error bars represent standard deviation from at least three independent experiments.

Figure 3.

TDG-mediated chromatin oligomerization is dependent on its N- and C-terminal domains. (A) TDG domains discussed in this work. (B, C) Precipitation assay to monitor nucleosome array oligomerization. Nucleosome arrays were incubated with the indicated protein, oligomers were removed by centrifugation, and the percentage of arrays remaining in solution was determined by gel electrophoresis. Error bars represent standard deviation from at least three independent experiments.

Figure 4.

TDG-mediated chromatin oligomerization is reversible. Insoluble chromatin oligomers were incubated with the indicated concentration of 601 DNA (A) or GADD45a (B), and the change in solubility was monitored following centrifugation. Error bars represent standard deviation from at least three independent experiments.

Figure 5.

DNA methylation inhibits TDG-mediated chromatin condensation. (A) Precipitation assay to monitor nucleosome array oligomerization. (Un)methylated nucleosome arrays were incubated with the indicated concentration of TDG, oligomers were removed by centrifugation, and the percentage of arrays remaining in solution was determined by gel electrophoresis. (B) Soluble fraction following treatment of (un)methylated arrays with different TDG variants (1 μM). Error bars represent standard deviation from at least three independent experiments.

Figure 6.

Proposed model for TDG-mediated chromatin remodelling (1). Upon recruitment, TDG preferentially binds to linker DNA between nucleosome resulting in decompaction of the chromatin fiber structure. The basic NTD contributes to nonspecific DNA binding in cis (i.e. to the same fiber as the catalytic domain) (37,38). (2) In the presence of nearby chromatin fibers, TDG’s NTD can also bind to DNA in trans (i.e. to a different fiber than the catalytic domain), facilitating oligomerization and condensation of the chromatin as local concentration of TDG increase. Because efficient oligomerization requires tethering of the NTD to chromatin (Figure 3C), we propose that DNA binding by the catalytic domain (which requires in cis DNA binding by the NTD), along with accompanying array decompaction, precedes oligomerization. (3) The CTD of TDG antagonizes chromatin condensation by weakening inter-fiber interactions between the NTD and DNA, potentially through direct contacts between the two disordered domains (38). This destabilizing affect allows for external regulators (e.g. GADD45a) to bind to and sequester TDG’s NTD away from DNA, resulting in disruption of inter-fiber interactions and re-solubilization of the chromatin. However, in the absence of the CTD’s destabilizing affect (ΔCTD), chromatin condensation becomes non-reversible due to tight inter-fiber binding of the NTD.