Lecture 8 - Weaver paper Flashcards
1
Q
What is the background of the Weaver paper?
A
- concerns the mechanisms whereby maternal effects or other forms of environmental programming are sustained over the lifespan of an animal
- Previously known:
- behaviour of mothers: licking, grooming, arched back attentive (high/low)
- High LG-ABN - offspring less fearful, most HPA responses
- Low LG-ABN - offspring more fearful, increased HPA responses
- different offspring have different levels of GR
- glutocortocoid is produced via the Hypothalamic-pituitary-adrenal (HPA) axis
- tactile stimulation acts by serotonin
- offspring of high LG-ABN mothers have enhaced GR expression that persists into adulthood
- behaviour of mothers: licking, grooming, arched back attentive (high/low)
2
Q
What is the Hypothalamic-pituitary-adrenal (HPA) axis? How does this related to the background of the Weaver study?
A
- Stress releases hypothalamic corticotropin-releasing factor (CRF)
- CRF acts on the pituitary-adrenal system
- This then releases glucocorticoid (steriod hormone - has lots of different activities in the organism)
- Elevated levels of GR (glutocortocoid receptor) enhances glucocorticoid negative feedback, resulting in a dampening down of this response
Already knew that the LG-ABN and HG-ABN animals have differnt levels of GR
3
Q
Where releases glutocortocoid receptor?
A
- Glutocortocoid is released from the adrenal gland
- GR are found on the vast majority of mammalian cells
- This study looked in the hypothalamus
4
Q
How is tactile stimulation though to act on GR expression?
A
- Acts via serotonnin
- Tactile stimulation activates the serotonin pathway
- Results in the activation of NGF1-A (TF) which binds to the binding site in the GR gene resulting in higher levels of GR
- offspring of high LG-ABN mothers have enhanced GR expression that persists into adult hood
5
Q
What was the aim of the Weaver paper?
A
How is the high expression of high LG-ABN maintained throughout life
6
Q
What did the first figure (Fig 1a.) demonstrate and how was this acheived experimentall?
A
- Knew: use of the GR gene 17 promoter is increased in offspring of high LG-ABN mothers (where NGF-1 TF binds)
- Sodium bisulfite analysis of cytosine methylation status of the exon 17 GR promoter in adult offspring
- looked at % methylation of these sites
- offspring of low LG-ABN mothers have a higher % of methylation
- Looked at the NGF1 binding site, 5’ and 3’ positions
- big difference in C methylation in low LG-ABN (high methylation) compare to high LG-ABN (low methlation) in 5’ site but not 3’ site
Presentation of Data: 5 of methylation at each site
- extract DNA -> PCR region of interest -> cloned individual PCR strands -> into plasmid vector -> sequence
- Individual strands become % at each site
-
lose a bit of information
- doesn’t make it clear whether at these sites they were 100% methylated or 0% methylation because all has been grouped together
- instead use filled circle visualisation
- shows some strands may be completely methylated/not at all
7
Q
What did Fig 1d. demonstrate and how was this acheived experimentally?
A
- Identified the behaviour of the mothers then switched offspring
- the effect of cross-fostering the offspring of low and high LG-ABN mothers on cytosine methylation at the 5’ and 3’ CpG dinucleotides
- cross-fostering occured within 12 hours of birth
- Significance of this experiment: Is it the behaviour that is important. Does it take on the methylation due to the behaviour of the mother rather than a genetic factor
- Get a profile of the foster mother not the birth mother
- Results: same at 3’, at 5’ L-L and H-L have high CpG methylation, H-H and L-H have low CpG methylation
- the behaviour of the mothers influences the methyaltion status
8
Q
What did Fig 1e. demonstrate and how was this acheived experimentally?
A
- Looked at the timing of the maternal effect on DNA methylation
- the maternal care of low vs. high mothers differs only in the first week of life
- post-natal changes in DNA methylation (aside from aging and cancer) were previously thought to be rare
- Did time series
- E20 before birth
- P - days after birth (1, 6, 21, 90)
- Results:
- E20 - low methylation for low and high
- By p1 - methylation for both becomes high (stress of birth?)
