Epigenetics Flashcards
What % of pregnancies does FGR (fetal growth restriction) occur in?
5%
What is FGR
Fetal growth restriction
A baby’s growth slows or stops in utero
Affects trajectory throughout pregnancy and throughout baby’s life
Causes of FGR
Chromosomal defects
Placental insufficiency – supply of nutrients from mother
Environment ~ multiple gestation (twins, triplets), smoking, alcohol, or abusing drugs, maternal illness or infections nutrition or stress
Explain how the placenta can be studied at lots of different levels
Size and structure
Transport capacity - Nutrients, toxins, IgG
Blood flow - Maternal, fetal
Metabolism - Nutrients, drugs
Whats one thing the placenta is important for
Amino acid transfer from mother to fetus, to allow proteins to be made
Explain risks of death or illness in FGR
Babies can be stillborn
At risk of developing lifelong disabilities (e.g. cerebral palsy)
At risk of developing non-communicable diseases in adulthood
What non- communicable diseases do small babies have a higher risk of?
Hypertension
Raised serum cholesterol
Impaired glucose tolerance
Type 2 diabetes
Obesity
Define gene
nucleotide sequence required to direct protein synthesis
Stages of blastocyst development
Oocyte
Zygote
2-cell
4-cell
8-cell
Morula
blastocyst (stage where differentiation is occurring - therefore different genes and proteins beginning to be expressed)
Explain gene expression in a developing embryo
Translation into protein is continued throughout the preimplantation period
Messages (mRNAs) inherited from the oocyte (maternally inherited) regulate embryo development early on
During early cleavage, the embryonic genome is gradually switched on to initiate de novo transcription.
What is epigenetics?
The study of heritable changes in gene activity that occur without a change in the DNA sequence
Explain how transcription factors control gene expression
TFs bind to promotor/control region (GRE = gene regulatory element)
Define epigenome
The Genome-Wide Epigenetic State, All of the Epigenetic modifications within the Cell’s Genome
What are epigenetic tags
Epigenetic marks or modifications
What are epigenetic modifiers
Enzymes that catalyse the addition or removal of epigenetic tags
What are the epigenetic mechanisms
- Chemical modifications of DNA e.g. methylation
- Post Translation Modifications of Histone Tails
- Histone Variants
What does CpG mean?
cytosine next to a guanine
Explain CpG methylation
Cytosine (CpG) is methylated to 5-methyl cytosine (5mC)
sits within groove id DNA, and provides a physical bloc that stops TFs from binding to DNA groove (Can also be a mark for methyl binding proteins)
Group attcached to DNA by DNA methyltransferase - DNMT
- Most common
- Stable
- No effect on base pairing
Which DNMTs are used in De-novo methylation?
Dnmt3a & Dnmt3b
Which DNMTs are used in maintenance methylation (whilst cell divides)
Dnmt1
What do Tet enzymes do?
Convert 5-mC (5-Methylcytosine) into 5-hmC
By what process does 5-hmC get converted into thymine?
Deamination
Explain CpG methylation occurrence
Occurs usually at a Cytosine followed by Guanine base
Palindromic Motif – C then G from 5’ to 3’ on both strands = CpG dinucleotide
Majority of CpGs are sparse and methylated
Silencing large regions of the genome
What percentage of CpGs are clustered in gene promotors
7% - these are refered to as CpG islands
What percentage of genes have CpG islands?
