Molecular Genetics

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Breaking News…

Major genetic risk factor works in addition to significant co-morbidities (obesity, age)

One of several instances where a Neandertal or Denisovan haplotype was linked to an increased in a particular human trait (immunity and pain)

Principles of human gene expression

Gene expression requires the recruitment of RNA polymerase II, a large and multi-subunit complex

• Initiation must occur at the beginning of genes, defined by cis- acting DNA elements called promoter regions.

• Termination must occur at the end of genes at sites called poly- adenylation sites (PAS).

• RNAP II recruitment to promoters requires the assistance of DNA binding proteins called transcription factors (TFs)

Kornberg R D PNAS 2007;104:12955-12961

Structure of core RNA polymerase II at 2.8-Å resolution

q core enzymes DNA→ RNA

O Fife "-

Figure 6-17 Molecular Biology of the Cell (© Garland Science 2008)

RNAPII promoter core elements carry TF binding sites

(Drosophila)

Kwak and Lis (2013) Ann Rev Genet

recruits&

binds

Transcription start site

What do human RNA Pol II promoters look like?

TATA box: - the best described type - Associated with sharp initiation sites - Correspond to tightly regulated, often tissue-specific genes

- NOT THE MOST FREQUENT (10-20%)

CpG islands: - THE MOST COMMON (~60% OF ALL PROMOTERS) - Associated with broad initiation sites - Correspond to broadly expressed “housekeeping” genes - Also include many inducible genes

!! 58% of human genes have at least TWO promoters - leads to expression of protein isoforms

1-Ispot isprecise

→minority

promotor is not recruited precisely

Alternative promoters in the Dystrophin gene

C: Cortex M: Muscle P: Purkinje R: Retinal CNS: Central Nervous System S: Sperm G: Ganglia

Frequent in human genes Leads to different protein isoforms at N-terminus

Other causes of protein isoform variation in the human proteome:

- Alternative termination (affects C-terminus) - Alternative splicing

Vaquerizas. Nature Rev Genet. 10:252, 2009

Transcription factors allow specific gene expression in time and space

1. Transcription factors are composed of modular DNA-binding domains

2. Most factors belong to multi-gene families; members share very similar DNA binding domains (ex: ZNF-C2H2 or HLH)

Characteristics of Site-specific Transcription Factors

Over 1000 TFs in the human genome!!

I '

ntetfactioh is

2-Ihcfinger (PRDM9)

#1: Zinc Finger Domains

https://www.youtube.com/watch?v=WyU2v7HT6bw

#2: Homeodomain proteins

https://www.youtube.com/watch?v=aHgbYEZMYlQ

Evo-Devo (Despacito Biology Parody) | A Capella Science (includes some nice videos of Homeobox) https://www.youtube.com/watch?v=ydqReeTV_vk

For fun: Role of TFs in development:

TF possess defined DNA binding modules that recognize specific DNA sequences

Vaquerizas. Nature Rev Genet. 10:252, 2009

Ubiquitous and tissue-specific TFs 100 TFs were expressed

in all 32 samples 75 TFs were expressed in only 1 of the 32 samples Tissue specific TF Drive the expression engaged in specific of housekeeping genes functions

Homeobox

D C

Everything else: ZNFs

TFs cluster in the human genome 4 clusters

NOT randomly distributed in our genome

diff clusters are like GPSfor ourbody /

Promoters are major Transcription Factor binding sites Transcription factors binding dictates tissue and time-specific expression patterns

TFs binding by ENCODE “Gene activity” model (Broad HMM) Red = active promoter Green = elongating RNA polymerase

Promoter region is also depleted in nucleosome (Nuclease hyper-sensitive)

BRCA2 CGI

Pbound by manyTF

Mostdense w/ TF

compared tothebody

p rich 1h TF & boundby many TF

almost always depleted

↳meansit IS open for TF

accessable for enzymes that might break DNA

Simplified promoter-centered transcription cycle

Chromatin opening by TF binding

PIC formation

Open complex formation

Promoter clearance

Escape from pausing

Elongation (coupled to splicing)

