Eukaryotic gene regulation PART II 2013
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Eukaryotic gene regulation PART II 2013
- 1. EUKARYOTIC GENE REGULATION II Jill Howlin PhD Canceromics Branch Department of Oncology, Lund University Medicon Village http://www.med.lu.se/english/klinvetlund/canceromics/research
- 2. Lecture structure Eukaryotic Gene Regulation I A Revision of the basics • What is a gene, chromosome etc. • Transcription • mRNA processing & Splicing • Translation… • DNA mutations Eukaryotic Gene Regulation II B Gene Structure and function • Structure of typical gene in the genome and its regulatory units • How gene structure influences regulation.. A Control of gene expression • Producing specificity: Transcription factors • Other forms of regulation of gene expression, non-coding RNA, epigenetic regulation: imprinting & methylation etc…. B Analysis of Gene Expression & Regulation • Analysis of gene expression in the genomics era: transcriptomics RNA-seq • Studying gene regulation, DNA-protein interaction
- 3. Source material: • Molecular Biology of the Cell. 5th edition. Alberts et al. • The Cell: A Molecular Approach. 2nd edition. Cooper GM. • Human Molecular Genetics. 4th edition. Strachan and Read • Virtual Cell Animation Collection http://vcell.ndsu.nodak.edu/animations/ (also available as free iphone/ipad app from itunes) • Wikipedia http://en.wikipedia.org/ & YouTube http://www.youtube.com • ENCODE explorer http://www.nature.com/encode/#/threads
- 5. PART IIA control of gene expression
- 6. Gene Regulation Why do we need gene regulation? All cells carry All the genetic information! - how is cell specificity determined? DNA = = =
- 7. Gene Expression • Specificity of function is determined by regulation of gene expression • Gene products will be produced only ‘when & where’ they are needed • A specific transcription factor is the ‘when and where’ determinant • Mediate temporal and spatial control of gene expression • Control the rate of gene transcription by influencing RNA polymerase binding to DNA • >2000 proteins with DNA binding domains in the genome (10%) protein coding transcripts
- 8. Transcription Factors • General and sequence-specific • Transcribed like any other protein coding gene • Intracellular & extracellular stimuli (growth factors, hormones, cytokines) • Developmental programs & signaling cascades • Feedback loop • Nuclear localization • Protein-protein interaction (co-regulations, chromatin remodelers etc.)
- 9. Transcriptional Regulation upregulation activation or promotion of the rate of transcription downregulation repression or suppression of the rate of transcription co-activator a protein that works with transcription factors to increase the rate of gene transcription co-repressor – a protein that works with transcription factors to decrease the rate of gene transcription co-regulators
- 11. Classes of Transcription Factors All have at least two functional domains or regions: Transactivation domain interacts with other proteins DNA-binding domain binds a specific DNA sequence several common protein structural motifs that TF use -Zinc finger proteins -Helix-turn-helix -Leucine zipper proteins -Helix-loop-helix proteins -Nuclear Receptors
- 12. Nuclear Receptors e.g.:ERalpha,AR • nuclear/steroid receptors have a third steroid-binding domain • bind ligand Zinc finger proteins HRE
- 13. HRE -hormone response element • short sequence in/near promoter region, 5’ to TSS • a pair of inverted repeats separated by 3 nucleotides HRE Transcription Factor Consensus Sequence ERE Estrogen receptor AGGTCANNNTGACCT GRE Glucocorticoid receptor AGAACANNNTGTTCT Estrogen receptor Estrogen AGGTCANNNTGACCT TSS
- 14. Position weight matrices used to depict the DNA binding preferences of transcription factors e.g.: Always ‘G’ at 3rd position
- 15. Transcriptional Co-regulators • interact with transcription factors to either activate or repress the transcription of specific genes • They can be referred to as coactivators or corepressors. They don’t bind DNA themselves • Co-regulators connect transcription factors with the complex of proteins that carry out transcription • Many co-regulators are proteins that modify histones (or recruit proteins that can) to allow the transcription factors access to the underlying DNA sequence
