Lecture 7 - Environmental factors Flashcards
1
Q
What environmental factors have been shown to affect the epigenome (prenatal or immediately post natal)?
A
Nutrition
- Folates
- Genistein
- Green tea
- Reserveratrol
- Malnutrition
Chemicals
- Xenobiotic chemicals - BPA
- Smoking
- Drugs
Behavioural cues
- Eusocial insects
- Weaver paper
2
Q
What is the agoutic gene metastable epiallele?
A
- Agouti gene encodes a paracrine signalling molecule that promotes hair follicales to change from black to yellow
- Yellow fur - seceptible to obesity, type II diabetes, cancer
- Under control of a developmental promoter in the middle of exon 2 of allele
- recessive a/a: black, A/A: yellow
- If agouti is expressed only in the middle part of the hair cycle, the hairs will have a black base, a yellow band in the middle and a black tip (typical agouti pattern)
3
Q
Give an example and definition of a metastable epiallele
A
Agouti (Avy - viable yellow) is a metastable epiallele: locus that can be epigenetically modified in a variable and reversible manner. This can change the phenotype of genetically identical cells
4
Q
Outline transcription from the agouti gene leading to the generation of the yellow phenotype
A
- transscription normally from a hair-cycle-specific promoter in exon 2 of the agouti A allele
- Ectopic agouti expression can occur depending on the methylation status of the IAP (intracisternal A particle - murine retrotransposon containing a cryptic promoter)
- brown colour shifts to yellow
- Ectopic (yellow) agouti expression: if IAP on Avy allele is not methylated, IAP acitve, shift trasncription of gene from deveolopmental promoter to ectopic promoter
- Developmental agouti expression: if IAP on Avy allele IS methylated
5
Q
Outline transcription from the agouti gene leading to the generation of the mottled phenotype
A
- IAP methylation can happen later in development and at different levels of methylation
- not all the cells may be methylated
- offsrping will have a mottled (pseudo-agouti) phenotype
- 5 categories: proportional to methylation of the IAP element
- extreme methylation - all yellow
- slightly mottled
- 50/50
- more brown (more psudo agouti phenotype)
- agouti phenotype
6
Q
Why are the agouti fur mice more prone to type II diabetes, obesity and cancer?
A
The ectopic agouti transformation happens in all cells not just the hair follicles
7
Q
What is the experimental evidence that meternal dietary methyl supplement can affect Avy coat colour?
A
- gave dietary supplements
- folic acid
- Vitamin B12
- choline chloride
- betaine
- to a/a dams two weeks before they were crossed with Avy/a sires, during gestation and lactation
- results: offspring shifted to more pseudoagouti phentype
- Observed to see if there was any change in the number of progenies in the 5 categories from WT
- WT control diet: Most are slightly mottled
- when supplemented: fewer yellow, psudoagouti and lightly mottled increases
8
Q
What is the experimental evidence that CpG methylation mediates the effect of supplementation on Avy coat colour?
A
- 9 methylation possibilities in the IAP promoter
- looked at sites 1-7, looked at % cells that were methylated
- All yellow - little methylation
- More mottled/pseudo agouti - methlayion increases
- more sites become methylated in supplemented
- Shift in coat colour due to an increase in CpG methylation
9
Q
What experimental evidence is there that the psuedoagouti phenotype shows hypermethylation of CpG sites?
A
- bisulfite sequencing
- shows hypermethylation of CpG sites in the pseudoagouti phenotype
- higher methylation levels of the CpG sites in the Avy IAP in genetically identical Avy/a littermates
- Bisulfite sequencing:
- C not methylated , becomes U when treated with bisulfite, T when sequenced
- Methylated doesn’t get converted
- Looked at sites 1-9
- Pseudoagouti - highly methylated (c stays as c)
- more yellow - C’s get fainter and fainter, T’s appear more and more, suggesting C is methylated in pseudo agouti but umethylated in yellow
10
Q
What was the experimental evidence of the effect of dietary supplements (mediated by CpG methylation) over multiple generations? Can the effect be inherited?
A
- gave supplements to pregnant F0 at E8.5-15.5
- crossed female F1 (Avy/a) to male (a/) without any supplements
- results: F2s of supplemented dams were more pseudoagouti (brown mouse - developmental agouti - more methylation of IAP sites 1-9)
- Effect appears to be heritable
11
Q
What is the effect of dietary supplements mediated by CpG methylaiton /
A
Unsupplemented mother
- Less methylation at the IAP possible methylation sites
- Expression from IAP cryptic promoter
- More yellow phenotype
- Ectopic agouti expression
Supplemented mother
- More methylation at the IAP possible methylation sites
- expression from the Agouti developmental promoter
- More psudo-agouti (brown) phenotype
- developmental agouti expression
12
Q
What is the importance of supplements affecting methylation status?
