Epigenetics

What is epigenetics?

• Changes that change DNA expression without changing the DNA sequence 

• Encompasses all aspects of gene regulation 

What are short term epigenetics?

Short term changes that respond to signals such as hormones that adapt the cell to its environment  

What are long term epigenetics?

Long term changes that can be heritable from cell to daughter cell and are important for establishing cell identity  

Is epigenetic marks heritable?

Whether the changes are heritable from parent to child is controversial 

What is the complexity of epigenetics?

• Lots of unanswered questions  

• Infinite complexity  

• Epigenome is different in different tissues and can change over lifetime 

What are the controversial theories of epigenetics?

• How far is it a link between early experience, lifestyle and health 

• Major determinations of who we are  

• Transgenerational affects  

What are the three mechanisms of epigenetics?

• Methylation of DNA where methyltransferase adds methyl to the cytosine and converts them to 5-methylcytosine  

• Modification of the histone proteins in nucleosomes  

• Affecting accessibility of DNA in the cell nucleus. To respond to regulatory affects the regulatory enzymes need to be able to access the DNA 

How do the three mechanisms work?

The processes tend to interact with each other  

How does DNA methylation work?

• Converts cytosine to 5-methyl cytosine  

• At the back of the base pair 

• Doesn't impact the base pairing or do anything to the genetic code 

• Methyl groups are on the outside of the double helix  

• Attract methyl DNA binding proteins that are used to regulate the cell  

Which cytosines are methylated?

• Not every cytosine is subject to methylation 

• DNA methyltransferase enzymes will only methylate cytosines that lie next to a guanine  

What are the features of CpG sites?

• There are around 28 million CpG sequences in the human genome  

• The sequences are important for gene regulation  

• Where there is a CpG sequence on one strand there will also be one on the other strand  

What are the features of the methylated CpG sites?

• Depends on the type of cell and role  

• Provides methylation signatures for understanding of what is happening in the cell 

How is DNA methylation maintained over replication?

• Newly synthesised DNA is not methylated  

• A maintenance methylase enzyme replicates the methylation pattern from the old strand and replicates it on the new strand  

• Produces the same pattern of methylation  

• Means that the cells identity remains intact 

How is the DNA packaged in the cell?

• The DNA in the cell nucleus is packaged with proteins and some RNA to produce chromatin  

• The main proteins are histones that are small basic proteins that have an affinity for the acidic DNA  

What are nucleosomes made of?

• Nucleosomes are eight histones made of four histones (H2A, H2B, H3 and H4) to produce a complex  

• 147bp of DNA is wrapped around them  

What are the N-terminal features of nucleosomes?

The N-terminus of each histone protrudes from the nucleosome and is the target of the enzymes that add epigenetic marks that modify the nucleosomes  

How are histones modified?

• Can involve many catalytic reactions  

• Lysine residues in the N-terminal tail there are enzymes that add acetyl and others that add methyl (1,2 or 3) groups  Wh

Which histone modifications are especially important?

• Modifications of the H3 and H4 are especially important  

• H3 Lysine 9 can be acetylated or methylated, serine 10 can be phosphorylated, lysine 14 can be acetylated  

What are writers?

• Writers are enzymes that add marks to the DNA or histones  

• Lots of enzymes and are important for gene regulation  W

What are readers?

Readers are proteins that respond to the epigenetic marks such as switching off gene expression based on the chromatin marks  A

What do the editors do?

Histone marks are always reversible so editors can demethylate or remove the acetyl groups from the histone  

What is the dynamic process of histone modification?

• Dynamic process as the marks is constantly written read and edited  

• Lots of genetic diseases are caused from mutations in the writer, reader or editor proteins  

How is DNA in the nucleus?

• 2m of DNA and other things are in the 10um cell nucleus so crowded   

• Regulatory molecules can only find the target sequence if it is accessible  

How is DNA accessibility impacted in the nucleus?

• Chromatin remodelling complexes that are driven by ATP and can move the nucleosomes along the DNA strand  

• Nucleosomes can be heterochromatin or euchromatin due to histone modifications  

• Compartments in the nucleus where genes are active and inactive so the position matters  

What is euchromatin?

There are spaces where there are no nucleosomes and the DNA is accessible to regulatory molecules 

What is heterochromatin?

• The nucleosomes are packed tightly together and the DNA is inaccessible  

• Mainly held in place by a protein called HP1 – reader  

What is different between euchroamtin and heterochromatin?

Difference is due to differences in the histones  

How is DNA methylation studied in the lab?

