Lecture 17 and 18: Epigenetics and Imprinting

Name three examples of epigenetic phenomena and explain their feature

Genomic imprinting, X chromosome inactivation, position effects, development, differentiation No changes in genotype! All change phenotype

Describe three different mechanisms whereby DNAme can affect transcription

Direct DNA methylation of CG rich promoter Inhibition of TF binding to enhancer sequences Recruitment of repressive methyl binding proteins

Name 2 proteins essential for de novo DNAme in oocytes and sperm

DNMT3A and cofactor DNMT3L

What is the link between de novo DNAme and the ICF syndrome? What regions of the genome are affected?

DNMT3B is mutated in ICF patients → some centromeric regions lose DNAme

ICF - immunodeficiency, centromeric instability and facial anomalies syndrome

Mature oocytes are methylated at 40%. What regions of the genome are methylated?

Regions: gene bodies of transcribed, active genes during oocyte growth

How do these regions of the oocyte genome acquire DNAme?

Transcribed regions are defined by RNA pol II as it catalyzes transcription Histone methyltransferase SETD2 acts as writer - deposits mark H3K36me3 as it travels along genes DNMT3A/B is then recruited to H3K35me3 marked regions via PWWP domain which reads the H3K36me3

DNMT3A/B methyaltes regions

What are CpG islands?

GC rich sequences present at the promoter region of ~70% of genes

Usually unmethylated to allow for gene transcription

What is the link between H3K4me3 and the methylated state of CGIs?

H3K4me3 mark inhibits DNMT3A and 3B methylases → keeps CGIs unmethylated

Mark is added by CXXC zinc-finger DNA-binding proteins (MLL1, MLL2, CFP1)

DNAme is erased during preimplantation stages as well as in primordial germ cells. What is a key difference between those two waves of demethylation?

DNAme marks at imprinted genes are erased in the germline In PGCs, they are maintained during preimplantation development (or else all imprinted expression would be lost)

What is the link between the maintenance of imprinted DNAme marks in preimplantation embryos and the histone mark H3K9me3?

Imprinted differentially methylated regions (DMRs) are recognized by zinc-finger proteins which act as methyl binding proteins Recruit the histone methyltransferase SETDB1 which deposits H3K9me3 on DNA methylated allele H3K9me3-marked regions are preferentially maintained

• DNMT1 cofactor UHRF1 binds mark

Several explanations were proposed to explain the lethality of parthenogenetic embryos. Explain two that were eliminated by the generation and analysis of gynogenetic embryos.

3 hypotheses

• extra-genetic sperm contribution or missing, or activation by fertilization required

  • ruled out: gynogenetic embryos are fertilized by sperm and then removed → sperm extra-genetic components do not rescue phenotype

• homozygosity of PG for most markers

  • ruled out: gynogenetic embryos can be made from two non-identical maternal pronuclei → does not rescue the phenotype

• non-equivalence of maternal and paternal genetic contributions

  • assumed correct - no ruling out

Name three characteristics of imprinted genes

Monoallelically expressed from single parental allele Often clustered in large chromosomal domains Associated with different epigenetic marks on the maternal and paternal allele, such as DNAme Abnormal expression can cause human imprinting disorders

How can DNA methylation regulate a paternally expressed imprinted gene?

Direct silencing the maternal copy through DNAme at CG-rich promoter → inherited from the egg and form a germline differentially methylated region in somatic cells

Silencing of maternal allele by cis-acting long non-coding RNA - expressed from maternal allele because its own promoter is silenced by a DNAme mark inherited from sperm

When do the germ cells acquire DNA methylation in the male germ line? What about in the female germline?

Male: de novo DNAme occurs in post-implantation germ cells (E15.5)

Female: maternal gDMRs established postnatally, de novo DNAme occurs during oocyte growth (2-3 weeks)

Which enzyme is absolutely required for this process?

DNMT3A

In this degree, probands A, B, C, and D in generation IV are all carriers of the same mutation, they are all heterozygous. Explain why A and B are affected by this imprinting disorder but not C and D

Name two mammalian species in which genomic imprinting has not been observed

• Only observed in mammalian species in which embyro grows at least partially in utero

• egg-laying monotremes - platypus and echidnas