Repost: Controls are Cool

Many of you know that as a post-doc in Uri Alon’s lab, Galit Lahav caused a small revolution in our understanding of how the p53 network responds to DNA damage.  By looking at single cells instead of populations, she showed that individual cells responding to the damage caused by gamma-irradiation show a series of stereotyped pulses (shown in this movie); different cells show different numbers of pulses, and as you increase the amount of damage, the number of pulses per cell increases.  Now the Lahav lab has identified another previously unsuspected feature of the p53 response (Loewer A, Batchelor E, Gaglia G, Lahav G.  2010.  Basal Dynamics of p53 Reveal Transcriptionally Attenuated Pulses in Cycling Cells Cell 142 89-100. PMID: 20598361). It turns out that p53 is being activated in normal growing cells all the time.  Because the cell cycle of cells in culture is unsynchronized, this activation can only be seen by looking at single cells. Since p53 may be the most studied protein on the planet, discovering something completely new and unexpected about its activities isn’t an everyday event.

The story started with an experiment that was originally intended as a control, looking at unstressed cells.  Unexpectedly, in these unstressed, undamaged cells they found p53 pulses that are indistinguishable in shape from the pulses seen in gamma-irradiated cells. The first clue to where these pulses come from was the observation that they’re correlated with specific stages of the cell cycle, primarily happening right after mitosis.  Loewer et al. used a Cdk inhibitor to show that when the cell cycle is stopped, the pulses go away.  And the pulses were also selectively stopped when the ATM/DNA-PK pathway, which monitors double-stranded DNA breaks, was inhibited. It appears that these pulses are triggered by transient DNA damage that is a routine part of the cell cycle.

Why respond with a full-size pulse to damage that must (in healthy cells) surely be minor and easily fixed? Loewer et al. argue that the mechanism for producing p53 pulses must be excitable, like an action potential in a neuron.  If you activate a pulse at all, it has to be a complete one.  They tested this hypothesis by damaging cells, then adding inhibitors of ATM/DNA-PK at different times to shut down the activation.  You can reduce the number of pulses this way, but you have to be quick: if you shut down ATM/DNA-PK after 15 minutes, you can stop most of the pulses.  But if a pulse escapes the shutdown, it’s a normal size and shape.

Now, in gamma-irradiated cells the point of activating p53 is to arrest the cell cycle and give yourself a chance to turn on DNA repair pathways, or, if there’s too much damage, to commit cellular hara-kiri (apoptosis).  Loewer et al. didn’t see either of these events in the unstressed cells, despite the p53 activation.  One key downstream gene is p21, and it turns out that p21 is not activated at all in these spontaneously pulsing cells. Why not? Loewer et al. show that to activate p21 you need both p53 pulses and sustained signaling from the ATM/DNA-PK pathway.  You can set off a p53 pulse with 15-30 minutes of ATM/DNA-PK activation, but you don’t get p21 activation unless the ATM/DNA-PK signal stays on for most of the duration of the pulse (60 minutes or so).

This is rather exciting, because — for the first time — it means that we have a way to pinpoint the difference between p53 that does activate p21, and p53 that doesn’t.  A lot of work has been done to identify post-translational modifications of p53 that are associated with increasing its transcriptional activity, but the ability to switch p53 from an inactive state to an active one by just waiting an extra few minutes before adding an inhibitor is new, and potentially very powerful.  Loewer et al. don’t attempt to do a comprehensive study — this will be a lot more work — but they clearly identify two acetylations on lysines near the C-terminus of the protein that were previously suspected to be associated with activation (K373 and K382) as present in p21-activating p53, and absent in the p53 that fails to activate p21. Using a deacetylase inhibitor, they also showed that artificially increasing the amount of acetylated p53 triggered the activation of p21.  Similarly, an inhibitory methylation (on K382) is removed in p21-activating p53.  So p53 activation takes place in two stages: there is an immediate response to the alarm bells from the DNA damage sensors (a p53 pulse gets triggered), but unless the alarm bells keep on ringing (allowing the activating post-translational modifications to be added and the inhibiting ones to be removed) the fire engines don’t start rolling.

Several new lines of investigation are made possible by these discoveries.  The fact that the p53 pulse is the shape it is suggests that p53’s main regulator, Mdm2, is upregulated even without the post-translational modifications.  Why is Mdm2 activated and p21 not?  Are the modifications Loewer et al. have already found the main ones, or are there additional modifications that make just as large a difference?  Do these basal pulses behave differently in cancer cells?  The whole story is something of a testament to the power of studying individual cells — and, of course, to the fact that it’s always worth looking carefully at your controls.

Loewer A, Batchelor E, Gaglia G, & Lahav G (2010). Basal dynamics of p53 reveal transcriptionally attenuated pulses in cycling cells. Cell, 142 (1), 89-100 PMID: 20598361