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November 20, 2009

Secrets of separation

Surprising complexity underlies the seemingly simple process of chromosome pair separation during cell division

image of chromosomes

Figure 1: Chromosomes assembled in frog egg extracts. Pairs of duplicate chromosomes are normally aligned in parallel along their entire length prior to cell division (center). However, when the regulatory processes underlying this organization are disrupted, the duplicates fail to be resolved (left) or come apart (right).  

When chromosomal DNA duplicates, the resulting identical ‘sister chromatids’ are tethered together by the cohesin protein complex until the onset of mitosis. Cohesin is then released in multiple stages, enabling the sister chromatids to condense into distinct rod-shaped structures as a precursor to separation from each other during cell division.

Several years ago, a team led by Tatsuya Hirano—now at the RIKEN Advanced Science Institute in Wako—identified human protein Wapl as a regulator of cohesin and likely participant in this process of ‘sister chromatid resolution’1. In order to better understand the function of this and other cohesin-modulating proteins, Hirano and postdoctoral fellow Keishi Shintomi have now performed a systematic series of experiments using extracts derived from frog eggs2. “In this experimental system, a protein of interest can be depleted and [the effects of] loss-of-function can easily be analyzed,” says Hirano. “Moreover, wild-type or mutant forms of the protein can be added back into the depleted extracts to test whether they might be able to rescue the loss of function.”

Hirano and Shintomi noted that Wapl and another protein, Pds5, normally load onto chromosomes in a cohesin-dependent manner, and that targeted depletion of either protein impairs release of cohesin complexes. Subsequent experiments helped them to identify specific segments of Wapl that mediate this activity; Wapl variants with mutations in these key domains lost the ability to bind cohesin or Pds5, and therefore failed to restore normal chromatid resolution in Wapl-depleted extracts.

The researchers also gained new insights into Sgo1, a protein that contributes to the staged release of cohesin by protecting a subset of complexes from dissociation until late mitosis. Although Sgo1 was thought to act primarily at the central portion of chromosomes, this protein was observed along the entire length of the chromosome in Wapl- or Pds5-depleted extracts, suggesting that it may also stabilize interactions between the arms of sister chromatids.

Collectively, their findings reveal a bounty of hidden complexity in this important process (Fig. 1). “At first glance, removing cohesin from chromosomes during mitosis looks like a simple task,” says Hirano. “But our current work shows that this process is regulated by an intricate network composed of many positive and negative regulators.” Having delved below the surface of this network, his long-term goal is ambitious: reconstructing the full assembly line of proteins involved in cohesin assembly and disassembly. “This is a big challenge,” says Hirano, “but nonetheless, it has to be done.”

References

  1. Gandhi, R., Gillespie, P.J. & Hirano, T. Human Wapl is a cohesin-binding protein that promotes sister-chromatid resolution in mitotic prophase. Current Biology 16, 2406–2417 (2006).
  2. Shintomi, K. & Hirano, T. Releasing cohesin from chromosome arms in early mitosis: opposing actions of Wapl–Pds5 and Sgo1. Genes & Development 23, 2224–2236 (2009). (Link)