Getting to grips with chromosome organisation and dynamics using ‘scopes, sequencing and silicon @IMBA_Vienna, @viennabiocenter
- Tweets 74
- Following 57
- Followers 401
- Likes 70
We propose a dual function of cohesin in homology search:
🔹Loop-forming cohesin defines search space, limited by TADs
🔹Cohesive cohesin tethers the break to its sister, to favor productive interactions
This ensures efficient & accurate homology search! 10/12
1
2
47
DSBs recruit two cohesin pools to distinct domains
🔹Loop-forming cohesin accumulates across ~1 Mb, controlling homology sampling range.
🔹Cohesive cohesin accumulates at DSBs, tethering the broken DNA ends to the intact sister chromatid. 9/12
1
2
43
Cohesion directs homology search towards sister chromatid
What happens if we disrupt sister cohesion? Upon sororin depletion (cohesion stabilizer):
✅ Loops remain intact
❌ Contacts between sister chromatids are lost
8/12
1
2
45
DSBs remodel sister chromatids in cis and trans
Sister-pore-C allows us to track how DNA architecture changes before and after DSBs. We found that DSBs locally increase loops and interactions between the broken ends and the intact sister chromatid. 7/12
1
2
46
A new tool: sister-pore-C
Homology-directed repair normally uses the sister chromatid as a template, but how does a DSB find its sister locus in 3D space? We developed sister-pore-C, a high-resolution method to map chromatin interactions within & between sister chromatids. 6/12
1
2
48
Cohesin-mediated loops control search range
When we disrupted cohesin regulators, search dynamics changed:
🔹Reducing loops by NIPBL depletion narrowed homology search
🔹Expanding loops by WAPL depletion made the search broader
Cohesin loops regulate homology search range! 5/12
1
2
52
RAD51 filaments sample local TADs
By mapping RAD51, a key repair protein, we found that homology search occurs within topologically associated domains near the break rather than across the entire genome.
How does chromosome organization influence the search range? 4/12
1
2
60
Site-specific induction of homology-directed repair
To study how chromosome architecture influences homology search, we induced DSBs at specific genomic sites in S/G2-synchronized human cells. Most breaks were repaired by homologous recombination. 3/12
1
2
70
Just had our annual lab christmas dinner, featuring curry and Schrottwichteln (a not-quite-secret-santa, rules here: geschenke.de/blogs/wichteln/…). Wishing you a festive end to 2024!
8
270
A great big congratulations to Sofia, who successfully defended her PhD thesis @IMBA_Vienna yesterday. Here’s one happy graduate complete with traditional lab-made defense hat 🎩 Well done Sofia!
@SofiaKolesni29 @viennabiocenter @TrainingVbc
1
2
37
9,575
… also at the symposium is new PhD student @neos_cruz, who presented his Master’s thesis in today’s 3-minute thesis competition - nicely done, Neos!
3
324
Massive congratulations to @pauldanielbatty, who has succesfully defended his PhD thesis @IMBA_Vienna @viennabiocenter, titled 'The mechanics of sister chromatid resolution'. Well done Paul!!
14
2,069
Together these results provide evidence that cohesin not only holds sister chromatids together, but also actively promotes their separation by its loop extrusion activity. Furthermore, removal of cohesion is not itself a prerequisite for high levels of sister resolution
8/9
1
1
4
1,793
To quantify genomic intervals over which sister chromatids resolve from each other, we performed sister-chromatid-sensitive Hi-C. WAPL depletion resolved sister chromatids over more than 10Mb, whereas depletion of NIPBL strongly reduced sister resolution vs wildtype
7/9
1
4
977
Sister DNAs in WAPL-depleted cells resolve around a single cohesin axis, which splits upon depletion of the cohesion cofactor Sororin. Thus, although cohesion prevents the separation of cohesin axes, it imposes only small constraints on sister chromatid resolution
6/9
1
1
2
1,292
Depletion of WAPL after chromosome replication promotes further resolution of sister chromatids into two parallel threads in G2 cells, resembling the morphology of wildtype chromosomes in mitotic prophase
5/9
1
1
7
1,271
This allows us to visualise substantial resolution of sisters in G2. Resolution is reduced by degradation of the cohesin loop-extrusion cofactor NIPBL, but not by degradation of condensins, indicating cohesin-mediated sister chromatid resolution already prior to mitotic entry
4/9
1
4
1,069
We visualise one sister chromatid per replicated chromosome by allowing cells to replicate for one cell cycle in the presence of F-ara-EdU, then one cell cycle in the absence of F-ara-EdU, followed by fluorescence labelling of F-ara-EdU using click chemistry
3/9
1
5
1,211
Pleased to introduce our latest preprint, out now: “Cohesin-mediated DNA loop extrusion resolves sister chromatids in G2 phase”. We present new quantitative assays probing the conformation of replicated chromosomes by imaging and high-throughput DNA sequencing. A tweetorial:
1/9
1
51
173
34,514
Sending constructs for whole plasmid sequencing. @plasmidsaurus requests a dinosaur drawing. Two PhD students who definitely weren’t watching ⚽️ while pipetting are happy to oblige:
3
14
The Gerlich lab is excited to have two new joiners - welcome, @viennabiocenter PhD students Inès & Dmitry!
1
11
Finishing off the IMP/IMBA recess - three days of scientific talks from across our institutes - with the Added Dimension talk. The speaker’s identity is closely guarded every year, who’s it gonna be!?
4
Proud to have brought together labs around the @viennabiocenter, and across the Atlantic, on this collaborative effort to understand how DNA looping and chromatin volume compaction cooperate to form mitotic chromosomes! #chromatin #mitosis #condensin #histone #phaseseparation
6/6
1
10
This deAc-driven compaction creates a discrete phase boundary on mitotic chromatin, allowing it to experience pushing forces. We demonstrated this by exposing droplets of fragmented mitotic chromatin to a monopolar spindle, which efficiently pushed droplets away from the pole
5/6
1
1
7
We observed that chromosomes in a hyperacetylated, more dissolved state are invaded by fluorescently labelled microtubules and tubulin subunits. In contrast, tubulin is well excluded by unperturbed mitotic chromosomes, which are correctly deacetylated
4/6
1
6
By injecting live mitotic HeLa cells with AluI, a prolific DNase, we digested mitotic chromosomes into small fragments. Strikingly, this fragmentation transformed mitotic chromatin into spherical droplets, which dissolved in cytosol when we inhibited histone deacetylation
3/6
1
3
11
We use short-term drug treatment + rapid degradation of endogenous protein to describe how 2 processes shape mitotic chromosomes: condensin loop extrusion imparts their cylindrical shape while histone deacetylation compacts chromatin + endows microtubule-resisting properties
2/6
1
1
5
A highlight of our summer at @MBLScience has of course been the collabs. Awesome to link up with groups from around the US / world - amongst others, @lifengchen, @liamholt, Sy Redding and their labs will all be sorely missed!
1
22
Weighing up the relative strengths and weaknesses of various chromosome conformation capture approaches
13
Proud bioinformatician @cchlanger presenting our new visualisation tool @hicognition at today’s @IMPvienna / @IMBA_Vienna PhD & PostDoc day 🧬
1
2
13