Understanding Circadian Rhythms

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00:00:07.12 So hi everybody, my name is Michael Rosbash,
00:00:09.25 and I'm an investigator of the Howard Hughes Medical
00:00:12.00 Institute, and a professor at Brandeis University in the department
00:00:15.19 of biology, where I've been for 40 years. So, the friendship
00:00:19.25 aspect of this title refers to the fact that I collaborated for
00:00:23.23 more than 20 years with my colleague, Jeff Hall,
00:00:26.07 who arrived at Brandeis at exactly the same time
00:00:28.28 I did. And we formed a fast friendship. Jeff is almost exactly
00:00:33.15 my age, and we had many interests in common. And
00:00:37.03 Jeff had worked as a postdoc for Seymour Benzer,
00:00:40.27 where he met Ron Konopka, where he became fluent
00:00:44.21 with the circadian story and also worked on that himself
00:00:49.15 in his own lab, before I got involved. So that's really how
00:00:52.25 this story began. And Jeff and I, as I indicated, had many
00:00:56.21 interests in common. One -- two of them are shown here,
00:01:00.20 tobacco and alcohol. We also both were great sports fans,
00:01:05.10 and also played basketball for a decade together in a
00:01:09.04 religious-obsession with the noon-time basketball game
00:01:13.01 during that decade. And it was after the basketball games I
00:01:16.00 in the locker room where everyone cools down and showers
00:01:18.12 that we would exchange scientific stories. And Jeff
00:01:21.04 would tell me about his research in circadian rhythms that was
00:01:24.26 ongoing. And it's really that friendship and those tales
00:01:29.22 which were the backdrop for this story. So to tell you
00:01:33.21 just a little bit about circadian biology, as shown here
00:01:37.02 is the wheel running activity of hamsters recorded
00:01:40.23 on an old machine and then plotted as a function of time
00:01:44.08 of day, from the top to the bottom. And all animals have some
00:01:50.02 circadian rhythms, from fungi to man. And they're commonly
00:01:54.22 assayed in constant darkness. And the rhythms, the
00:01:58.17 intrinsic rhythms of animals, are not precisely 24 hours
00:02:01.26 -- they're about 24 hours, a little slower, a little faster.
00:02:05.06 Hence, the term "circadian" or "circa dia," about a day.
00:02:08.10 And shown here, is the rhythm of these rodents in constant
00:02:13.05 darkness, which is a little faster than 24 hours, as indicated by
00:02:17.03 every day starts a little bit earlier than the day before.
00:02:20.11 And in a light-dark cycle, however, shown at the bottom
00:02:24.29 panel, where there's 12 hours of light and 12 hours of darkness,
00:02:28.25 then the animal locks on to the light-dark cycle in the incubator
00:02:32.29 as we lock onto a light-dark cycle from the external world.
00:02:37.15 The set rising and setting of the sun, and temperature cycles
00:02:41.15 can also do this as they accompany the light-dark in the external
00:02:45.02 light-dark cycle. You'll notice here also, that these animals
00:02:49.03 are nocturnal, their activity coincides with the night, they
00:02:52.10 sleep during the daytime, and that's a characteristic of any
00:02:56.06 but not all rodents. Now the star of our show is Drosophila
00:03:01.12 melanogaster, as shown here using a primitive counting
00:03:05.16 apparatus to keep time. This is drawn by my daughter, who is
00:03:09.26 a graphic designer. And here's a picture of Drosophila
00:03:16.26 locomotor activity rhythms, the analog if you like, of the
00:03:20.25 hamster's wheel running activity. And what's shown are the
00:03:24.13 fact that these animals, insects -- most insects, in general,
00:03:28.01 are active at dawn and dusk, with a morning peak
00:03:32.00 and an evening peak of activity. In this case, in an
00:03:34.18 incubator, so the morning peak coincides with the appearance
00:03:39.16 of light, whereas the evening peak coincides with the
00:03:43.01 transition from light to dark. And the arrows refer to
00:03:46.23 the fact that the activity ramps up in advance of those
00:03:50.25 discontinuous incubator transitions, indicating that there's
00:03:54.13 really a timing mechanism that can anticipate the change
00:03:58.07 in the external world's conditions. And anticipation
00:04:01.21 is the name of the game in circadian rhythms. It's
00:04:04.05 more advantageous to know what's going to happen
00:04:07.00 than to know what has just happened. And so that's
00:04:10.27 really what the circadian system contributes to the animals,
00:04:16.22 one of several things. So genetics, in terms of addressing
00:04:21.06 this kind of problem, has had some negative history, especially
00:04:24.18 in the first half of the 20th century. For example, the eugenics
00:04:29.01 movement, in which people of low intelligence, handicapped,
00:04:34.22 or even of some disfavored races at the time, were
00:04:39.08 inhibited from procreating, in some cases exterminated
00:04:43.18 as you all know from Nazi Germany. So this movement
00:04:47.01 gave behavioral genetics, gave intrinsic properties of
00:04:51.18 animals or people, a bad name. And similarly, the psychologist
00:04:58.00 B.F. Skinner, who had a long and distinguished career at Harvard,
