Imprinted genes in mammals

Question 1

How do you get a mule?

Answer 1

• Breed a female horse (mare) with a male donkey (jack)

Question 2

What is the difference between a mule and a hinny? (3)

Answer 2

• Mules are very placid and good farming animals but hinnies are unsuitable for labour
• Both are generally sterile
• Early indication that the maternal and paternal genomes are not equal

Question 3

How do you get a hinny?

Answer 3

Breed a female donkey (jenny) with a male horse (stallion)

Question 4

How do you get a liger? (3)

Answer 4

• Breed a female tiger with a male lion
• Ligers over twice as heavy as either of their parents
• Males are sterile, females are occasionally fertile

Question 5

How do you get a tigon? (3)

Answer 5

• Breed a female lion with a male tiger
• Moderately sized
• Males are sterile, females are occasionally fertile

Question 6

Why are ligers huge? (4)

Answer 6

• Lions have a competitive mating strategy meaning a female lion can be pregnant from multiple male lions at once
• The maternal genes want all offspring to be the same size as they are all hers, but the paternal genes want its own cub to be the biggest/strongest and outcompete the others in the womb from different fathers
• Male growth factor genes encourage growth and female growth factor receptor genes oppose this by receptor downregulation, which balances out resulting in normal sized offspring
• Tigers don’t have a competitive mating strategy so no need for downregulation of sensitivity to growth factors from the male lion, meaning that ligers (tiger mum and lion dad) end up massive because no growth factor inhibition

Question 7

What was the first evidence for genomic imprinting? (4)

Answer 7

• Injecting maternal and paternal pronuclei into an enucleated oocyte and implanting into a foster mother resulted in viable embryos
• Gynogenetic and androgenetic diploids were inviable
• Inviability could be due to some offspring bring YY however even the XX individuals didn’t survive so authors concluded it couldn’t be due to the sex chromosomes
• Due to genomic imprinting meaning that both maternal and paternal genes are essential for embryo development

Question 8

What is a gynogenetic diploid?

Answer 8

Injection of 2 female (haploid) pronuclei into a mouse oocyte

Question 9

What is an androgenetic diploid?

Answer 9

Injection of 2 male (haploid) pronuclei into a mouse oocyte

Question 10

What is an example of an imprinted gene? (2)

Answer 10

• Insulin-like growth factor 2 (IGF2)
• IGF2 is on chromosome 7 (autosome) meaning it is not a sex-linked trait

Question 11

How is IGF2 imprinted?

Answer 11

Maternally imprinted so maternally silenced and paternally expressed

Question 12

What is the result of targeted knockout of IGF2? (5)

Answer 12

• Breed WT female with IGF2/- male mouse
• Offspring who are IGF2/IGF2 are normal sized (both sexes) but offspring who are IGF2/- are growth-deficient (both sexes) because they have inherited a null copy from their father and the maternal copy is silenced
• Breed IGF2/- female with WT male mouse
• All offspring are normal size because the maternal IGF2 is silenced so doesn’t matter if they inherit the normal or null copy from the mother
• Phenotype is determined by the paternal gene

Question 13

What were the conclusions from the IGF2 imprinting paper DeChiara et al., 1992 (Cell)? (6)

Answer 13

• Transmission of the IGF2 mutation through the male germline results in heterozygous progeny that are growth deficient
• When the disrupted gene is transmitted maternally the heterozygous offspring are phenotypically normal
• The difference in the growth phenotypes depends on the type of gamete contributing the mutated allele
• Homozygous mutants are indistinguishable in appearance from growth deficient heterozygous siblings
• Only the paternal allele is expressed in embryos while the maternal allele is silent
• Key finding: in mice the paternal and maternal members of some autosomal gene pairs are functionally non-equivalent

Question 14

What is euchromatin? (4)

Answer 14

• Open and active chromatin
• Associated with marks such as H3K14 acetylation by histone acetyltransferases (HAT) such as p55
• H3K14ac is a docking site for bromo-domain proteins which stimulates nucleosome accessibility for transcription
• Acetylation can be removed by histone deacetylases (HDAC) such as Rpd3

Question 15

What is heterochromatin? (4)

Answer 15

• Condensed and repressed chromatin
• Associated with marks like H3K9me3 by histone lysine methyltransferases (KMT) such as SUV39H1
• H3K9me3 is a docking site for heterochromatin protein 1 (HP1) which promotes chromatin condensation, impairing nucleosome accessibility
• Methylation can be removed by histone demethylases such as TET demethylase

Question 16

What is Air? (2)

Answer 16

• An antisense transcript which uses base pairing to recruit silencing machinery to the chromatin
• Important in imprinting

Question 17

What are CpG islands? (3)

Answer 17

• C/G pairs in the promoter upstream from gene transcriptional start sites
• Methylation of CpG islands is associated with silencing of that locus as the transcriptional machinery can’t access the promoter
• Demethylated CpG islands generally means the gene is expressed, still methylation within the gene body

Question 18

What does maternally imprinted mean? (2)

Answer 18

• Allele of a particular gene inherited from the mother is transcriptionally silenced (i.e. CpG island in the promoter is methylated)
• Direct observation of the phenotype governed by the paternal allele

Question 19

What is paternal imprinting? (2)

Answer 19

• Allele of a particular gene inherited from the father is transcriptionally silenced (i.e. CpG island in the promoter is methylated)
• Direct observation of the phenotype governed by the maternal allele

Question 20

What are examples of paternally imprinted genes in mice? (3) ***

Answer 20

• IG-DMR (12)
• H19-Igf2 (7) (H19 is expressed maternally, IGF2 is expressed paternally, imprinting control centre is paternally methylated)
  (- Rasgrf1 (9))
  (Genomic regions rather than single genes, chromosome number in brackets)

Question 21

What are examples of maternally imprinted genes in mice? (7) ***

Answer 21

• Nespas (2)
• Peg3 (7)
• Snrpn (7)
• Kcnq1 (7)
• Grb10 (11)
• Igf2r (17)
• Gnas (2)
  (Genomic regions rather than single genes, chromosome number in brackets)

Question 22

How was uniparental reproduction achieved in mice (bimaternal/bipaternal)? (4)

Answer 22

• Cultured embryonic stem cells in a medium which causes global DNA demethylation
• 3 paternally imprinted loci were deleted one by one using CRISPR Cas9 to generate bimaternal mice
• 7 maternally imprinted loci were deleted using CRISPR Cas9 to generate bipaternal mice
• Issues with this long term due to deletion of major gene regions