Epigenetic control of development Flashcards
LO
LO
Lecture 1
- Epigenetics
- What does epigenetics mean
- Epigenetic modifications
- DNA methylation
- Histone modifications
- Non-coding RNAs
- Early Development
- MZT
Lecture 2
- Development & epigenetics
- TE/ICM differentiation
- establishment of Tissue specific gene expression
- Effect of early life environment
- Maternal environment
- Paternal effects
What type of control do genes have in development?
They have temporal control
What type of tissue specific control do genes have?
Even though cells have the same DNA, what makes them different?
- Cells all have the same DNA
- It’s in the mRNA
- Make sure right genes are switched on in the right cells
What does epigenetic gene regulation ensure?
That genes are expressed in the right place at the right time
What is the definition of epigenetics?
Definition: Processes that induced long term stable changes in gene activity without a change in gene sequence
Why does a cloned cat show the importance of epigenetics?
A cloned cat shows the importance of epigenetics: although same DNA they are epigenetically distinct. Hence why carbon copy of animals shows physical differences
(look up photo of rainbow and Carbon copy)
What are the major epigenetic processes?
- DNA methylation
- Histone modification
- non-codign RNAs
Tell me about where DNA methylation occurs?
- Cytosine is methylated to 5-methyl cytosine
- 90% of methylated cytosine is found as a dinucleotide CpG
How does DNA methylation affect gene transcription?
- Methylation prevents the RNA polymerase binding and therefore transcription of gene occurring
What are the enzymes involved in DNA methylation and what is the role of each?
DNA methyl transferases (DNMTs)
- DNMT1: maintenance DNMT (copies marks from old strand onto the newly synthesised ones)
- DNMT3a and 3b: de novo DNMTs (establishes new methylation marks in the first place)
What are the DNA demethylase enzymes?
TET enzymes (ten-eleven translocation)
What do the TET enzymes do?
DNA demethylases: TET enzymes
Tet converts 5- methyl Cytosine (5mC) to 5-hydroxymethylcytosine (5hmC)
What does the state of histone govern?
The accessibility of DNA to RNA pol
What is chromatin a combination of?
Chromatin= DNA + histones
Whats a nucleosome and what is its structure?
- They are the basic unit of chromatin
- Have 4 different histone subunits: 2x H2a, 2xH2b, 2xH3 and 2x H4
- Histones are positively charged in order to interact with the negatively charged DNA to create a tight binding site
What are nucleosome further packaged to form?
Nucleosomes are further packaged to form a 20nm fibre or solenoid
A solenoid
What is the type of binding in a solenoid and what does this determine?
The binding may be loose or tight (depends on state of histones)
What 2 domains does the histone have?
- Globular domain (C- terminal)
- Amino tail domain (N-terminal, lots of lysine’s)
The histone tails are subject to modification
What does the position and type of modification of the tail determine?
Whether binding is loose or tight DNA is in an open or condensed form Gene is active or repressed
What is histone acetylation associated with?
Gene activity
What happens in histone acetylation to promote increased gene activity?
- The acetyl groups are present on the histone ‘tails’
- The histones are no longer tightly packed
- The promoter and target gene are now accessible to transcription factors and RNA polymerase
- Acetylation of the lysine’s in the tail neutralises their charge (lysine’s are positively charged)
- This reduces the affinity of the tail for the DNA
- Opens up DNA allows RNA pol to bind and the gene to be active
What enzyme is involved in lysine acetylation?
HAT
What is the histone methylation effect dependent on?
Which lysine is methylated
Tell me the types of histone methylation and what their methylation is associated with?
H3K4 (histone 3 and lysine number 4): methylation associated with open structure & gene activity
H3K9/K27 methylation: closed structure & gene silencing
What is the histone code?
What are the three types of epigenetic modifiers?
- Writers
- Readers
- Erasers
What is the primary role of each of the epigenetic modifiers?
Writers: add groups
Erasers: remove groups
Readers: molecules that read and interpret the mark
Give some examples of writers and what they add?
Writers: add groups
- Histone acetyl transferases (HATs) add acetyl groups
- Histone methyl transferases (HMTs) add methyl groups
- DNA methyl transferases (DNMTs) add methyl groups to DNA
Give some examples of erasers and what they remove
Erasers: remove groups
- Histone deacetylases (HDACs) -remove acetyl groups
- Histone demethylases (HDMs)- remove methyl groups
- DNA demethylases – remove methyl groups from DNA
Provide some examples of readers
- Reader of Histone acetylation - SWI/SNF
- Clears nucleosomes from promoter region
- Reader of histone methylation at K9/K27 –HP1
- HP1 – heterochromatin protein 1
Epigenetic modifiers
Histone acetylation- writer and eraser interaction
Name a complex which has a key role in epigenetic writers and development
What are types and what does it have a role in?
