Alberto Marin

Alberto Marin

@albertomarin.bsky.social

Assistant Professor, Department of Cancer Biology Perelman School of Medicine, University of Pennsylvania

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Alberto Marin
@albertomarin.bsky.social

We found that loops on the broken chromatid promote the initial encounter between the break and the sister chromatid by steering the break toward TAD boundaries. Loops on the sister chromatid drive the subsequent 1D scanning that is the rate-limiting step for homology identification. 8/n

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Alberto Marin
@albertomarin.bsky.social

Cohesin effects were more pronounced when we simulated searches along a large TAD (~1 Mb), where diffusion alone is inefficient. Hence, cohesin might be especially important in stimulating HR for breaks occuring in large TADs, that likely require more extended searches. 6/n

Alberto Marin
@albertomarin.bsky.social

A surprising finding: removing loops in the broken chromatid had a small effect in search dynamics. It is the loops in the sister chromatid that play a dominant role in lowering search times! We looked at individual search instances to understand this. 7/n

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Alberto Marin
@albertomarin.bsky.social

Here, we used polymer simulations to model the homology search on a replicated TAD. Our simulations recapitulate key experimental findings (RAD51 ChIP-Seq profiles, chromosome contacts and HR efficiency) and reveal new insights into how cohesin promotes homology searches. 4/n

Alberto Marin
@albertomarin.bsky.social

First, housekeeping cohesins accelerates the search by over 4x compared to 3D diffusion alone; adding break-interacting cohesins (break-anchored loops and cohesive clamp) boost the search by an additional 2x. Thus, searches are approx. an order of magnitude faster when cohesins are present! 5/n

Alberto Marin
@albertomarin.bsky.social

After DNA replication, cells activate a mechanism to fix DNA breaks known as homologous recombination (HR), which restores a broken site using its replicated copy as template. HR is powerful, but it presents a unique challenge: the broken locus needs to search for and find its replicated locus. 2/n

Alberto Marin
@albertomarin.bsky.social

Recent works by us and others have shed light into these genomic searches, revealing that they are stimulated by the chromatin looping activity of the cohesin complex. But many questions remained unanswered. What are the dynamics of the process? How does loop extrusion stimulate the search? 3/n

Alberto Marin
@albertomarin.bsky.social

Last but not least, I am incredibly grateful to my mentor, Taekjip Ha, for giving me the freedom to take risks and for guiding me throughout the project. And co-mentor, Ralph Scully, for all his support and mentorship. 11/n

Alberto Marin
@albertomarin.bsky.social

Also, check out the related work by @fedeteloni.bsky.social et al, from @gerlichlab.bsky.social who looked at the role of cohesive cohesin as well. www.science.org/doi/10.1126/... And the insightful perspective by Jiazhi Hu! 9/n www.science.org/doi/10.1126/...

science.orgCohesin guides homology search during DNA repair using loops and sister chromatid linkagesAccurate repair of DNA double-strand breaks (DSBs) is essential for genome stability, and defective repair underlies diseases such as cancer. Homologous recombination uses an intact homologous sequenc...
Alberto Marin
@albertomarin.bsky.social

We discovered that instead of searching randomly, cells use an active 1D scanning process: the repair machinery leverages a looping protein called cohesin to ‘slide’ the break along the DNA and find the matching sequence. 6/n

Alberto Marin
@albertomarin.bsky.social

Now, after DNA replicates, sister chromatids are held together approximately every 1 Mb so the search is confined to ~1 M nucleotides. But finding the right match is still a huge challenge – especially if the sisters aren’t perfectly aligned. 4/n

Alberto Marin
@albertomarin.bsky.social

Simply relying on random 3D diffusion – letting the broken DNA wander through the nucleus – would be inefficient. Even for a 1 Mb region, the broken DNA would take far too long to find its matching sequence by chance alone. 5/n

Alberto Marin
@albertomarin.bsky.social

Homologous recombination is key for protecting the genome, but it’s also challenging because the broken DNA must find its matching copy within billions of nucleotides. How can a cell achieve this? This is known as the “homology search” problem. 3/n