A
DNA methylation
- cytosine gets a methyl group transferred to it (5mC) by a methyltransferase (dnmt) with S-adenosyl methione as the methyl donor
- Dnmt is limited by SAM
- SAM has one-carbon metabolism by which it gets more methyl groups
13
Q
Outline the one-carbon metabolism of SAM (how it gets its methyl groups)
A
- SAM made from methionine (where gets the methyl group from)
- Methionine is made from homocysteine
- Other factors feed into this
- Betaine and choline - can come from diet
- get converted to Dimethylglycine then to methioine
- Folic acid and other types of folates can feed in
- leads to an incresase in DHF which end results in an increase in methionine
- All supplements can feed into cycle to produce more SAMm which leads to increased activity of Dnmt and increased methylation (more psudo-agouti phenotype)
- Betaine and choline - can come from diet
14
Q
How can SAM be recycled in the one-carbon metabolism?
A
- SAM can be recycled to become homocysteine by becoming SAH
- So long as other supplements are introduced into the cycle to permit becoming SAM again
- chlorine/betaine
- B12/B6 also feed in as part of methionine synthase
- Folate/folic acid invovled in the THF cycle - more SAME
- Supplements can change balance
- alcohol can inhibit this production
- environmental factors can pro/inhibit production of SAM
15
Q
What is the environmetal evidence that Genistein has an effect on Avy coat colour?
A
- Genistein is a phytoestrogen from rich soy diets
- mechnism is not clear - Genistein doesn’t fit into the one carbon cycle
- gave genistein to a/a dams two weeks before they were crossed with Avy/a sires, during gestation and lactation
- Result: offspring shifted to more psuedo-agouti phenoype
- more methylation - get shift towards more methylation with psudo-agouti supplemented mother
16
Q
What is the experimental evidence that Xenobiotic chemicals (Bisphenol A (BPA) ) has an effect of Avy coat colour?
A
- Bisphenol A (BPA) exists in polycarbonate plastics
- shouldn’t exist in the body: get rid, supress, keep at v low levels to deal with
- exposed a/a dams to BPA two weeks before crossing with Avy/a sires, during gestation and lactation
- result: offspring shifted to a more yellow phenotype
- Looked at methylation levels
- significantly lower in those mothers who were exposed to BPA
- sites 4, 5, and 6 were particularly suceptible
- if added supplements can this mitagated the negative effect of the BPA? Yes.
17
Q
What is the result of low-dose exposure to BPA in rats during development?
A
- In rats, low-dose exposure to BPA (during development) increases suceptibility to cancer
- changes the DNA methylation pattern of important cell signalling genes
- Limitations: this is a murine specific retrotransposon, nothing similar in humans
18
Q
What is the experimental evidence that maternal BPA affects other metastable epialleles?
A
- Cabp IAP metastable allele
- can’t use coat colour as indication of methylation
- can look at methylation status of 9 potential methylation sites on the allele
- Maternal BPA exposure decreases offspring methylation at the Cabp IAP metastable epiallele
- Dietary supplements counteract BPA
- As seen in the Avy metastable epiallele
19
Q
What other environmetnal effects have been explored using the Agouti mice?
A
- Reservatrol
- Green tea
- Malnutiriton (and human)
- Smoking (and human)
- Antidepressants
- Ionising radiation
- shifts fur coat from yellow to brown
- positive effect at low doses
- Body resists by expressing certain genes
20
Q
Why is reservtrol referred to as the elixir of life?
A
- resveratrol is a phytochemical
- resveratril activates Sir2 in yeast and has been shown to increase life span
- resveratrol mimics the effects fo calorie resriction via the activation of SIRT1
21
Q
What are the effects on SIR1 by a high and low calorie diet?
A
- Hyper calorie diet: LowNAD/NADH ratio (H4K16ac, H3K9ac), SIRT1 (NAD-dependent histone deacetylase, mammalian ortholog of yeast Sir2) inactive
- Calorie restircted diet: High NAD/NADH ratio, SIRT1 active by binding of NAD, removes H4K16ac, H3K9ac promoting closed complex of chromatin, supression of expression. Metabolism related phenotype, increased longevity. Dietary phenols can also result in this e.g. Resveratrol - activates Sirt1 without calorie restriction
22
Q
What is the experimental evidence that mice put on a high calorie die have increased performance and survival?
A
- Middle aged mice put on standard or high calorie diet or high calorie diet with resveratrol
- mice on HC diet gained weight and gradually declined
- by 60 weeks the survival rate of the HC and HCR became indistinuishable
- HCR mice also gradually improved their motor skills on a rotorod - although the body weight of the HCR was similar to the HC
- resversatrol increased:
- insulin sensitivity
- improved liver histology
- increase mitochondria number
- decreased acetylation of genes that are known to be SIRT1 targets
- shifted the gene expression pattern of HCR to SD
- resversatrol increased:
There is a link to SIRT1 but the mechanisms are not known.
23
Q
What is the experimental evidence that some other polyphenols can have an effect on DNA methlation and cancer
A
- Active ingredients in green tea are polyphenol
- inactivate dnmt1 (results in hyomethylation)
- shown to affect DNA methyation SAM:SAH ratio
- in humans, one study shows drinking 6 cups of GT per day hypomethylated genes involved in gastric cancer
- oral and gastric surfaces are most susceptible to treatment as they come into contact with high concentration of green tea before it is metabolised
24
Q
What was the result on the methyation of genes involved in metabolism as a result of the dutch hongerwinter (1944-45)?
