Epigenomics I& II. docx.docx

Table of Contents {#table-of-contents.TOCHeading} ================= [Epigenomics 3](#epigenomics) [Epigenetics 3](#epigenetics) [Epigenomics: 3](#epigenomics-1) [How the DNA is packaged into chromatin: 3](#how-the-dna-is-packaged-into-chromatin) [Epigenetics and Phenotypes relationship: 3](#epi...

Table of Contents {#table-of-contents.TOCHeading} ================= [Epigenomics 3](#epigenomics) [Epigenetics 3](#epigenetics) [Epigenomics: 3](#epigenomics-1) [How the DNA is packaged into chromatin: 3](#how-the-dna-is-packaged-into-chromatin) [Epigenetics and Phenotypes relationship: 3](#epigenetics-and-phenotypes-relationship) [Altering chromatin structures: 3](#altering-chromatin-structures) [Histones 3](#histones) [Histones Modification and Histone Code: 3](#histones-modification-and-histone-code) [Active and Repressive marks: 4](#active-and-repressive-marks) [Histone acetylation: 4](#histone-acetylation) [DNA methylation 4](#dna-methylation) [The role of DNA methyltransferases in DNA methylation 4](#the-role-of-dna-methyltransferases-in-dna-methylation) [Methylation of DNA is reversible: DNA Demethylation: 5](#methylation-of-dna-is-reversible-dna-demethylation) [TET proteins function in: 5](#tet-proteins-function-in) [Hypomethylation: 5](#hypomethylation) [Hypermethylation: 5](#hypermethylation) [Changes of DNA methylation during development 5](#changes-of-dna-methylation-during-development) [Epigenetic inheritance 5](#epigenetic-inheritance) [Identical twins 5](#identical-twins) [Environmental factors can control epigenetically controlled development: Queen versus worker bee. 5](#environmental-factors-can-control-epigenetically-controlled-development-queen-versus-worker-bee.) [Epigenetics II 6](#epigenetics-ii) [Relevance of Epigenetics: 6](#relevance-of-epigenetics) [1. Imprinting disorders 6](#imprinting-disorders) [2. X-inactivation patterns: 6](#x-inactivation-patterns) [3. Cancer: 6](#cancer) [4. Developmental origins of health and disease: 6](#developmental-origins-of-health-and-disease) [DNA methylation and cancer 6](#dna-methylation-and-cancer) [Epigenetic Mutations 6](#epigenetic-mutations) [1. Inherited disorders 6](#inherited-disorders) [2. Disorders of imprinting 6](#disorders-of-imprinting) [Fragile x syndrome: 6](#fragile-x-syndrome) [Genomic imprinting 6](#genomic-imprinting) [Non-imprinted and imprinted genes: 7](#non-imprinted-and-imprinted-genes) [Beckwith-Wiedemann Syndrome 7](#beckwith-wiedemann-syndrome) [Skewed X inactivation 7](#skewed-x-inactivation) [Female carrier of Duchenne muscular dystrophy 7](#female-carrier-of-duchenne-muscular-dystrophy) [DOHAD- developmental origins of health and disease 8](#dohad--developmental-origins-of-health-and-disease) [Epigenetics VS epigenomics 8](#epigenetics-vs-epigenomics) [Role of methylation: 8](#role-of-methylation) [Detection of methylation by bisulfite conversion and sequencing 8](#detection-of-methylation-by-bisulfite-conversion-and-sequencing) [Methylated DNA Immunoprecipitation sequencing 8](#methylated-dna-immunoprecipitation-sequencing) [ChIP-chip and ChIP-seq 9](#chip-chip-and-chip-seq) [ChIP-on chip wet -lab portion: 9](#chip-on-chip-wet--lab-portion) [ChIP-seq: 9](#chip-seq) [Encode 9](#encode) Epigenomics =========== Epigenetics ----------- *Is the study of changes in the regulation of gene activity and expression that are not dependent on gene DNA sequences.* - Changes to DNA that regulates whether genes are turned on or off. - The epigenetic regulatory mechanisms control gene expression across cell types such as nerve cells, RBC, smooth muscle, fat cells , intestinal epithelial cells, striated muscle cells, bone tissue with osteocytes and loose connective tissue with fibroblasts. - These changes are made by adding or subtracting various chemical tags on DNA nucleotides and histones such as: - Methylation - Acetylation - Phosphorylation Epigenetic studies factors that cause: - Stable & heritable, yet reversible changes in the way genes are expressed without changing their original DNA sequence. Epigenomics: ------------ *Is the more global analyses of epigenetic changes across the entire genome.* How the DNA is packaged into chromatin: --------------------------------------- The epigenetic changes modify the packaging of DNA or the accessibility of genes for the transcription machinery.  