BIOLOGY 305 1st Edition Lecture 24 Outline of Last Lecture I. Introduction to Eukaryotic RegulationII. Transcriptional Regulation of RNA Pol II GenesIII. The Yeast Gal SystemIV. Regulatory Transcription FactorsV. Transcription Factors as ActivatorsVI. Euchromatin, Heterochromatin, and InsulatorsOutline of Current Lecture I. Introduction to EpigeneticsII. 5 Classic Epigenetic PhenomenaA. Position Effect Variegation (PEV)B. Polycomb and ThrithoraxC. X-chromosome InactivationD. ImprintingE. VernalizationF. EpiallelesIII. Stem CellsIV. Biotechnologies and EthicsV. Vocabulary and Sample QuestionsCurrent LectureI. Introduction to EpigeneticsWhat is epigenetics?1) Heritable genetic information that excludes the DNA sequence2) Contained in chromatin modificationsEpigenetics is the study of changes in gene expression and inheritance patterns caused by mechanisms other than changes in underlying DNA sequence. Epigenetic marks, these inheritedalterations, are changes in DNA methylation patterns and in chromatin structureTotal Information within Cells is comprised of:DNA sequence > methylation > Histone modifications > Gene regulatory networks > physiological statesThese are factors that can affect genetic information and are shown in order from most stable to least stable (transient)One genome (DNA as stored information)  chromatin (organized information)Which = many epigenomes and thus, many possible variationsII. 6 Classic Epigenetic Phenomena1) Position Effect Variegation (PEV)2) Polycomb and Trithorax3) X-chromosome Inactivation4) Imprinting5) Vernalization6) EpiallelesDuring mitosis, cells must maintain a memory of the structure of their chromatin domains in order to pass it on to daughter cells.How are DNA methylation patterns inherited?Maintenance Methylation Mechanism:1) Pre-replication, both DNA strands are methylated2) Post-replication, hemi-methylated DNA is formed3) Methyltransferase recognizes hemi-methylated DNA and methylates the other strandHow are Histone Modifications inherited?Not well known but likely to be similar to maintenance DNA methylation:If Histone H4 is acetylated, so must it’s daughter cellsDuring DNA methylation replication, modified histones may be randomly distributed with new unmodified ones in the daughter strands and direct their modificationA. Position Effect Variation (PEV)PEV is an obscure event in Drosophila. There are strains of flies that have patches of red and white patches. A gene called ‘white’ is mutated but a fragment of the chromosome is inverted. A portion is white the rest of the chromosome remains as “heterochromatin”Heterochromatin tends to spread; turning off the white patchNote: No change in DNA sequence occurs, chromatin modification only = epigeneticMore Red phenotype = mutations disrupt heterochromatin, thus are called suppressorsMore White phenotype = mutations disrupt euchromatin, thus are called enhancers B. Polycomb and TrithoraxPolycomb: Proteins maintain repression, remodeling chromatin = epigenetic silencingTrithorax: Maintain activation = maintains genetic expressionC. X-chromosome InactivationWhy are differences in X chromosome number tolerated between males and females?Recall: Different species have different mechanisms that ‘equalize’ gene expression of the X chromosome between gendersNon-tolerated differences can be observed by looking at chromosomal diseases in aneuploidyEx: XO females with Turner vs. XXY males with KlinefelterRecall Dosage Compensation:In dosage compensation, not just a cluster of genes but an entire chromosome is subjected to coordinate regulation of genesAll mechanisms involve heritable alterations in chromatin structureHuman: One of the female X chromosomes is turned off while the other is onFlies: Male X chromosome is up-regulatedWorm : Female XX chromosomes are suppressed by 50%How does this happen epigenetically?Embryo forms  x-inactivation occurs randomly  new cell progeny retains x-activation  this state is maintained epigeneticallyBecause x-inactivation is random: Females mammals are ‘mosaics’Cats: some have the spotted calico phenotype because the color gene lies on XWith some X-linked diseases, heterozygous phenotypes of females can varyThe difference in histone modification and chromatin change causes phenotype change, not related to DNA sequenceActivation of Dosage Compensation is controlled by counting: the n-1 ruleall Xs in excess of one in somatic cells will be inactivated in females as a Barr bodyXY: male with XaXX: female with Xi+XaXXY: male with Xi+XaXXX: female with Xi+Xi+Xa D. ImprintingGene expression according to the parent of originA normal zygote will not form if a oocyte fertilized with sperm doesn’t contain a maternal and paternal pronucleusBut contribution of the male and female gamete is not equal:The principle is that the paternal (or maternal) allele is masked and so the phenotype is not consistent with the genotype, yet inheritance is kept the sameImprinting at a Molecular Level:Control elements – differentially methylated on the paternal and maternal allelesImprints are not mutations; they are chromatin marks on those genesA specific mechanism allows occurs in the germ-line and maintained somatically Ex: Igf2 ImprintingIn the maternally inherited allele, DNA methylation occurs before Exon 3, masking expressionIn the paternal allele, there is no DNA methylation and transcription can proceedEx 2: Locus lgf2/H19 ImprintingIn maternal alleles, insulator elements is activeAs a result, an enhancer cannot help in the transcription of Igf2instead, H19 gene is transcribed and expressedIn paternal alleles, methylation occurs in the Insulator element and deactivates itThe H19 allele is also methylated and inactivatedThe enhancer element can contribute to the transcription of Igf2Imprinting at a cellular level:Imprints are erased and re-established in the germlineThe zygote is imprinted accordingly and leads to all the somatic celsin the somatic cells:Imprints are erased and then new maternal imprints are made in thefemale germline while new paternal imprints are made in the male germline (methylations are shown in red) Imprinting at a Pedigree level:A paternally imprinted disease gene is shown. The allele inherited from the father is NOT expressed yet the alleles are all inherited normally. Don’t expect the phenotype to be consistent with it’s genotype:Inheriting a disease-causing mutation form the father will appear to