Alberto Marin
@albertomarin.bsky.social
Postdoctoral fellow in the labs of Taekjip Ha and Ralph Scully at Harvard Medical School. Investigating homologous recombination in time and space.
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!
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!
February 13, 2025 at 1:16 AM
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!
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!
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.
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.
February 13, 2025 at 1:16 AM
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.
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.
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.
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.
February 13, 2025 at 1:16 AM
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.
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.
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.
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.
February 13, 2025 at 1:16 AM
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.
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.
/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.
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.
February 13, 2025 at 1:16 AM
/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.
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.
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.
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.
February 13, 2025 at 1:16 AM
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.
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.
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!
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!
February 13, 2025 at 1:16 AM
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!
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!
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?
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?
February 13, 2025 at 1:16 AM
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?
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?
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.
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.
February 13, 2025 at 1:16 AM
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.
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.
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.
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.
February 13, 2025 at 1:16 AM
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.
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.