- 16. Accessingthe DNA roleofchromatininregulatinggeneexpression The structure and function of chromatin is influenced by: 1. Histone modifications 2. DNA modifications
- 17. Heterochromatin & Euchromatin Packaged chromatin has 2 basic variants: 1. Heterochromatin – closed & inactive either constitutive or facultative 2. Euchromatin - open & accessible XX-example in females Specific histone modifications differentiate the main types of chromatin (e.g.: H3k9me2/3) Heterochromatin Feature Transcriptionally active chromatin Transcriptionally inactive chromatin Chromatin conformation Open, extended conformation Highly condensed conformation; particularly apparent in heterochromatin (c&f)
- 18. Nucleosome structure • 147bp DNA wrapped around a complex of histones • "beads on a string” • Histone N- and -C terminal tails protrude from the nucleosome Core of 8 histones (H2A, H2B, H3, H4) X 2 Histone H1 Linker DNA Core DNA Histone tails
- 19. Histones • small (130 aa) basic proteins, positively charged • Enzymatic modifications of specific amino acid in the tails can determine • nucleosome behavior • DNA accessibility Modification Effecting enzymes Reversing enzymes Acetylation (HATs) histone acetyltransferases (HDACs) histone deacetylases Methylation (HMTs) histone methyltransferases Histone demethylases Phosphorylation Histone kinases Histone phosphatases Large families of enzymes ‘’Writers’’ ‘’Erasers’’
- 20. Histone modifying enzymes http://www.actrec.gov.in/histome/The histone Infobase Examples of ‘’Writers’’ Examples of ‘’Erasers’’ Amino acid Possible modifications Lysine Methylation, Acetylation, Ubiquitination, Arginine Methylation Serine Phosphorylation Threonine Phosphorylation • Lysine methyltransferases EZH1/2, SETD1A/B • Lysine acetyltransferases NCOA1/3, CBP/P300 • Lysine deacetylases HDAC1/2/3..; SIRT1/2/3.. • Lysine demethylases KDM1A/2A, JHDM1D, JMJD5
- 21. • Histone deacetylases (HDACs) are negative regulators of transcription • Histone acetylases (HATs) are positive regulators of transcription In general… + - e.g.: , CBP/P300 e.g.: , HDAC1/2
- 22. The Histone Code Specific histone modifications determine function H3K4me3 Histone 3 Lysine Position 4 from the N-terminal Modification type Tri-methylated ac: acetylation ph: phosphorylation ub: ubiqutination Reading the notation….. Amino acid Possible modifications Lysine Methylation, Acetylation, Ubiquitination, Arginine Methylation Serine Phosphorylation Threonine Phosphorylation
- 23. Barth and Imhof ; Trends in Biochemical Sciences Volume 35, Issue 11 2010 618 – 626c Active gene TSS gene body • H3K36me3 is found in actively transcribed gene bodies • H3K4me3 found in actively transcribed promoters, particularly just after the transcription start site
- 24. Barth and Imhof ; Trends in Biochemical Sciences Volume 35, Issue 11 2010 618 - 626 Inactive gene • even distribution of silencing modifications H3K9ME2/3, H4K20ME3 • H3K27me3 is enriched in the promoter gene body TSS
- 25. Reading the Histone Code • Specific histone modifications act as binding sites for other proteins ‘’Readers’’ • Families of proteins with the right type of domain recognize particular modifications Bromodomain containing proteins (BRD4, BRD2) recognize acetylated lysines Chromodomain proteins (CHD1, CDY) recognizes methylated residues Examples of ‘’Readers’’
- 27. UCSC Genome Browser encodehistonetracks
- 28. DNA modifications • DNA can be modified by methylation of adenine and cytosine bases Methylated Base Methylation Sequence C5-methylcytosine (5-mC) CpG C5-hydroxymethylcytosine (5- hmC) CpG, CpHpG1, CpHpH1 H = Adenine, Cytosine, or Thymine "p" in CpG refers to the phosphodiester bond
- 30. Epigenetics Epigenetics - regulation of gene expression that is not at the level of the DNA sequence The so called ‘Histone Code’ can be considered part of a wider Epigenetic Code, as can DNA methylation
- 31. Imprinting • expressed differently according to the parent of origin • always either the version from the mother or father Prader-Willi syndrome : missing gene activity that normally comes from only from the paternal copy Angelman syndrome missing gene activity that normally comes from only the maternal copy Igf2 is imprinted on the chromosome inherited from the mother H19 is imprinted on the paternal chromosome. genes that escape epigenetic reprogramming chromosome 15q partial deletion,
