Science Signaling Podcast: 12 February 2013

Technical Details

Length: 14 min

File size: 8.0 MB

File Format: mp3

RSS Feed: http://stke.sciencemag.org/rss/podcast.xml

Listen to Podcast: http://podcasts.aaas.org/science_signaling/ScienceSignaling_130212.mp3

Educational Details

Learning Resource Type: Audio

Context: High school upper division 11-12, undergraduate lower division 13-14, undergraduate upper division 15-16, graduate, professional, general public and informal education

Intended Users: Teacher, learner

Intended Educational Use: Learn, teach

Discipline: Cell biology, human biology, molecular biology,

Keywords: Science Signaling, anoxia, cell proliferation, cell stress, DNA replication, HIF-1α, hypoxia, hypoxia-inducible factor 1α, transcription factor

Transcript

Host – Annalisa VanHookWelcome to the Science Signaling Podcast for February 12th, 2013. I’m Annalisa VanHook, and today I’ll be speaking with Gregg Semenza and Maimon Hubbi about a paper that appears in the current issue of Science Signaling (1) on a nontranscriptional role for a protein that’s most well known as a transcription factor that helps cells survive hypoxia.

Hypoxia is a state of limited oxygen availability, and it affects a cell's ability to generate energy. When oxygen becomes limiting, cells initiate a variety of means to cope with the situation. One of the events that hypoxia triggers is activation of hypoxia-inducible factor 1, or HIF-1. HIF-1 is a heterodimeric protein that’s composed of two subunits—an α subunit and a β subunit. Under normal conditions, the α subunit is degraded, but under hypoxic conditions, HIF-1α is stabilized, and the HIF-1 complex acts as a transcription factor to promote the expression of genes that help the cell cope with the low oxygen conditions.

In a study that appears in this issue of Science Signaling, Maimon Hubbi, Gregg Semenza, and their colleagues report that HIF-1α also functions independently of its transcriptional regulatory role to modulate the cellular response to hypoxia. Hubbi and Semenza spoke to me from the Johns Hopkins University School of Medicine in Baltimore.

Interviewer – Annalisa VanHookHello, Dr. Hubbi, Dr. Semenza. Welcome to the Science Signaling Podcast.

Interviewee – Gregg SemenzaThank you.

Interviewee – Maimon HubbiThanks.

Interviewer – Annalisa VanHookHIF-1α is best known as a transcription factor—or a subunit of a transcription factor—that promotes the expression of genes that help a cell survive oxygen deprivation. In this paper, you report that HIF-1α also functions independent of this role in regulating gene transcription. How did you identify that nontranscriptional role for HIF-1α? Gregg?

Interviewee – Gregg SemenzaWe’ve been interested in identifying proteins that interact with HIF-1α for quite some time. We’ve been studying this for about 10 years or so based on the assumption that interacting proteins would either regulate HIF-1α or, alternatively, they might be regulated by HIF-1α. And when we first started these studies more than a decade ago, the technology that we utilized was the yeast two-hybrid assay. And using that technique, we identified a protein known as MCM7 that interacted with HIF-1α. And then more recently, we’ve taken a proteomics approach and incubated human cell lysates with a bacterially expressed GST-HIF-1α fusion protein and used that to pull down interacting proteins, which were then identified by mass spectroscopy. And one of those proteins that were identified by that method was a protein called MCM3. So we had identified these two MCM proteins by these different methods. And there are a total of six MCM proteins—MCM standing for “minichromosome maintenance” proteins—that were first identified in yeast and shown to function as a DNA helicase that’s required for DNA replication. And we found that the interaction of HIF-1α with MCM7 stimulated the oxygen-dependent ubiquitination and proteasomal degradation of HIF-1α, whereas the interaction with MCM3 inhibited the ability of HIF-1α to activate transcription. And then we also showed that MCM2 and MCM5 were negative regulators of HIF-1 activity as well. And so this work, which we published in Molecular Cell in 2011 with Maimon as the first author (2), established a direct connection between HIF-1α and components of the DNA replication machinery, and it really set the stage for our more recent study in Science Signaling.