Termination

Recycling

Fuda et al. Nature 461:186,2009

TF: Transcription Factor PIC: Pre-Initiation Complex RNAP: RNA Polymerase GTFs: General Transcription Factors

RNAP complex

GTFs

Pausing factors

Elongation factors

RNA Transcription: the movie https://www.youtube.com/watch?v=WlMV_l88Lus

TF opens the chromatin, evicts the nucleosome revealing the promoter region. -once you reveal the promoterRegion you can recruit additional transcription factors I 1

general TF

RNA pot complex actualTF machihang

→quality control

Forget about promoters… It’s all about enhancers! Characteristics of enhancers

1. Clustered binding sites for different TFs

2. Located far from the TSS

3. Recruit RNA Pol II and other complexes

4. Stimulate transcription, orientation- independent in a regulated manner

boundbyTF

start site

#I goal

↳ of target gene

Gene expression often requires enhancer elements:

Looping

Enhancers work at a distance through DNA looping

• Enhancers historically identified in model organisms as distant mutations that disrupt normal gene expression

• Distant sequences found to correspond to clustered binding sites for different TFs

• Cis-acting DNA sequences acting at a distance to facilitate RNA PolII recruitment

mediates looping

interactions

The Interferon-E Enhancer is a cluster of TF binding sites

Panne.The enhanceosome. Current Opinion in Structural Biology. 18:236, 2008

A B M

Type of signaling molecules that are involved in immune& Inflammatory signal

responses . (vital Infections )

The interferon-E gene (IFNB1)

Short gene / No CpG island promoter / No clear TF binding site around promoter

IFNB1 is located in a gene desert that is nonetheless enriched in TF binding sites (Regulatory jungle!!)

Enhancers (Orange / yellow)

no activity

✓

ZOOM out

enhancers-- control tower at airport send info to promoter

- -

multiple enhancers

all targeting multiple regions'FNB 't w/ different TF 's bound

Global Characterization of Enhancers

Enhancers are located mostly 5- 500 kb upstream or downstream from promoter they regulate

One gene can possess many enhancers!!

Large-scale identification of enhancers during cardiac differentiation Wamstadt et al., (2012) Cell

How many enhancers are there ?

characterized location & # of enhancers on everystep.

TSS

Enhancers are difficult to recognize at the DNA sequence level • Correspond to strong clusters of TF binding motifs • Variable size (200 bp – 2 kb) • Best characterized by epigenetic profiles (open chromatin, RNA Pol II

binding, specific histone modifications)

Enhancers act at a distance • Neighboring promoters is often, but not always the target

Enhancers are dynamically regulated in a time- and cell-type- specific manner

• Regulation mediated through chromatin modifications • By contrast, promoters are often not dynamically regulated!

Characteristics of Enhancers

runway i always clear

beginning of runway : promoter (always clear) ↳ almost always open .

control tower : makes the decision, radioing signal in the distance to the promoter

Enhancers often coordinate precise spatiotemporal gene expression patterns during development

Consequences of enhancer mutations include developmental malformations (polydactyly, limb formation)

Robson et al., (2019) Molecular Cell

3diff regions b/c 3diff enhancer's cell type specific activity

tail tip forebrain time Hemigway's cat's

also in humans

extra I

claw

How many enhancers? De Laat and Duboule (2013) Nature

Enhancers enhancersOras-acting 331

regulatory sequences

Kim and Shiekhattar (2015) Cell

Updated understanding of enhancers

Enhancers resemble promoters! • Bound by TFs • Recruit RNAP (in both directions) and initiate transcription (generating eRNAs) • Mediator, with the help of cohesin rings, secures the loop • Somehow… the enhancer-promoter contact facilitates expression

recruited bothways

mediated ring

complex protein

stabilizes the loop

RNAP doesn't know

right gene direction.

(signal)

Extensive 3D networks of promoter-enhancer and promoter- promoter contacts underlie gene expression

Li et al., (2012) Cell

Matharu and Ahituv (2015) PLoS Genetics

Science October 25 2013 http://www.sciencemag.org/content/342/6 157/1241006.long

HOW do you → putaface together ?