Epigenetics and Phenotypes relationship: ---------------------------------------- All three have an impact on the phenotype of a person. Altering chromatin structures: ------------------------------ There at least three different processes that can alter gene transcription through changes in chromatin: 1. Modification of the histone proteins 2. Chromatin remodelling 3. DNA methylation. Histones -------- Nucleosomes are arranged as an octamer of histone proteins with protruding N-terminal ends. - 147 base pairs of coiled DNA are wrapped around the histones. - 2 of each of the core histones H2A, H2B, H3 and H4, with H1 (linker protein) bound to DNA between nucleosomes. ### ### Histones Modification and Histone Code: - Histone code (pattern of histone modification) determines how histones behave and define the chromatin state. - These modifications are **post-translational modification.** - Many of histone tags work together to control histones (such as Acetyl, methyl, phosphate, and ubiquitin) - These chemical modifications are not just adding a group, they change the nature/behaviour of that particular amino acid, and that has an impact on functionality - These modifications of the histone tail act as **epigenetic marks** that control the expression or replication of chromosomal regions (read by transcription factors) - These marks are heritable. ### Active and Repressive marks: - The histone tails are made up of different amino acid, each of these amino acids can have a covalent modification added to it and the type of covalent modification depends on the amino acid. - Active marks which are represented in the upper section usually turn genes on and make DNA easier to read. - Repressive marks which are represented in the lower section usually turn genes off and make DNA harder to read. - These markers are not translatable to all the other organisms. ### Histone acetylation: - It is very difficult to change DNA charge due to the huge phosphate group. - DNA is negatively charged whilst histones are positively charged. - They have high affinity to each other, and DNA is tightly wounded. - The acetylation happens on lysine which is a positively charged amino acid, this neutralizes the positive lysine charge. - This then decreases the histone affinity for DNA, resulting in a less tightly wounded DNA that is open and transcriptionally active. Acetylated lysine residues -\> leads to transcriptional activation (gene expression) - HAT is an enzyme that adds acetyl groups. Deacetylated lysine residues -\> transcription repression (gene silencing) - HDAC is an enzyme that removes acetyl groupls - Histones near active genes are hyperacetylated. DNA methylation --------------- - It is the best understood example of epigenetic gene regulation. - Most genes have GC rich areas of DNA in their promoter region called CpG islands (p stands for phosphate) - Methylation of C residues within the islands leads to gene silencing/ repression. - The covalent addition of the methyl group at the 5-carbon of the cytosine ring making it 5-methylcytosine (5mC) - The methylation prevents binding of transcription factors and leads to condensed chromatin, which represses transcription leading to gene silencing. - Demethylation however expands the chromatin and permits transcription. - DNA methylation is essential for the normal control of gene expression in development. - About 1.5% of human DNA is 5mC which is a pretty high percentage as we only have around 2% of protein encoding DNA, making it nearly all of them. - In somatic cells, 5mC is almost exclusively in CpG sites except for embryonic stem cells. - Which further highlights their totipotency as they can be anything. - Addition of methyl groups is controlled by the enzyme family called DNA methyltransferases (DNMT) - 3 DNMTs are required for establishment and maintenance of methylation patterns DNMT 1, 3a and 3b. - 2 other DNMTs have more specialised functions such as DNMT2 and DNMT3L ### The role of DNA methyltransferases in DNA methylation  - DNMT 3a and 3b mediate establishment of new (denovo) DNA methylation patterns. - DNMT1 is responsible for the maintenance of established patterns of DNA methylation. - It follows the replication fork adding methylation marks to newly synthesized DNA (maintenance of DNA methylation) - Hemimethylated DNA (hemi means half) carries the information which nucleotide on the new strand should be methylated. - DNMT3b may assist DNMT1 in maintaining normal gene hypermethylation in diseased cells (e.g., cancer cells) ### Methylation of DNA is reversible: DNA Demethylation: - it is the process of removal of the methyl group. - important for epigenetic reprogramming - demethylation is catalysed by ten-eleven translocation -TET (family of 5mC hydroxylases - including TET1,2,3 - The process is promoted by binding CpG rich regions to prevent unwanted DNA methyltransferase activity and by converting 5mC to C via hydroxylase activity. ### TET