Epigenetics exam 3

X chromosome controlling element (Xce)

Choice of X to inactivate influenced

Environmental enrichment alters epigenome

In hippocampus - environmental enrichment rapidly increases acetylation of multiple lysine (K) residues at core histones H3 & H4 • In cerebral cortex - fewer lysine residues altered - involves both histone methylation & acetylation

TsiX

is an antisense RNA of Xist thought to regulate activity of Xist in mice at onset of X inactivation - active on active X and inactive on inactive X

Nucleoside analogues

- substitute for cytosine - incorporated into DNA - bind to DNA methyltransferases (DNMTs) Mode of Action: Nucleoside analogue DNMT inhibitors act as substitutes for cytosine • Converted into deoxynucleotide triphosphates and incorporated in place of cytosine into replicating DNA • DNA methyltransferases (DNMTs) become trapped on nucleoside analogues, resulting in formation of heritably demethylated DNA

Model for endocrine-disrupted-induced epigenetic trans-generational disease.

-Both male and female offspring develop disease (breast cancer, immune abnormalities, kidney & prostate disease) -However, trans-generational vinclozolin effects transmitted only through males. -Endocrine disrupted alters DNA methylation and reprogrames embryonic development. -Genome-wide study identified 16 promoters with altered DNA methylation in sperm of F3 male rats descended from F0 females exposed to vinclozolin

RNA-mediated Kit paramutation requires Dnmt2 methyltransferase

-Dnmt2 methylated tRNAs. -Homozygous Dnmt2 loss-of0function mutation prevents kit white tail phenotype. -tRNA methylation siginificantly reduced in sperm of Dnmt2-/- males.

Quantitative real-time PCR (qPCR)

-Enables both detection and quantitative of one or more specific sequences in DNA sample: absolute slumber of copies and relative amount normalized to endogenous control DNA. -Amount of PCR product estimated in each amplification cycle using fluorescent markers. -BY plotting fluorescence against cycle number, real time PCR instrument generates amplification plot that represents accumulation of product over duration of PCR reaction -Threshhold cycle (Ct) is cycle number at which fluorescent signal of reaction crosses threshold. -Ct used to calculate initialDNA copy number, because Ct value is inversely related to amount of starting template. -in Comparing two real-time PCR reactions, one with twice as much starting template as the other, the reaction with 2X starting amount will have. CT one cycle earlier.

Epigenotype & metastability

-Epigenetic states are metastable: transmitted through cell divisions, remain unchanged unless sufficiently disturbed, enviornmental factors such as toxin exposure & nutrition during critical developmental stages can alter epigenetic profiles. -Changed epigenetic states exhibit inertia: perpetuated in absence of conditions that established them. -Reversability of epigenetic states has important implications for disease treatment (epigenetic therapy) and health maintenance (nutrition)

RNAi therapy for Viral infections:

-Hepatitis B virus (HBV): siRNA drug now in Phase 2 trial for treatment of HBV patients. -Hepatits C virus (HCV): causes liver failure and liver cancer Adeno-associated viral vector RNAi therapy in Phase 1 trial of 12 HCV patients -Ebola: in animal models, siRNA drug rescues animals from certain death. In humans phase 1 trial put on hold by FDA in 2014 due to problematic immune responses in volunteers given highest dose.

Jirtle. Genistein study.

-Jirtle and other scientists manipulated maternal genistein( Phytoestrogens in soybeans). - Experimental treatment made genistein level comparable to high soy human diets (asian, vegetarian) -Genistein supplementation in mother: increased methylation levels in offspring, increased darkness of pigmentation, and reduced obesity &cancer risk. Implications: -In utero dietary genistein affects gene expression and alters susceptibility to obesity in adulthood in Avy mice by permanently altering the epigenome. -Epigenetic effects of genistein may play a role in lower incidence of cancer in asians compared to westerners, as well as increased cancer incidence in asians emigrating to the US.