- 32. Genetic conflict hypothesis Beckwith-Wiedemann Syndrome http://learn.genetics.utah.edu/content/epigenetics/imprinting/index.html
- 33. Epigenetic Inheritance transmittance of information from one generation to the next without alteration of the primary DNA sequence Various complex cellular mechanisms allow for retention of epigenetic marks Retention of epigenetic marks is seen during chromosomal replication where modifications of new histones and DNA are patterned on those of the old histones and DNA
- 34. Transgenerational Epigenetic Inheritance • From organism to offspring not just cell to cell • Can be hard to prove true transgenerational inheritance • Intergenerational effect • Examples: male-line transgenerational effects Swedish Överkalix Study variation in the food supply during the early life of paternal grandparents was related to variation in mortality rate specifically diabetic deaths in their grandchildren Rat liver fibrosis study Liver injury to male rats led to epigenetic changes in their sperm, which transmit increased protection from fibrosis into their male offspring Mother -1st Fetus -2nd Offspring's reproductive cells -3rd Simultaneous exposure to environmental agent Nature Reviews Genetics 14, 228-235 (March 2013)
- 35. END OF PART IIA
- 36. PART IIB Analysis of Gene Expression & Regulation
- 37. Evolution of Genomics Technologies Kahvejian et al., (2007) Nat Biotechnology 26:1125
- 38. Studying Gene Regulation….inthepost-genomicsera Gene expression profiling /transcriptomics • DNA/oligonucleotide microarray based assays • RNA sequencing analysis Mapping DNA-protein interactions Chromatin IP followed by microarray based analysis or sequencing • ChIP-Chip • ChIP-seq
- 39. Gene expression profiling…..microarraytechnology • Microarray hybridization technology developed in the late 1990’s • Hybridization is the process of establishing a non-covalent, sequence-specific interaction • two perfectly complementary strands will bind to each other readily Hybridization strong weak Relative gene expression high low upregulation downregulation
- 40. Results of GEX 1000’s Individual genes Hierarchical clustering Control Experimental Red genes are ‘upregulated’ in the experimental samples… green ‘downregulated’ in the control
- 41. Gene expression profiling using microarrays has been successfully used to subtype breast tumors based on their gene expression patterns Sorlie et al., 2001, PNAS Individual genes Individual tumor samples
- 42. Sequencing Sanger (First Generation) • Developed by Frederick Sanger and colleagues in 1977 • Days to weeks per genome..costly • Remain useful for small scale projects & longer reads Method: chain-termination incorporation of radioactive or dye-labeled nucleotides Next-Generation Sequencing (costs less by four orders of magnitude) • Hours for a genome @ $10-20k Method: reversible chain-termination - Ilumina system
- 43. Sanger Sequencing chaintermination Readout capillary gel + A G C TPCR measure the length and color of the resulting individual fragments
- 44. Next Generation Sequencing www.genomesunzipped.org No individual fragments to measure because the dye termination is reversible DNA is immobilized in dense clusters Massively parallel, sequencing by synthesis: Illumina video clusters of library to be seq
- 45. Types of NG Sequencing experiments RNA • whole transcriptome -total RNA seq • mRNA seq (poly-A tailed RNA) • specific target-enriched DNA • whole genome • whole exome • specific target-enriched -normal cellular nucleic acid, disease tissue, tumor tissue
- 46. mRNA-Seq Poly-AdependantRNAanalysisthroughcDNAsequencing TSS, alternative splicing, novel transcripts & gene fusion events
- 47. total RNA-Seq wholetranscriptome NotPoly-Abasedcapture,rRNA depletedtotalRNA both coding and non- coding RNA species
- 48. NGS target enrichment (RNA & DNA) DNA : selectively sequence particular coding regions of the genome e.g.: X chromosome, custom selection of oncogenes & tumor suppressors RNA: particular RNA species, e.g.: Kinome –all kinases in the genome, custom selection of genes