proteins function in: - Transcriptional activation and repression (TET1) - Tumour suppression (TET2), example fixing wrong methylation patterns or reverse methylation - DNA methylation reprogramming process (TET3) ### Hypomethylation: - Too little methylation - More active transcription - "Turns on" genes promoting cell growth. - Chromosome instability (highly active DNA is more likely to be duplicated, deleted, and moved) ### Hypermethylation: - Too much methylation - Turns off genes that keep cell growth in check - Turn off genes that repair damaged DNA - Turns off genes that initiate programmed cell death. ### Changes of DNA methylation during development Epigenetic inheritance ---------------------- - it is when epigenetic tags remain and can be passed down to future generations. - when the zygote is formed many epigenetic tags are removed from the chromosomes of the parents - it also describes heritable alterations in which the DNA sequence itself is unchanged. - Parents experience, manifested in the form of epigenetic tags, that can be passed down to future generations. Identical twins ---------------  The differences over time looking at the comparative genomic hybridisation onto metaphase chromosomes for methylated DNA: - The 3-year-old twins have a similar DNA methylation distribution. - The 50-year-old twins show abundant changes (hypermethylation and hypomethylation events= green and red signals Environmental factors can control epigenetically controlled development: Queen versus worker bee. ------------------------------------------------------------------------------------------------- - Larvae that develop into workers or queen bees are genetically identical. - The royal jelly acts on a key gene -Dnmt3- which codes for a DNA methyltransferase that influences the queen genes. - If Dnmt3 is on, the queen genes are silenced the larvae are default worker bees - The royal jelly turns Dnmt3 off, the queen genes are activated, the larvae becomes queen bee Epigenetics II ============== Relevance of Epigenetics: ------------------------- ### Imprinting disorders - Can result from abnormal methylation of key genes in growth and development. ### X-inactivation patterns: - Can explain disease expression in X-linked disorders. ### Cancer: - Epigenetic mechanisms responsible for silencing of tumour suppressor genes or activation of oncogenes may be useful in diagnosis and monitoring or as therapeutic targets. ### Developmental origins of health and disease: - Impact of environment/diet during foetal development, infancy and childhood on disease risk in adulthood, thought to be mediated by epigenetic mechanisms. DNA methylation and cancer -------------------------- - Tumour suppressor genes are often silenced in cancer due to hypermethylation. - However, the genomes of cancer cells are hypomethylated, meaning we have a greater activity of genes that could be involved in rapid cell division (cancers trademark). - There is the exception of the hypermethylation events at genes that are involved in cell cycle regulation, tumour cell invasion and DNA repair. - For certain cancers, hypermethylation is detectable early and could be a biomarker of the disease. Epigenetic Mutations -------------------- ### Inherited disorders - Inherited defects that have an effect through epigenetic mechanisms - Fragile X syndrome (silencing of FMR1 gene loss of protein) - Rett Syndrome (MECP2 protein is a transcriptional activator) ### Disorders of imprinting - The defects in methylation or centres controlling methylation e.g.: - Prader-Willi syndrome (parental deletion, maternal imprinted ch15) - Beckwith-Wiedemann syndrome (uniparental parental disomy , ch11) Fragile x syndrome: ------------------- - It is a X-linked dominant disease. - Causes intellectual disability, behavioural and learning challenges, various physical characteristics including long narrow face and prominent ears. - Most common single gene cause of autism (estimated 5% of patients diagnosed with autism spectrum disorder. - Most common cause: is the expansion of a CGG triplet repeat region in the 5' UTR of the FMR1 gene (familial mental retardation 1) - More than 200 repeats result in the silencing of the gene. - The repeats result in C methylation and transcriptional silencing. - It is subject to anticipation as the repeat length increases each generation. - The frequency is quite high 1:4000 males and 1:6000 females. Genomic imprinting ------------------ - It is what causes genes to be expressed in a parent-of-origin specific manner. - An inheritance process involving DNA methylation and histone methylation. - It might be a mutation changing