Epigenetics -HS
Types of epigenetic modifications
Also modifciation in cytoplasm- non coding regions for rna silencing Non-coding rnas are in cytoplasm HM and DM in the NUCLEUS
Bisulfite modification
Bisulfite conversion is a popular technique used to study DNA methylation that involves converting cytosine to thymide on dna (uracil on rna) .- nh2 residue converted to an O Gold standard of methylation analysis Chemical change to dna seq Similar to restriction enz Prevented from happening if C is methylated So if u have a C after doing BM, cyt was methylated Next step = pcr Pcr uses thy BM then sanger seq Or second genertation seq - look at ENTIRE gemone for the dna pattern - look for parts of dna w/ methyl grp or not - could be the SAME pattern in the genome of the species
Bisulfite Sequencing
Bisulfite sequencing (also known as bisulphite sequencing) is the use of bisulfite treatment of DNA before routine sequencing to determine the pattern of methylation. Then Pcr amplifies product If c was not methylated, all C is converted to T If there's 50% methylation, there's a SMALL amount of C but a LOT of T If theres 100% methy, there's a cyt peak For each C that is followed by G
Nucleosome = 8 Histones + DNA
Chromatin= nucleosomes In each nucleo= 8 histones - A nucleosome is a basic unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound in sequence around eight histone protein cores Histones = GLOBULAR PROTEINS--A histone octamer is the eight protein complex found at the center of a nucleosome core particle. It consists of two copies of each of the four core histone proteins (H2A, H2B, H3 and H4). Linker histone H1 is an essential component of chromatin structure.
Epigenetics: DNA methylation
DNA methylation PREVENTS GENE EXPRESSION BY: Preventing the BINDING OF TRANSCRIPTION FACTORS to DNA Recruiting complexes that SILENCE GENE EXPRESSION TF specifies which gene needs to be expressed If promoter region is not methylated - TF binds to TF recognition sequence and cause transcription If promoter region is methylated (has methyl grps attached)- TF can't promote the expression - methyl grp may prevent TF from recognising its recognition site Methylated region- brings on other molecules that mess with transcription
ChIP (Chromatin Immunoprecipitation)_3
Dissociate DNA from histones/ digest histones- leaves enriched amount of dna in tube QUANTIFY THE ENRICHMENT for your gene of interest in the histone-bound fraction (vs pre-precipitation) Dont care about the histones- they will mess w/ the next step In tube there is Only dna that was bound to histones w/ the modification Then u calculate enrichment - ull know the amount of gene in input- then measure the output- like medip If there's 100% acetlyation in histones bound to dna, ull have same amount of dna in in and output If NO acetylation- there's be no dna in output
Epigenetic studies could help us understand...1
EG helps us understand things that did not make sense b4 U'd assume twins will look and beh similarly As they age, there's disparity in their beh/ heath- depending on diff things like their env But changes due to epigen - env
What is "Epigenetics"? 2
Epigenetic changes: Heritable, but POTENTIALLY REVERSIBLE, changes in gene expression that occur WITHOUT CHANGES IN THE DNA SEQUENCE- reversible b/c ur NOT changing the dna sequence the study of changes in organisms caused by modification of gene expression rather than alteration of the genetic code itself. Epigen goes against the dogma of genetics- dna transcribed into mRNA- translated into proteins -will affect phenotypes
Epigenetic studies could help us understand...2
Epigenetic studies could help us understand... If twin has Rett syndrome, 100% chance their twin will have it If first degree relative has shz, theres a high chance ull get it If u have MZ twin who has sch, there's only a 50% chance ull have it Not a single gene responsible for schz - many genes can increase ur chances of having it- but for rett syndrome, there's a single gene involved - mutation of the MECP2 gene- has high EG control
What is "Epigenetics"? 3
Epigenetics is the study of heritable changes in gene expression (active versus inactive genes) that do not involve changes to the underlying DNA sequence — a change in phenotype without a change in genotype — which in turn affects how cells read the genes. Epigenetic change is a regular and natural occurrence but can also be influenced by several factors including age, the environment/lifestyle, and disease state. E Epigen messes w/this There should be a protein expressed, but epigen affects this
Epigenetic studies could help us understand...3
Genetic vulnerability predisposes to disease.... ...but the disease DEVELOPS ONLY UNDER SPECIFIC ENVIRONMENTAL INFLUENCES
What is "Epigenetics"?
Genetics: the study of heredity and the variation of inherited characteristics- determined by ur parents.