Face Formation

Did thephenotype E s

change ? E E =

Enhancers are often mutated in common, complex disorders

¥ T

E ÷ ⇒ ¥ •

Super enhancers tend be occupied in only one cell-type

Super enhancers tend be mutated in disease

Heatmap of activity. Red = strong Tissues are arrayed vertically Enhancers (1000s) are arrayed horizontally enhancers control gene expression then can

ask, which genes do the super enhancers control? The answer identify the most critical gene for every celltype. If important, controlled by super enhancers.

specific

- important to understanding human diseases

Remember FTO?

(Herman and Rosen 2015)

• Identified as the TOP hit in GWAS for obesity • FTO encodes for an RNA demethylase • FTO is expressed in hypothalamic neurons

that control appetite and energy expenditure. Transgenic and knockout studies have demonstrated an effect of FTO copy number on adiposity

• However, SNP lies in intron of FTO… • SNP disrupts a TF binding site • This site functions as an enhancer

for nearby IRX3/IRX5 genes • IRX3/5 are TFs that control the

differentiation of adipocyte precursors and control energy expenditure

Epigenetics: what’s in a name?

• “epi”: from greek: “above, beyond”.

• “genetics”: heritable source of biological information.

Epigenetics: : a series of reversible and self-reinforcing modifications of eukaryotic chromatin (F.C. June 2013)

all dueto chemical modifications of DNA

Nuclear transfer experiments

Transplant nuclei into enucleated fertilized eggs

Above genetics?

? ?Embryonic lethality

oocytes haploid nucleus

embryos all die

Conclusions from nuclear transfer experiments:

• Inheriting a diploid genome is not sufficient for development

• A viable embryo can only develop if a maternal and a paternal genome are brought together

• This suggests that there is “extra” information attached to each parental genome that is not encoded in the DNA

E m br yo nic let hal ity

Epigenetic layers and players

Two main layers • DNA methylation:

- Tag = methyl (CH3) group - @ CG dinucleotides - Associated with silencing - Prevalent

• Histone modifications: -Tags = methyl, acetyl, phosphate + many other - @ histone tails - Associated with silencing or activation depending on tag

• Both tagging systems are conserved and critical for development • Tags are often deregulated in disease (cancer, autoimmunity) • Tagging can be influenced by environment or reprogrammed

Writers, Erasers, Readers

“Writers” add post-translational modification to histone tail

• Acetyltransferase • Methyltransferase • Kinase…

“Erasers” remove post-translational modification

• De-acetylase • Demethylase • Phosphatase…

“Readers” bind to specific post- translational modification and trigger a biological output

• Transcription / splicing • Chromatin compaction &

organization • Replication / Recombination

DNA methylation

• 5-methyl-cytosine results from the transfer of a methyl group (CH3) to cytosine residues

• Catalyzed by DNA MethylTransferases (DNMTs)

• ~75% of all CpG dinucleotides are methylated in our genome

1 2

4 3

5 C

DNA replication CH3

CH3

CG CH3

CH3

CG GC

CH3

DNMT1

DNMT1 ensures replication-associated maintenance DNA methylation with 95- 99% fidelity: epigenetic memory

DNA methylation patterns are mitotically heritable from cell to cell

mother-daughter

DNA Methyltransferases

Bind to DNA and catalyze the formation of 5- methylcytosine (“the fifth base”) using S-adenosyl-L- Methionine as co-substrate

SAM = universal methyl group donor

DNMT3A 1 912

variable1 854DNMT3B

PWWP ADD I IV VI VIII IX X DNMT (catalytic)

Binds to Chromatin (H3K4) only if

unmodified

DNMT1: maintenance DNA methyltransferase • Associates with DNA replication fork to copy parental

patterns onto daughter strands

DNMT3a/b: de novo DNA methyltransferases • Establish DNA methylation patterns “fresh” during early differentiation • Mutations in DNMT3B cause ICF syndrome (Immunodeficiency Centromeric and Facial anomaly) • Also bind to chromatin