Polycomb repressive complex (PRC)
- PRC1 and PRC2 work together
- PRC2 is thought to be the most important as it has catalytic activity
- Catalytic subunit of PRC2 is EZH2 - a HMT (writer)
- Mediates trimethylation of H3K27
- Readers of H3k27me3: HP1
- Ensures the right genes are expressed in the right cell types
The polycomb repressive complex 2 (PRC2) is a transcriptional repressor complex best known as a “writer” of H3K27 methylation, a chromatin mark associated with transcriptional repression
What are the two types of non-coding RNAs?
Tell me about each type and give examples
Non-coding RNAs (don’t code for proteins)
- Small non-coding RNAs
- Less than 200 bases
- eg miRNAs, piRNAs, tRFs
- know the most about miRNA
- Long non-coding RNAs (LncRNAs)
- Greater than 200 bases
What is miRNA and what is its role?
- small single-stranded non-coding RNAs of 21-25 nts in length
- Bind to mRNA and induce their degradation or inhibit their translation
Tell me the steps to miRNAs function
- miRNA gene is transcribed by RNA pol II into primary miRNA
- this is then processed by a complex called Drosha into a pre-miRNA
- the pre-miRNA is then transported into the cytoplasm of the cell where it is then cleaved by a dicer into a mature miRNA
- this is loaded onto a complex known as risk which then takes the miRNA to its target messenger
- if binds to 3’ to messenger RNA then it induces it degradation in that cell (of the messenger)
- if binds to 5’ of messenger then it inhibits translation
DNA methylation is associated with:
a. gene silencing
b. gene activation
a. gene silencing
Histone acetylation leads to:
a. closed condensed structure
b. open relaxed structure
b. open relaxed structure
Methylation of lysine H3K4 is associated with:
a. gene silencing
b. gene activation
b. gene activation
A HAT is a:
a. eraser
b. writer
c. reader
b. writer
A HDM is a:
a. writer
b. reader
c. eraser
c. eraser
HDACs:
a. add methyl groups
b. add acetyl groups
c. remove methyl groups
d. remove acetyl groups
d. remove acetyl groups
HP1
a. reader
b. writer
c. eraser
a. reader
DNA methyl transferase is considered:
a. reader
b. writer
c. eraser
b. writer
Tell me about gene expression in early embryos and during early cleavage
- Messages (mRNAs) inherited from the oocyte (maternally inherited) regulate embryo development early on.
- During early cleavage:
MZT: maternal-zygotic transition (maternal messenger RNA are in control, but at 2 cell stage they are degraded and the zygotic RNA comes into control)
ZGA: zygotic genome activation
Tell me about MZT levels
- Initially, the destruction of maternal mRNAs is accomplished by maternally encoded products – destabilise the mRNAs
- Zygotic transcription leads to the production of miRNAs (miRNAs-430/270)
Does MZT occur at different times across species?
Yes
Tell me about cleavage
Cleavage:
- Once the zygote is formed, it begins mitotic divisions to produce more cells
- Cells are totipotent (as can differentiate into any adult cells and also form the placenta)
Tell me about compaction, differentiation and cavitation during development
what cells are involved in each stage?
Compaction:
- cells on the outer part of the morula become bound tightly together with the formation of desmosomes and gap junctions
Differentiation:
- epithelial trophectoderm (TE; will form extraembryonic tissues such as placenta and cord)
- undifferentiated Inner cell mass (ICM; will form embryo proper)
Cavitation: to form the blastocyst
What is Oct4 a key regulator of?
Pluripotency
Tell me about Oct4 and other factors involved in maintaining pluripotency
- POU domain transcription factor
- Works in conjunction with nanog and sox2 to maintain pluripotency
- High levels of maternal expressed Oct4 in oocyte and zygote then switch to zygote oct4 (MZT)
- Levels of oct4 on ICM drop down to allow cell differentiation
- Oct4 comes down only when you need to differentiate cells
Tell me about CDX2 and TE differentiation
- CDX2 is a caudal-related homeobox transcriptional factor (TF) essential for TE formation during preimplantation development
- Overexpression of CDX2 in embryonic stem cells (ESCs) is sufficient to differentiate ESCs into TE cells
- Cdx2 null embryos: no TE cells, no implantation
Summary
- Epigenetic regulation of genes
- Define Epigenetics
- Understand what mechanisms epigenetics include
- DNA methylation
- Histone modification
- Non-coding RNAs
- Role of epigenetics in development
- MZT/ZGA
- ICM/TE differentiation
LO II
- ICM/TE differentiation
- Tissue specific expression
- Effect of the environment on the epigenome and development
- in vitro culture
- smoking
- Diet
Tell me about the link between CDX2 cells and TE (tropoectoderm) cells differentiation
- CDX2 is a caudal-related homeobox transcription factor (TF) essential for TE formation during preimplantation development
- Overexpression of CDX2 in embryonic stem cells (ESCs) is sufficient to differentiate ESCs into TE cells
- Cdx2 null embryos: no TE cells, no implantation
- CDX2 also downregulates Oct4 and nanog in outer cells
What signalling pathway determines TE/ICM fate?
The Hippo signalling pathway
What two factors are associated in the Hippo signalling pathway?