- 49. DNA-protein interactions • where in the genome does a particular TF bind? • or where is a particular histone modification located? ChIP Chromatin Immuoprecipitation 1. DNA & protein complex cross-linked using formaldehyde 2. Bound chromatin is sheared by sonication 3. Antibodies bind either TF or Histone modification & immunoprecipitate the DNA– protein complex 4. Cross-linking is reveresed & DNA asssoc. with the TF/histone modification is purified transcription factor ‘X’ histone modification anti-H3K36me3 antibody anti ‘X’ antibody identify DNA fragments
- 50. ChIP-Chip Determine genome-wide DNA binding sequence for a specific TF ChIP-seq • DNA fragments identified by hybridization to a microarray • oligonucleotide probes from entire genome or selected regions e.g.: promoter regions, specific chromosomes • Like GEX, limited by sequences on the microarray • DNA fragments of interest are sequenced directly • Not limited by known sequences on an array
- 52. UCSC genome browser specificTFtracks
- 53. END
Hinweis der Redaktion
- My name is ….I originally come from Dublin, Ireland where I obtained my PhD in 2004. I’ve worked at Lund University for about 6 years, first as a post-doc in CRC in Malmö and now as a junior group leader at the Dept. of Oncology, Canceromics Branch which are situated in Medicon Village. My main interest is then unsurprisingly in cancer research and cancer genomics in particular breast cancer and melanoma.
- The lectures are divided over 2 days …we have 2 X 2 hour slots ..we wont need all that time probably a little over an hour each time..1hour ½ today, 1hour next timeToday we will firstly revise the very basics and then go a little deeper than you’ve gone before and introduce some new conceptsNext week we will continue with more detailed concepts and final address the experimental study of gene regulationFrom the last years student feedback I know that some really people like the revision part, others find it boring but we can also see from the exams the some of the basic stuff wasn’t well known or understood so its likely that’s there is a mixture of levels and background knowledge in the class so I think its good that we make sure we are all on the same page at the beginning. The purpose of the lectures is to give you a very structured overview of this particular topic…its not that you could just read about it yourself you certainly could but by coming to a lecture you get it all appropriately summarized. I tend to make sure that I put the most important content and text on my slides in case there is some difficulty in understanding me I am one of if not the only non-swedish speaker that you have as a lecturer and I don’t what anyone to be at a disadvantage because of that.I am happy to get feedback and or questions now or via emailI will discuss the exam and questions next week.
- The same genome is responsible for making the entire cadre of cell types, each of which has its own functionIn order for us to have different types of cells, there has to be some mechanism for controlling the expression of our genes.In some cells, certain genes are turned off while in other cells they are switched on…Or and this is more likely that the similar groups of genes are expressed to subtly different degrees…this is referred to as GENE EXPRESSION
- Having control over gene expression means that gene products will be produced only ‘when & where’ they are needed and to the extent that they are neededA specific transcription factor can be thought of as the ‘when and where’ determinant for a particular gene or set of genesMediate temporal and spatial control of gene expressionThey function to mediate temporal (at a particular time)and spatial ( at a particular place) control of gene expressionThe work in the simplest terms by controlling the rate of gene transcription by influencing RNA polymerase binding to DNA: controlling the rate of gene transcription by eitherhelping or hindering RNA polymerase binding to DNA Because RNA polymerase II, which transcribes mRNA, cannot bind to promoters in eukaryotic DNA without the help of transcription factorsDepending on the database you are looking at there is thought to be more than 2000 proteins with DNA binding domains in the genome which means that approx 10% of the protein coding transcripts are potential TFs…HUGH FAMILYIn eukaryotes, unlike prokaryotes, the ground state of expression is restrictive in that, although strong promoters might be present, they are inactive in the absence of some sort of recruitment to the promoter by transcription factors.
