Week 15 - Epigenetics, microsatellites, ministallites

Some strains of mice have:

Agouti allele (A)

Agouti Viable Yellow (Avy)

Agouti Viable Yellow (Avy) allele

helps scientists see how epigenetics works

because different methylation levels of this Avy allele cause different coat colors in mice

Agouti gene

regulated by a hair cycle-specific promoter during development

- time-specific

- tissue specific (hair follicles)

Agouti allele (A)

dominant; encode a protein that give yellow (pheomelanin) pigment in the hair shaft of mice

two recessive (aa) alleles

not produce the agouti protein

they have solid black (eumelanin) fur (non-agouti)

Pheomelanin

Individual mice hair will have a light band of yellow pigment on an otherwise dark hair shaft

Eumelanin (non-agouti)

Black color

Genotype of mouse -AA

Two dominant Agouti alleles, pheomelanin produced

Genotype of mouse -Aa

One dominant Agouti allele, one recessive allele ,pheomelanin produce

Genotype of mouse -aa

Two recessive non-Agouti alleles, pheomelanin NOT produced.

Eumelanin (black) pigment

Avy insertional mutation

- constitutively (all the time, not just during specific stages of hair development)

- ectopically (in all tissues, not just in hair cells)

Intracisternal A Particle (IAP)

transposon; 5.4 kilobases (kb) in size

inserted upstream of the normal hair-specific promoter of the Agouti allele's

cryptic promoter

drive:

1. ECTOPIC Agouti gene expression

2. CONSTITUTIVE Agouti gene expression

combination of this two expression patterns lead to mice with

• yellow coat color, and• metabolic health issues (obesity, type 2 diabetes, tumor susceptibility, reduced lifespan

normal expression is functionally masked

IF Agouti expression is driven from the cryptic promoter in IAP

Since expression from this promoter is constitutive and ectopic

no homozygous AvyAvy

results in lethality before birth

Ectopic expression

because pheomelanin is made in tissues like the liver and brain, where it's not normally made

Constitutive expression

because pheomelanin is made continuously, not just during a specific time in development

CpG sites are present in the IAP cryptic promoter

There are NINE CpG sites in IAP cryptic promoter

If none or only one of the CpG sites is methylated

the Avy allele is expressed constitutively and ectopically

leading to excessive pheomelanin production across all tissues

5 CpG sites are methylated

reduced Avy allele expression

leading to DECREASED pheomelanin productio

most or all of the CpG sites are methylated

the Avy allele is silenced (no expression)

No pheomelanin from this allele

HYPOMETHYLATION

ectopic and constitutive expression of Avy

Pheomelanin in all cells all the time

HYPERMETHYLATION

Avy is silenced .... No pheomelanin made from this allele

BUT the A allele is expressed normally

Pseudoagouti

possess a specific genotype but exhibit a brown-colored coat that appears similar to a wild-type agouti mouse

difference between the healthy brown mouse and its overweight yellow twin

not a change in the DNA sequence (mutation) of the Agouti allele

Instead, the difference stems from methyl group

Epigenetic Variation Without Genetic Differences

Twins may develop different diseases (e.g., cancer, diabetes) due to lifestyle or environmental factors altering their epigenome

CpG Methylation and Gene Silencing

Hypermethylation of tumor suppressor genes (e.g., BRCA1, MLH1)silence them, increasing cancer risk

Hypomethylation of oncogenes promote cancer

Pregnant mom fed diet rich in methyl groups

IAP promoter for the Avy allele is methylated at all 9 CpG sites because of mom's high methyl diet during embryogenesis

result of Pregnant mom fed diet rich in methyl groups

Avy allele is silenced at the IAP pro = no pheomelanin produced from this promoter in any cell

