What’s Chromatin Got to Do With It?

Alisa Moskaleva


We know that cells lead intricate lives of growth, change, and division. We also know that DNA has not only the four letters A, T, C, and G, but also an intricate grammar of modifications on DNA-associated proteins, termed chromatin, that changes over time. We can surmise that there is a connection between the life cycle of a cell, called the cell cycle, and its chromatin. But how does the cell cycle influence chromatin? Yang Xu and colleagues shed new light on this question in a paper in the latest issue of the Cell Cycle.


Before a cell can divide, it must first condense its chromatin into packages called mitotic chromosomes, so that its genome may be evenly divided between its two daughters. One of the chromatin modifications that promotes this condensation is the deubiquitination of histone H2A. It’s been known for six years that a protein called Ubp-M can deubiquitinate histone H2A. Now Xu and colleagues explain what causes Ubp-M to deubiquitinate histone H2A before mitosis and not at other times in the cell cycle.


Xu and colleagues focused on a phosphorylation on the 552nd amino acid, a serine, of Ubp-M. This serine is in a motif that a kinase called CDK1 likes to phosphorylate. CDK1 is to the cell cycle what a conductor is to the symphony orchestra: it coordinates all the events, so that they happen in the right sequence and at the appropriate time. By knocking down CDK1 and using chemical inhibitors, Xu and colleagues established that CDK1 indeed phosphorylates Ubp-M on its serine 552.


Phosphorylation changes interactions between proteins. To find the function of the phosphorylation of serine 552, Xu and colleagues looked at the interaction between Ubp-M and a nuclear exporter called CRM1. This is a particularly interesting interaction because Ubp-M spends most of the cell cycle in the cytoplasm, even though it must go to the nucleus to deubiquitinate histone H2A. Therefore, Ubp-M is actively exported from the nucleus, and Xu and colleagues used an inhibitor of CRM1 to show that CRM1 participates in this export. Interestingly, a mutant version of Ubp-M that cannot be phosphorylated on the 552nd amino acid does not get exported as much. This mutant version also decreases cell proliferation and reduces the number of cells that enter mitosis. However, the mutation has no effect on the ability of Ubp-M to deubiquitinate histone H2A. Since CDK1 becomes more active before mitosis, Xu and colleagues propose that it phosphorylates Ubp-M on serine 552 and increases the fraction of Ubp-M in the nucleus, thus promoting chromatin condensation and mitosis.

Serine 552 of Ubp-M is present in primates but is not conserved in the mouse or rat homolog of Ubp-M. Though this particular example of temporal control using phosphorylation and localization occurs in only a few animal species, the principle is likely more general. Moreover, Ubp-M may contain other more conserved phosphorylation sites that function in the same way. And it is intriguing to speculate what special function this phosphorylation may serve in primates. Regardless, Xu and colleagues flesh out a direct connection between the cell cycle and chromatin modification to a rare level of detail.

DNA Damage, Cell Division, and Human-like Mushrooms

Alisa Moskaleva

Biologists often use yeast, fruit flies, mice and other so-called model organisms to understand humans, but how about using humans to understand mushrooms? In a recent paper, Carmen de Sena-Tomás and colleagues show that when it comes to the recognition of DNA damage, mushrooms are quite human-like. In addition to underscoring the relatedness of all living things, their discovery may help biologists understand how all eukaryotes control cell division.

Humans served as a model organism here because as part of the Human Genome Project in June 1995 a team led by Yosef Shiloh discovered the genetic cause of ataxia telangiectasia. This rare childhood disease is characterized by uncoordinated movement, weakened immune system, and, importantly, increased predisposition to cancer. The team named the gene ATM for “ataxia telangiectasia mutated.” A year later, Karlene Cimprich and colleagues published the discovery of a related gene that goes by the name ATR for “ataxia telangiectasia mutated and Rad3 related”. Mutations in ATR cause Seckel syndrome, an extremely rare disease that among its many symptoms also has predisposition to cancer. Cancer arises more frequently in patients with ataxia telangiectasia and Seckel syndrome because they suffer from more mutations to tumor suppressors and oncogenes as a result of DNA damage that goes unrecognized and unrepaired. The protein products of ATM and ATR genes recognize DNA damage and also prevent mitosis in the presence of high levels of damaged DNA, which can be lethal or, if the daughter cells survive, can produce still more carcinogenic mutations.

If physicians hadn’t documented these human diseases and geneticists hadn’t gone looking for the causative genes, it is doubtful whether biologists would have ever stumbled upon what is now known as the DNA damage checkpoint, consisting of ATM, ATR, and downstream proteins. Since the discovery in humans, the DNA damage checkpoint has been found in many organisms including yeast and mice. Carmen de Sena-Tomás and colleagues decided to look at Coprinopsis cinerea, a model system of mushrooms.

