L26&27 Epigenetics Flashcards
Epigenetics
Epi - upon
Genetics - DNA
the interaction of genes with their environment which brings the phenotype into being
Why is epigenetics so important?
- key to cell differentiation, stem cell maintenance, cloning, totipotency, pluripotency
- many different disease states including cancer
- long-term transgenerational effects of exposure during development
From DNA packaging to genome regulation
see onenote slides
- human cell >3 metres of DNA
- histones package DNA
- histone proteins from a nucleosome
- modifications alter DNA accessibility
- heritable changes
From host defense to gene silencing
- cytosine residues methylated
- DNA is silenced
- chromatin structure altered
- heritable
- altered in disease/cancer
Inheritance of DNA CpG methylation
see onenote
maintenance methylase
From junk DNA to lncRNAs and regulation
see onenote
- lncRNAs bind chromatin modifying proteins
- target specific regions of the genome
- lncRNAs also affect mRNA stability/splicing/translation
Increasingly complex epigenetic landscape
see onenote diagram
- ncRNA
- RNA modification
- DNA modification
- chromatin modificiation
DNA mutations and epigenetic diseases
see onenote
- typically genes that affect methyltransferases, demethylases, ICRs
- environmental factors are the biggest effectors
Lots of genes responsible for epigenetic regulation
One mutation can effect many genes as it is part of the higher level gene regulation
Epigenetic phenomenon - x-inactivation
see onenote slides
- chromosome wide silencing mechanism
- conserved across all mammals
- regulate balance in gene expression between male and female cells
- silences single x-chromosome in female cells
X-inactivation
- epigenetic mechanisms
- lncRNA
- histone modification
- DNA methylation
X-chromosome inactivation
see onenote slides
- XIC = x chromosome inactivation centre
- XIC controls expression of xist gene
- xist = x-inactive specific transcript
- xist produces non-coding 17kb RNA
- “coats” the entire local x-chromosome - cis-acting
What determines x-chromosome inactivation
see onenote
Tsix antisense transcript of xist
- regulates lots of genes around itself
X-inactivation mechanism
see onenote slides
It requires:
- initial xist rna expression and coating
- association of chromatin modifying proteins
- DNA methylation 5’ of x-chromosome genes
- modification of histones by methyltransferases
- other chromatin modifying proteins
deletion of A-loop prevents the chromatin modifier PRC2 binding
- A loop important for forming tightly packed DNA
- trimethylates histone H3 on lysine 27
- more active gene state
What controls XIST expression?
see onenote
- tsix
TSIX is the anti-sense strand of the XIST gene
see onenote
- tsix promotes xist promoter CpG methylation
- active expression of tsix on active x-chromosome inactivates any xist
Knockdown of tsix causes skewed x-chromsome inactivation
see onenote
- PGK1 and MECP2 are x-linked genes
- when tsix is inhibited X is inactivated, can’t stop xist
- Interaction between xist and tsix only occurs in one chromosome, the other chromosome will become the one that the other one isn’t (if the other x is inactivated then the other one won’t be)
TSIX asymmetry governs choice
- tsix must be down regulated for xist expression on the future inactive x chromosome
- tsix expression must remain for xist down-regulation on the future active x chromosome
x-inactivation evolution
- variable mechanisms in mammals
- marsupials don’t have xist or xact and have a different lncRNA altogether
X-inactivation and disease
see onenote
- skewed x-inactivation with deleterious alleles
- ATRX patients
- incontinentia pigmenti IKBKG mutations
- fragile X (marker of skewed inactivation)
Can preferentially inactivate an x that carries a disease allele
- Cells can’t make sentient choice
- Cells with deleterious alleles outcompeted by cells with the good allele
- Less cells in body with deleterious alleles
- Depending on proportion of cells with either good or bad allele, indicates severity of the disease
- Cell with preferentially inactivate x with deleterious mutation
Genomic imprinting
- genomes we inherite from our mothers and father aren’t functionally equivalent
- certain genes are expressed in a parent-of-origin specific manner
