Lecture about paper: Epigenetic programming by maternal behavior

Question 1

There are natural variations amongst maternal care of rats. What variations are there?

Answer 1

High and low licking and grooming (and arched back nursing)

Note: from now on licking and grooming is LG.

Question 2

Fill in:

A normal stress system has a high or low sensitivity for negative feedback.

Answer 2

A normal stress system has a high sensitivity for negative feedback.

Question 3

Explain the genetic pathway that occurs in a baby mouse that has high LG.

Answer 3

High LG results in a healthy stress system where:

• Serotonin (5-HT) is produced that binds to its receptor (5-HT7).
• downstreams cAMP is activated, which activates PKA.
• PKA phosphorylates effector proteins/activators (e.g. CBP) of transcription factors
• CBP binds and activates the transcription factor NFGIA
• NGFIA is able to bind to the noncoding I_7­ promotor
• transcription of GR (glucocorticoid receptor)

Question 4

What is the result of the increased expression of GR as a consequence of high LG?

Answer 4

Low corticosterone levels, low anxiety, high licking or grooming.

Question 5

Explain the genetic pathway that occurs in a baby mouse that has low LG.

Answer 5

In baby mice with low LG, the noncoding I_7­ promotor is methylated. This prevents that the transcription factor NGFIA can bind to this promotor and this prevents/reduces the expression of GR.

Question 6

What is the result of the decreased expression of GR as a consequence of low LG?

Answer 6

High corticosterone levels, high anxiety, low licking and grooming

Question 7

What is parental or epigenetic imprinting?

Answer 7

Epigenetic changes that are already present in the DNA of the cell’s organism. This causes genes to be expressed in a parent-of-origin-specific manner.

Question 8

Exon I_7­ has several areas that can be methylated. But when is methylation only possible?

Answer 8

Methylation is only possible when cytosine is followed by guanine (CpG island)

Question 9

Two regions of the I_7­ exon are of interest in this article. The 5’ (site 16) and 3’ (site 17) end of the CpG island, seen in the picture. Are there differences in methylation in rats with low or high LG?

Answer 9

Yes, it is seen that the 5’ end or region/site 16 is highly methylated in rats with low LG and almost not methylated in rats with high LG.

3’ end or region/site 17 is methylated in both high and low LG rats. This would mean that methylation of this region, doesn’t interfere with binding of the transcription factor.

Question 10

What is seen here?

Answer 10

Methylation is also seen in other (15) CpG regions of the I_7­ exon, for high and low LG rats. But methylation is highest for the low LG rats.

Question 11

Why can’t you just conclude based on the fact that you see differences in methylation in the I_7­ exon, that this is the cause of differences in stress levels of rats?

Answer 11

Beause in between epigenetic changes and behavior, are more steps, like mRNA, proteins etc.

Question 12

What is done in this study to confirm (or deny) that epigenetic changes are the cause of changes in stress level and are caused by the behavior of the mother?

Answer 12

Cross fostering, where the litter from high LG mothers are placed with low LG mothers. If the methylation is dependent on the behavior of the mother, you would expect that the situation created by a low LG mother is restored by the high LG mother.

Question 13

Can the conclusion of this study be based on solely cross fostering of litter from high LG mother placed with low LG mother?

Answer 13

No, you need to check for causality because high LG mother rats might not recognize adopted litter, so you need a control where the litter of high LG mother is placed with another high LG mother.

Question 14

What can you conclude based on this picture, where the effect of cross fostering on methylation patterns is measured?

Answer 14

Based on the graph of region 16/5’ CpG, you can state that methylation is dependent on the mothers behavior.

Question 15

What’s seen in this picture where they measure methylation patterns during embryonic and pup development?

Note: no cross-fostering is performed here

Answer 15

• E20 = embryonic day 20, P1 = first dat of life out of the womb, P90 = adult
• The region 17/3’CpG is not interesting, region 16/5’CpG is interesting.
• You see that in region 16, there’s no methylation during pregancy (E20). Once they’re born (P1) all of the rats have methylation and on P6 there’s a difference between the two groups of high and low LG pups → probably due to the mother’s behavior.

Question 16

What is de novo methylation?

Answer 16

Automatic methylation immediately after birth

Question 17

What’s the next step after cross fostering and measuring methylation levels during and after birth?

Answer 17

Investigating whether the level of methylation have an affect on protein level and whether this has an effect on hormone (cortisol) levels.

Question 18

What’s seen in regard to protein density when you compare low and high LG pups?

Answer 18

That the GR protein density from rats raised by low LG mother is way less compared to rats raised by high LG mothers.

Question 19

Can you conclude based on this picture that epigenetic modifications influence protein density?

Answer 19

No, because there are more ways to influence protein density besides epigenetic modification.

Question 20

What’s used to study whether epigenetic modifications do influence protein density (and to confirm for causality)?

Answer 20

Demethylating everything by TSA (note that TSA is administered to rats via a certain tube on the brains while they’re alive → gives stress, so therefore a control (vehicle) is also used.

Question 21

What’s seen in regard to protein density when TSA is used to delete (as much as possible) methylation?

Answer 21

That removing methylation does influence protein density. The vehicle/control still has lower levels of protein density (probably due to methylation), while the the removal of methylation by TSA increases protein density in both groups.

Question 22

So we just confirmed that protein density is affected by methylation (by comparing it with protein density where TSA was used to remove methylation). What is the last step in this study?

Answer 22

To measure hormone concentration during stress

Question 23

What is expected in rats raised by low or high LG when stress is induced by putting them in a small plastic box where movement is restricted?

Answer 23

• In rats with high LG in vehicle and TSA, it’s expected that they have a healthy stress system. So stress should go down, when the plastic box is removed.
• In rats with low LG in vehicle and TSA, it’s expected that they don’t have a good stress system. So stress is expected to stay high, even when the box is removed.

Question 24

What is seen in rats raised by low or high LG when stress is induced by putting them in a small plastic box where movement is restricted?

Answer 24

See picture

Question 25

What explanation is there for low LG rats that exhibit high stress levels even after the stressful event has gone away?

Answer 25

Low LG rats have more methylation → less protein (GR) → less molecules for sensitive negative feedback system → higher and longer stress

(Baseline of stress of low LG rats is as high as the peak stress of high LG rats → almost twice as high)

Question 26

What is the expectation if the methylation is removed in low LG rats and the same stress experiment is performed?

Answer 26

The expectation is that the amounts of GR will increase and that the stress system will work the same as in high LG rats. This is the case, low LG rats have a normal stress sytem when methylation is erased.