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Human and Medical Genetics > L26&27 Epigenetics > Flashcards

L26&27 Epigenetics Flashcards

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1
Q

Epigenetics

A

Epi - upon
Genetics - DNA

the interaction of genes with their environment which brings the phenotype into being

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2
Q

Why is epigenetics so important?

A
  • key to cell differentiation, stem cell maintenance, cloning, totipotency, pluripotency
  • many different disease states including cancer
  • long-term transgenerational effects of exposure during development
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3
Q

From DNA packaging to genome regulation

A

see onenote slides

  • human cell >3 metres of DNA
  • histones package DNA
  • histone proteins from a nucleosome
  • modifications alter DNA accessibility
  • heritable changes
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4
Q

From host defense to gene silencing

A
  • cytosine residues methylated
  • DNA is silenced
  • chromatin structure altered
  • heritable
  • altered in disease/cancer
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5
Q

Inheritance of DNA CpG methylation

A

see onenote

maintenance methylase

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6
Q

From junk DNA to lncRNAs and regulation

A

see onenote

  • lncRNAs bind chromatin modifying proteins
  • target specific regions of the genome
  • lncRNAs also affect mRNA stability/splicing/translation
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7
Q

Increasingly complex epigenetic landscape

A

see onenote diagram

  • ncRNA
  • RNA modification
  • DNA modification
  • chromatin modificiation
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8
Q

DNA mutations and epigenetic diseases

A

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

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9
Q

Epigenetic phenomenon - x-inactivation

A

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
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10
Q

X-inactivation

- epigenetic mechanisms

A
  • lncRNA
  • histone modification
  • DNA methylation
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11
Q

X-chromosome inactivation

A

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
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12
Q

What determines x-chromosome inactivation

A

see onenote

Tsix antisense transcript of xist
- regulates lots of genes around itself

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13
Q

X-inactivation mechanism

A

see onenote slides

It requires:

  1. initial xist rna expression and coating
  2. association of chromatin modifying proteins
  3. DNA methylation 5’ of x-chromosome genes
  4. modification of histones by methyltransferases
  5. 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
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14
Q

What controls XIST expression?

A

see onenote

  • tsix
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15
Q

TSIX is the anti-sense strand of the XIST gene

A

see onenote

  • tsix promotes xist promoter CpG methylation
  • active expression of tsix on active x-chromosome inactivates any xist
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16
Q

Knockdown of tsix causes skewed x-chromsome inactivation

A

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)
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17
Q

TSIX asymmetry governs choice

A
  • 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
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18
Q

x-inactivation evolution

A
  • variable mechanisms in mammals

- marsupials don’t have xist or xact and have a different lncRNA altogether

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19
Q

X-inactivation and disease

A

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
20
Q

Genomic imprinting

A
  • 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
21
Q

Epigenetic mechanisms

A
  1. DNA methylation
  2. lncRNA
  3. histone modification
22
Q

Angelman and Prader-Willi

A

see onenote

Prader-Willi
- paternal deficiency

Angelman
- maternal deficiency

23
Q

Paternal and maternal genomes are both required for normal development

A

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
24
Q

Cyclical re-imprinting

A

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

25
Q

Imprinting clusters

A

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

26
Q

IGF2 and H19

A

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

27
Q

Mice created without fathers

A

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

28
Q

H19 lncRNA controls gene expression by recruiting MBD1

A

see onenote

MBD1 - methy-CpG-binding domain protein 1
- Able to bind chromatin modifying proteins, binds igf2, further silences it

29
Q

IFG2 imprinting and disease

A

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
30
Q

SNRPN imprinting and disease

A

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

31
Q

So why have imprinting?

A
  • 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

32
Q

Genome wide epigenetic landscape

A
  • 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

33
Q

What can epigenetic marks tell us?

A

see onenote

34
Q

ChIP-Seq

A

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

35
Q

Genome wide epigenetic scans

A

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
36
Q

Genome wide scans in ESCs

A

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
37
Q

Non-coding RNAs and epigenetics

A

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

38
Q

the expanding world of ncRNA

A

see onenote

39
Q

lncRNA and cancer

A

see onenote

MEG3 - maternally expressed gene
- Mis-expressed in cancer

Important for understanding in how to best treat diseases

40
Q

Diet and inheritance

A

see onenote

41
Q

Variation in coat colour in isogenic yellow agouti mice

A

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
42
Q

Maternal environment affects methylation

A

see onenote

  1. maternal exposure to endocrine disruptors and toxic compounds
    - low methylation in offspring
  2. normal conditions
    - variable methylation
  3. maternal supplements with methyl donors and cofactors
    - high methylation in offspring
43
Q

Diethylstilbestrol (DES)

A

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

44
Q

Hypospadia; an epigenetic disease?

A

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
45
Q

Transgenerational inheritance or direct exposure?

A
  • 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

Human and Medical Genetics (32 decks)

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