00:05:01.16 had a tremendous amount of influence and believed that one
00:05:04.21 could change the behavior of virtually every individual
00:05:08.02 by simply subjecting them to constraining conditions,
00:05:11.19 which would then change their behavior. Meaning
00:05:15.14 that intrinsic behavioral features were not really, were not really
00:05:20.25 fixed. And so, of course there's positive history, especially
00:05:26.06 in the latter half of the 20th century that offsets that preceding
00:05:29.23 negative history. We know that we can breed plants
00:05:32.27 in order to enhance certain characteristics, that's also
00:05:37.18 true for animals, cattle, dogs, etc ... And of course,
00:05:42.03 the 1973 Nobel Prize was awarded to three scientists
00:05:46.03 who really worked on animal behavior as intrinsic
00:05:51.20 to those particular species. So the landscape shifted,
00:05:56.09 I would say, as the 20th century progressed.
00:05:59.08 And so, the question for you and for me, is
00:06:03.09 why use genetics to try and address the problem like
00:06:06.11 circadian rhythms? And I want to emphasize that this is
00:06:09.08 different from the nature/nurture problem, we're not
00:06:11.29 trying to debate how much of circadian biology is inherited,
00:06:15.26 and how much can be environmental. Nor whether
00:06:18.25 you're rhythm is different from my rhythm because of genetic
00:06:21.25 features or environmental features. We're using genetics
00:06:25.15 as an entree into this process. Can we figure out what
00:06:29.11 keeps time by finding mutants which disrupt that process?
00:06:33.22 And therefore will provide access to the genes and the proteins
00:06:37.26 which underlie the process. And to emphasize this distinction,
00:06:42.28 let me point out to you that even learning, the ultimate
00:06:45.26 in nurture, if you like, relies on proteins and therefore,
00:06:51.26 relies on genes. So genetics, genes, proteins are
00:06:55.27 inherent in everything. So I turn now to the studies of Konopka
00:07:00.19 and Benzer, who identified clock mutants of Drosophila
00:07:05.08 melanogaster in this landmark paper published in 1971.
00:07:09.25 So they fed fruit flies mutagens to increase frequency
00:07:14.25 of mutations and then screened those flies for aberrant
00:07:19.25 circadian properties. And they came up with three mutants
00:07:23.19 in single gene, all three were alleles of one gene,
00:07:28.09 which either gave rise to an absent clock, that is no
00:07:33.24 rhythm what so ever, a short period clock, which ran
00:07:37.04 fast, or a long period clock, which ran slow. And all
00:07:41.16 three of those, as I said, were single mutations, single
00:07:45.14 nucleotide changes in the same protein, which they named
00:07:48.28 Period, which suggested of course that this protein had some
00:07:52.08 important role in the timekeeping process. But it was really
00:07:56.02 -- and here's an example of the wild type circadian pattern
00:08:02.00 on the left, or the per short pattern on the right. And the
00:08:06.11 vertical line refers to the fact that the lights were turned
00:08:09.08 off. And you'll notice that the wild type fly, on your left,
00:08:12.18 keeps very good time during the course of the subsequent
00:08:18.01 days, that is almost exactly 24 hours. Whereas the per short
00:08:22.07 mutant strain, every day advances the period by several
00:08:26.25 -- advances the beginning of activity by several hours
00:08:29.07 hours by comparison to the day before. Very similar to
00:08:32.07 the hamster phenotype, as I said before, only more
00:08:35.05 profound, since this animal has a 19 or 20 hour period
00:08:39.12 as contrasted with the almost precise 24 hour period
00:08:43.05 of the wild type. So, how does one get at this problem?
00:08:46.21 And there was really almost a 15 years between the
00:08:51.20 publication of Konopka and Benzer and the appearance of
00:08:55.24 recombinant DNA, which could be applied to this problem
00:08:59.17 where the Period gene could be cloned and sequenced
00:09:02.19 to try to ask what might this gene be doing?
00:09:05.26 And so I was working on recombinant DNA in my laboratory
00:09:09.07 on studies on yeast gene expression, Jeff Hall had not
00:09:13.29 yet adopted this technology. So during one of these
00:09:16.17 basketball games, I suggested to Jeff that we get
00:09:19.12 together, that we collaborate and that we try to clone
00:09:22.14 this gene, our two laboratories, to see if we could
00:09:24.26 figure out what it was doing. And we did that at the same time
00:09:29.05 Mike Young's laboratory at the Rockefeller University also
00:09:32.19 accomplished the same task. And unfortunately, this protein
00:09:36.00 was a pioneer protein, as the expression goes, it
00:09:39.19 had no relatives in the database. These were early days
00:09:42.13 for DNA sequencing, there were a limited number of proteins
00:09:45.23 that had been sequenced and identified, and so we
00:09:48.21 were left still with the question of what this protein
00:09:51.12 did. And it was Paul Hardin in his explorations of the Period
00:09:57.22 gene's expression, where he discovered that the Period
00:10:01.06 messenger RNA underwent strong circadian oscillations