TEAD4 (TF)
Co-activate Yap (an associated protein)
What are the two models that are used for thr hippo signalling pathway?
Inside/out model
Cell polarity model
Tell me about the inside/out model
- Cell-cell adhesion means Hippo activated
- Yap is phosphorylated and now cannot move into the nucleus of the cell
- Meaning that the cells on the inside stay in an undifferentiated state
- That’s why only cells on the outside differentiated and express Cdx2
Tell me about the cell polarity model
- Polarised have an apical and basal edge
- Unphosphorylated Yat is only allowed into the nucleus to phosphorylate Cdx2
- AMOT (angiomotin) interacts with polarity proteins to suppress LAT (linker of activated T cells) activity
- Extensive cell adhesion activates LATS kinase to phosphorylate AMOT, in turn phosphorylates YAP
In the ICM, what does Oct-4 maintain?
Pluripotency
Tell me how Oct-4 maintains pluripotency, what involved thats important for this process?
- Different cell types are produced due to the developmental control genes
- These genes are controlled by dna methylation and histone regulation
Tell me about differentiation and DNA methylation in maternal and paternal genome and how this changes from fertilisation to when they are able to differentiate
- Following fertilization demethylation of the genome occurs
- Methylation marks on the paternal genome removed 12-24 hours after fertilization
- Removal of methylation marks on maternal genome complete by about 48 hours after fertilization
- Totipotent cells at double black line
Tell me about the potency in early embryonic cells, what’s switched on?
These cells are marked by both active and repressive markers
- H3K4me3
- H3K27me3
- RNA Pol II
- PRC2 complex
- PRC1 complex
They are in a bivalent domain
Tell me how bivalent domains are resolved
- Keeps repressive marks if not needed for particular cell type
- Genes permanently switch off when DNMT and HP1 binds
Embryo and DNA methylation
Change in potency during development
Tell me about tissue specific expression
- Embryonic cells
- Neural cells
- Cardiac muscle
- No methylation across promotor region of gene (NT)
- Contractile muscle are never required in this cell type (neurons), meth in promotor region, bound by HP1
Early life environment can alter the epigenome
When in the epigenome most susceptible?
In early life
What type of processes can change epigenetic processes?
In vitro culture and assisted reproductive technologies can change epigenetic processes
In, In vitro culture, tell me about the early phenotype and the offspring phenotype
Early phenotype
- In vitro culture can affect mRNA expression in blastocysts
- Expression of 5/11 genes involved in epigenetic and chromatin regulation was reduced
Offspring phenotype
In vitro culture can affect male offspring phenotype:
- insulin resistance
- aberrant testes gene expression
- less sperm with reduced motility
- reduced fertility
In vitro culture…
Large offspring syndrome
Abnormally large offspring observed after in vitro production or manipulation of farm animal embryos
Are there epigenetic risks?
There is a substantial body of evidence that adversity in early life can lead to epigenetic changes associated with increased risk for disturbances of childhood mental health, more disordered developmental trajectories, poorer educational achievements, and lifelong risks of chronic disorders of health and well-being.
Risk of rare diseases such as Beckwith-Wiedemann and Angelmans syndrome increased 3.5-fold
What two factors can be genetic risks?
Pollutants e.g., smoking
Nutrition
Tell me about pollutants e.g., smoking and how this is a epigenetic risk
Tell me about nutrition and the epigenome
Methylation diet
Maternal protein restriction induces a change in PPAR a methylation in foetal liver
Tell me about the Dutch hunger winter
- A period of severe food shortage in the Netherlands in 1944.
- Energy intakes dropped from 1800 to between 400 and 800 kcal per day (equivalent 100 - 200g pasta).
- Obesity, CVD, T2D (children born from pregnant mothers during this time was more at risk to these factors)
- Alterations in DNA methylation has been shown in adult children from mothers who
- Were exposed to famine during pregnancy compared to their non-exposed siblings
Tell me effects of over nutrition
Over nutrition can also alter DNA methylation in the offspring
What does maternal over nutrition lead to?
Maternal over nutrition leads to change in DNA methylation and increases adiposity in the child
Tell me about the paternal effects
- However recent studies have also suggested that paternal body composition or diet can also induce persistent metabolic changes in the offspring, suggesting sperm mediated epigenetic inheritance.
- Studies in experimental models are beginning to identify the mediators of paternal intergenerational transmission
What can the paternal environment affect
Are the long term effects of paternal high fat diet mediated through non-coding RNAs?
Does the sperm RNA transmit the signal about paternal environment to the offspring?
Which RNA species contributes to the acquired metabolic disorder?
tRFs- tRNA-derived small RNA fragments
A mechanism to explain paternal effects?
An adaptive process?
The first 1000 days
Summary II
- Epigenetic regulation of genes
Define Epigenetics
Understand what mechanisms epigenetics include
DNA methylation
Histone modification
Non-coding RNAs
- Role of epigenetics in development
MZT/ZGA
ICM/TE differentiation
establishment of Tissue specific gene expression
- Effect of early life environment on the epigenome
IVF
Maternal nutrition
Paternal effects