Dimorphism

-Larvae fed on royal jelly develop into queens; standard diet produces sterile workers. -All three subfamilies of DNA methyltransferases (DNMT1, DNMT2 and DNMT3) present in honey bees.: RNAi silencing of Dnmt3-silenced and royal jelly-fed larvae, level of DNA methylation reduced and genes associated with growth unregulated (Juvenile hormone)

Lipid siRNA nanocarriers

-Most advanced in clinical development -IN Phase 1 trial, siRNA-containing lipid nanaparticles (LNPs): successfully knocked down target gene expression in livers of patient with trans theyretin amyloidosis -efficient release of siRNA depends on bio materials used in nanocarrier: 1mg.kg siRNA dose required to acheive 38% knockdown of transthyretin with 1st generation lnps, dose of only 0.3mg/kg achieved 85% knockdown with 2nd generation LNPs, Transthyretin level reduced by 80% in phase 2 trial. -Phase 3 patisiran trial completed SEptember 2017: Alnylam will now apply for FDA approval.

Nutritional effects on the Epigenetic prism

-Nutrigenomics-application of "omic" sciences to human nutrition, especially the relationship between nutrition and health. -Growing evidence that diet can result in striking, heritable modifications of the epigenotype.

The Fetal Origins of Adult disease hypothesis "BARKER hypothesis"

-Nutrition during fetal development and infancy affects susceptibility to chronic diseases in adulthood: stimulus or insult at critical, sensitive period of early life results in long-term changes in physiology or metabolism -Barker and Clark proposed that under-nutrition affects metabolism by changing concentration of fetal and placental hormones such as insulin and insulin-like growth factors -Recent findings suggest epigenetic basis for the fetal origins of adult disease affect

RNAi-mediated trans-induction of paramutation

-Paramutagenic alleles usually associated with repressive chromatin state low and expression. -Hypothesized that dsRNA is formed bytranscription from sense and antisense strands of tandemly- repeated DNA -Activated siRNA Pathway, resulting in transcriptional gene silencing of paramutable allele. -mop1 gene at maize b1 locus encodes RNA-dependent RNA polymerase essential for paramutation. -Loss of function mop1 mutations: prevent paramutation and deactivate mutator transposons.

Maternal Microchimerism

-Schlerderma: maternal microchimerism was more frequent in women with schleroderma (72%) than in healthy women (22%) -Pityriasis lichenoided (PL): uncommon inflammatory disease that causes scaly rash, maternal microchimerism found in 11 of 12 young males with zPL and 4 of 7 healthy controls, and abundance of maternal cells significantly higher in PL individuals -Myositis: degenerative muscle disease and in one study, maternal microchimerism present in muscles biopsies from all 10 patients with myositis compared to 2 of 10 patients with other muscles. -In laboratory mice, lupus experimentally induced by injecting parental cells into offspring.

Fang et al. Green Tea

-Studied the affects of major polyphenol in green tea, Epigallocatechin-3-Gallate (EGCG), on expression of four methylation-silenced genes in human colon, esophageal and prostrate cancer cell lines. -EGCG inhibited activity of DNMTs -Increasing concentrations of EGCG reduced and increased level of gene expression. EGCG also inhibits cancer growth.

Therapeautic RNAi strategies

-Synthetic siRNAs: reprogram RNAi machinery to silence target sequence. r -Synthetic miRNAs: reprogram RNAi machinery to restore or strengthen miRNA regulatory networks. -Analog inhibitors: antisense oligonucleotides (antagomirs) sequester deleterious miRNAs, upregulate tumor suppressor genes.

RNAi-based delivery: Viral Methods

-Viral delivery: long-term, stable repression of target gene expression., viral vector contains promoter and precursor siRNA or miRNA -Adenoviral &adendo-associated viral (AAV) vectors (RNA viruses): episomal expression -LentiViral vectors (RNA Viruses): reverse transcribe small RNA expressing gene into patients genome. -Utilizes short hair pin RNAs(shRNAs) or primary transcript RNAs(pri-mirnas): shrnas expressed as double stranded hairpin and cleaved by diver into siRNAs. pri-mirnas processed by Drosda and cleaved by dizer into miRNAs.

Practical implications of fetal microchimerism. 3. Donor sex and parity on graft-versus-host disease (GVHD)

-could help avoid donors who have microchimerism that might pollute donation of cells to people that don't match that cell-type.

Practical implications of fetal microchimerism: 2. Prenatal genetic testing and gender determination

-could help with avoiding amnio centesis. -but, these cells could be from previous pregnancies.