Interviewer – Annalisa VanHookIn this Science Signaling paper, you identified a nontranscriptional function of HIF-1α. What is that nontranscriptional function, and is that also regulated by hypoxia?

Interviewee – Gregg SemenzaSo, it’s known that hypoxia inhibits the proliferation of most cell types, because if oxygen is limiting, then it makes no sense to generate more cells that will consume more oxygen. Based on that, we hypothesized that just as the components of the DNA replication machinery inhibit the function of HIF-1α, then, in a reciprocal manner, HIF-1α might inhibit the function of the DNA replication machinery under hypoxic conditions.

Interviewer – Annalisa VanHookDoes HIF-1α inhibit cell division under hypoxic conditions? Maimon?

Interviewee – Maimon HubbiYeah, so after we found the interaction with HIF-1α and the MCM proteins, I looked at other proteins that are involved in DNA replication and asked whether they can bind to HIF-1α and found that Cdc6, which is important for loading the MCM helicase onto DNA, also interacted with HIF-1α. And then I wondered whether it had an effect on the interaction with the MCM complex, and it seemed to increase the binding. So you could hypothesize that that leads to increased MCM loading, but, at the same time, Cdc6 needs to come off for activation to proceed, so activation was blocked, and that’s exactly what we saw in our experiments. So that established that HIF-1α actually binds to these proteins and blocks DNA helicase activity and DNA replication.

Interviewer – Annalisa VanHookHIF-1α inhibits the activity of a DNA helicase that’s required for replicating DNA, which is obviously important for cell division, and so that’s how HIF-1α inhibits cell division under hypoxic conditions.

Interviewee – Maimon HubbiYeah, that’s exactly right.

Interviewer – Annalisa VanHookAre both the transcriptional regulatory activity of HIF-1α and this activity of inhibiting DNA replication necessary for the cells to survive hypoxia?

Interviewee – Gregg SemenzaSo that’s a good question…

Interviewer – Annalisa VanHookGregg Semenza

Interviewee – Gregg Semenza…but it’s really a difficult one to answer experimentally because it’s not been possible to eliminate the interaction of HIF-1α with components of the DNA replication machinery by making mutations without inhibiting its transcriptional activity, which would be necessary to answer that question. So I think that’s one that I think still remains to be determined.

Interviewer – Annalisa VanHookRight. You would have to identify the portion of HIF-1α that’s interacting with the replication machinery and then specifically alter the protein in those places.

Interviewee – Maimon HubbiYeah, that’s right…

Interviewer – Annalisa VanHookMaimon Hubbi

Interviewee – Maimon Hubbi…and one other issue is that HIF-1α seems to bind to so many of these proteins involved in DNA replication, and they bind to different sites on HIF-1α, so it’s a little bit difficult to make a point mutation or a small deletion that would just affect the binding of HIF-1α to the DNA replication machinery.

Interviewer – Annalisa VanHookHow about HIF-1α–independent mechanisms for shutting down cell division in response to hypoxia? Or does HIF-1α mediate all of the responses to hypoxia?

Interviewee – Maimon HubbiSo, in most cell types, HIF-1α is essential. There has been work in certain cancer cell lines, where there is a HIF-independent effect on cyclin D, and that also causes cell cycle arrest in certain cancer cell lines.

Interviewee – Gregg SemenzaThe other issue is that under very low oxygen conditions—so, near anoxic conditions—

Interviewer – Annalisa VanHookGregg Semenza

Interviewee – Gregg Semenza…there are other mechanisms that will block cell proliferation, but that’s, I think, distinct from sort of physiological levels of hypoxia.

Interviewer – Annalisa VanHookEssentially, then, HIF-1α can inhibit cell division in response to hypoxia in two ways: It can activate the transcription of genes that can shut down the cell cycle, but it can also directly affect the cell cycle by shutting down DNA replication. Is that direct response—that fast response—more important to the response to hypoxia than the gene transcription regulatory role? Maimon?