PRIMARY TARGETS: Repeated DNA elements ◼ Silencing of “junk” DNA – Genome Defense ◼ Loss of DNA methylation leads to:

◼ Reactivation of transposons ◼ Increased genomic rearrangements

Xu et al., Nature 402, 187-91 (1999)

a5meC

WT ICF

SECONDARY TARGETS: Gene regions ◼ Regulation of gene expression ◼ Loss of DNA methylation leads to:

– Embryonic lethality – Failure of X-inactivation (females) – Autoimmunity (Sytemic Lupus)

Morgan et al., 1999, Nature Genet.

Agouti Expression Methylation

The human genome

Retroelements:

Why is DNA methylation important?

DNA methylation shows bimodal distribution

Most of the genome is heavily methylated

The “exception to the rule” correspond to highly expressed CpG island promoters

Esteller, 2007

Heavily methylated (~80%)Unmethylated

“CpG islands” (CGIs): • GC-rich, CG-dense regions of the genome

• rarely methylated even though they are an ideal target

• serve as promoters for ~60% of human genes

• the protection of CGI promoters from DNA methylation and epigenetic silencing is key to their function

DNA methylation undergoes dynamic reprogramming through development

As sperm enters the oocyte ( before the 2 genome fuse) it is attacked by eraser that will strip off most of its methyl group ↳ removing methyl groups allows genome to turn back on ( leads to a reboot of genome)

entire genome

f transcribed

DNA methylation patterns represent a form of “molecular ID” for various cell types

Cancer cells show abnormal DNA methylation patterns

Can be used for early diagnostic

Cancer Epigenome project

Human Epigenome Roadmap Initiative Integrative analysis of 111 reference human epigenomes Nature volume 518, pages 317–330 (19 February 2015)

DNA methylation patterns can be modified by exposure to certain environments

• Nutrition (folate levels, famine) • Toxins, poisons • Aging

Alterations in DNA methylation profiles are linked to human diseases

• DNA methylation changes are perhaps the most prevalent of all changes in human cancers

• Human imprinting disorders (Prader-Willi / Angelman syndrome, Beckwith-Wiedeman syndrome)

• Autism-spectrum disorders • Human reproductive failure (sterility, spontaneous abortion,

stillbirth)

→ leads to abnormal DNA methylation .

- If I0W

can interfere w/

DNA methylation ( leafy green)

Histone modifications represent a second layer of epigenetic control

X-ray structure of the nucleosome (Luger et al., 1997)

• DNA is wrapped around histones in our cells

• Histones carry accessible tails

• Histone tails can undergo various modifications

• Modifications are: ▪ conserved from yeast to human ▪ versatile (expression – silencing)

N-term tails

front / side

µ,,one ya,,, undergo anem,ca, mo,,g,among ,

)

Histone Acetylation Acetylation by Histone AcetylTransferases (HATs) promotes chromatin accessibility and gene expression

De-acetylation by Histone Deacetylases (HDACs) promotes chromatin compaction and gene silencing

Conventional view: • Active genes tend to be hyper-acetylated • Inactive genes show hypo-acetylation

leads tochromatin -

opening

Longest in history

N-term tail

Wang et al., 2009 Cell

Modern view: Acetylation is dynamic during transcription

Wave of acetylation and de- acetylation during transcription elongation along gene bodies

Promoter region always acetylated

(if active)

Histone Acetylation

HI HATS HATS are adding methyl groups as the polymerase is moving thru.

If promoter is active the In the gene body .

nucleosomes around the promoter Net result no acetylation ble '

II't:& ,

"ftp.Yhmpeas?wntin5on8rbeasaecPefyirYgwups/Ditakei- ott .