- Ok so what are TFs: Important to note I’m referring now to TF that are not just the basal TF machinaryThere are two types of TFs: general and sequence-specificSo what turns them on? They are just genes too! Regulate the regulatorsTFs themselves are regulated by external signals that can be either environmental or cellular derived, such as the signal from growth factors, hormones & cytokines NB: Lars Ronnstrand lectures within the context of developmental cues or signaling cascadesSome of the controls they have include:Feedback loop:One interesting implication of this is that transcription factors can regulate themselves, usually repressing themselvesnuclear localization signals determined by their state of activation which is also mediated by protein-protein interaction, lead to coregsSo in short a particular cell at a particular place and time will be influenced by some form of external stimulus setting in motion a cascade of events leading to transcription of a particular set of genes and ultimately the expression of a particular set of proteins
- So what are the protein protein interactions:• coactivator– a protein that works with transcription factors to increase the rate of gene transcription • corepressor– a protein that works with transcription factors to decrease the rate of gene transcriptioncan be collectively refereed to as coregulators as this is more accurate --the same proteins depending on the particular complex can activate or repress: Other proteus such as chromatin remodelers, histone acetylases, deacetylases, kinases, and methylases, which we will get to also playcrucial roles in gene regulation but as they lack DNA-binding domains so not called TFsIf we say upregulation or gene upregulation this is the activation or promotion – increase the rate of gene transcription downregulation repression, or suppression – decrease the rate of gene transcription
- Ok so we can see a short clip on regulated txn
- There is a large number of specialized transcription factors:- prob up to 2000, identified with significant variation in their structures but, All have at least two functional domains or regions:These highly conserved sequences have been used to categorize the known TFs into various "families," Transactivation domain interacts with other proteins (protein-protein interactions) and functions to alter the rate of transcription.DNA-binding domain recognizes a specific DNA sequence (motif) and binds to it,There are several common protein structural motifs that TF use to bind DNA although the precise sequence of the DNA-binding domain determines the DNA sequences specific recognition: theses include-TFs of the same family share the same defining domain structure. Nuclear receptors are a special case because they also have a ligand binding domain and we can take a closer look at these as an example
- So in addition to the two shared by all transcription factors: they have a steroid-binding or ligand binding domain, through which they bind their ligand, and have their activity regulated.Examples in include the estrogen receptor, androgen & glucocorticoid receptorsMost ‘drugable’ TF targets e.g.: tamoxifen
- The responses elements for hormones are usually a short sequence within or near the promoter region, most commonly a pair of inverted repeats separated by three nucleotides (indicating the receptor binds as a dimer)Nuclear receptors bound to hormone response elements recruit a significant number of other proteins (transcriptional coregulators) that facilitate or inhibit the transcription of the associated target gene recruit other proteins (transcriptional coregulators) that facilitate or inhibit the transcription of the associated
- Not just nuclear receptors but Position weight matrices (PWMs) are widely used to depict the DNA binding preferences of transcription factors. (TFs)
- So what are transcriptional coregulators?transcriptional co-regulators are any proteins that interact with transcription factors to either activate or repress the transcription of specific genes They don’t bind DNA themselves
- In order to understand howTF and coregulators get to te DNA to switch on/off txn, we gave to revisit chromatin structureAnd its role in gene regulationThe structure and function of chromatin is influenced by:Histone modifications (post translation protein modifications:-DNA modifications (additions to the DNA such as Methyl groups)See typical nucleosome structure
- Chromatin can be defined as DNA plus the proteins, like histones, that package it within the cell nucleus. This packaged chromatin can be divided into 2 basic variants:Heterochromatin is closed and in an inactive conformation it is situated at the chromosome ends close to telomeres and around the centrosomeHowever it can be classds as either constitutive (usually repetitive sequences)or facultative : facultative heterochromatin can, under specific developmental or environmental signaling cues, lose its condensed structure and become transcriptionally active.[1] Heterochromatin is often associated with the di and tri-methylation of H3K9Facultative chromatin is not consistent across all sequences so a region that in one cell that is packaged in facultative heterochromatin (and the genes within are poorly expressed) may be packaged in euchromatin in another cell (and the genes within are no longer silenced)ConverselyEuchromatin is open & accessible by TFs and is always potentially transcriptionally activeAs a good example is the XX chromosomes in females where one X is essentially facultative heterochromatin and mostly silent and the other is packaged as euchromatin and expressed.