Nucleosome = 8 Histones + DNA 2
H1 links nucleosomes into higher order structures Dna makes 2 loops around nucleosomes Histones have protein tails- important for the MODIFICATIONS that occur- involves opening/closing chromatin Additional histone on the dna Histones are proteins so have CHARGES Dna has a neg charge Modifications will alter net electric charge in nucelosome to determine if the nucleosome will open or close Histones help to organise the dna so its not tangled Helps TF find their reco site quicker
Epigenetics: Histone modifications
HISTONE ACETYLATION-> Addition of acetyl grp at Lysine residues* Leads to activation of gene transcription- repulsion of charges to ALLOW NUCLEOSOME OPENS and TF can transcribe Catalysed by HATs- HISTONE ACETYL TRANSFERASES But Deacetylation is catalysed by HDACs-> There are 3 classes of HDACs & 11 ISOZYMES METHYLATION-> at LYSINE & ARGININE residues in histone tail Leads to GENE ACTIVATION OR INACTIVATION (DEPENDING ON THE RESIDUE) of gene
histone modifications
HISTONE MODIFICATION C1 Can use IHC- same for dna methylation- look for anywhere in brain there is inc/decrease of specific modfication Specific for epigenetics = chip
How do histone modifications lead to reduced gene expression?
Heterochromatin- nucleosomes tightly packed- dark area - harder to get expression from genes here Euchromatin- open form- acetyl groups are opening chromatin Open -> active form, accessible to transcriptional machinery- E - acetyl groups are opening the protein allowing methylation/phosphorylation- allows TF bind Closed -> inactive form, inaccessible to transcriptional machinery- remove acetly grp - changes electric charge pf chromatin and and makes it condensed
Chromatin = DNA + Histones
Histones: Allow TIGHT & ORGANIZED packing of the DNA in the nucleus MODIFY THE "TIGHTNESS" of the DNA packaging Epigen is based on what occurs in the nucleus Seq of dna is wrapped around the histones 2 main modifications are based on dna and histones - dna methylation and histone modification
DNA methylation
INHIBITS gene transcription Involves methylation of the CYTOSINE AT CpG DINUCLEOTIDES "CpG" dinucleotides ARE ENRICHED at the gene promoter region*(CpG islands)- promoters have a high cpg content Most of CpGs OUTSIDE CpG islands are METHYLATED Most "C"s (of CG couples) at CpG islands are UNMETHYLATED Catalysed by DNA methyltransferases (DNMT) DNMT1-> copies DNA METHYLATION patterns DURING MITOSIS DNMT3a & DNMT3b -> DE-NOVO methyltransferases- influence from env
DNA methylation 2
If methylation is in coding region, rather than promoter, there may actually be MORE expression The CpG dinucleotides are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5' → 3' direction. *Most cpg is in promoter region- gives u an additional way to fine tune gene expression Promotor region is often unmethylated Methylating this area messes with expression Only makes a difference if there's a need for that gene to be expressed -if there was no TF binding to the gene anyway, then methylation doesn't make a diff
ChIP (Chromatin Immunoprecipitation)_1
In biology, histones are highly alkaline proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes. They are the chief protein components of chromatin, acting as spools around which DNA winds, and playing a role in gene regulation Not the dna chips for seq Same principle as for medip Use IP to enrich the amount of gene that is linked to a histone w/ a specific modification ChIP aims to determine the specific location in the genome that various histone modifications are associated with, indicating the target of the histone modifiers.[1] DNA and associated proteins on chromatin in living cells or tissues are crosslinked (this step is omitted in Native ChIP). The DNA-protein complexes (chromatin-protein) are then sheared into ~500 bp DNA fragments by sonication or nuclease digestion. Cross-linked DNA fragments associated with the protein(s) of interest are selectively immunoprecipitated from the cell debris using an appropriate protein-specific antibody.
