Week 6: Sororin Mediates Sister Chromatid Cohesion By Antagonizing Wapl

Question 1

What was already known prior to this paper

Answer 1

1. Sororin is required for formation of stable cohesin-DNA interactions (Depletion of sororin results in reduction of cohesin complexes on DNA)
2. So researchers wanted to know if sororin is required for sister chromatid cohesin and if so, how?

Question 2

Wapl vs sororin

Answer 2

1. Wapl: breaks apart cohesin-DNA interactions
   1a. W/o Wapl, sororin can bind cohesin
2. Sororin: promotes stable cohesion-DNA interactions
   2a. DNA replication and cohesin acetylation promote binding of sororin to cohesin
   2b. Sororin displaces Wapl from it’s binding partner pds5

Question 3

Cohesin complexes

Answer 3

1. 3 cohesin subunits
2. ATPases smc1 and Smc3
3. Kleisin Scc1/rad21/mcd1

Question 4

Cohesin in the cell cycle

Answer 4

1. Cohesin complexes are loaded onto DNA before replication
2. Cohesin is established during replication
3. In mitosis, cohesion is dissolved by removal of cohesin
   3a. Step 1: prophase by polo-like kinase 1 and Wapl (at ends)
   3b. Step 2: separase in anaphase (not before because SAC inhibits APC/CDC20)

Question 5

What did FRAP identify:

Answer 5

1. Cohesin binds to DNA much more stably after than before DNA replication….suggests cohesion depends on unidentified event during DNA replication that stabilizes cohesin on DNA

Question 6

What is essential for cohesion establishment (Delete)

Answer 6

1. Acetylation of cohesin by eco1

Question 7

Sororin

Answer 7

1. A substrate of APC/CDC20
2. Active during mitotic exit and G1
3. Essential for cohesin; needed for cohesin to bind to DNA in G2 (needed for stable interactions)
4. When phosphorylated in prophase, it falls off

Question 8

Figure 1A/B

Answer 8

1. To test if Sororin is needed for sister chromatid cohesion during the S phase of the cell cycle.
2. Experiment: HeLa cells were synchronized in S phase and treated with RNAi to deplete Sororin. The cells were labeled with BrdU to confirm S phase status, and cohesion was assessed using FISH targeting chromosome 21.
3. Results: Sororin depletion led to increased separation of sister chromatids, indicating loss of cohesion. This shows that Sororin is essential for maintaining cohesion during DNA replication, supporting its key role in stabilizing cohesin early in the cell cycle.”

Question 9

Figure 1F

Answer 9

1.Sororin-LAP localises to centromere in mitosis
2. Stained: GFP, CREST (kinetochores), DAPI (DNA)
3. Sororin LAP accumulated on chromatin between early S-phase and G2 phase and mostly disappears from chromosomes in prophase (dissociation)
4. Results: sororin associates w cohesin at centromeres (localized at kinetochore)

Question 10

Figure 2A/B

Answer 10

1. To test whether Sororin’s association with chromatin depends on cohesin.
2. Experiment: HeLa cells expressing GFP-tagged Sororin (Sororin-LAP) were depleted of the cohesin subunit Scc1 using RNAi. The cells were then fixed and stained to observe the chromatin-bound levels of Sororin.
3. Results: Depletion of Scc1 significantly reduced the amount of Sororin on chromatin, indicating that Sororin’s binding to chromatin is dependent on the presence of cohesin. This supports the idea that Sororin functions in conjunction with cohesin to maintain chromatid cohesion.”

Question 11

Figure 4B

Answer 11

1. “Objective: To examine the effects of depleting Sororin, Wapl, or both on chromosome cohesion in mitotic cells.
2. Experiment: Xenopus egg extracts were depleted of Sororin, Wapl, or both proteins. Mitotic chromosomes were stained with XCAP-E (condensin marker) and Bub1 (kinetochore marker) to assess the separation between sister chromatids.
3. Results: Depletion of Sororin caused a partial loss of cohesion (increased distance between chromatids), while Wapl depletion resulted in tightly connected chromatids. When both Sororin and Wapl were depleted, the phenotype resembled Wapl depletion alone (tightly connected chromatids), suggesting that Sororin’s primary role is to counteract Wapl’s cohesion-dissolving activity.”

Question 12

Figure 4E

Answer 12

1. “Objective: To visually assess the effects of depleting Sororin, Wapl, or both on chromosome morphology during mitosis.
2. Experiment: Mitotic chromosomes from HeLa cells treated with RNAi to deplete Sororin, Wapl, or both were spread on slides and stained with Giemsa for morphological analysis.
3. Results: Chromosome spreads showed that Sororin depletion caused separated sister chromatids (cohesion defect), Wapl depletion led to tightly held chromatids, and codepletion of both resembled the Wapl-depleted phenotype. This supports the idea that Sororin’s main function is to inhibit Wapl, maintaining sister chromatid cohesion.”