Thanks for your help with the first trials. Initially cells had to be irradiated quickly, before the LEDs desoldered 😂

Thanks for your contribution, was great to collaborate on this.

Very happy we pushed this across the finish line. Huge thanks to all the co-authors: Simon, Shuyao @shashuyao.bsky.social, the Greulich lab @franzigmuc.bsky.social, Susi, Karin @baybioms.bsky.social, Sandra, the Kubicek lab, the Uhlenhaut lab, and especially Bernhard @kusterlab.bsky.social. (11/11)

Overall, we present a resource of nearly 50 photo-crosslinked, i.e. direct physical, DNA interactomes and identify 688 peptide-DNA crosslinks. These crosslinks mark known but also unknown protein-DNA interaction sites, some overlapping with variant sites linked to genetic or somatic disease. (10/n)

DNA photo-crosslinking takes 1 minute on ice, while formaldehyde requires 10 minutes at room temperature. We record a time series of photo-crosslinked DNA interactomes during acute BAF inhibition and identify fast-acting restrictors of chromatin accessibility missed by conventional methods. (9/n)

We use these quantitative powers to profile three genotoxic drugs and find very distinct reactions in the DNA interactome of cancer cells. This reveals drug-specific vulnerabilities that can be exploited for combination treatments. (8/n)

This also works in primary cells, where we see recruitment of inflammation-associated transcription factors to DNA in macrophages challenged with LPS. (7/n)

To identify transcription factors or chromatin modifiers responding to changes in a cell, DNA interactomes can be compared between conditions. Treating breast cancer cells with increasing estrogen doses, we find the estrogen receptor and other regulators recruited to DNA dose-dependently. (6/n)

In contrast to formaldehyde crosslinking, photo-crosslinking only captures direct interactions (‘zero-distance’). The proteins we find in close proximity to DNA show very high degrees of intrinsic disorder. In fact, intrinsic disorder rises the closer proteins localize to DNA. (5/n)

XDNAX extracts are very clean and we discover previously elusive peptide-DNA crosslinks within them. They pinpoint protein-DNA interaction sites to individual amino acids. (4/n)

To analyze protein photo-crosslinked to DNA by proteomics, we develop XDNAX: protein-crosslinked DNA extraction. (3/n)

Unlike natural DNA, 4ST can be activated by wavelengths far outside the absorption range of most natural biomolecules. This avoids photodamage, allowing us to use high-power LEDs, with ~1000-fold higher intensity than conventional bulbs, for activating 4ST in living cells (www.uven.org). (2/n)

UV crosslinking works great for protein-RNA complexes, but DNA is much less photo-reactive and photo-crosslinking not commonly used. Analogous, we find that the photo-activatable nucleotide 4ST (DNA) is about ten times less reactive than the commonly used 4SU (RNA). (1/n)