Detection of microchimerism

-difficult to detect foreign fetal cells because they are at a low frequency and genetically similar to mother especially if the baby is a female. -y chromosom facilitates detection of fetal cells in mother. -two methods used to detect y chromosome: FISH: fluorescence in situ hybridization; fluorescente-labeled probes Bins to Y chromosome DNA sequences. PCR: using primers that specifically amplify Y chromosome DNA sequences -Detection of fetal cells because of enrichment.:blood separated into plasma and nd cellular components. Specific cellular components retained for analysis. Most commonly detected cells: CD34+ and CD38+ among lymphoid and myeloid stem cells

Paramutation

-directed heritable change in one allele (paramutable allele) induced by second allele (paramutagenic allele) in heterozygote. -changed epigenetic state of paramutated allele stably inherited, even if paramutagenic allele not transmitted.

Feto-maternal trafficking

-fetal cells cross placent enter maternal circulation and infiltrate variety of tissues. -Fetal cells detected in maternal circulation as early as 4 weeks post conception. Post partem frequency of fetal micrchimeric cells in circulation rapidly decreases within 40 days.

Practical Implication of Fetal microchimerism: 1. Forensic Y-chromosome typing

-in sexual assault cases, male fetal cells could be mistaken for other male cells. And give false positive results

Chimera

-individual whose body consists of populations of cells derived from more than one individual.

RNAi therapy

-involves sequence-specific post transcriptional and transcriptional gene silencing. -small interfering rnas: specific targeting and suppression of disease-causing genes and foreign fenetic elements -MicroRNAs: gene network regulation disrupted in diseases such as cancer.

Male fetal cells in women without sons

57% in abortion group tested positive for microchimerism. 22% in miscarriage groups tested positive.

Histone deacetylation and cancer

Acetylation (which is good) of lysine amino acid on histone tails associated with open chromatin which allows access to gene promoters - histone acetylation mediated by histone acetyl transferases (HATs) • In cancer, epigenetic silencing of tumor suppressor genes is associated with histone deacetylation - histone deacetylation mediated by histone deacetylases (HDACs)

Polymerase Chain Reaction (PCR)

Allows amplification of minus quantities of DNA for sequencing, microsatellite DNA profiling, SNP Analysis, bisulfite sequencing and many other applications Requirements: -DNA Template: a single copy -Primer pair: single-stranded DNA sequence (18-30 bp) that binds to sequences on either sides of target sequence, on opposite strands. -Four deoxyribonucleoside triphosphates (dNTPs) A, T, G, C/ -Thermostable DNA polymerase such as Tao -Successive rounds of denaturation, primer annealing, and extension. -in Exponential amplification phase, double amount of PCR product with each cycle.

Chromatin immunoprecipitation (ChIP)

Allows in vivo identification of - transcription factor (TF) binding sites - histone modifications - DNA methylation - any target on chromatin against which antibody can be raised • Involves chemical cross-linking of DNA-protein interactions with formaldehyde to "fix" TFs to cognate binding sites in genome • Crosslinked chromatin then fragmented and specific antibodies used to immunoprecipitate TFs • ChIP-chip: amplification or pooling of ChIP samples, fluorescent labeling of ChIP-enriched DNA and hybridization to genomic microarrays • ChIP-seq: involves direct sequencing of ChIP-enriched DNA after amplification and size selection of DNA fragments

Genetic sex determination (GSD)

Autosomal gene • M gene in some housefly populations Chromosomal • Haplodiploidy (Hymenoptera) - male from haploid unfertilized egg - female from diploid fertilized egg • Female heterogamety - ZW/ZZ (birds & butterflies) • Male heterogamety - XX/XO (arachnids, some insects) • ratio of Xs to autosome set - XX/XY (most insects, mammals) • ratio of Xs to autosome set (Drosophila) • Y-linked male determining Sry gene (mammals)

The X-inactivation process

Before inactivation, Xist RNA expressed in unstable form and may be blocked from coating chromosome • Blocking factor lost from either maternal or paternal allele and transcription of Xist RNA upregulated on that X chromosome • Xist RNA coats Xic, followed by histone methylation • Spreading of Xist RNA and inactivation across entire X chromosome through heterochromatin protein 1-mediated heterochromatin formation, histone deacetylation and DNA methylation • Spreading of inactivation may involve LINES acting as "way stations" or "booster elements" • LINEs make up 29% of human X chromosome sequence and are especially abundant in regions flanking Xist

Paths to microchimerism

Cells transferred from one individual to another (CEll trafficking) through. -Transplantation -transfusion: anucleate RBCs short lived , nucleated WBC can persist longer in recipient. -pregnancy: maternal cells o fetus and Vice versa.