Pregnant mom fed normal mouse food

IAP promoter for Avy allele is unmethylated at all 9CpG sites because of the mom's LOW-methyl diet during embryo genesis

result of Pregnant mom fed normal mouse food

Avy allele is constitutively and ectopically expressed = excessive pheomelanin produced all the time in ALL cell types

high-methyl donor diet

leads to increased methylation across the entire mouse genome, not just at the agouti locus

all methyl donors

folate, choline, and betaine

Folate and Neural Tube Defects

Folate (a methyl donor) supplementation during pregnancy reduces neural tube defects by ensuring proper methylation of genes critical for development

Developmental Origins of Health and Disease

prenatal environment (e.g., maternal nutrition) "programs" offspring metabolism, immunity, and disease risk

genes are not destiny

Even with identical DNA, environmental factors like diet can "rewire" gene expression through epigenetics, shaping health outcome

BPA-containing diet

a shift from more methylation to less

more yellow phenotype

result of BPA-containing diet

in these offsprings, the Avy allele is ectopically and constitutively expressed, leading to severe health consequences

Stress Response Pathway

Stress activates the hypothalamus → pituitary gland → stimulates the adrenal glands

adrenal glands

release:

Cortisol (in humans)

Corticosterone (in many animals)

glucocorticoid hormones

stress hormones

How the Body Turns OFF the Stress Response

Cortisol binds to the glucocorticoid receptor (GR) in the brain

sends a signal to the hypothalamus: "Stress is over — stop the response"

GR gene

makes the glucocorticoid receptor (GR protein)

Effect of DNA Methylation: CpG island at the GR promoter

GR gene is highly methylated:

The gene is turned OFF

Less or no GR protein is made

Without GR protein

Cortisol cannot send the "all clear" signal

The stress response stays ON longer

This can lead to increased stress and anxiety

Epigenetic transgenerational behavioral imprinting

Experience-dependent; non-germline mediated epigenetic transmission

Pups that received low levels of licking and grooming

methylation of GR gene

experience higher levels of stress and anxiety

result of Pups that received low levels of licking and grooming

pups became adults an had offspring, they showed the same behavior toward their babies. As a result, the GR gene in their offspring stayed methylated

Pups that received high levels of licking and grooming

demethylation of the GR gene

LOWER levels of stress and anxiety

result of Pups that received high levels of licking and grooming

pups became adults and had offspring, they showed the same high licking and grooming behavior toward their babies. Asa result, the GR gene in their offspring stayed demethylated

GR gene (NR3C1)

Children who receive sensitive, responsive caregiving show reduced methylation of NR3C1, leading to better stress hormone (cortisol) regulation

Mothers with trauma histories

often exhibit NR3C1 hypermethylation, which they can transmit to their children

99.9% of the DNA sequence

shared among all humans

remaining 0.1%

what makes us unique; about three million base pair

3M bp is mainly located in

moderately repetitive (repeat) DNA regions

Tandem paralogues

duplicated genes that are located next to each other on a chromosome

Paralogues

genes that originated from duplication of an ancestral gene

Tandem

duplicated genes are arranged side-by-side

rDNA

ribosomal RNA (rRNA) genes arranged one after another in tandem repeats

Tandem repeats

DNA sequences that are repeated many times in a row in ahead-to-tail arrangement on a chromosome in non-coding regions

satellite DNA

Short sequences of DNA that are tandemly repeated as many as 10 million times in the DNA; much of it is located in the telomeres

microsatellite repeat

# of repeats = 5 -100

minisatellite repeat

# of repeats = 2 to several hundred

macrosatellite repeat

n > 100 bp long

number of these repeats

different(polymorphic)between individuals.

