1. Imprinting in the news Colon Cancer- 3 rd most deadly cancer in America Loss of Imprinting (LOI) of the Igf2 gene correlates with colon cancer With family history- 5X more likely to show LOI Polyps detected- 3X more likely to show LOI Personal history- 22X more likely to show LOI Source- AP News, March 14, 2003 Result= Maternal Igf2 gene is turned on Potential blood test to screen for colon cancer

2. Genomic Imprinting Definition- Differential expression of two parental alleles –Only occurs in eutherians (placental, nonmarsupial) mammals –Not in other vertebrates Of 20-some identified genes, most are involved in 1. Fetal growth Igf2, IgF2r, H19, Grb1 2. Brain development Prader-Willi syndrome (PS), Angelman syndromes (AS), Peg1/Mest

3. Categories of imprinted genes 1. Fetal growth genes- Insulin-like growth factor-like II (IGF2) response pathway IGF2 Igf2r Grb10 H19 Gnas Rasgrf Mash2 –Why?- Embryo develops in a parasite-like relationship with mother.

4. Categories of imprinted genes 2. Brain development- –Prader Willi Syndrome- paternal chromosome deletion –Angelman Syndrome- maternal chromosome deletion –Why?- Any ideas?

5. Prader-Willi Syndrome 1 in 15,000 live births mostly sporatic deletion at 15 q11-q13 diagnosis at 2 years obesity, short, small hands/feet, unusual facial features, mild mental retardation compulsive overeaters

6. A typical Prader-Willi patient Prader Willi Syndrome- Due to paternal chromosome deletion

7. 1 in 25,000 live births mostly sporatic 80 % have deletion at 15q11-q13 –Specifically mutation of UBE3A gene Angelman Syndrome

8. Speech impairment None or minimal use of words Receptive and non-verbal communication skills higher than verbal ones Movement or balance disorder, usually ataxia of gait Behavioral uniqueness: any combination of frequent laughter/smiling; apparent happy demeanor easily excitable personality, often with hand flapping movements; hypermotoric behavior; short attention span

9. Angelman Syndrome Normal –Angelman Syndrome- maternal chromosome deletion Or 2 paternal chromosomes Or an imprinting defect

10. Definitions Androgenotes- two paternal genomes Gynogenotes- two maternal genomes Both of these (andro- and gyno-genotes) result in an imbalance (increase or decrease) of expression of imprinted genes –Result is developmental abnormalities Functional studies- Chimeras + Implant into pseudopregnant female chimera

11. Functional studies- Chimeras Androgenetic- oversized, small brains Gynogenetic- growth retarded, large brains (cortex) –Conclude- imprinted genes contribute to rapid expansion of the cortex

12. Turner syndrome 1/2000; females only short stature, failure to mature sexually Often learning difficulties, skeletal abnormalities, hearing loss, liver dysfunction, heart and kidney abnormalities, infertility, and thyroid dysfunction Females with single X chromosome –If X from father- better verbal and social skills than if X from mother –Conclude- Some imprinted genes on X escape inactivation only if from father Only 1 X chromosome

13. Parent Offspring Conflict Hypothesis (Haig hypothesis) Conflict between male and female over allocation of maternal resources to offspring Dad uses imprinting to direct all resources to immediate offspring (not future litters) Mom uses imprinting to allocate resources to multiple litters –Thus, predict paternally expressed genes would promote growth, maternally expressed genes should slow it down –Prediction mostly hold true Example- Igf2 (paternally expressed)-if defective=40% reduction in growth

14. Parent Offspring Conflict Hypothesis (Haig hypothesis) Example – The Igf2 gene and its receptor Igf2r Igf2 (paternally expressed)-if defective=40% reduction in growth Igf2r (Igf2 receptor)- if defective=increase growth Igf2 - /Igf2r - = normal Another test- Ask if imprinting fails to occur in a monogomous species The Beach mouse is entirely monogomous ….but imprinting still occurs, contrary to model

15. Is the imprint erased during embryogenesis?

16. Observation-Aparthenogenetic embryos generated from from immature embryos proceed to late developmental stage that from using mature embryos- Answer- Probably Two general mechanisms proposed: 1.Passive process via direct methylation of Dnmt1 2.Active process via specific demethylation

17. Is the imprint erased during embryogenesis? Evidence in support –Biallelic expression of imprinted genes occurs in primordial germ cells –Igf2r imprinting control region (ICR) is methylated in E8 embryos, but unmethylated E12.5 embryos –Dnmt1 is at high levels during Igf2r maternal imprint Answer- Probably

18. How are imprinted genes silenced? S. Tilghman, Cell 96:185

19. How are imprinted genes silenced? S. Tilghman, Cell 96:185 Dnmt-/- mice- Many imprinted genes (e.g. H19) reactivated..but, Igf2 and Igf2r are silenced Mechanism- Methylation interferes with transcription factor binding Problems with model- 1. Promoters of silent Igf2 and Igf2r alleles are unmethylated 2. One gene, Mash2, is unaffected by loss of methylation

20. How are imprinted genes silenced? S. Tilghman, Cell 96:185 Mechanism- Promoters compete for a single enhancer Problem with model- Both H19 and Igf2 are expressed if H19 gene replaced with luciferase Igf2H19

21. How are imprinted genes silenced? S. Tilghman, Cell 96:185 Epigenetic marker binds to unmethylated DNA Mechanism- Methylation serves two purposes 1.Inactivate a gene (e.g. H19) 2.Prevent binding of epigenetic marker so that Igf2 is activated Igf2H19 Epigenetic insulator prevents enhancer from “talking to” Igf2 Evidence in support: if delete insulator element- both Igf2 and H19 expressed

22. Evidence for chromatin boundary mechanism Deletion of ICR- both genes expressed Identify protein (called CTCF) that binds ICR CTCF cannot bind methylated DNA Thorvaldsen and Bartolmei, Science 288:2145, 2000

23. How are imprinted genes silenced? S. Tilghman, Cell 96:185 Mechanism- Antisense transcription of unmethylated chromosome blocks sense strand transcription Mechanism- Antisense RNA blocks sense strand transcription

24. Normal expression patterns = ON Prader-Willi Syndrome

25. Normal expression patterns = ON Prader-Willi Syndrome In Prader-Willi deletion, maternal and paternal copies are silent In Angelman, many genes are activated, but UBE3A is silenced

26. Prader-Willi Syndrome Proposed mechanism of SNPRN imprinting In Prader Wille, the Switch Initiator Site (SNSIS) is mutated in paternal chromosome, such that it can’t bind BD RNA Imprintor gene RNA (BD RNA) SNSIS SNRPN gene Female allele Imprintor gene RNA (BD RNA) SNSIS SNRPN gene Male allele Dnmt 1 2 3