50%
Explain how methylation of CpG islands is used in control of gene expression (in a normal cell)
Housekeeping genes that are transcribed a lot in the cell are not methylated- so that RNA Pol can make contact with the promoter, and gene transcription can occur
Also genes that we don’t want transcribed all the time e.g. time dependent, cell dependent/ need to be able to switch genes on and off. The CpG islands in these gene promotors can be methylated so RNA Pol cannot bind (this can be turned on and off depending on when you want the genes transcribed, in terms of development and response to stimulus)
X inactivation also due to methylation: Two x chromosomes in female, cant have all switched on at same time, methylation used to switch off the X chromosome not being used
Explain what happens when there is faulty methylation of CpG islands
cancer we get methylation of gene promotor of e.g. protective genes against cell replication
Sites can also be methylated due to environmental influences
Or we can get inappropriate removal of the methylation at certain gene sites, so that we get gene expression e.g. cancer oncogenes being transcribed
FDG – faulty methylation of genes that control growth
Methylation of cpg islands involved in:
– Cell-Specific Differences in Transcription
– Developmental Differences in Transcription
– Genomic imprinting - certain genes controlled throughout development due to their methylation pattern
What are the two processes where DNA methylation is critical?
variable in different tissues and involved in regulating tissue-specific gene expression patterns
permanently ‘imprinted‘, therefore maintained and memorised in (nearly) all tissues
Which genes don’t undergo demethylation when fertilisation occurs
Imprinted genes
Explain a graph that shows methylation against developmental time
Methylation marks are erased in primordial germ cells (PGCs).
Oocyte and sperm continue to re-aquire methylation marks until/during maturation yet in different time frames and to different extents.
Following fertilization demethylation of the genome occurs (accept imprinted genes) - Demethylation of paternal genome occurs at a fatser rate than maternal
Re-methylation begins at the blastocyst stage in a cell- type specific manner (ICM vs TE)
Carried out by Dmnt3a and Dmnt3b
What factors lead to epigenetic drift
Intrinsic and environmental factors
Explain the principle of the battle of the sexes in imprinting
Genes that promote fetal and placental growth are maternally imprinted (to secure the survival of the mother)
Genes that inhibit fetal and placental growth are paternally imprinted (to secure passing on of the father‘s genes at cost of the mother)
- usually balanced correctly to make normal size baby
What does maternal imprinting do?
maternal imprinting limits use of maternal resources
by baby in utero
What does paternal imprinting do?
paternal imprinting maximizes use of maternal resources by baby in utero
Define imprinting
A process that leads to the heritable silencing of a gene on one of the parental chromosomes.
Explain regulation of imprinting
Methylation of regulatory regions on one of the parental chromosomes (differentially methylated regions, DMRs or imprinting control regions, ICRs)
Explain imprinting
Inherit 2 copies of each gene - one maternal, one paternal
only one expressed, other silenced by methylation (this is the one that is imprinted)
Does imprinting refer to the gene that is silenced or the gene that is expressed?
Silenced
Altered placental imprinted gene expression leads to…
FGR
What do DMRs and ICRs stand for
DMR: differentially methylated regions
ICRs: imprinting control regions
Explain how paternal imprinting leads to normal fetal and placental growth
Genes that inhibit fetal and placental growth are paternally imprinted (methylated)
= switch off inhibition of growth to increase growth
maternal genes are not methylated (and these reduce growth)
so therefore balance
Explain what happens in hypomethylation in paternal imprinting
Paternal DMRs/ICRs are not methylated, and thedore both maternal and paternal genes are causing reduced growth, and therefore theres reduced growth
Explain what happens in hypermthylation in paternal imprinting
Not just the paternal ICR/DMR, but also the maternal ICR/DMR, are methylated, and there is therefore increased growth
Give an example of a gene that is imprinted and how it works
The imprinted insulin –like growth factor 2 (IGF2) gene
-Matches placental nutrient supply to fetal demand
-Altered IGF2 expression related to FGR
Insulator binds maternal unmethylated ICR1
–H19 gene expressed
–Blocks IGF2 expression
Paternal methylation at ICR1
–prevents CTCF binding
–IGF2 is expressed
explain how a change in the H19/Igf2 imprint loop leads to loss or gain of Igf2 expression
Loss:
Neither maternal or paternal methylated at ICR1, H19 expressed, CTCF insulator can bind, and the enhamcer cannot reach Igf2 to help express it
Gain:
Noth maternal and paternal methylated at ICR1, H19 is not expressed and the CTCF insulator cannot bind, meaning the enhancer can bind to Igf2 and help express it
What is Igf2
a major fetal GF involved in differentiation, organogenesis and metabolic regulation
Give an example of a disorder caused by loss of imprinting at the Igf2/ICR1/H19 domain
Silver-Russell syndrome
what are two imprinting disorders and what do they cause?
