Week 3 - Epigenetics

Question 1

What is epigenetics?

Answer 1

Epigenetics is the study of how cells control gene activity without changing the DNA sequence eg. behaviours and environment

Question 2

DNA methylation

Answer 2

Methyl marks added to certain DNA bases repress gene activity

Question 3

Histone modification

Answer 3

A combination of different molecules can attach to the tails of proteins called histones. These alter the activity of the DNA wrapped around them.

Question 4

Cytosine methylation

Answer 4

• Occurs at CpG dinucleotides
• Catalysed by a family of DNA methyltransferases (Dnmts)
• DNAm at regulatory regions can inhinit transcription

Question 5

How do you measure DNA methylation?

Answer 5

Sodium bisulphite
* DNA is first denatured (made single stranded) and then treated with sodium bisulphite
* Sodium bisulphite selectively changes unmethylated cytosines into uracils through deamination, while leavng methylated cytosines unchanged
* Followed by PCR

Question 6

Chromatin and histone modifications

Answer 6

• Chromatin has a basic unit which is made up of an octamer of histone proteins (2 of each H2A, H2B, H3 and H4)
• Can be regulated through acetylation, methylation, phosphorylation, ubiquitylation

Question 7

Histone modification and transcriptional activity

Answer 7

• Gene activation correlated with acetylation
• Gene silencing correlated with methylation

Question 8

Histone modifiers

Answer 8

1. HAT’s - histone acetylases
2. HDAC’s - histone deacetylases
3. HMT’s - histone methylases

Question 9

How to measure histone marks using chromatin immunoprecipitation (ChIP)

Answer 9

• Widely used technique for determining the in vivo location of binding sites of various transcription factors, histones and other proteins
  Process
• Cross-linking of the chromatin-bound proteins by formaldehyde, followed by sonication of nuclease treatment to obtain small DNA fragments. Immunoprecipitation is then carried out using specific antibodies to the DNA binding protein of interest. DNA is then released from the proteins then released using various methods.

Question 10

Regulatory noncoding RNA’s (ncRNA’s)

Answer 10

• ncRNA is functional RNA that is transcribed from DNA but not translated into a protein
• They regulate gene expression at transcriptional and post transcriptional level, histone modification< DNA methylation targeting and gene silencing
  The below are different types of ncRNA’s:
  1. miRNA and siRNA - 18 to 25 nucleotides, post-transcriptional gene silencing, RNA interface
  2. piRNA, snoRNA - 20 to 300 nucleotides, RNA modification, telomeres, chromatin, transcription
  3. long ncRNA - 300 - 1000 nucleotides, X-inactivation, DNA imprinting, transcription, generating other ncRNAs

Question 11

Measuring miRNAs

Answer 11

• miRNA sequencing
• miRNA arrays
• miRNA realtime PCR

Question 12

Roles of epigenetics

Answer 12

• X-inactivation
• Genomic imprinting
• Protecting genome from transposition
• Tissue, gene developmental stage-specific expression
• Genome-environment interaction

Question 13

X-chromosome skewing

Answer 13

Occurs when the X inactivation of one x chromosome is favoured over the other leading to an uneven number of cells with each chromosome inactivated

Question 14

Genomic imprinting

Answer 14

• The process by which only one copy of a gene is expressed (mother or father) while the other copy is suppressed
• Imprinted genes - we inherit only one working copy depending on the gene, either the copy from the mother or the copy from the father is genetially silenced
• The epigenetic marks - the epigentic marks usually stay in the same position for the life of the organism but are reset during sperm and egg formation
  Imprinting is required for normal development
• Normally, an individual has one active copy of the imprinted gene
• Having two active or inactive copies can lead to severe developmental abnormalities, cancer and other problems

Question 15

Levels of DNA methylation in repetitive DNA sequences

Answer 15

• 3 billion base pairs of DNA - a small percentage codes protein. Most of the rest is made up of several types of non-coding repeated elements
• Interspersed repetitive elements are in single copies and distributed widely throughout the genome. Constitute about 45% of genome which includes transposons
• Repetitive elements are usually methylated to suppress their activity - an active transposon is potentially disasterous in cancer
• This is why about 60 - 90% of CpGs are methylated in mammals