(tri)-methylation of lysine 4 (H3K4me3) is associated with open chromatin (promoters) in our genome

Histone methylation imparts position-specific effects

Taverna et al., Cell 2006

H3K4me3 is specifically recognized by the TAF3 subunit of TFIID

Vermeulen et al., Cell 2007

3methyl groups added

(tri)-methylation of lysine 9 (H3K9me3) is associated with constitutively silent regions of our genome (centromeres)

H3K9me3 is recognized by HP1 (heterochromatin protein 1) Jacobs and Khorasanizadeh, Science (2002)

HP1 binding triggers chromatin compaction Canzio et al., Mol Cell (2011)

Histone methylation imparts position-specific effects

( '

chromodomain #

( rigid) r'leader

Histone methylation imparts position-specific effects

(tri)-methylation of lysine 27 (H3K27me3) is associated with facultative silencing of developmental genes. H3K27me3-mediated repression is caused by Polycomb Repressive Complexes 1 and 2

Silencing involves recognition of H3K27me3-marked nucleosomes and chromatin compaction

Need to be lysine specific

(optional)

→ Needy to be turned on at the right cell type at the right time . Of

development

Bound by both PRCI& PRC2

Enhancers characteristics

Enhancers are defined by TF binding binding and H3K4me1-marked nucleosomes

Rada-Iglesias et al., Nature 2010

- “Active” enhancers are associated with H3K4me1 AND H3K27Ac

- “Poised” (inactive) enhancers are associated with H3K4me1 AND H3K27me3

OFF

sites

0 0

BoundbyTF

acetylation ↳openregion

BTW: can DNA methylation be “read”? Or: how does DNA methylation achieve silencing?

Deaton and Bird (2011) Genes Dev

DNA methylation can prevent binding of (some) TFs DNA methylation is “read” by Methyl-Binding Domains (MBDs) MBDs recruit HDACs to compact chromatin.

Important concept: co-operation between DNA-based and chromatin- based epigenetic regulation systems

There are DNA methylation ores acetyl groups from nucleosomes.# Rem

readers -_ MBD : These proteins have specific

which closeschromatin domains that recognize

making it had for TF only methylated - & RNA transcripts cytosine to bind

→become inactive.

ChIP-seq ENCODE data from human ESCs

H3K4me3

H3K9Ac

H3K36me3

H3K9me3

H3K27me3

DNA CH3

Active promoter marks Likely CpG island

Elongation mark: active gene

Why so much DNA methylation here?

DNA methylation absent from

promoter Control of “junk” DNA!!

Revealing the chromatin landscape

DNAmethylationatthe body IS NOTsignificant

only silenced whenat promoter But helpful to silence LINES& SINES

A broader view

H3K4me3

H3K9Ac

H3K36me3

H3K9me3

H3K27me3

• Active promoters (CpG islands) are easy to spot! • So are transcribed genes (H3K36me3)

/active

stronger acetylation more active the

gene is

H3K4me3

H3K9Ac

H3K36me3

H3K9me3

H3K27me3

Spotting developmentally regulated (silent genes)

“Poised” genes carry activating AND silencing marks at the same time…

Wall of H3K27me3 silencing over key neuronal gene

A case of an undecided promoter

From

embryo so notdifferentiated yet which is why NEUROG I =silenced

key gene in neuron

differentiating Readyto go

→responds quickly

High DNA methylation

High Histone methylation

H3K27me3 H3K9me3

Low Histone methylation

H3K4me1 H3K4me3

Low Histone acetylation

H3K9,K14

Repressed chromatin

Low DNA methylation

High Histone methylation

H3K4me3 Low Histone methylation

H3K27me3 H3K9me3

High Histone acetylation

H3K9,K14

Active CpG promoters

High DNA methylation

High Histone methylation

H3K36me3 Low Histone methylation

H3K27me3 H3K9me3 H3K4me

Histone acetylation Transient

Transcribed gene bodies

Enhancers sequences

DNA methylation Low (active) High (inactive)

High Histone methylation H3K4me1 H3K27Ac (active) H3K27me3 (inact.) Low Histone methylation

H3K9me3 Histone acetylation Depends on activity (p300)

Chromatin activity states

What is epigenetics good for?