- First I want to remind you of the nuc structure from last week:octamer : consists of about 146 bp of DNA[12] wrapped in 1.67 left-handed superhelical turns around the histone octamerhistone octamer, consisting of 2 copies each of the core histones H2A, H2B, H3, and H4. Adjacent nucleosomes are joined by a stretch of free DNA termed "linker DNA" Histone have protruding N- and C- terminal tails tails but generally acquire modifications on their N-terminal tails
- Histones themselves which make up nucleosomes are small (130 amino acid) basic proteins that have a high a affinity for negatively charged DNA due to their strong positive charge.Histones were originally thought to function as a static scaffold for DNA packaging dynamic proteins, undergoing multiple types of post-translational modifications that regulate chromatin condensation and DNA accessibility.Modifications of specific amino acid in the Histone tails can control the behavior of the nucleosome and accessibility of the underlying DNAMyriad of enzymes controlling the modifications and I just list 3 types of modification here.Writers add the modification while erasers delete it
- These familes of enzymes are very large and if you want to the extent of them there are database such as histone infobase try to collect all te info on themFor now I can just highlight a few examples that are grouped according to the amino acid that hey put the modification on. Different aa’s have different possible modifications
- If we just consider the acet modifications writer and eraser system in general it follows that HATs or Ac of the histones promote txn, and hdacs, or deacet of histones inhibit txnAs you can imagine its not really this simple as different modification of specific histone residues occur in combination and have different effects
- The idea of the Histone code is really a hypothesis that is based on the idea that rather than through simply stabilizing or destabilizing the interaction between histone and the underlying DNA…. Specific histone modifications also act to recruit other proteins by specific recognition of the modified histone via protein domains The nomenclature for other modifications include ac:acetylation, ph:phosphorylation, ub:ubiqutination
- If we look at the example of an active gene:2 modifications particularly assoc with active genes: H3K4Me3 in promoter and H3K36Me3 in bodyAt the transcriptional start site TSS there is a nucleosome- depleted region (NDR) within the promoter. Active modifications such as methylation of H3K79 are present in the body of these genes. H3K36me3 is found in actively transcribed gene bodies.
- If we look at the example of an inactive gene:Inactive genes, as shown in (c) and (d), have a fairly even distribution of silencing modifications, such as H3K9 methylation and H4K20 methylation, whereas H3K27 methylation is enriched in the promoter. These modifications can be bound by heterochromatic proteins (blue) and, thus, this chromatin area can condense, as seen in heterochromatin. NB: to say modifications may alter the electrostatic charge of the histone resulting in a structural change in histones or their binding to DNA
- As well as ‘writers’’ , ‘’erasers’’ enzymes which I mentioned earlier there are also ‘’readers’’ of the histone codeIt follows that Histone modifications can be read by other poteinsExamples include theBromodomaincontaining proteins e.g.: BRD4, BRD2 that recognize acetylated lysines or chromodomainsconytaining proteins CHD1, CDY that recognizes methylated residues such as the bromodomains (recognizes acetylated lysines) orchromodomains(recognizes methylated residues)
- If we take an example of just Histone3List function of each mod if known, the writer eraser and reader
- I spoke about the encode data that was released last year and one of the things this tried to achieve was to completely map histone modification across the genome of many humnacel linesSnap shot of the public database: can look at your favorite gene in this case Im showing you progesterone receptor target gene of ER
- As well as Histone mod there is also DNA mods:DNA can be modified by methylation of adenine and cytosine bases The "p" in CpG refers to the phosphodiester bond between the cytosine and the guanine, which indicates that the C and the G are next to each other and NOTCG nucleotide pairsAbout 60% of human genes have CpG islands (CGIs) at their promoters