Question 16

Ageing DNA methylation changes

Answer 16

• During ageing there is a continuous accumulation of epigenetic changes which might give rise to multiple age related pathologies
• Monozygotic twins exhibit an increased rate of phenotypic discordance particularly for age-related diseases among other siblings
• Steve Howarth (2013) developed an age predictor based on DNA methylation values of 353 individual CpG sites.
• Clock starts ticking early during development where foetal tissues as well as embryonic and induced pluripotent stem cells reveal a DNA methylation age (DNAm age) between -1 and 0 years.

Question 17

Epigenetic markers for age-related diseases

Answer 17

• DNAm age acceleration associated with incidence, future onset and mortality across several types of cancer
• DNAm age was reported to be a useful biomarker for predicting physical and mental fitness in elderly individuals, and was shown to be associated with cholesterol, insulin, glucose and triglycerides
• DNAm changed associated with diabetes, neurodegenerative diseases, heart disease etc.

Question 18

Diseases and conditions associated with the epigenetic clocks

Answer 18

• By repressing DNAm age on chronological age, epigenetic clocks can determine whether biological age acceleration occurs in certain diseases or in response to environmental factors
• By using this approach, age acceleration measurements in blood were associated with body mass index (BMI), obesity, physical fitness, Huntington’s disease, Parkinson’s disease, sleep and smokinh

Question 19

Developmental origins of health and disease (DoHD) hypothesis

Answer 19

Hypothesis
* Multiple factors during pre-pregnancy, pregnancy, and the early post-natal period influence long-lasting disease susceptibility in offspring
* Markers of offspring health and even lifespan can be influenced by numerous maternal and paternal factors and it is likely that many of these parental effects are transferred by multiple molecular mechanisms including epigenetic regulation.

Question 20

Barker Hypothesis

Answer 20

• David Barker (early 1990’s) noticed that the poorest regions of the UK were the ones with the highest rate of heart disease
• He found a link between small birth size (poor prenatal nutrition) and heart disease in middle age

Question 21

Thrifty Phenotype Hypothesis

Answer 21

Hales and Barker (1992) - maternal undernutrition could retard the growth of fetal beta cells in utero and subsequently led to T2DM and metabolic cydrome in adult life; this hypothesis is known as the thrifty phenotype hypothesis because a fetus can be forced to assume its most thrifty phenotype tailoredto a maternally undernourished environment

Question 22

Mismatch Theory

Answer 22

• Developmental plasticity - attempts to tune gene expression to produce a phenotype best suited to the predicted later environment
• When the resulting phenotype is matched to its environment, the organism will remain healthy
• When there is a mismatch, the individuals ability to respond to environmental changes may be inadequate and risk of disease increases
• The degree of mismatch determines the individuals susceptibility to chronic disease

Question 23

Parental diet with obesity and diabetes in offspring

Answer 23

A 264% increases in odds of child obesity when the mothers suffer from obesity before contraception

Question 24

Maternal diabetes

Answer 24

Diabetes during pregnancy can have long-lasting metabolic effects on offspring

Question 25

Study in Pima Indian’s

Answer 25

Maternal diabetes increased development of T2DM, higher systolic blood pressure and haemoglobin A1C and generate greater rates of obesity in offspring

Question 26

Gestational weight gain and maternal obesity

Answer 26

Mothers with overweight or obese BMI’s during early pregnancy had children that were significantly more likely to be overweight at 3 years of age.