- if the allele inherited from the mother is imprinted, it is silenced and only the allele from the mother is expressed
- results in monosomy for ~100 genes (almost) all essential for normal fitness
- must be faithfully reproduced during cell divisions and erased in germ line
Epigenetic mechanisms
- DNA methylation
- lncRNA
- histone modification
Angelman and Prader-Willi
see onenote
Prader-Willi
- paternal deficiency
Angelman
- maternal deficiency
Paternal and maternal genomes are both required for normal development
see onenote
Two female sets of chromsomes
- Encodes for small placenta, invests lots of growth in embryo
- Placental insufficiency, embryo dies
Two male sets
- Encodes for large placental growth
- Not much investment in embryo, it dies
Male can fertilise many females, all they care about is that the foetus gets the most nutrient possible - large placenta
Female
- Maximise genetic fitness
- Restricted placenta so it won’t suck all the resources from the mother and she can have more children
Cyclical re-imprinting
see onenote slides
Demethylation and re-methylation in male specific manner during spermatogenesis
Demethylation and re-methylation in female specific manner during oogenesis
Every cell in our body contain both male and female imprinting but in germ line, either male or female methylation specific manner otherwise the germ cells aren’t viable
Imprinting clusters
see onenote
Only need to regulate few clusters
Red - maternal, only expressed from mum
Blue - paternal, only expressed from dad
- Show opposite patterns
- Need a single copy of each of these genes for normal development
Snrpn cluster - maternally methylated
Igf2-H19 cluster - paternally methylated
IGF2 and H19
see onenote
Igf2 and h19 under control of one enhancer element down stream of h19 (DMR - differentially methylated region)
DMR
- boundary element
- controlled by methylation
h19 expressed from maternal chromosone only
- DMR not methylated
- CTCF binds to DMR
Igf2 expressed from paternal chromosome only
- DMR methylated
- h19 silenced by methylation
deletion of DMR removes imprinting of IGF2 and H19
Mice created without fathers
see onenote
Female
- No expression of ifg2 (insulin growth factor 2), dies, embryonic lethal
No consensus sequence for ctcf to bind to => mouse able to produce single copy of igf2 and h19 on a completely female chromosome background
- deleted DMR and H19 to create male chromosome from an initial female chromosome
H19 lncRNA controls gene expression by recruiting MBD1
see onenote
MBD1 - methy-CpG-binding domain protein 1
- Able to bind chromatin modifying proteins, binds igf2, further silences it
IFG2 imprinting and disease
see onenote
Wilms tumours
- CTCF doesn’t bind to female chromosome DMR
- no H19 produced, only iGF2
Bladder cancer
- CTCF binds to both female and male chromosome DMR
- no IGF2 produced, only H19
SNRPN imprinting and disease
see onenote
Angelman
- Secile
- Eat lots, gains lots of weight
- Severe mental retardation
Prader wili
- Very active
- Severe mental retardation
- Insatiable appetite
- Has almost no fat stores
Loss of balance of male and female genes being expressed
So why have imprinting?
- must have an evolutionary advantage
- genetic/epigenetic tug of war
- overall strategy for fitness advantage for paternal and maternal genome is different
Dad doesn’t mind if it is detrimental to mother’s health as long as offspring is okay, he can mate with other females as well
Mum needs to provision carefully how much resource is given to each offspring so she can have more offspring
- Restrict growth of embryo and placenta, what is needed for child to survive but no more than that
Needs to maximise her own reproductive output
Genome wide epigenetic landscape
- genes throughout the genome regulated by epigenetic marks
- NOT sex specific (differs from imprinting - imprinting is sex specific and x-inactivation)
- same mechanisms apply
- plastic and subject to environmental changes
- heritable
Epigenetic occurs in every single cell which determine what kind of cell the cell will become, which gene is switched on/off
- highly plastic
- immediate response (in a single generation) to changing environment
1. in utero exposures/nutrition
2. exogenous chemicals
3. altered cell states (cancer)
Important what our grandmas saw in terms of chemicals
Smoking, drinking during pregnancy
What can epigenetic marks tell us?