• PCR product's don't work as there is no methylation – need original genomic DNA  

• Some restriction enzymes that won't cut DNA if methylation so use the pattern to determine the region  

• Bisulphite modification

What does bisulphite modification do?

• Converts C to U but only if they aren't methylated  

• Don't convert 5-methyl cytosine  

How does bisulphite impact the PCR product?

• Treating with sodium bisulphite if there methylation then nothing happens  

• Unmethylated are converted to uracil  

• PCR then U's are represented as T's  

• Produces different sequences based on if the cytosine was methylated or not  

How do methylation chips work?

• Methylation chips are microarrays that have probes for ½ million CpG containing sequences  

• Treat the genomic DNA with sodium bisulphite and hybridise to the microarray  

• For the CpG containing locus there are two probes, one for the C-containing methylated sequence and one for the U-containing unmethylated region  

• Look at the ratio of signal from the probes and gives the proportion that have the sequence methylated 

What is X-inactivation?

• In an embryo counts the number of X chromosome and at random epigenetically inactivates all but one  

• In the embryo once decided which X is inactivated the clones of the cell have the same X active  

• Means that a normal woman is a mosaic for which X is active  

• If there is a X-linked condition there are consequences  

What do lncRNAs do in X Inactivation?

• XIST is encoded by a gene on the X chromosome  

• Only expressed from the inactive X  

• XIST RNA coats the inactive X 

• Spreads across it and switches off expression  

How is the X chromosome visualised?

• Curls up as condensed chromatin at the periphery of the lymphocytes  

• Known as the Barr body  

• For someone with three X chromosomes there are two Barr bodies  

How does X-mosaicism impact disease?

• Heterozygous for X-linked ectodermal dysplasia  

• Skin cells don't have sweat glands  

• Using iodine, sweat and starch powered  

• Where sweat the starch doesn't stick  

• Can see which cells have the X active  

How does X inactivation work in cats?

• Tortoiseshell cats are heterozygous orange and blacked hat are X-linked  

• Always female as require two X chromosomes  

What causes cancer?

• Cancer cells inactive genes that act as breaks on cell division  

• Tumour suppressor genes  

How do epigenetics impact cancer?

• In tumour suppressor genes both copies are inactivated  

• Normally done by deleting a gene or mutating a sequence  

• Can also be done with the sequence intact and silenced epigenetically  

Why is imprinting important for development?

• Must have one from maternal and paternal  

• Doesn't matter if there are the right number of chromosomes  

• If one isn't maternal and the other paternal then the embryo won't develop  

• Female produce a ovarian teratoma and males produce hydatidiform mole  

How are sperm and egg different?

• Sperm and egg have different genetic marks  

• Normally during embryo the imprints erase however there are several imprinted genes that aren't erased and in the adult  

How does imprinting impact gene expression?

• Genes are expressed differently if they are the maternal or paternal chromosomes 

• Normally expressed on one chromosome and not the other  

• Mutation in the imprinted gene will only be impacted if it is in the chromosome that has the expressed copy  

• Provides a unusual pedigree pattern  

How is imprinting reversible?

• Gametes have a specific imprint  

• For the imprinted gene it persists into the somatic cells  

• For other genes it is erased and replaced with imprints for the tissue  

• Somatic cells have a record of which chromosome came from which parent  

• When producing sperm and egg the imprint is erased  

How does imprinting cause disease?

Leads to conditions where a mutation in the imprinted gene can cause different conditions  

What are the features of Angelman and Prader-Willi?

• Deletion of a cluster of differentially imprinted genes at chromosome 15q11-q13 causes very different conditions  

• Loss of the paternal chromosome causes Prader Willi   

• Loss of the maternal chromosome causes Angelman  

What is the imprinting summary?

• There are maybe 100 imprinted genes in the human genome 

• The paternal and maternal chromosomes show different DNA methylation patterns  

• Imprinted genes normally come in clusters that contain both maternally and paternally imprinted genes  

• Imprinted regions often encode large non-coding RNAs as well as proteins that are important for gene expression  

• Speculative if imprinting has any function  

How does imprinting impact metabolism?

• Nutrition in early infancy sets the epigenetic profile such as metabolism and impacts the disease risk for the rest of the life  

• Quite a lot of evidence for it – but how far  

Are epigenetics inheritable?

• Normally epigenetic marks are erased and reset from parents to child  

• Some environmentally imposed epigenetic marks might be inherited  

• Evidence in plants, worms and mice