Histones
In biology, histones are highly alkaline proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes. They are the chief protein components of chromatin, acting as spools around which DNA winds, and playing a role in gene regulation Chromatin is a complex of DNA, and protein found in eukaryotic
MeDIP (Methyl-DNA Immunoprecipitation)_1
Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest Use Abs to Isolate dna w/ methy grp attached
Non-coding RNA silencing
MicroRNAs- main form Produce POST-TRANSCRIPTIONAL GENE SILENCING Involved in DEVELOPMENT, DIFFERENTIATION AND DISEASE PIWI-INTERACTING RNAS Control TRANSPOSABLE elements DIRECT DNA METHYLATION at TRANSPOSABLE ELEMENT- determine if the transposon will move or not LONG NON-CODING RNAS May DIRECT EPIGENETIC ENZYMES to SITES IN THE GENOME- have a SIMILARITY to gene of interest b/c they need to bind to the gene- LOW COPY NUMBER- NO SIMILARITY b/w species
Epigenetics: Histone modifications
Phosphorylation-> at Serine & Threonine residues Associated with chromatin inhibition or activation Ubiquitination-> at Lysine residues SUMOylation-> at Lysine residues Addition of phosphate to ser or thr Depending where this occurs there may be inc/decreased gene expression Addition of ubiquitin grp at lysine
Methylation-sensitive restriction enzymes
Restriction enzymes have a recognition site- some RSs have C-G where methylation occurs ACIL enz may or may not cut-depends on if there's a METHYL grp If dna is unmethylated and u incubate it w/ acil, the enz will CUT gene of interest- will give u 2 fragments of smaller sizes - the smaller fragments will have the correct molecular weight b/c ull know WHERE the enz has cut it- so u can tell that ur gene was NOT methylated - 2 smaller mw bands ACIL can recognise if the gene is methylated- if it is, it will not cut it- the methyl grp DISRUPTS the activity of the enz- u will end up w/ a SINGLE product that has the mw of the FULL product - single band- end up w/ a singl product w/ the molecular weight of orginal product
Epigenetic studies could help us understand...4
The LONG-TERM EFFECTS of psycho-therapies (long after they stopped) Why they're successful
Epigenetics: Histone modifications 2
The MORE STABLE of the modifications FIVE types of HM *histone 3 and 4 are the main ones important for determining if these modifications will affect the chromatin Acetylation ALWAYS leads to increase in gene expression If histones are deacetylated, it is more difficult for TFs to reach ther reco sequence Some compounds in foods/drinks to cause deacetylation of dna Methylation =Addition of meythl grp to lys or arg If u methylate LYS 2,9, 14,17,23,37,20,12 and 3,u get DECREASE in gene expression If in 4 or 36, there's an INCREASE in GE Methylation at gene histone 27 - removing methylation here causes RAPID DIFFERENTIATE
How does DNA methylation lead to reduced gene expression?
The methyl group PREVENTS BINDING OF TRANSCRIPTION FACTORS (or transcriptional machinery) to their recognition sites at the DNA The TRANSCRIPTIONAL MACHINERY IS EXCLUDED from the PROMOTER REGION by the METHYLCYTOSINE-BINDING PROTEINS (MeCP2, MDB1-4)- methyl grp binds other proteins which do not allow TFs to bind* The methylcytosine-binding proteins can RECRUIT LARGE PROTEIN COMPLEXES including HISTONE DEACETYLASES which further SHUT DOWN GENE EXPRESSION *Some of these proteins can change the histones Dna does not have residues but bases
Types of epigenetic modifications
The three types of epigenetic modifications CAN INFLUENCE (INDUCE) EACH OTHER FURTHER repressing gene transcription
MeDIP (Methyl-DNA Immunoprecipitation)_4
Then use QPCR or use sequencing if looking at multiple genes Then look at % of enrichment We know HOW MANY PIECES of dna we has when we started in homogenate and how may are left at the end If everything was methylated, ull have same amount of dna If hardly anything was methylated, ud have MUCH LOWER amount of dna Use PCR b/c we're not Looking IN GENERAL at anything that has methyl grp but just at GOI Digest Antibodies (by enzymes?) & quantify enrichment for gene (promoter) of interest
Does gene expression ALWAYS lead to decrease in expression
not always- it inhibits gene expression most of the times eg in PROMOTER region however if methylation happens in the coding region, it can lead to MORE gene expression It depends- yes if the gene was supposed to be expressed No if the gene was NOT REQUIRED to be expressed
If a gene is methylated in skin it will not be be methylated in heart
there will be different activation of methylation enz- the skin and heart are influenced by diff env factors - exposed to diff dnmt (3a and 3b) and dnm1 b/c when stem cells in the skin and heart divide, the dna pattern will be copied which will be diff in skin and heart- so skin cells behave differently to heart A heart cell is diff from skin cell b/c they express diff genes The expression of the gene is diff in both tissues b/c the EPIGENETIC MODIFICATIONS for the gene is diff in each tissue If expression is diff, epi modifications have to be diff