Hybridization-based markers

MINISATALLITES: are hybridization-based markers

Variable number tandem repeat (VNTR)

Minisatellites; used for DNA fingerprinting by comparing the DIFFERENCES (POLYMORPHISM) in VNTRs between individual

DNA fingerprinting

cutting out the VNTRs from the genome with restriction enzymes, running it on a gel, transferring it to a membrane, and then detecting it with complementary sequences called probes

DNA fingerprinting is actually two techniques

1. Restriction fragment length POLYMORPHISM (RFLP) of VNTR

2. Southern blot

PCR-based markers

MICROSATELLITES: are PCR-based markers

Short Tandem repeats (STRs)

Microsatellites; used for DNA fingerprinting by amplifying the STRs and comparing the DIFFERENCES(POLYMORPHISM) in STRs between individuals

diploid organisms

inherit one set of chromosomes from each parent.

VNTRs

(10 to 100 base pair length) located in non-coding regions of the genome rather than within genes

STRs

usually located in non-coding regions of the genome rather than within genes.

alleles

different VNTR or STR versions at the same genomic location

polymorphism are detected by molecular techniques such as

RFLP, Southern blots, PCR

Minisatellites

Variable number tandem repeats (VNTRs)

Detect this polymorphism by RFLP followed by Southern blot

Microsatellites

Short Tandem Repeat (STR)

Detect this polymorphism by PCR

Minisatellites are similar between individuals, but they can differ in

DNA sequence

Number (length) of repeats

VNTR repeats are located at different loci

when comparing individuals, the same loci must be analyzed for all individuals being tested

VNTRs in the genome are flanked by recognition sequences for restriction endonucleases

the VNTRs can be isolated or cut out from the genome by specific restriction endonuclease enzyme also called restriction enzymes (RE

RestrictionFragment Length Polymorphisms (RFLPs)

a molecular technique that identifies variations in homologous DNA sequences by analyzing the different lengths of DNA fragments produced after digestion with restriction enzymes

DNA profiling by VNTR

- Extract genomic DNA from cells or tissues.

- Digest the DNA with restriction enzymes flanking the VNTR region.

- This produces thousands of DNA fragments, so Southern blotting is used to identify the VNTR-containing fragment.

- A labeled DNA probe complementary to the VNTR sequence binds specifically to that fragment for detection

DNA probes

20 nt probe is labelled with radioactive phosphorus (32P)

DNA Fingerprinting is used for

Paternity and Maternity

Criminal identification and forensics

Diagnosis of inherited disorders

Paternity and Maternity

Person inherits their VNTRs from their parents

Parent-child VNTR pattern analysis is used to solve paternity identifications cases

Criminal identification and forensics

DNA isolated from blood, hair, skin cells or other genetic evidence left at the scene of a crime can be compared

Use DNA fingerprints to link suspects to biological evidence - blood or semen stains, hair, or items of clothing

Diagnosis of inherited disorders

Diagnose inherited disorders in both prenatal and new-born babies

e.g. Cystic fibrosis,haemophilia, Huntington'sdisease, sickle cell anaemia,familial Alzheimer's etc

paternity

child DNA will exactly match the DNA of either of the two parents

50% of the bands in a child would match each parent

All bands in the child must come from one of the two parents

the child cannot have DNA that does not match with one parent or the other

Advantage of DNA profiling by RFLP - Southern blot

Many VNTRs are scattered throughout repetitive regions of the genome, allowing even very small chromosomal fragments to be profiled

Disadvantage of DNA profiling by RFLP - Southern blot

Requires relatively very large amount of DNA

Requirement of radioactive probe makes the analysis expensive and hazardous

Time consuming, laborious, and expensive

Simple Tandem Repeats (STR)

minisatellite; 1-9 nucleotides

flanked at both ends (5' and 3') by unique sequences

unique sequences

used to design primers to amplify out the STR sequence

STRs are detected by PCR

Types of STR repeat units

shorthand way of representing STRs

(ex.) Di (AT)8

simple Perfect Repeats

No mismatch in the repeat; Identical nucleotides and length

Simple Imperfect Repeats

Repeat with a mismatch / interruption; Two or more adjacent simple repeats

Compound or complex Repeats

Multiple different repeats; Two or more adjacent simple repeat

Variable nucleotide length & possible intervening non-STR nucleotide