- rapid drop in methylation post birth, in High LG-ABN, this is maintained
- Low LG-ABN stays high
- 3’CpG dinucleotide stays high for both
- Thought that the default is high methylation
- low levels of GR, weak dampening down reponse
- strong fight/flight response
9
Q
What pathway is active post birth in high LG-ABN offspring to bring down the methylation of the 5’ CpG dinucleotide for NFG1-A?
A
Activation of the seretonin pathway results in demethylation of the NGF1-A 5’ binding site
10
Q
How was it shown whether the maternal effect on DNA methylation correlates with increased histone acetylation (H3K9-AC) and/or increased interaction between NGF1-A and the promoter?
A
- ChiP - looking at the in vivo interaction between TF and DNA
- A) H3K9 is a strong active mark
- B) binding of the TF
- I: input of ChiP; A: Immunoprecipitation (Ab); N: negative control (IgG not expected to pull down
Higher levels of active histone mark in High LG-ABN as have lower levels of DNA methylation (assocatied with inc. histone acetylation)
- IP see 2 X PCR for the GR exon and the b-actin promoter (positive control, expect this promoter to have acetylated histones but would expect the same between La dn H offspring)
Looking at the TF
- Er-a: exon promoter, control for input, to check the specificity of the Ab and the process, expect not to bind NGF TF
- Bit in brackets - what being stained
- See increased binding of the TF in high LG-ABN compared to low LG-ABN
11
Q
How can the NG1 TF binding be linked with a change in histone acetylation?
A
- already knew that NGF TF interacts with CBP (CREB binding protein (a histone acetyltransferase))
- Activation of the TF recruit CBP to increase acetylation levels, resulting in more open chromatin, directing further and more stronger binding of NGF1
But why are the methyl groups removed?
- 5hmC is a route by which get demethylation
- or could be competition between open chromatin and ability of methyltranferase to act on this region
12
Q
How was it shown whether the impact of these early experiences is reversible, and what was the result?
A
- Histone deacetylase activity is inhibited by Trichostatin A (TSA)
- pushing towards a more active confirmation
- does this affect inhiition of transcription by DNA methylation?
- MeCP2 binds region of methylated DNA and recruits the Sin3a co-repressor and a histone deacetylation, which recruits histone lysine methyltransferase to result in a change in chromatin stucture
- When methylation is high (low LG-ABN) have low H3K9ac
- When methylation is low (high LG-ABN) high high H3K9ac
- Can this be shifted low to high?
Results - immunoprecipittion of Ab/input GR
- Vehicle - need to dissolve trichostatin A in something, need to check the solvent isn’t having an effect
- Look at acetylation levels and binding of TF in the vehicle in low LG and between the vehicle in high LG
- Acteylation increases in the low LG-ABN and in the high LG-ABN but only to a significant level in the low LG-ABN
- Look at level of NGF1-A to see if correlates
- see same pattern
- Also looked at DNA methylation as a result
- DNA methylation decreased at 5’ site between vehicle and low LG-ABN significantly (not in high LG-ABN)
- Also decreased in the 3’ site for both high and low LG-ABN
- Looked at whole region
- General reduction in methylation between the vehincle and treatment but not always significant
13
Q
Why does TSA treatment result in loss of DNA methylation?
A
- chromatin modifications are dynamic
- balance between acetylation and methylation
- If inhibit deacetylation it is pushed towards demethylation by an undefined mechanism
14
Q
How was it shown whether GR levels are increased by TSA treatment?
A
- Used western blot to look for GR levels
- Antibodies to detect
- GR
- a tubulin (loading control - although TSAis a broad spectrum epigenetic drug so may have an effect)
- Inreased levels of the receptor in high Lg-ABN not in low LG-ABN
- with TSA get incresaes in both both not significant aside from in the low- LG-ABN
15
Q
How was it demonstrated the effect of TSA treatment affecting HPA responses to stress?
A
- Measured levels of stress by the steroid hormone corticosterone levels
- Observed with and without TSA
- Stress increased by restraining
- Measured before, during and after restraint
- Results:
- In all animals see a dampening down effect
- Before restraint, the low LG-ABN already had increased levels of the steroid hormone
- similar profile to teh others in the timing of the dampening down
- Looked at levels of GR alongside
- high GR in low maternal
- even with high GR still get a response (important in nature)
