Lecture 4- Genetic components of the mammalian molecular clock

Question 1

DBP

Answer 1

“classic” clock output gene, rhythmic expression of which was identified by Shibler (150x stronger in day vs night)

Question 2

Pleiotropic effects of clocks

NB pleiotropy is where one gene has one or more seemingly unrelated phenotypic effects

Answer 2

sleep/ wake, cell cycle progression, DNA damage , metabolism, energy metabolism, immune responses

Question 3

How much of the genome is under circadian control?

Answer 3

10%

Question 4

Disruptions in clock genes are related to..

Answer 4

Sleep disorders, mental health problems, obesity, cancer CV problems

Question 5

What makes the SCN unique?

Answer 5

The only somatic clock that receives light input (NB neurons types in the SCN are not unique)

Question 6

How many cells are in the SCN?

Answer 6

20,000

Question 7

SCN output

Answer 7

behavioural, hormonal, peripheral opscillators

Question 8

Examples of cells that do not possess clocks?

Answer 8

Embryonic stem cells and germline cells

Question 9

What happens to the cellular clock if a cell reverts back to its embryonic state?

Answer 9

Cellular clocks are lost

Question 10

Mutagenesis and genome wide screening

Answer 10

… have been to show that the basic building blocks of clocks are highly conserved between animals

Question 11

Other than mutagenesis and GWS, what methods have been employed to find clock genes?

Answer 11

Behavioural studies in mice and flies

Question 12

How was the gene PERIOD identified?

Answer 12

PERIOD1,2,3 identified by mutagenesis in Drosophila, then cloned by homology in mammals

Question 13

How was the gene CLOCK identified?

Answer 13

Mutagenesis in mammals

Question 14

How was the gene BMAL1 identified?

Answer 14

As an interacting protein (with CLOCK) in mammals

Question 15

How was CRY identified?

Answer 15

CRY1,2 identified by clock relevant mutagenesis in Drosophila

Question 16

How was Casein Kinase 1 ε (CK1ε) identified?

Answer 16

As a spontaneous tau mutation (hamster)

Question 17

CK1ε function

Answer 17

Binds to and phosphorylates PER proteins, both of which leads to their degradation and prevents them from being able to enter the nucleus and suppress the transcription of clock genes

Question 18

BMAL1 and CLOCK w/r/t PER and CRY

Answer 18

bind to the Ebox elements of the nuclear DNA, but PER and CRY do not, instead they bind directly to BMAL1 and CLOCK

Question 19

Phosphorylation of PER and CRY…

Answer 19

… targets them to the 26s proteasome for degradation (as well as causing them to detach from BMAL1 and CLOCK)

Question 20

Where are clock regulatory elements found?

Answer 20

In promoter regions of downstream genes

Question 21

REV-ERB proteins

Answer 21

Nuclear receptors and transcriptional repressors of BMAL1

Question 22

ROR proteins

Answer 22

Nuclear receptors and transcriptional activators of BMAL1

Question 23

Mutations in CK1ε

Answer 23

Alter cycle length (HOW)

Question 24

What are the core clock genes?

Answer 24

CLOCK, BMAL1, PER, CRY, REV-ERBalpha, ROR

Question 25

PER KO studies

Answer 25

Gradual loss of rhythmicity in PER1/2 KOs but not in PER3 KOs; PERs are essential but PER3 is unlikely to be involved in locomotor behaviour, more likely sleep/wake regulation; paired PER1/3 and PER2/3 KOs have indicated partial redundancy between PER1 and PER2

Question 26

CRY KO studies

Answer 26

in DD, KO of CRY1 or CRY2 = free running wherease a double KO = arrhythmic –> partial redundancy but are essential

Question 27

CLOCK KO studies

Answer 27

CLOCK is non essential and mice w/o exhibit normal circadian rhythm

Question 28

Which TF makes CLOCK partially redundant?

Answer 28

NPAS2

Question 29

Only clock gene with no redundancy and is completely essential

Answer 29

BMAL1

Question 30

Tau (C->T) mutation in CK1ε

Answer 30

More active degrader of PER2 –> speeds up clock by ~3hours

Question 31

CK1delta inhibitors

Answer 31

increase the period of circadian clocks

Question 32

Modulation of REV-ERBalpha

Answer 32

resets the circadian clock in a phasic manner

Question 33

Lithium

Answer 33

increases amplitude and period of circadian clock

Question 34

Non-pharma improvement of circadian rhythm

Answer 34

regular meal times, scheduled exercise, light therapy