At first glance, mushrooms and humans are nothing alike. Though both are multicellular eukaryotes with differentiated tissues, mushrooms are threadlike scavengers that crisscross the soil with their networks of hyphae, feed by releasing digestive enzymes into their surroundings, sprout the recognizable stalk-and-cap fruiting bodies, and practice a curious version of mitosis. A mushroom cell has two haploid nuclei that do not fuse, replicate separately, and divide synchronously, so that each daughter cell once again has two haploid nuclei. Carmen de Sena-Tomás and colleagues wondered if the DNA damage checkpoint known to regulate human mitosis could be regulating mushroom mitosis as well.

They found orthologs of ATR and a target of ATR called Chk1 in the recently sequenced Coprinopsis cinerea genome. When they perturbed the function of the mushroom ATR and Chk1, they found increased sensitivity to DNA damage and increased number of abnormal mitoses where the two haploid nuclei would divide at different times and daughter cells would receive more or fewer than two nuclei. These results lead to two important conclusions. First, ATR and Chk1 in mushrooms recognize DNA damage, just like in humans and many other organisms. Second, ATR and Chk1 prevent abnormal mitosis.

Carmen de Sena-Tomás and colleagues speculate that ATR and Chk1 prevent abnormal mitosis by sensing whether or not the mushroom cell has completed DNA replication and repair. Since the DNA damage checkpoint is so similar in mushrooms and humans, it is tempting to propose that all eukaryotes use ATM and ATR to prevent mitosis before the completion of DNA replication and repair. Though tempting, this is not established. Carmen de Sena-Tomás and colleagues have no direct evidence. The most direct evidence comes from experiments with purified ATR and damaged DNA, which may or may not accurately represent the situation inside living cells. Moreover, there is opposing evidence from yeast that the DNA damage checkpoint does not react to the low levels of DNA damage present in most cells before mitosis. And further evidence points at proteins distinct from ATM and ATR and collectively known as the DNA damage tolerance pathway as the guardians of mitosis from DNA damage. That’s why the effect of the DNA damage checkpoint on mitosis in mushrooms discovered by Carmen de Sena-Tomás and colleagues is important. It doesn’t provide a complete answer about what happens in all eukaryotes, but it should reinvigorate the debate.

Stick a PIN in it

Nicole Crown

Post-translational modification (PTM) of proteins is an essential cellular process used to regulate protein function and stability.  PTMs are assumed to have an impact on protein structure and conformation, but the effects of specific protein conformations on biological processes are difficult to understand and test in vivo.  The importance of protein conformation is of particular interest in DNA repair as many of the same proteins are involved in context specific repair processes.  For example, the Mre11-Rad50-Nbs1 complex is an essential DNA double-strand break sensor that is found at every type of double-strand break.  It has been proposed that this complex can, in theory, adopt up to 216 conformational states and acts as a “molecular computer” to detect damage and regulate repair pathway choice (i.e. should the break be repaired via nonhomologous end joining or homologous recombination)1In vivo studies of the roles that particular protein conformations play are rare, yet critical for understanding the regulation of DSB repair.

New work from Steger and colleagues2 shows that Pin1, a prolyl isomerase that catalyzes cis/trans isomerization, binds multiple DNA repair proteins, including CtIP, a key player in double-strand break end resection.  The authors show that the interaction between Pin1 and CtIP is induced by phosphorylation of CtIP at two S/T-Proline sites, and that Pin1 binding does indeed cause a conformational change in CtIP.  This conformational change leads to polyubiquitylation of CtIP and subsequent degradation. Overexpression of Pin1 causes hyporesection and a decrease in homologous recombination; similarly, depleting Pin1 causes hyperresection and decreased NHEJ.  These data and others presented in the paper lead to a model in which CtIP is phosphorylated, causing Pin1 to bind and isomerize CtIP.  This isomerization leads to ubiquitin-mediated CtIP degradation and appropriate end resection.  Pin1 overexpression leads to reduced CtIP activity and therefore hyporesection and decreased homologous recombination, whereas Pin1 depletion leads to increased CtIP activity, hyperresection and decreased NHEJ.

In this particular case, the researchers were lucky to find a specific protein, Pin1, that induces a known conformational change, cis/trans isomerization, by acting directly on the substrate protein. However, most conformational changes may be caused indirectly by the effects of post-translational modifications.  While the identification of Pin1 as a critical regulator of DNA repair is a large step forward in understanding the role protein conformation plays in function, this same understanding for other proteins will require more cross-disciplinary studies that are able to modify protein conformation in vivo and determine the biological outcomes.

1. Williams, G. J., et al. (2010). “Mre11-Rad50-Nbs1 conformations and the control of sensing, signaling, and effector responses at DNA double-strand breaks.” DNA Repair 9(12): 1299-1306.

2. Steger, M., et al. (2013). “Prolyl Isomerase PIN1 Regulates DNA Double-Strand Break Repair by Counteracting DNA End Resection.” Molecular Cell 50(3): 333-343.