00:10:04.27 in time, so messenger RNA levels increased and decreased
00:10:09.06 by about a factor of 10. With a precise 24 hour period
00:10:12.23 even in constant darkness, and remarkably, shown in
00:10:16.16 blue, the per short messenger RNA, different, I remind you
00:10:22.07 by only one single nucleotide from the wild type or normal
00:10:26.11 strain, had only 20 hours between their peaks, just like
00:10:30.11 its behavior as compared to this 24 hour difference in
00:10:35.17 the wild type. So the RNA reflected the behavior and
00:10:38.23 we proposed that the protein must feed back onto its
00:10:42.23 messenger RNA somehow, and that that was intrinsic
00:10:46.15 and important for the timing process. But we didn't know
00:10:49.07 at what level this occurred. We didn't know whether this
00:10:52.14 was transcriptional in origin, yet the focus on gene expression
00:10:57.10 was something I was very familiar with and knew that the
00:11:00.16 half lives of mRNAs in higher organisms were generally
00:11:05.24 in the hours or even tens of hours range. Something that
00:11:09.11 was perfect for being related to circadian timing.
00:11:13.10 And then a publication in 1988 by Steve Crews and
00:11:16.22 colleagues identified a Drosophila transcription factor
00:11:19.28 named Single Minded, which had homology with the period
00:11:23.13 protein. And that set us off on an expedition to rapidly
00:11:29.28 see if transcription was part of the story.
00:11:33.02 And sure enough, within the next two years, we were able
00:11:35.18 to show through several independent kinds of investigation,
00:11:39.05 that the Period protein was nuclear and it affected transcription
00:11:43.13 and therefore, we could propose that the feedback
00:11:46.11 loop was direct and was transcriptional on origin.
00:11:50.17 And over the subsequent six years or so, the circadian
00:11:54.13 field really exploded. And both identified the positive transcription
00:11:59.08 factor, responsible for the synthesis of these regulators,
00:12:03.16 led by Takahashi and his colleagues in mammalian studies.
00:12:08.10 Followed very quickly by genetic studies in my lab and
00:12:12.06 others. And in addition, people made the leap between flies
00:12:16.23 and mammals, showing for example, that the Period protein
00:12:19.14 was conserved and had a relative, a very close relative in mammals
00:12:25.04 which did very similar, if not identical job. So there was
00:12:29.20 really a grand synthesis, if you'd like, and this story
00:12:33.20 has held up for the 17 years since 1998. And so
00:12:41.15 the question, the summary if you will, is what do I draw
00:12:45.11 as a personal conclusion from this journey of mine?
00:12:49.27 And I would say, both from the nature and nurture
00:12:52.29 standpoint, I've been incredibly lucky, from the nurture
00:12:57.19 point of view, I went to Brandeis almost by accident.
00:13:00.11 It was not a very well-considered decision, and Brandeis
00:13:04.10 has been a wonderful place to do work. I've had great
00:13:07.18 people who have come to my lab. I met Jeff Hall, to collaborate
00:13:11.25 and my colleagues in the administration have been remarkably
00:13:15.10 supportive and allowed me to undertake this thrilling
00:13:20.24 enterprise. The NIH and in particular, HHMI, which has
00:13:28.08 funded me for the last 25 years, have really been a wonderful
00:13:32.25 source of support. HHMI funds people and not projects.
00:13:35.28 Meaning I could more or less do whatever I wanted.
00:13:39.17 And was able to accomplish this. I've had a very supportive
00:13:42.19 family, that despite my obsessions loved me and allowed me to
00:13:48.07 pursue this. And finally, let me emphasize that even from
00:13:51.29 the nature point of view, whatever talents we individually
00:13:55.11 bring to a problem, those talents, what we inherit from our
00:13:59.04 parents, as we say, those parents are not -- those genes are not
00:14:03.13 by choice, we are all victims of accident. In my case,
00:14:08.05 a happy victim. And so it's really good fortune that underlies
00:14:14.01 this story. Thank you very much.

This Talk

Speaker: Michael Rosbash

Audience:

Educators of H. School / Intro Undergrad
Student
Educators of Adv. Undergrad / Grad
Researcher
Educators

Recorded: November 2015

Talk Overview

Michael Rosbash tells the story of how a collaboration with friend Jeff Hall led to the discovery of the genetic mechanisms that regulate circadian rhythms. Circadian rhythms are the physiological and behavioral changes, such as sleep and wakefulness, which occur in response to a 24-hour cycle of light and darkness. Using Drosophila as a model, Rosbash and Hall cloned the circadian timekeeping gene called period. They later demonstrated that period blocks its own transcription through an oscillating negative feedback loop.

Speaker Bio

Michael Rosbash

Michael Rosbash is Professor of Biology at Brandeis University and Investigator of the Howard Hughes Medical Institute. Rosbash uses genetics to understand the underlying mechanisms of circadian rhythms. In 1984, Rosbash, along with collaborator Jeff Hall, cloned the period gene in Drosophila, which led them to discover the transcriptional negative feedback loop of circadian clocks… Continue Reading