- DNA meth and hist mod are not two separate entities but rather interact and influence one another.e.g.: transcriptional repression by histone deacetylation may be mediated by DNA methylation
- The so called ‘Histone Code’ can be considered part of a wider Epigenetic Code as can DNA methylationEpigenetics refers to any regulation of gene expression that is not at the level of the DNA sequencereset during meiosis Conventional view is that Reprogramming always resets the epigenome and this occurs in the the early embryo: reset during meiosis Soon after egg and sperm meet, most of the epigenetic tags that activate and silence genes are stripped from the DNA.In terms of gene regulations ---Why Epignetics? DNA code not enough? (more static)Flexability-response to changing environment
- The major exceptions to the rule of reprogramming= ImprintingImprinting describes the situation where the marks are not erased by reprogramming and determine parent specific expressionImprinted genes are genes are expressed differently according to the parent of origin: Regardless of whether they came from mom or dad, certain genes are always silenced in the egg, and others are always silenced in the sperm.It is always the same member of a pair of genes that is imprinted and hence inactive; for some genes this is the version inherited from the mother, and for other genes it is the paternal version. 2 developmental disorders due to imprintingPrader-Willi syndrome : missing gene activity that normally comes from only from the paternal copy Angelman syndrome missing gene activity that normally comes from only the maternal copy
- Why do we need imprinting at all? In terms of gene regulations ---Why Epignetics? DNA code not enough? (more static)Flexability-response to changing environment One of the theorys put forward is that of Genetic conflict hypothesisPaternal imprinting favors the production of larger offspring, and maternal imprinting favors smaller offspring. So it's in the interest of the father's genes to produce larger offspring. The larger kittens will be able to compete for maternal resources at the expense of the other father's kittens. On the other hand, a better outcome for the mother's genes would be for all of her kittens to survive to adulthood and reproduce. Mother conserves resourcesActivation of the maternal Igf2 gene during egg formation or very early in development causes Beckwith-Wiedemann Syndrome (BWS). While children with BWS have a variety of symptoms, the most common and obvious feature is overgrowth. Babies with BWS are born larger than 95% of their peers. They also have an increased risk of cancer, especially during childhood.
- Epigenetic inheritance is the transmittance of information from one generation to the next that affects the traits of offspring without alteration of the primary structure of DNA It turns out tat its not just our DNA that can be inheritedHow are epigentic marks maintained?Various complex cellular mechanisms allow for retention of epigenetic marks Not fully understood!!!marks Retention of epigenetic marks is seen during chromosomal replication where modifications of new histones and DNA are patterned on those of the old histones and DNANew cells acquire the same Methylation patternHowever this is at a cell to daughter cell level and normally reprogramming resets the epigenome of the early embryo Retention ofepigentic marks through mitotucdevision is not completekly understood…copied or resetablished
- When the epigenetic marks are passed not just from cell to daughter cell but from organism to its offspringHard to prove due to direct exposure and transient nature of epi marks the repond to enviromental or other stimuli and some are just intergenerational effectsQuestion of transgenerational stability always arrises?? Might be inherited and then erased due to enviromentExamples of what are believed to be true transgenerational inheritance include:The Swedish studyDiabetes mortality increased if the paternal grandfather was exposed to a suplus of food Rat liver Studyinjury to male rats in two different models of fibrosis (carbon tetrachloride or bile duct ligation), leads to epigenetic changes in their sperm, which transmit increased protection from fibrosis into their male offspring, made apparent when the offspring's livers are injured in a similar mannerThe mediating molecular mechanism remains elusive!!
