Lectures 4-6 Flashcards
Define Chronobiology
A field of biology that examines timing processes, including periodic (cyclic) phenomena in living organisms, such as their adaptation to solar and lunar-related rhythms.
These cycles are known as biological rhythms.
What are the 4 main selective day/night pressures that drive universal biological timers?
Sunlight
Temperature
Predation
Food availability
What two things does have a biological timer allow?
The adaptation to and prediction of changes in our environment.
Name 4 physiological perimeters that are under rhythmic patterns/cycles.
Body Temperature.
Melatonin Secretion
Cortisol Secretion (high during day and low at night).
Urine Production. (High during day and low at night).
What is interesting about the clock mechanism and the differences in it throughout the body?
The core clock mechanism is similar across the body.
However, the genes that control these rhythms are very different; they are tissue specific clock genes.
Where is the SCN in the circadian hierarchy?
At the top.
List 9 things that a disrupted clock increases the chances of.
Sleep Disorders
Metabolic Syndromes
Obesity
Diabetes
Cardiovascular Risks
Stroke
Arthritis
Hypertension
Cancer
What does circadian mean?
About a day.
What was the seminal Jean-Jacques de Mairan (1729) study?
He observed that plants show circadian rhythms and wanted to test if they were solely driven by light or were internally driven.
So he left one plant in a window to receive light information and another in the darkness to observe the effects.
He found that in both conditions there were circadian rhythms; they were internally driven.
What studies into circadian rhythms were done by Nathaniel Kleitman?
Cave Studies - he is known as the father of modern clock research.
He took a student to a cave in Kentucky and they stayed there for more than a month.
They withdrew ALL time cues; no clocks, no light, no temperature cues etc.
They also imposed a 28 hour clock on themselves.
After a month, they revealed that there was about a 24.3 hour rhythm that was maintained in the absence of light.
Who was Jurgen Aschoff and what influential study did he do?
He was one of the 3 founders of chronobiology.
In his study he:
Changed ex-WW2 bunkers into sleep study ‘labs’ as he could measure and control a lot of variables within these.
He tested the following:
- Ion concentration in Urine (CA and K)
- Volume of urine
- Body Temp
- Sleep/wake cycle
The results showed for the first time that there was a ~24 rhythm in humans.
Who were the 3 scientists who won the noble prize in chronobiology and why?
Jeffrey C. Hall
Michael Roshbash
Michael W. Young
They discovered the period gene in drosophila that was in charge of it’s circadian rhythm.
This discovery (and noble prize) solidified Chronobiology as an important field.
What has genome analysis shown us about the basic building blocks of the molecular clock?
They are conserved between all animals.
What did the 1971 study by Konopka and Benzer discover?
The first ever mutant fruit fly with a different periodicity was discovered; called it the period mutant as they couldn’t clone genes at this point (no PCR technology).
*This gene was cloned for the later nobel prize discovery.
What famous discovery happens in 1984 and was done by Hall, Roshbash and Young?
They cloned the Period gene from Drosophila.
What was the finding of the 1988 study by Ralph & Menaker?
They identified the Tau hamster that had a 20 hour period instead of 24 (gene wasn’t cloned yet).
This was the first found mammalian mutation that had a 20 hour period.
What was the discovery of the 1990 study by Ralph & Menaker?
They identified that the SCN was the central pacemaker of the circadian rhythms by transplanting the Tau mutant SCN into a receiver.
This led to a change of rhythms to 20 hours.
They also did the reverse and found that tau mutants could regain a normal rhythm after a WT SCN transplant.
This shows that the SCN carries all the timing information needed to drive internal rhythms.
What did the 1994 Sehgal & Young study discover?
They found the binding partner for period in drosophila; it was called timeless.
What did the 1997 Takahashi study reveal?
The first clock gene found in a mammal.
They found mutating this gene strongly changed the circadian phenotype.
(Did this using mutagenesis in which you mutate all genes of a single type and see if there are phenotypic effects on the rhythm).
What did Hogenesch find in 1998?
He identified the Bmal1 and Npas2 genes which are part of the positive arm of the circadian clock.
They initiate the clocks’ start.
What did Kloss, Price and Young find in 1998?
They identified a Kinase called double time (in drosophila).
This was important because Per and Cry were repressors so they couldn’t start the clock.
To reset the clock, Per and Cry needed to be degraded - which is does by Kinase Mediated Phosphorylation.
