8L. Epigenetics

Question 1

Meaning of epigenetics

Answer 1

The study of heritable mechanisms that affect the transcriptional state of a gene that cannot (!) be explained by DNA sequence

• Expand the capacity of the genome to create diversity
• Clonal and irreversible changes in genes
• Silencing: epigenetic loss of expression (equivalent to inactivating mutation)
• Generally lead to monoallelic expression of that gene

Question 2

Relationship of genome, epigenome and phenotype

Answer 2

• Epigenome: the complement of chemical compounds that modify the expression and function of the genome (if genes are switched on/off f.ex)
• Genome: the genetic material of an organism (DNA/genes)
• Phenotype: (fra nett): total characteristics displayed by an organism under a particular set of environmental factors, regardless of the actual genotype of the organism
• Genome+epigenome+environment -> phenotype
• *Se bilde powerpoint

Question 3

Epigenetic mechanisms

Answer 3

1) Transcriptional
- DNA methylation
- Chromatin remodelling (Active: accesible, condensed: inaccesible)
2) Posttranscriptional
- RNAi
- miR
- LncRNA
3) Post-translational
- Histone modifications (histone code hypothesis

Question 4

Transcriptional epigenetics: DNA methylation (enzymes, pattern, consequences)

Answer 4

3 DNA methyltransferases (DNMT1, -3A & -3B)

Normal pattern:
Unmethylated CpG island and CpG island shore -> methylated gene body -> methylated repetitive sequence
- Abnormal pattern: opposite of above

Consequences:

a. Hypomethylation: genome instability
b. Hypermethylation: promoter silencing
c. Deamination: mutation
d. UV: increased UV-induced mutations
e. Carcinogen: carcinogen-induced mutations

Question 5

Transcriptional epigenetics: Chromatin remodeling

Answer 5

1) Acetylation (generally make access (activ.) chromatin)
       \+ Acetyltransferases
       - Deacetylases
2) Methylation (generally inactivating chr, except H3K4)
       \+ PKMT (lysine) or PRMT (arginine)
       - Demethylases
3) Phosphorylation
       \+ Kinases
       - Phosphatases
4) Ubiquination
       \+ Ligases
Other: nucleosome positioning

Question 6

X chromosome inactivation, role of XIST

Answer 6

X-inactive specific transcript

• Spliced, noncoding RNA
• Association of Xist RNA w/X-chr = condensed chr
• Required to initiate silencing of X chr

X chr inactivation process

• Xist RNA synthesized from XIC locus on X chr
• Xist coats X chr -> inactivated chr
• Xist recruits chromatin regulators to silence gene expression on the inactive X chr

Question 7

Autoimmunity and X inactivation

Answer 7

Predominance of females with autoimmune diseases - suggest X chr involvement
- Due to skewed X inactivation: expression of X-linked antigens in thymocyte development in thymic medulla

Question 8

Genomic imprinting definition

Answer 8

The non-equivalent expression of genes based on parent-of-origin (determined which is silenced - not random)

• Imprinted allele is silenced (e.g maternal imprinting = paternal allele expressed only)
• Epigenetic event
• Established in the germline

Question 9

Evidences of nonequivalence of parental genome

Answer 9

Nuclear transplantation demonstrates the non-equivalence of parental genomes:

• Fertilized egg (M+F) = normal
• Gynogenote (F+F) = Failure of development
• Androgenote (M+M) = Failure of development

Mola hydatidosa
- Complete mole: egg fertilized by 2 sperm, and maternal material absent/lost (=only paternal genetic material)

Question 10

Mechanisms of genomic imprinting

Answer 10

2 major mechanisms (wikipedia):

1) DNA methylation
2) Histone modification

Question 11

Causes of Prader Willi and Angelman syndromes

Answer 11

Prader-Willi syndrome (PWS) - missing paternal allele

• Maternal UPD
• Paternal deletion of 15q11-13 (most common)
• Wrong imprinting (<2 %)

Angelman syndrome (AS) - missing maternal allele

• Paternal UPD
• Maternal deletion of 15q11-13 (most common)
• Wrong imprinting (8 %)
• UBE3A mutation

*Methylated allele is silenced (H3-K9-diMe), while the other allele is expressed (no DNA methylation - H3/4-Ac + H3-K4-diMe)

Question 12

Possible role of genomic imprinting

Answer 12

Embryonic, placental and neonatal growth regulators

• Paternal genes promote growth (“paternally expressed imprinted genes”)
• Maternal genes suppress growth (“maternally expressed imprinted genes”)
• Parental conflict of interest theory - male wants its offspring to be as strong as possible, while female wants to distribute resources between several offspring (with potentially different fathers)