- So now we move in to looking at the technologies that allow us to study these types of gene regulationMicroarrays started in themid to late 90s followed by ChIP & NGSThes are the ones tat explode and the ones we will talk about now
- In particular I will cover ……
- As I said Microarray hybridization technology developed in the late 1990’sIt’s a principle based on complementary hybridization is the process of establishing a non-covalent, sequence-specific interactiontwo perfectly complementary strands will bind to each other readilyOptical picture and determine relative intensities
- Heat map Disease vrs normalCancer vrs normalOne cell type vrs anotherOne drug treatment vrs another
- Sorlie landmark study on molecular subtypes in breast cancerNot all breast cancers are the same
- To over come limits of array based experimentation…So before I describe RNA seq first I need to tell you about sequencing methods and how they workSangerchain-termination incorportation of radioactive/dye labelled nucleotidesRadioactive not used anymore needed 4 separate reactionsNGSTodays sequencing methods mean that whole genomes can be rapidly evaluatedJust like Sanger sequencing relies on the ddNTP to stop the PCR reaction, Illumina’s reversible terminator sequencing all rests on the reversible terminator bases (RT-bases). Just like ddNTP, these bases stop PCR reactions when they are incorporated; they have additional molecules, including a base-specific dye, attached to the standard base which stops the PCR enzyme adding more bases (A bases have red dye, C bases have blue dues, G yellow and T green):there exists a cleavage enzyme that chops all the extra molecules off, and turns the RT-base into a normally functioning nucleotide. We multiply up the template stand, i.e. the bit of DNA that we are sequencing, and stick on a few bases of ‘adaptor sequence’; this sequence sticks on to complementary bits of DNA stuck to a surface, which holds the DNA in place while we sequence it:
- Radioactivity: A dark band in a lane indicates a DNA fragment that is the result of chain termination after incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP)Dye-termination: In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with fluorescent dyes, each of which emit light at different wavelength-Developed by Leroy Hood In First Generation (Sanger) sequencing, we run a PCR reaction in the presence of a bunch of ddNTPs, with each different base pair dyed a different colour. We then measure the length and colour of the resulting fragments of DNA, and use that to work out the sequence; a bit of DNA 35 base pairs long ending in a blue ddNTP tells us that the sequence has a “C” at the 35th position. www.genomesunzipped.org
- Just like Sanger sequencing relies on the ddNTP to stop the PCR reaction, Illumina’s reversible terminator sequencing all rests on the reversible terminator bases (RT-bases). Just like ddNTP, these bases stop PCR reactions when they are incorporated; they have additional molecules, including a base-specific dye, attached to the standard base which stops the PCR enzyme adding more bases (A bases have red dye, C bases have blue dues, G yellow and T green):there exists a cleavage enzyme that chops all the extra molecules off, and turns the RT-base into a normally functioning nucleotide. We multiply up the template stand, i.e. the bit of DNA that we are sequencing, and stick on a few bases of ‘adaptor sequence’; this sequence sticks on to complementary bits of DNA stuck to a surface, which holds the DNA in place while we sequence it:
- http://www.nature.com/nrg/series/nextgeneration/index.html
- Prior to mRNA-seq, microarrays were unchallenged as the experiment of choice for transcriptome analysisLimitations -Poly-A based capture!!
- With total RNA seq can discover coding and non-coding RNA species, incl some long/small RNA and pseudogenesNeed to remove ribosomal RNA since ribosomal RNA represents over 90% of the RNA within a given cell, studies have shown that its removal via probe hybridization increases the capacity to retrieve data from the remaining portion of the transcriptome.
- Extra enrichment step after library generation ..prior to seq results in substantatial reduction in cost..can analyze more samples
- Ok so now we understand how to find out what genes are expressed and when but what about if we want to know how and why those genes were turned on/offDNA & protein crosslinkrd using formaldehyde Bound chromatin is sheared by sonication or enzymatic process
- The purified DNA fragments identified by hybridization to a microarray, so the same technology as GEXBut in this case oligonucleotide probes from entire genome or selected regions e.g.: promoter regions, specific chromosomesNGS is not Limited by sequences on anarray
- So what does this type of data look like?