A critical means to achieve dosage compensation for X-linked genes

X-inactivation requires counting: - transient pairing interactions between homologues? - mediated by the X-inactivation center

Once X’s have been counted, one is chosen for inactivation and inactivation proceeds in cis

The inactive X is coated by Xist RNA

ES Intermediate differentiated

Differentiated

X-inactivation as a first paradigm of epigenetics

Straub and Becker, 2007

Random XCI occurs around the blastocyst stage

! The X chromosome shows imprinted X inactivation in the placenta: Xp is always silenced

Plath et al. Annual Reviews 2002

Mechanisms of X chromosome inactivation - Recap

Early stage – both X active

Counting via pairing at the X inactivation center (xic)

Random inactivation

Condensed Barr body anchored to lamin binding region (LBR)

Modified from Jegu et al., Nature Reviews Genetics 2017

HDAC recruitment via XIST Eviction of RNAPII

Recruitment of PRC complex Deposition of H3K27me3

Addition of DNA methylation Further compaction by H3K9me3

Modified from Engreitz et al., 2016

Mechanisms of X chromosome inactivation - Recap

Active de-acetylation H3K27me3 H3K9me3 + DNA me

Permanent silencing (Xist no longer needed)

Temporary silencing

Visualizing X-inactivation

Wu et al., (2014) Neuron

Nuclear transfer experiments

Transplant nuclei into enucleated fertilized eggs

Genomic imprinting as a second epigenetic paradigm

? ?Embryonic lethality This phenomenon is due to genomic imprinting

What is genomic imprinting ?

§ Genes subject to genomic imprinting show parent-of- origin-specific gene expression

• Two classes of genes: Ø Some genes are expressed only when inherited from the maternal lineage while others are expressed only when inherited from the paternal lineage

Pat

Mat

SNRPN chr. 15

Pat

Mat

H19 chr. 11

Imprinted genes “remember” their parental origin

Where is genomic imprinting found ?

How many genes are imprinted ?

Currently, ~80-100 genes are known to be imprinted

Many imprinted genes regulate fetal growth and differentiation as well as placental growth and function Many brain-specific imprinted genes also exist and regulate cognitive functions

What type of genes are regulated by imprinting ?

Eutherian mammals ~ placental mammals (imprinting is also found in marsupials – not in monotremes)

How was imprinting discovered ? 1. Nuclear transfer experiments in mouse 2. Human Parthenogenetic Tumors

• Hydatidiform moles – Androgenetic placental tumor

• Ovarian teratoma – Gynogenetic benign tumor

3. Early gene knockouts

IGF2 (insulin growth factor II) knockout. • No detectable phenotype when deleted chromosome transmitted maternally • Obvious phenotype (reduction in pup size and loss of viability) when deletion transmitted paternally

IGF2 is paternally expressed!

4. Non-mendelian inheritance in human syndromes Prader-Willi syndrome (PWS) / Angelman syndrome (AS)

• In PWS: failure to thrive at birth followed by hyperphagia, obesity, mild intellectual disability, autism spectrum • In AS: profound intellectual disability, little language skills, repetitive hand motion, inappropriate smiling • Due to deletions, duplications or mutations at 15q11-q13

Lack of expression of paternal genes

Lack of expression of maternal genes

Uniparental disomies are an interesting tool to identify imprinted genes

Pat Mat

Maternal disomy

Paternal disomy

Mat Mat

Pat Pat

Modified from Nicholls and Knepper (2001)

How did imprinting evolve ?

Genetic conflict theory v Imprinting evolved as a result of the asymmetric involvement of mother and father in feeding the fetus during its in utero growth (and during lactation period). Imprinting co-evolved with placentation.

• Paternally expressed genes tend to increase fetal growth by increasing placental size and promoting transfer of nutrients from the mother to the fetus • Maternally expressed genes tend to restrict fetal and placental growth

Ø Only the right combination of maternally and paternally expressed genes can balance the supply and demand for the growth of the fetus.