the activity of histone acetylase or DNA methyl, which then causes a change in epigenetic pattern but the epigenetic pattern itself is NOT a change in the DNA sequence. - Epigenetic marks are established which are imprinted in the germline and are maintained in the somatic cells of an organism through mitotic division. - DNA methylation may develop differently for each sex during gamete formation. - Offspring inherit an inactive (methylated) copy and an active (demethylated) one called genomic imprinting (dependent on which parent they get it from) - In mammals about 200 genes might differ in this way some might be active in the egg and not in the sperm - Found in Fungi, plants, and animals. Non-imprinted and imprinted genes: -------------------------------------------------------- Beckwith-Wiedemann Syndrome --------------------------- - It is a disease associated with 1:10000-14000. - Their features: embryonic and placental overgrowth, predisposition to childhood tumours. - Cause: genetic and epigenetic changes in a region of about 1 mega base on chromosome 11p, encompassing 15 genes, most of them being imprinted, - Another cause is that patients have both chromosome 11 copies received from the father -\> too many active genes from the father and not enough maternally expressed genes from the mother leading to an imbalance of expression. - IGF2 and CDKN1C are key genes: - IGF2: normally this gene is only expressed by the parental chromosome. - CDKN1C, is 700 kb away and is normally maternally expressed. - An increased expression of IGF2 and suppression of CDKN1C are believed to be the major cause of the disease. **X-inactivation -- monoallelic expression:** - If both X chromosome genes are expressed in females-\> unequal expression in males and females - X inactivation occurs early in the development of most female mammals (64-128 cell blastocyst stage in mouse zygote) - Most of the genes on one X chromosomes are inactivated by methylation in every cell leading to a whole chromosome inactivation. - Overall transcription dosage of chromosome X genes is equal in males and females "**dosage compensation".** - X chromosome inactivation silencing is random , each cell makes an independent choice leading to a mosaic pattern Skewed X inactivation --------------------- - Can sometimes be skewed=non-random - Proposed that an abnormal or mutation carrying X chromosome: - Is preferentially inactivated, and/or - Has some growth proliferation disadvantage so cells in which it is active are fewer in number in adult females. - Although these two reasons are not proven yet - Relevance to human disease: - It can provide evidence that an X-linked disorder might be occurring in a family. - Can explain phenotypic variability in females "carrying" X-linked disorders. Female carrier of Duchenne muscular dystrophy --------------------------------------------- - DMD is characterized by progressive muscle degeneration and weakness. - X-linked recessive disease - Inherited from the mother or caused by a new mutation. - Caused by absence of the protein dystrophin that maintains muscle fibers cell membrane. - In a photo of dystrophin antibody staining in female carriers some fibers stain positively for dystrophin (brown) whilst others are negative - Contributes to a mosaic pattern(because it is a random choice of which X chromosomes genes are silenced) reflecting X-individuals in individual cells. DOHAD- developmental origins of health and disease -------------------------------------------------- - Epigenetics VS epigenomics -------------------------- - Epigenetics is the study of changes in regulation of gene activity and expression that are not dependent on gene DNA sequence, often refers to the study of single genes or sets of genes. - Epigenomics refers to a more global analyses of epigenetic changes across the entire genome ### Role of methylation: - - - - Detection of methylation by bisulfite conversion and sequencing --------------------------------------------------------------- - It was a previous gold standard: combine sodium bisulfite conversion with PCR and Sanger sequencing. - Cytosines in bisulfite-converted sequences indicate that the cytosine in the original genomic DNA fragment was methylated as the bisulfite does nothing to both 5-methylcytosine and 5-hydroxymethycytosine (conversion rxn= does not turn into uracil) - Need to sequence both to tell which was methylated in the original DNA and which was not. - Although it was the best method for studying methylation limiting aspects include: - Requirement for primer design which introduces biases. - Limited to surveying a few loci from each