Imprinting in the news Potential blood test to

Imprinting in the news Potential blood test to screen for colon cancer • Colon Cancer- 3 rd most deadly cancer in America Loss of Imprinting (LOI) of the Igf 2 gene correlates with colon cancer Result= Maternal Igf 2 gene is turned on • With family history- 5 X more likely to show LOI • Polyps detected- 3 X more likely to show LOI • Personal history- 22 X more likely to show LOI Source- AP News, March 14, 2003

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 • Igf 2, Ig. F 2 r, H 19, Grb 1 2. Brain development • Prader-Willi syndrome (PS), Angelman syndromes (AS), Peg 1/Mest

Categories of imprinted genes 1. Fetal growth genes- Insulin-like growth factor-like II (IGF 2) response pathway • • IGF 2 Igf 2 r Grb 10 H 19 Gnas Rasgrf Mash 2 – Why? - Embryo develops in a parasite-like relationship with mother.

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

Prader-Willi Syndrome • • • 1 in 15, 000 live births mostly sporatic deletion at 15 q 11 -q 13 diagnosis at 2 years obesity, short, small hands/feet, unusual facial features, mild mental retardation • compulsive overeaters

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

Angelman Syndrome • 1 in 25, 000 live births • mostly sporatic • 80 % have deletion at 15 q 11 -q 13 – Specifically mutation of UBE 3 A gene

Angelman Syndrome • 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

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

Functional studies- Chimeras + Definitions Implant into pseudopregnant female • 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 chimera

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

Turner syndrome Only 1 X chromosome • 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

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- Igf 2 (paternally expressed)-if defective=40% reduction in growth

Parent Offspring Conflict Hypothesis (Haig hypothesis) Example – The Igf 2 gene and its receptor Igf 2 r • Igf 2 (paternally expressed)-if defective=40% reduction in growth • Igf 2 r (Igf 2 receptor)- if defective=increase growth • Igf 2 -/Igf 2 r- = 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

Is the imprint erased during embryogenesis?

Is the imprint erased during embryogenesis? • Observation-Aparthenogenetic embryos generated 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 Dnmt 1 2. Active process via specific demethylation

Is the imprint erased during embryogenesis? • Evidence in support – Biallelic expression of imprinted genes occurs in primordial germ cells – Igf 2 r imprinting control region (ICR) is methylated in E 8 embryos, but unmethylated E 12. 5 embryos – Dnmt 1 is at high levels during Igf 2 r maternal imprint • Answer- Probably

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

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

How are imprinted genes silenced? Mechanism. Promoters compete for a single enhancer Igf 2 H 19 Problem with model. Both H 19 and Igf 2 are expressed if H 19 gene replaced with luciferase S. Tilghman, Cell 96: 185

How are imprinted genes silenced? Epigenetic marker binds to unmethylated DNA Igf 2 H 19 Mechanism. Methylation serves two purposes 1. Inactivate a gene (e. g. H 19) 2. Prevent binding of epigenetic marker so that Igf 2 is activated Epigenetic insulator prevents enhancer from “talking to” Igf 2 Evidence in support: if delete insulator element- both Igf 2 and H 19 expressed S. Tilghman, Cell 96: 185

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

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

Prader-Willi Syndrome Normal expression patterns = ON

Prader-Willi Syndrome Normal expression patterns = ON In Prader-Willi deletion, maternal and paternal copies are silent In Angelman, many genes are activated, but UBE 3 A is silenced

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