see onenote
ChIP-Seq
see onenote
- chromatin immunoprecipitation used to identify histone bound regions
- isolated DNA deep sequenced
- mapped to reference genome
Using antibody to pull chromatin out
- Raise antibody to recognise a particular modification that may associated to an active promoter/enhancer
Fragment dna, they are still wrapped around histone
Make antibody for that histone with the specific mark, pull out chunks of genome that is bound to the histone with the particular mark
Genome wide epigenetic scans
see onenote
- critical advancement, majority of disease mutations don’t occur in protein coding regions
Skin cell => nerve cell
- Nerve cell genes will be poised in the skin cell
Histone marks
- exon/intron boundaries
- Enhancer
- Large scale repressive regions of the genome
- Is the region of the gene active/inactive?
- Can be used to discover genes, diseased genes, regions important for driving genes
- Mutations usually occur in intergenic space
Genome wide scans in ESCs
see onenote
- identified active ESC (class 1) genes
- and poised (class 2) genes that drive later embrogenesis
NODAL
- Has active histone marks
- Also has repressive histone marks at the promoter of that gene
- We used to think they’d only have either active or repressive, not both
- Active histones parked in the right place ready to go, gene can be switched one easily
- If we only had repressive histones, would need to then attach active histones, would be slow, and vice versa
- Genes which have both active and repressive markers - poised genes
Non-coding RNAs and epigenetics
see onenote slides
- increasingly complex genome
- 1.5% of genome protein coding, are the rest junk?
- majority of genome is transcribed
- ChIP Seq and transcriptome sequencing identifying new RNA classes
- in total ~30% of all lncRNA in ESCs associate with chromatin modifiers
- 23% with PRC2 alone
- most associate with multiple modifiers
Blocking production of lncRNA which is required to maintain epigenetic signature, could result in disease, lncRNA can be used as diagnostic marker
the expanding world of ncRNA
see onenote
lncRNA and cancer
see onenote
MEG3 - maternally expressed gene
- Mis-expressed in cancer
Important for understanding in how to best treat diseases
Diet and inheritance
see onenote
Variation in coat colour in isogenic yellow agouti mice
see onenote
Coat colour - epigenetic trait
- Folic acid helps with normal DNA methylation
- No folic acid, can’t methylate, yellow
- Too much acid, over methylated, brown
Maternal environment affects methylation
see onenote
- maternal exposure to endocrine disruptors and toxic compounds
- low methylation in offspring - normal conditions
- variable methylation - maternal supplements with methyl donors and cofactors
- high methylation in offspring
Diethylstilbestrol (DES)
see onenote slides
- synthetic estrogen
- drug given to women beween 1930s to 1970s to prevent miscarriage and premature birth
- over 10 million pregnancies treated worldwide
- caused reproductive cancers and abnormalities
Abnormalities last for multiple generations
- 3rd generation have increased rate of hypospadia, reproductive cancer
Hypospadia; an epigenetic disease?
see onenote
- increased incidence of hypospadias in sons of mothers exposed to DES
- increased by exposure in utero to any estroegenic chemical
- most common birth defect in Australia 1:120 live male births
- rapidly increasing faster than genetic changes
- environmental endocrine disruptors pervasive in our environment
Transgenerational inheritance or direct exposure?
- true heritable transgenerational effects must persist to the F3
- effects must be passed on without direct exposure
- DES: hypospadias, cervical and uterine cancer rates persist to F2, F3 being born now
- reprogramming of the germline occurs in the gonads, most affected by endocrine disruption
exposure to unfavourable conditions in utero has the ability to affect not only the exposed fetus but multiple generations to come