Interviewee – Maimon HubbiSo, in our experiments, we found that HIF-1α was able to inhibit DNA replication on a very fast time scale—within just a few hours of exposure to hypoxia. So that’s a timescale that’s earlier than what you’d expect from something involved in gene transcription, but you’re right that later on during chronic hypoxia, that’s when changes in gene transcription might also have an effect. So there seems to be a difference in the acute response hypoxia and chronic response to hypoxia and also the response to physiological levels of hypoxia and anoxia.

Interviewee – Gregg SemenzaAnd, in general, when we look at responses to hypoxia, it’s very common to see acute responses that don’t require changes in gene expression and then changes in gene expression that result in more durable changes in the phenotype of the cell. What’s different here is that, you know, in most of the other examples, HIF-1 is controlling the response to the chronic hypoxia, but the acute response is mediated in some other way, whereas in this case, HIF-1α is acting in a way that it can mediate an acute response to hypoxia through a nontranscriptional mechanism. And that’s what makes this a fairly, so far, unique role for HIF-1 in the response to hypoxia.

Interviewer – Annalisa VanHookHIF-1α, I think, most people think of first and foremost as a transcription factor, but now you’ve shown that it has this other function independent of its transcriptional regulatory activity. There are other proteins that double as transcription factors and do something else in the cell as well. In the case of HIF-1α, which do you think came first: its ability to regulate gene transcription or its ability to inhibit DNA replication? What I am kind of asking is is HIF-1α a transcription factor that acquired the ability to inhibit DNA replication, or was it a cell cycle inhibitor that evolved the ability to activate transcription?

Interviewer – Gregg SemenzaThat’s a very good question, and I think that the way to address that would be to look at some of the very simple metazoan species. For example, it is known that pretty much in all metazoan species, HIF-1 regulates metabolism through its role as a transcription factor, and it would be very interesting to see whether cells from simple metazoan species would also have this property of arresting DNA replication through this mechanism. So that’s a question that’s probably pretty easily answered experimentally, but we don’t have any data addressing it yet.

Interviewer – Annalisa VanHookHow common are proteins that act both as transcription factors and have some other role in the cell, either in the nucleus or in the cytoplasm, but just something in addition to their transcriptional regulatory role?

Interviewee – Gregg SemenzaWell, this is probably no different for transcription factors than for any other proteins. You know, we only have less than 25,000 protein-coding genes that have to do everything in the human body, and so it’s very likely that most proteins have more than one role. And, you know, there’s certainly a growing number of examples of that. We showed, for example, that a glycolytic enzyme, pyruvate kinase M2, also functions as a transcriptional coactivator of HIF-1. And there are many examples like this where proteins with enzymatic activities are being shown to act as transcription factors, where transcription factors are being shown to have other roles within the cell. And when you think about it, HIF-1α levels are induced by hypoxia, and, you know, by interacting with other proteins, this provides a mechanism to regulate the activity of many other proteins within the cell, so I suspect that this paradigm that we’re establishing here of HIF-1α interacting with proteins and having a nontranscriptional effect on cell responses to hypoxia, we’ll find many other examples that fit this paradigm in the future.

Interviewer – Annalisa VanHookThanks to both of you for speaking with me.

Interviewee - Gregg SemenzaOur pleasure.

Interviewee - Maimon HubbiThank you.

Host – Annalisa VanHookThat was Maimon Hubbi and Gregg Semenza, authors of a paper published in the current issue of Science Signaling. That paper is by Hubbi and colleagues, and it's titled “A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication” (1).

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And that wraps up this Science Signaling Podcast. If you have any questions or suggestions, you can write to us at sciencesignalingeditors{at}aaas.org. This show is a production of Science Signaling and of AAAS—Advancing Science, Serving Society. I'm Annalisa VanHook, and on behalf of Science Signaling and its publisher, the American Association for the Advancement of Science, thanks for listening.