Question 13

Relationship between genomic imprinting and cancer

Answer 13

Cancer can be due to LOI (loss of imprinting = loss of original pattern of imprinting/abnormal imprinting):

1) Abnormal imprinting of oncogenes
2) Abnormal imprinting of tumor suppressor genes

Methylation:

• Global hypomethylation -> mitotic recombination & genomic instability -> cancer
• CpG island hypermethylation/promoter hypermethylation in tumor suppressor genes -> loss of TSG expression -> cancer

Histone modification

Question 14

Position effect

Answer 14

Fra nettet: The alteration in the expression of a gene or genetic region due to its relocation within the genome as a result of inversion or translocation (virker riktig)

• Explains role of transposable elements in gene expression
• If gene is inserted into/close to a transposon -> variable expression depending on distance
• If gene is inserted into an active site -> expression

Question 15

Epigenetic changes caused by aging and by in vitro fertilisation

Answer 15

Twin study:
- 3 yr old: several epigenetic tags in same place
- 50 yr old: many different epigenetic tags
Suggest aging affects epigenetics - somatic events
1) Stochastic establishment of epigenetic state
2) Environmentally-induced epimutation
3) Random, age-related epimutation

Question 16

Transgenerational effects

Answer 16

Transgenerational epigenetic inheritance
- From parent to child
Germline events: 
1) Epimutation in germ cells
2) Incomplete erasure of parental marks

Can be due to RNA transfer - white spotted phenotype in offspring both with and without the white spotted gene
Or due to protein transfer - e.g Cre recombinase - green mouse even though lacking Cre gene

Question 17

Erasers, writers and readers in histone modification

Answer 17

Erasers:

• Demethylases
• Deacetylases
• Phosphatases

Writers:

• Methyltransferases
• Acetyltransferases
• Kinases
• Ligases

Readers:
- Proteins with domains such as bromo, chromo and tudor

Question 18

Histone code hypothesis

Answer 18

Different combinations of histone modifications, especially located near or within a gene’s promoter, may be VERY SPECIFIC to the transcriptional state of that gene

Question 19

Chromatin states

Answer 19

• Active (mostly acetylated)
• Permissive (more methylated)
• Repressed (repressor added)
• Inactive (many repressors, condensed, more phosp)

Question 20

Posttranscriptional epigenetic regulatory mechanisms

Answer 20

RNAi: RNA interference
- Gene silencing (of specific genes and perhaps endogenous viruses, transposons, retroviruses)

miR: MicroRNA (endogenous source)
Block translation of mRNA (interact with 3’ UTR region on mRNA)

LncRNA (long noncoding RNA)
Mechanism: molecular interaction with nucleic acids and protein

Question 21

XCI + types

Answer 21

X chromosome inactivation (“Lyonization”) -> Barr body

1) Random XCI -> both normal & mutant product made
2) Skewed XCI “fortunate” -> mostly normal product
3) Skewed XCI “unfortunate” -> mostly mutant product
4) Male (XY): only mutant product

Question 22

RNAi (Posttranscriptional epigenetic regulatory mechanisms)

Answer 22

RNAi: RNA interference

• Gene silencing (of specific genes and perhaps endogenous viruses, transposons, retroviruses)
  1) Trigger dsRNA cut to siRNA by a DICER (endonuclease)
  2) siRNA (exogenous source) combines with RISC (RNA induced silencing complex), bind perfectly (!) to mRNA -> mRNA cleavage

Question 23

miR (Posttranscriptional epigenetic regulatory mechanisms)

Answer 23

miR: MicroRNA (endogenous source)

• Block translation of mRNA (interact with 3’ UTR region on mRNA)
• 20-23 nucleotide long
• Hairpin RNA cut by DICER -> miR
• miR combines with RISC, bind imperfectly (!) to mRNA -> inhibit translation (or degrad. if near-perfect binding)

Question 24

LncRNA (Posttranscriptional epigenetic regulatory mechanisms)

Answer 24

LncRNA (long noncoding RNA)

• Mechanism - molecular interaction with nucleic acids and protein:
  a) LncRNA - DNA (pretranscriptional/transcriptional): make complex configurations that can associate with regulatort proteins to affect neighboring regions
  b) LncRNA - RNA: LncRNA hybridize to sense RNA -> modulate sense mRNA expression
  c) LncRNA - protein: either work as molecular decoys (“traps”) preventing protein function - or - as scaffolds for assembly of protein complexes (e.g Xist - X chr inactiv.)