bisulfite-treated sample. - Low throughput - **MethylC-sequencing** involves preparing a special DNA library and using a chemical treatment called **bisulfite sequencing** (BS-sequencing). - **Bisulfite conversion** changes unmethylated cytosines into another base (uracil), but **methylated cytosines** stay unchanged. This allows researchers to see which cytosines are methylated. - **MethylC-seq library preparation** takes about 2 days and provides a detailed, **genome-wide map** of where methylation occurs at **single-base resolution**. This means you can see exactly which cytosines are methylated across the entire genome. - After the **high-throughput DNA sequencing**, researchers can estimate the **frequency of DNA methylation** at each cytosine, as long as there's enough sequencing coverage. - Using this method, methylomes (methylation maps) for species like **mouse, human, and Arabidopsis** typically cover **90--95%** of all cytosines in the genome. Methylated DNA Immunoprecipitation sequencing --------------------------------------------- - MeDIP-seq= methylated DNA immunoprecipitation - It is a large-scale chromosome or genome-wide purification technique to enrich for methylated DNA sequences - After the methylated DNA is purified they can be input to high throughput DNA detection methods such as: - High resolution DNA microarrays (MeDIP-chip) - Next generation sequencing (MeDIP-seq) ChIP-chip and ChIP-seq ---------------------- - chromatin **immunoprecipitation** (ChIP) followed by **microarray hybridization** (ChIP-chip) or **high-throughput sequencing** (ChIP-seq). - Both techniques allow for genome-wide discovery of **protein-DNA interactions** such as RNA polymerases, transcriptional co-factors and transcription factor bindings and histone modifications. -  These approaches have led to many important discoveries related to transcriptional regulation, epigenetic regulation through histone modification, nucleosome organisation, & interindividual variation in protein-DNA interactions. - **ChIP-chip** emerged soon after year 2000 for organisms with small genomes (e.g. yeast). - **ChIP-seq** emerged later with next generation sequencing technologies and is an attractive alternative. It has **advantages over ChIP-chip** such as: ChIP-on chip wet -lab portion: ------------------------------  - The **protein of interest (POI)** is chemically \"cross-linked\" (attached) to the specific **DNA sequence** it binds to. This happens in an experimental setting (in vitro), often using **formaldehyde**, which temporarily locks the protein to the DNA. The cross-link can be reversed later with heat. - **Cells are broken open** (lysed), and the DNA is cut into smaller pieces using either **sonication** (sound waves) or enzymes like **micrococcal nuclease**. - An **antibody** that specifically targets the POI is used to pull out the **POI-DNA complex**. This process is called **ChIP** (chromatin immunoprecipitation). - The **cross-link** between the protein and DNA is reversed, the DNA is separated from the protein, and the DNA is purified. - The DNA is then **amplified and labeled** to make it easier to detect. - Finally, the labeled DNA is **hybridized** (matched) to a **microarray (chip)** that has many DNA sequences. This helps identify which DNA sequences the protein was bound to. ChIP-seq: --------- - It works in principle the same way as chip-chip - Oligonucleotide adaptors are then added to the small stretches of DNA that were bound to the POI to enable massively parallel seq instead of hybridizing to a chip.  Encode ------ - ENCyclopedia Of DNA Elements. -  ENCODE Consortium: international collaboration of research groups -  ENCODE project began as a pilot project on 1% of the genome. -  In 2007 the effort was scaled to whole- genome assays, followed by expansion to similar assays in mouse. -  Project creates a comprehensive catalog of gene elements and functional elements in the human and mouse genomes by: - Measuring RNA expression levels - Identifying proteins that interact with RNA and DNA (e.g. modified histones, transcription factors & RNA-binding proteins). - Measuring the levels of DNA methylation, and - Identifying regions of DNA hypersensitivity. The ENCODE Consortium analyses the data it produces in an integrative manner: Organises annotations of the most salient analysis products. **Ground level annotations:** usually directly derived from the experimental data.\ **Middle level annotations:** integrate multiple types of experimental data and multiple ground level annotations. **Top level annotations:** integrate broad range of experimental data and ground and middle level annotations.