IGF2 : insulin growth factor II, paternally expressed • in the embryo, promotes fetal growth • in the placenta, increases placental growth, facilitates diffusion and transport of nutrients

Ø knockout of Igf2 results in smaller placenta (65% of wt) and smaller pups (~60% of wt)

IGF2R : insulin growth factor II receptor, maternally expressed in placenta and embryo • reduces the amount of circulating IGF2

Ø knockout of Igf2r results in increased placental size (~140% of wt) and fetal overgrowth

Examples supporting the genetic conflict theory

IPL : imprinted in placenta, maternally expressed (in spongiotrophoblast) • reduces placental growth

Ø knockout of Ipl results in larger placenta

PEG3 : paternally expressed gene 3, expressed in brain. • contributes to complex behaviors such as caring for offspring, including feeding (lactation)

Ø knockout of Peg3 results in lack of pup rearing (decreased licking, feeding and warming of the pups, leading to high rate of early mortality)

From Constancia et al., (2004) Nature 432:53-57

Imprinting might have evolved from the asymmetric involvement of mother and father in these processes

Imprinting controls resource acquisition during fetal and post-natal development

• Imprinting is entirely epigenetically determined

• Imprinting is sensitive to nutritional inputs

• Imprinting is critical for human reproductive health

Why do we care?

Imprinting centers show opposite: • DNA methylation profiles • Chromatin structures • Replication timing • Transcription profiles

Epigenetics regulates genomic imprinting

Methylated (DNA) Deacetylated histones Histone H3 K9 methylation Replicates late S phase “OFF”

Unmethylated (DNA) Acetylated histones Histone H3 K4 methylation Replicates early S phase “ON”

Reik and Walter (2001)

Imprinted genes are often found in clusters

Each cluster is controlled by one cis-acting Imprinting Center (IC)

Deletion of an IC results in loss of imprinting for the whole cluster

How does it work??

15q11 Prader-Willi / Angelman cluster

Robertson Nat Rev Gen (2005)

“Long range effects” propagate signal from IC to genes in the cluster

Very similar to enhancers! • On the paternal chromosome, the IC is a powerful enhancer for

multiple genes in the cluster • On the maternal chromosome, this paternal enhancer is silenced.

Instead, a maternal enhancer reaches back to UBE3A / ATP10C

The H19 / IGF2 locus is controlled via enhancer competition and CTCF blocking

Mom Dad

• Enhancer works on H19 • IGF2 excluded by looping and silenced

• Looping is reconfigured because IC1 is DNA methylated

• H19 is silenced and IGF2 accesses enhancers

Epigenetic imprints are acquired in the germline

Maternal Paternal

Erasure

Establishment

Maintenance

Modified from Falls et al., 1999

Imprinting illustrates the heritable and reversible character of epigenetic memory

Imprinted genes show a complex epigenetic life cycle !

Imprinted genes escape zygotic reprogramming: They “remember”

where they came from

Birth

In utero development

Throughout life

Puberty

Imprints are acquired at different times in males and females

During meiosis I block Prior to meiosis

Imprinting is sensitive to nutritional inputs

• What Mom eats or is exposed to can affect pregnancy outcome as well as future lifelong health risks and reproductive potential of offspring

• Early embryonic development is epigenetically very labile and sensitive to environmental and nutritional effects

Diet (folate intake, maternal obesity, under-nutrition)

Lifestyle

Environment

Imprinting is critical for human reproductive health

Failure to properly set up epigenetic imprints in gametes can lead to reproductive wastage: sterility, moles, spontaneous abortion, stillbirth

Prader-Willi syndrome • Hypotonia (birth) • Failure to strive • Mild cognitive impairment • Hyperphagia – obesity • Hypogonadism

Beckwith-Wiedeman syndrome • Fetal and postnatal overgrowth • Predisposition to tumors

Silver-Russel syndrome • Severe intra-uterine

growth restriction • Dwarfism

Several childhood disorders linked to chromosomal rearrangements and/or incorrect establishment of epigenetic imprints are caused by aberrant expression of imprinted genes

Epigenetics Genomic Imprinting

Germline development and reprogramming

Childhood Disorders, Intellectual Disabilities

Reproductive wastage

Cancer

Genes

Environment

Diet

Lifelong increased

disease risks in offspring

Trans- generational

effects? ?