Alberto Marin
@albertomarin.bsky.social

15/n Also, check out the pre-prints by @fedeteloni.bsky.social from @gerlichlab.bsky.social and by @charlesyeh.bsky.social from @jcornlab.bsky.social with other cool insights about the homology search! www.biorxiv.org/content/10.1... www.biorxiv.org/content/10.1...

biorxiv.orgCohesin guides homology search during DNA repair via loops and sister chromatid linkagesAccurate repair of DNA double-strand breaks (DSBs) is essential for genome stability, and defective repair underlies diseases such as cancer. Homologous recombination uses an intact homologous sequenc...
Alberto Marin
@albertomarin.bsky.social

12/n Our model, in a nutshell: cohesin drives homology search via 1D scanning. During HR, a RAD51 filament locally scans the sister chromatid, but this search could be unproductive (e.g., because the donor is far). Cohesin loops would then facilitate long-range scanning to help find a donor!

Alberto Marin
@albertomarin.bsky.social

13/n Incredibly thankful to my mentor, Taekjip Ha, who supervised and mentored me on this project, to my co-mentor, Ralph Scully, who designed the mESC exps and mentored me on HR, and to co-first authors Adam Rybczynski and Namrata Nilavar, who helped make this possible!

Alberto Marin
@albertomarin.bsky.social

10/n We noticed that the RAD51 domain is constrained by TAD boundaries, suggesting a role for cohesin in mediating the homology search. Indeed, acute degradation of the cohesin unloader WAPL, known to yield elongated loops, resulted in a broader search.

Alberto Marin
@albertomarin.bsky.social

11/n Does loop-extruding cohesin (Nipbl) regulates HR? In the short-range HR-GFP reporter, Nipbl depletion mildly reduces HR (45%). But in the long-range reporters, Nipbl depletion substantially reduces HR (75%). Thus, Nipbl is critical for HR when long searches are required.

Alberto Marin
@albertomarin.bsky.social

8/n We used an HR-GFP reporter system. Upon inducing a DSB, a broken GFP gene can be repaired by HR via use of a 5’-truncated donor, giving a functional GFP. We reasoned that, upon DSB induction, RAD51 should show a peak at the donor, indicative of a successful homology search.

Alberto Marin
@albertomarin.bsky.social

9/n So, we generated monoclonal mES cell lines with the donor at +441 kb and +563 kb from the break. When we induced the DSB…we found a beautiful RAD51 peak at the exact location of the donor! The RAD51 chromatin domain is thus capturing the homology search.

Alberto Marin
@albertomarin.bsky.social

6/n Motivated by this finding, we performed a second time-course experiment, where we looked at the dynamics of RAD51 recruitment, the core HR factor. RAD51 dynamics mimic break-anchored loop formation, suggesting a causal relationship between loops and HR.

Alberto Marin
@albertomarin.bsky.social

/n But why does RAD51 spread to such long distances (~0.5 Mb) from the break? End resection was constrained to ~ 5 kb, so the broad RAD51 domain does not come from the RAD51-ssDNA filament. We hypothesized that the RAD51 domain reflects the homology search. We tested this.

Alberto Marin
@albertomarin.bsky.social

4/n Time-course Hi-C, enabled by our light-activated very fast CRISPR, showed that break-anchored loops are late repair events. Loops form after γH2AX domain, ruling out previous models where break-anchored loops propagate γH2AX at early stages. What is the role of such loops?

Alberto Marin
@albertomarin.bsky.social

5/n To answer this, we induced DSBs in cell-cycle-synchronized populations. Cells in late S/G2 phases, which are HR-proficient, showed break-anchored loops. But cells synchronized in G1, which are HR-deficient, did not show the loops. Break-anchored loops are HR events!

Alberto Marin
@albertomarin.bsky.social

2/n The homologous recombination (HR) pathway repairs DNA lesions by copying intact sequence information from a DNA template… but how does a damaged DNA find a correct DNA template in the 3D genome? We provide some answers to this Q, known as the homology search problem.

Alberto Marin
@albertomarin.bsky.social

3/n Using our multi-target CRISPR system – which induces hundreds of DNA double-strand breaks (DSB) on demand – and Hi-C, we found that Cas9 breaks act as anchors for chromatin loops. Cohesin is recruited to the breaks and is required for break-anchored loop formation.

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