Silver-Russell syndrome
-Prenatal growth failure
-Loss of imprinting at the IGF2/ICR1/H19 domain
Beckwith-Wiedemann syndrome
-Prenatal over growth (macrosomia)
-Abnormally large offspring observed after in vitro production or manipulation of farm animal embryos.
-Gain of methylation of IC1 on the maternal chromosome
what effects did in vitro culture have on mouse embryos
reduced methylation and specific imprinted sites, had effect on gene expression and number of cells at the blastocyst stage
Explain an experiment that compared different media used for embryo culture in IVF
Market-Velker et al. 2010
- mouse models
- showed that mediums effected methylation of imprinted genes
- in vivo:
H19: 82% hypermethylation
Snrpn: 92% ^
Peg3: 100% - commercial media that effected hypermethylation least: KSOM
H19: 75%
Snrpn: 73%
Peg3: 93% - comercial media that effected hypermethylation most: Whitten’s
H19: 61%
Snrpn: 58%
Peg3: 54%
Explain how placental-specific IGF-II is a major modulator of placental and fetal growth
Constância et al 2002
Mouse fetuses in which the Igf2 gene has been completely deleted weigh ≈60% of wild-type fetuses.
Deletion of the Igf2 gene transcript (P0) specifically expressed in the placenta leads to fetal growth restriction
compared gestational age to Placental weight and also compared gestational age to fetal weight
compared F/P weightvratios to gestational age
(all on graphs)
saw that:
Deletion of the Igf2 gene transcript (P0) specifically expressed in the placenta.
Leads to reduced growth of the placenta.
Followed several days later by fetal growth restriction.
= Greater fetal/placental ratio
explain why the fetus weight didnt decrease to start with when Igf2 is deleted
smaller placenta is able to compensate and provide for fetus, by end of gestation, fetus is also smaller
Explain simply how a relaxed vs condensed structure of chromatin effects TFs
Relaxed structure = activation of transcription Access for Transcription Factors
Condensed structure = inhibition of transcription No access for Transcription Factors
Explain the degree of compaction of linear DNA
– 1:6 for nucleosomes
– 1:36 for the 30 nm fibre
– >1 :10 000 for the metaphase chromosome
What is chromatin
-Complex of DNA & Histone Protein in Chromosomes
-Basic Structural Unit = Nucleosome
Explain the structure of the nucleosome core particle
DNA
~147 base pairs
-Wraps 1.67 left-handed super helical turns
-Negatively charged
Histone Core Octamer
-Histone = Small highly conserved basic protein (positive charge)
-102–135 amino acids
= 2 copies of each core histone: H2A, H2B, H3, H4
= 2 H2A-H2B dimers & 1 H3-H4 tetramer
-Histone H1 linker molecule
Histone N-terminals = histone tails
- Extend out of the core nucleosome
- Subject to modifications
What histone modifications mainly effect histone structure
Histone 3 & 4
Explain histone tail modifications
Histone N-terminus = histone tail
Subject to modifications at different positions on different amino acids
most common is lysine (K)
modifications include:
-Acetylation
-Methylation
-phosphorylation
Explain acetylation of histones
Acetylation site on histones, leads to activation through repelling interaction between histones, pushing them apart
What are the three types of histone modifiers:
Writers
erasers
readers
What do the histone modifiers writers do? give examples
add groups e.g. HATs, HMTs (histone methyl transferases)
What do the histone modifiers erasers do? give examples
Erasers: remove groups e.g. HDATs KDM (lysine demethylase)
What do the histone modifiers readers do?
molecules that read and recognise the mark, and bind
What does methylation level of DNA also impact?
how stuck DNA is to histones
When it comes to histone modifications, what makes good drug targets?