A
- some genes involved in metabolism are differentially methylated in individuals conceived during the dutch famine compared to siblings
- offspring of women exposed to the famine during early pregnancy were more likely to develop a metabolic syndrome in adult hood
- cancer, type II diabetes, growth issues, metabolic issues
- Hypomethylation at te promoter of IGF2, a maternally imprinted gene implicated in growth and development (esp. impotant in males) has been observed in those exposed during the peri-conceptional period, relative to unexposed siblings (correct by looking at the liklihood of that site to be methylated in siblings)
25
What is the experimental evidence that undernutrition can have effects that are transmitted to subsequence generation?
* under nutrition during the late stages of gestation can cause low birth weight
* linked to higher risjs of developing type II diabetes and obesity
* this effect can be transmitted to subsequent generations
* pregmant mice were fed ad lib and then later during pregnancy they were move to an UN or normal diet.
* crossed F1s from different litter to get the F2
* only time they were diet restrcited was at the late stages of gestation in F0
* F2 males are insulin intolerant (both mother and father were from starved F0)
* all subsequent generations were also insulin intolerant by the age of 4 months
* tested the methylation status of CpG islands of imprinted genes though to be involved, but no change. Hisont modification or upstream factors?
26
What is the evidence that smoking causes hypomethylation and expression changes in human lung tissues?
* somking causes hypomethylation and expression changes in human lung tissues
* mice exposed to heavy levels of smoking
* looked at methylation of AHRR (tumour supressor, only want activated when needed e.g. when come across toxins)
* Benzypyrene: really bad. When in contact with AHRR it protects the body, in non smokers there is no need for the AHRR gene (methylated and not expressed)
* Smokers: AHRR hypomethylated and expressed
* also correltation between AHRR hypomethylation and smoking intensity
* AHR's have a role in the metabolism of dioxins, benzopyrene and other environmental toxins
27
what is the involvement of epigenetic and psychiatric diseases, specifically depression?
* compared Bdnf (brain derived neurotrophic factor) in no stress, chronic stress, and under treatment with an antidepressent
* chromatin remodelling and histone modification
* No stress: basal level of Bdnf
* Chronic stress: No Bdnf, closed form of chromatin - no bdnf linked to depression
* Antidepressant: Expression of Bdnf
* Some HDAC inhibitors (e.g. sodium borate) have been used as antidepressants in animal models
* get more aceylation
* genes (e.g. Bdnf) are expressed
28
How is epigenetics linked to eusocial insects?
*Apis mellifera* Honey Bees:
* the larvae that develop into workers and queens are genetically identifcal
* those destined to become queens are grown in queen cups (full of royal jelly - contains HDAC inhibitors and a compound that impacts DNA methylation) and will develop ovaries and a larger abdomen for egg laying
* worker is sterile
* queens also behave differently
**Genetics**
* over 500 genes show differential expression pattern between queen and worker brains
* link between DNA methyation and the development of castes
29
What is the experimental evidence that there is a link between DNA methylation and the development of castes? (Eusocial insects and epigenetics)
* knock down (RNAi) of DNMT3 results in queens rather than workers
* Queens more developmentally advances than workers (+3 days)
* several genes show a decrease in CpG methylation in queens (e.g. dynactin 63 - conserved gene responding to feeding)
* looked at brains - doesn't replicate, any changes due to epigenetic changes not DNA replication mistakes
* minics the high methylation rates in worker larvae
* and low methyaltion rates in queen larvae when reared inside colonies
* Reduced methyation therefore appears to mimic the effect of royal jelly
* larvae fed royal jelly for the longest shown decreeased DNMT3 activity and expression, lower methylationof dynactin 62 and an increase of queens
30
How does DNA methylation regulate caste fate in bumblebees?
Chemical inhibition of DNMT3 promotes reproduction by workers in colonies with no queens
31
What occurs when the queen is removed from a Harpeganthos colony?
* Workers fight to take her place
* become egg layers
* live longer (life span of queen: 10 years, life span of workers: 1 years, gamergate: 3 years
* epigenetic changes to:
* longevity genes
* telomerase
* sir proteins
* brain shrinks
32
Outline Caste development in Camponotus floridanus
* genetically identifcal siblings develop different caste identifies determined during larval developmet
* developmental plasticity
* Major and minor workers
* Dictated by environmental stimuli
* social context
* nutrition
* physical (temperature)
* Neuroendocrine pathways (IIS and JH signalling) transduce environmental signals
* Epigenetically regulated:
* DNA methyaltion (DNMT3 inhibition promotes redproduction by works in colonies with no queens - honey bees)
* Changes in H3K29ac (reveasls caste-specific transcriptome states)
* changes correlated ith CBP (major HAT for H3K27ac) and mRNA expression
* Neuronal regulation and aging genes affectecd
33
How can the epigentic drift in aging identical twins be explained?
* Could be indirectly explained
* Over years individuals are exposed to different environments leading to epigenetic changes