Question 27

Dutch Hongerwinter 1994 Study

Answer 27

• Children born or raised in this time were small, short in stature, and had many diseases including anaemia, diabetes and depression
• Women living in this time had children 20 - 30 years later with the same problem

Question 28

Maternal undernutrition hypothesis

Answer 28

Energy deprivation retards growth of the specific system that is affected. Eg. since the second trimester is a period of rapid nephron and bronchiole growth, undernurtrition during these periods could lead to negative, organ-specific health outcomes in adult offspring such as microalbuminuria or obstructive airway disease

Question 29

Epigenetics as a mechanism

Answer 29

• Epigenetic regulation and particulalrly DNA methylation seems to potentially influence this process
• Offspring exposed to the Dutch famine about 60 years prior displayed persistent to redcuced DNA methylation of the differentially methylated region of the IGF2 gene in comparison to same sex unexposed siblings
• Adult offspring of mothers exposed to the Dutch famine during early pregnancy exhibit lowers performance on a selective task at age 56 - 59. This suggests an increased rate of age association deterioration and cognitive decline in offspring exposed to early gestation

Question 30

Paternal diet and children

Answer 30

• Paternal and grandpaternal diet may lead to both positive and negative phenotypes in offspring. Studies of Swedish birth cohorts found:
  1. Fathers who experienced low food availability during their ‘slow growth period’ (ages 9 - 12) had offspring that showed lower CVD mortality
  2. Increased survival if their paternal grandfather experienced low food availability during their slow growth period. Excess food availability had the opposite effect.
  3. Additionally, subjects displayed increased diabetes mortality when the paternal grandfather lived through high food availability during their slow growth period
  4. Food restriction can have a very different effect depending on the timing - fathers who were undernourished in gestation due to the Dutch famine had offspring that were more obese.

Question 31

Maternal dietary interventions

Answer 31

• Maternal high-fat and/or high energy diets can lead to negative and potentially life-span reducing phenotypes in offspring
• Female mice fed a high-fat diet prior to mating and during pregnancy and nursing had offspring with increased adiposity, glucose intolerance and hypertension even after weaned onto regular diets.
• The offspring exhibited non-alcoholic fatty acid liver disease, oxidative damage in the liver of these offspring, insulin resistance at 8 weeks of age prior to the development of increased adiposity and impaired vascular function
• Dams fed a diet of supplmented with the antioxidant 2-micropteethylamine during pregnancy had offspring who experienced signifcantly increased average life spans

Question 32

Post-natal diet on lifespan (and catch-up growth)

Answer 32

• Calorically restricted in mice early lifespan shows an 18% increase in median lifespan.
• Limiting post-natal growth increases longevity and protects against the life shortening effect of an obesity-inducing diet later on
• By contrast, lifespan is shortened if the post-natal period of growth is accelerated to make up for the reduced growth in utero, and that, in addition, these mice are susceptible to the adverse effects on longevity of an obesity-inducing diet afteer weaning.

Question 33

Environmental toxicants

Answer 33

Pre-natal exposure to multiple environmental toxins pose significant threat to the offspring.
1. Cigarette smoke - In humans, maternal smoking has been shown to play a role in offspring development of diseases such as obesity and others that reduce life expectancy
2. Polychlorinated biphenyls (PCB’s) - PCB’s found in insulating material, plastic softeners and electrical equipment. Female mice offspring exposed perinatally to endocrine disruptors (PCB’s, herbicides etc.) show multigenerational transmission of effects.

Question 34

Prenatal maternal social stress and mental health

Answer 34

• Increased levels of stress - related behaviours in offspring
• Greater rates of inflammation in adult offspring independent of adult depression
• Predicts insulin resistance and higher BMI in adult offspring

Question 35

Season of birth

Answer 35

• Shown to have an influence on longevity and health eg. US study of 1880 - 1895 born centenarians - odds of becoming a centenarian increased if the month of birth was in the second half of the year as opposed to the first half of the year
• Relative age effect (RAE), aka. birth date effect is used to describe a bias, evident in sport and academia where participation is higher amongst those born earlier in the relevant selection period.

Question 36

Positive environmental impacts on offspring health

Answer 36

Maternal exercise used to improve offspring health span

Question 37

Prenatal exercise

Answer 37

Maternal exercise significantly improves both insulin sensitivity and glucose homeostasis in offspring. Mice born to mothers that exercised during pregnancy showed lower fat mass and greater lean mass
- In humans, maternal exercise is associated with increased fetal heart rate vulnerability and decreased heart rate, indicative of improved cardiac autonomic control