Histone modifiers (enzymes) - writers, erasers, readers
Explain features of histone tails
-Post-Translational Modifications
-Influence Gene Expression
-Activating Marks
-Repressive Marks
Explain effects/features of histone tail modifications
-Create or Prevent binding of Chromatin Remodelling Factors
-Influence Nucleosome mobility & function
-Scaffold for the recruitment of regulatory proteins
Marks/changes to the histone tail are sometimes referred to as…
The histone code
Explain how methylation of different residues of histone tails can lead to activation or repression
H3 tail:
- methylation of K4 = activating
- methylation of K9 and K27 = repressing
What is a common histone tail mark
Acetylation of K27 - activating
often used to check if chromatin is in active state
Give examples of activating/repressive marks and what the mark codes stand for
Active:
H3k4me2
H3K9ac
H3K27ac
Repressive:
H3k27me3
H3k9me3
H3 - histone 3
K—number - lysine residue and position of residue
me2 = dimethylation
me3 = trimethylation
ac = acetylation
What do CxxC domain proteins do?
Read where CG islands are in the genome and bind, ensure genome gets transcribed by modifying epigenetic structure, adding or removing marks
When can CxxC domain proteins bind CpGs?
When in unmethylated state
What are MBPs
Methyl binding proteins
Give an example of a CxxC domain protein, that is not an enzyme, and the enzyme complex it associates with
CFP1 (not enzyme) physically associates with the enzyme complex SET1
Adds mark – methylation at histone H3 position 4 in tail
causing a permissive state - switching on gnes
Give an example of a CxxC domain protein that is an enzyme
KDM2A = lysine specific demethylase 2A
Removes mono/di methylation on histone H3 at lysine 33
These marks block the transcriptional machinery from binding
Modifying chromatin structure
Opening up - Making ‘landing sites’
causing permissive state - switching on genes
Give an example of a CxxC domain protein which causes a restrictive state (switching off genes)
KDM2B binds at CG islands
KDM2B associates with the polycomb protein repressive complex = PRC1
KDM2B guides PRC1 to the CG island creating the restrictive chromatin state
What proteins are responsible for gene silencing
Polycomb group proteins (PcG)
What are the two main complexes of PcGs
Polycomb-repressive complex 1 and 2 (PRC1 & PRC2)
work independently or together
PRCs mediate gene silencing and x-inactivation
Explain what PCR2 does
PCR2 catalyses tri-methylation at H3K27 via EZH2
Functional link with HDAC and DNMT
Whats do methyl binding proteins do?
bind to methylated sites, read DNA and identify sites where genome needs to be shut down
Give an example of an MBP
meCP2
Give an example of a X inactivation disease
Retts syndrome
Explain features of Retts syndrome
neurodevelopmenal disorder that affects girls.
Characterized by normal early growth and development followed by a slowing of development, loss of purposeful use of the hands, distinctive hand movements, slowed brain and head growth, problems with walking, seizures, and intellectual disability.
Life expectancy ~ 40 years
No effective treatment
gene mutated = meCP2 (an MBP) - specifically abundant in neurones
explain how Retts syndrome is an X inactivation disease
Mutated gene = meCP2
gene found on the X chromosome so no males with Retts as they die
Explain the two different domain mutations that lead to Rett syndrome
Missense mutation in MBD (methylated DNA binding domain) - faulty protein and cannot identify methylated DNA and therefore cannot bind
(residues 100-150 roughly)
misssense in a second domain that binds the protein partner which is the HDAC complex, therefore this cannot bind and as this is what silences genes, this doesn’t occur (residues 302-306)
Explain how mutations in meCP2 actually lead to symptoms in Retts syndrome
Histone H1 affected
Epigenome is disorganised
Specific gene expression up; others /down/unchanged
Effects transposons
Mouse model showed that this caused:
- Reduced brain size
- smaller neurones - less complex
- no cell death