What did Menaker and Takahashi discover in 2000?
That the gene behind the tau mutation was Caesin Kinase 1 epsilon (CK1e) which was the ortholog of double time in drosophila.
This is amazing because it shows that the mammalian and drosophila clock use the same system.
What are the 2 genes used in the negative arm of the mammalian and drosophila clock?
Mammalian: Period and Cryptochrome.
Drosophila: Period and Timeless.
What are the 2 alternative names for BMAL1?
ARNTL or MOP3
What is another name for NPAS2?
MOP4
What are the 6 methods used to search for clock genes, starting from least to most modern?
Mutagenesis and behavioural tests in flies and mice.
Cloning of homologous genes.
Searching for interacting partners of known clock proteins.
Accidental Discoveries
Genome-wide RNAi/compound screening in cells.
Machine learning.
Which were the main clock genes discovered using the cloning of homologous genes?
mCrys
mPers
Which were the main clock genes that were discovered using mutagenesis and behavioural tests in flies and mice? (6)
dPeriod
Timeless
mClock
Doubletime
Cycle
Fbx13
Which were the main clock genes found by searching for interacting partners of known clock proteins?
TIME for PER
BMAL1 for CLOCK/NPAS2
What are the two main genes that were found by “accidental” discovery?
CK1epsilonTau.
DBP.
Detail briefly the discover of the first clock gene.
It was Period in drosophila by Konopka and Benzer, 1971.
They used actograms and found 3 mutant lines:
Per0 = arrhythmic.
PerS = short rhythmicity.
PerL = long rhythmicity.
What is the difference in the amount of Period genes in drosophila and mammals?
Drosophila only have 1 period gene.
Mammals have three mutations:
Per1
Per2
Per3
What did the study into mPer and their KO’s reveal?
METHODS:
- Had a WT mouse, a mPer1 KO, a mPer2 KO and a mPer3 KO.
- Tested their phenotype via actograms in sleep wake and then free running in darkness.
RESULTS:
- mPer1/2 lead to a gradual loss of rhythmicity (mPer2 KO is quicker to lose it).
- mPer3 is important in timing sleep but not in rhythms - it’s KO led to normal rhythms.
What did the Double Mutant mPer study show? Describe it and draw conclusions.
METHODS:
- Researchers used compound mutations to cross different combinations of mPer:
- mPer1/mPer3
- mPer2/mPer3
- mPer1/mPer2
- Used light/dark cycle and then constant darkness and measured behaviour with actograms.
RESULTS:
mPer1/mPer3:
- Gradual Loss of Rhythmicity
mPer2/mPer3:
- Gradual Loss of Rhythmicity.
mPer1/mPer2:
- IMMEDIATE Loss of Rhythmicity.
CONCLUSION:
- The inclusion of Per3 into a cross with Per1/2 doesn’t change anything; their rhythms are the same in KO with or without Per3.
- Thus showing that Per3 is not important for rhythms.
- Per1 and Per2 must have a partial redundancy and are VITAL as losing them means immediate loss of rhythm.
Describe the study into the Clockdelta19 mutant.
METHODS:
- Planned to screen mice constantly until they found one with a mutation in clock gene.
- Took 200 mice to find a mutant that they wanted.
- Mutated a single allele or both alleles to see what happened.
RESULTS:
- WT mouse has 23.6 period.
- A single allele mutation in Clockdelta19, the period is longer at 224.8 hours.
- When both alleles are mutated then it has an even longer period of 27.1.
CONCLUSION:
- This occurs because CLOCK loses the ability to activate downstream genes but it can still bind to BMAL1 but there is NO transactivation domain present.
Describe the finding from which researchers found the Clock wasn’t necessary for
Researchers thought that if you knock out CLOCK then you would get a strong phenotype, due to the results of the single and double allele mutations in clock changing rhythmicity.
However, if you knock out CLOCK, the animals have almost perfect rhythmicity.
This shows a big differences between mutant and knock out.
Knock Out will have other genes ‘step in’ and compensate for it.
In this case, it is Npas2 as it can equally bind to Bmal1 and activate the transmembrane domain.
In the mutant, the Clock gene can still bind to Bmal1 but it cannot activate downstream genes due to lack of transactivation domain.
Now, Npas2 cannot do anything as it isn’t filling the EMPTY role of CLOCK; it’s not able to preferentially step in.
