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Science is a collaborative adventure, and this moment really feels like a celebration of the collective effort, curiosity, and passion that drives research.

in a PDS5 knockdown? STAG2 seems to be a liiiiittle enriched there (Fig. 4c) and AF is more confident in STAG2 than in STAG1. Yet we didn't test STAG2.
Nevertheless, in the (local) absence of PDS5, CTCF might use the KTYQYR motif to interfere with cohesin as our data suggests..

seems to happen also experimentally. We didn't explore PDS5 binding to KTYQR computationally or experimentally. It might be that KTYQR-PDS5 binding is stronger than KTYQR-STAG binding and this is thus the dominant mode seen in vivo. Are you aware of Hi-C and/or binding (as in your Fig. 4) data..

Hi @elphegenoralab.bsky.social and @eecutts.bsky.social . True, the KTYQR motif has been identified by both of your cited articles as PDS5 binding regions. We didn't test PDS5 in our assays, only cohesin-NIPBL. Seeing that AF suggested KTYQR-STAG binding was surprising to us and more so that that..

We started

Thank you to all that contributed!! Some on bluesky: @jacotorre.bsky.social @ceesdekker.bsky.social, especially to the fantastic team at the IMP Vienna Gabriele Litos, Iain Davidson, and Jan-Michael Peters!!

We hope that by disentangling the multiple contributions of the CTCF N-terminus to the stalling of cohesin at CTCF sites, we are another step closer to understanding how cohesin and CTCF control and shape genomes across the tree of life 🌳

To summarize:

KTYQR is distinct from YxF: it reduces cohesin's LE activity overall, but doesn't impact cohesin's directionality.

Together, the two motifs account pretty much for the impact of the full NTR!! We hypothesize that connecting the two on one protein chain and some adjacent sequences modulate further.

A computational AlphaFold screen suggested the KTYQR motif, located N-terminally of the YxF, that appears to also bind STAG1.

Even though YxF has a large impact on cohesin's LE activity, it doesn't fully account for the impact of the full N-terminal region of CTCF. So we searched more 🔎

And indeed, FCS experiments showed that STAG1's DNA affinity increases upon addition of the YxF motif.

Curiously, we observed that cohesin also changes direction much less frequently in the presence of YxF! Taking inspiration from Shaltiel et al, Science, 2022, we hypothesized that this is due to a higher DNA affinity of some cohesin subunit in the presence of YxF. Our main candidate: STAG1-kleisin

We started with the known YxF motif. In brief, the YxF motif:
- reduces cohesin's ATPase rate
- reduces LE initiation
- reduces the fraction of complete LE steps (in Magnetic Tweezers)
So these few amino acids alone already tinker quite a lot with cohesin!

Observing LE by Magnetic Tweezers provides a much more detailed view, however. We can see individual steps made by cohesin. Previously, we already observed a plethora of phenomena: cohesin often makes full LE steps, as seen by a height decrease of the magnetic bead. But these are also often reversed