Thymine DNA glycosylase mediates chromatin phase separation in a DNA methylation-dependent manner.

Thymine DNA glycosylase (TDG) is an essential enzyme involved in numerous biological pathways, including DNA repair, DNA demethylation, and transcriptional activation. Despite these important functions, the mechanisms surrounding the actions and regulation of TDG are poorly understood. In this study, we demonstrate that TDG induces phase separation of DNA and nucleosome arrays under physiologically relevant conditions in vitro and show that the resulting chromatin droplets exhibited behaviors typical of phase-separated liquids, supporting a liquid-liquid phase separation model.

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

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.

Acetylation of the histone H3 tail domain regulates base excision repair on higher-order chromatin structures.

Despite recent evidence suggesting that histone lysine acetylation contributes to base excision repair (BER) in cells, their exact mechanistic role remains unclear. In order to examine the influence of histone acetylation on the initial steps of BER, we assembled nucleosome arrays consisting of homogeneously acetylated histone H3 (H3K18 and H3K27) and measured the repair of a site-specifically positioned 2'-deoxyuridine (dU) residue by uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE1). We find that H3K18ac and H3K27ac differentially influence the combined activities of UDG/APE1 on compact chromatin, suggesting that acetylated lysine residues on the H3 tail domain play distinct roles in regulating the initial steps of BER.

Chromatin Structure and the Pioneering Transcription Factor FOXA1 Regulate TDG-Mediated Removal of 5-Formylcytosine from DNA.

Although a functional relationship between active DNA demethylation and chromatin structure is often implied, direct experimental evidence is lacking. We investigated the relationship between chromatin structure and thymine DNA glycosylase (TDG) using chemically defined nucleosome arrays containing site-specifically positioned 5-formylcytosine (5fC) residues. We show that the extent of array compaction, as well as nucleosome positioning, dramatically influence the ability of TDG to excise 5fC from DNA, indicating that the chromatin structure is likely a key determinant of whether 5fC is removed from the genome or retained as an epigenetic mark.

Polycomb repressive complex 2 binds RNA irrespective of stereochemistry.

The Polycomb Repressive Complex 2 (PRC2) interacts promiscuously with G-quadruplex (G4) RNA structures. Herein, we tested the limit of this promiscuity by exploring the interaction of PRC2 with G4 RNAs comprised of l-ribonucleic acids (l-RNA), the enantiomer of naturally occurring d-RNA. Remarkably, we find that PRC2 binds similarly to both d- and l-G4 RNAs, suggesting that these interactions are independent of stereochemistry.

Plug-and-Play Approach for Preparing Chromatin Containing Site-Specific DNA Modifications: The Influence of Chromatin Structure on Base Excision Repair.

The genomic DNA of eukaryotic cells exists in the form of chromatin, the structure of which controls the biochemical accessibility of the underlying DNA to effector proteins. In order to gain an in depth molecular understanding of how chromatin structure regulates DNA repair, detailed in vitro biochemical and biophysical studies are required. However, because of challenges associated with reconstituting nucleosome arrays containing site-specifically positioned DNA modifications, such studies have been limited to the use of mono- and dinucleosomes as model in vitro substrates, which are incapable of folding into native chromatin structures.