What did the Clock Shock Experiment show us?
That if you remove BOTH Clock and Npas2 then you lose rhythmicity.
You cannot afford to lose both to maintain rhythms.
What compensates for loss of Clock in peripheral circadian oscilators?
Npas2.
What are the two most important take aways from the ‘Clock Shock’ Paper?
There can be important differences between a mutant gene product (Clockdelta19) and the absence of a gene product (Clock-/-).
Clock and Npas2 are redundant in the SCN and peripheral tissues.
What did the study into Cry1 and Cry2 KO mice reveal?
When free running in darkness:
- Cry1 KO leads to a shorter periodicity.
- Cry2 KO leads to a longer periodicity.
If both Cry1 and Cry2 are KO then there is an immediate loss of rhythm.
This study shows that they are essential for circadian clock function but they also have some redundancy.
What is arguably the most important clock gene in the circadian rhythm? Why?
BMAL1.
There is NO redundancy and the KO leads to immediate loss of rhythmicity.
What is the importance of CK1epsilon and what is it?
It is an important kinase that regulated clock pace; it targets Per protein for degradation.
It’s also the ortholog of doubletime.
Briefly outline the finding of CK1epsilon tau mutant effect on the clock.
Tau mutant had an internal rhythm of ~4hrs faster than the WT.
Thought that this was a LOF of the enzyme.
Researchers asked: how could a kinase LOF speed up the clock? If anything it would lead to a longer clock because it would inhibit the breakdown of the negative arm proteins (Per and Cry).
Then a later study came.
Describe the PART 1 findings of the Meng et al., 2008 into tau mutations in mice.
Created a tau mutation version of the hamster in a mouse as they are easier to study .
Found that a SINGLE base pair mutation changed the whole rhythm to 20 hours.
Shows the power of a tau mutation.
Outline the PART 2 Meng et al., 2008 study into tau mutant PER2::Luc mice.
METHODS:
- Used a PER2::Luc (luciferase) Tau mutant mouse.
- Tracked the rhythm of the molecular clock in the SCN via PER2 gene expression.
RESULTS:
- Found that the molecular clock is accelerated in the Tau mouse, not just the phenotypic rhythm.
- This can be seen in the bioluminescent expression of Luciferase.
Outline the results of the PART 3 Meng et al., 2008 study into tau mutant PER2 degradation in mice.
If there is a tau mutation, the degradation of PER2 is faster.
Therefore it is a GOF mutation because the enzyme has become MORE active.
The CK1epsilon tau mutation targets Per2 only, not Cry1; it doesn’t matter which mutation you introduce, the degradation of Cry1 was not increased.
Outline the results of the Meng et al., 2010 study into inhibition of casein kinase 1 enzymes.
There is a dose dependent response between the amount of CK1delta INHIBITOR and the length of the clock.
*You need CK1delta for clock rhythm to be maintained.
What are the two nuclear hormone receptors responsible for regulating BMAL1?
RORs and REV-ERBalpha/beta.
What is RORs?
A nuclear hormone receptor that regulates BMAL1
What is REV-ERBalpha/delta?
A nuclear hormone receptor that regulates BMAL1
Describe the process of the Transcriptional Translational Feedback Loop.
In the Morning:
Clock and BMAL1 (the positive arm) bind to the E-box (enhancer box) found in the promoter regions of Per and Cry.
Resulting in Per and Cry being transcribed into proteins, from which they come together and bind to dimerise.
In the Evening:
They come back to the nucleus and they bind to Clock and BMAL1 and form a protein complex.
From here, this supresses the ability of Clock and BMAL1 to activate downstream genes and you then close the loop.
For the next day to start, the repressors, Per and Cry, have to be degraded and this is by kinase mediated phosphorylation.
For Per, it’s CK1.
For Cry, it’s GSK3beta and AMPK.
After these are degraded, the next cycle will start again.
To make this more robust, BMAL1 is fine-tuned via the nuclear hormone receptors.
Name 5 pieces of evidence that support the TTFL model.
Mice lacking functional Pers, Crys, Clock/Npas2 or Bmal1 are arrhythmic.
Mutations affect cycle length (e.g., CK1, Clock).
CLOCK and BMAL1 increase Per/Cry/Rev-erb-a/b expression; PER and CRY inhibit their expression.
CK1 activity determines clock pace.
Rhythmic expression in the SCN and peripheral tissues/cells.