Support for epigenetic model: II. MZ twins diverge in epigenetic profiles with age

Comparison of DNA methylation and histone acetylation patterns in 40 sets of MZ twins ranging in age from 3 to 74 found - young MZ twins epigenetically very similar - older MZ twins often highly discordant in epigenetic profiles • Supports view that small epigenetic differences become magnified as genetically identical individuals age

Microchimerism

Condition in which small population of cells within one individual originated from another genetically distinct individual.

Intergenomic conflict

Conflict between genetic elements in different individuals. -paternally expressed imprinted genes in embryo versus genes in mother. -sexually antagonistic genes, genes that benefit one sex but harm the other.

Intragenomic conflict

Conflict between genetic elements within the individual -transposable elements versus the rest of the genome. -paternally versus maternally inherited imprinted genes with embryo -meiotic drive alleles versus wild-type alleles. -nuclear genes versus cytoplasmic genes genes in cellular endosymbiotants and organelles.

DNA microarrays

Consist of large number of single -stranded DNA segments (usually exon sequences) permanently affixed to glass slide in precise row by column array • Typically used to measure changes in gene expression levels in response environmental perturbation, drug treatment, disease state, etc • mRNAs isolated from control and treatment tissues and reverse transcribed into cDNA • Fluorescent tags attached to cDNA probes - e.g., green tag for control tissues versus red tag for HDAC inhibitor -treated tissues • cDNA probes bind to complementary DNA sequences in microarray - intensity of fluorescent signal varies with degree of sequence homology • Genes transcribed in control tissue fluoresce green while those transcribed in HDAC - treated tissue fluoresce red

Real-time PCR fluorescence detection systems

DNA binding agents such as SYBER Green 1. When SYBER Green dye is added to sample, it immediately binds to all double-stranded DNA. 2. During PCR, DNA polymerase amplifies target sequence. Which creates double-stranded PCR product, or amplificou. 3. SYBER Green dye then binds to each new copy. 4. As PCR progresses, more application is created. 5. Since SYBER Green dye binds to all double-stranded DNA, results in an increase in fluorescence intensity proportional to amount of PCR produced. Advantages: -High sensitivity and inexpensive Disadvantages: -Binds all double-stranded DNA so can be imprecise if level of non-specific amplification is high

Antidepressants alter behavior and epigenome

Defeated mice exhibited elevated H3-K27 dimethylation in P3 and P4 promoters of brain-derived neurotrophic factor (Bdnf) gene imipramine increases H3 acetylation

Epigenome-Wide Association Studies (EWAS)

EWAS: Systematic assessments of a specific epigenetic mark, usually DNA methylation, across the genome in groups of individuals that are different for a given environmental exposure, trait or disease with the goal of identifying differences associated with that exposure or phenotype. EWAS studies indicate that variation in epigenetic states contribute significantly disease phenotypes and phenotypic variation • Variation in the methylation state of the IGF-1 promoter accounts for 10% of variation in human height

The epigenetic model of MZ twin discordance

Early chance differences in epigenetic states between twins (e.g., level of cytosine methylation) become magnified through development and aging MZ twin discordance for complex, chronic non-Mendelian disorders such as schizophrenia could arise from chain of unfavorable epigenetic events in affected twin

RT-qPCR allows quantitation of varying levels of gene expression

Epigenetic mechanisms such as DNA methylation, histone modifications and RNAi act by modulating levels of gene expression • RT-qPCR most sensitive technique currently available for mRNA detection and quantitation • Most rigorous method (Comparative CT) employs endogenous control - internal control gene, such as housekeeping gene b-actin - quantification of mRNA target can be normalized for differences in amount of total RNA - DNA concentration represented as fold difference between target gene and endogenous control - Fold difference = 2 change of CT

Genome-wide association studies (GWAS)

Examination of DNA sequence variants to determine whether variants are associated with a phenotypic or disease state • Typically involves assaying >100,000 SNPs distributed across entire genome • Normally compare two groups - Control - Individuals with disease or phenotype • For each SNP, compare allele frequency in control and disease using odds ratio test • Create Manhattan plot showing negative logarithm of P-value as a function of genomic location

Factors associated with increased fetomaternal cell trafficking

Fetal and placental abnormalities : fetal aneuploidy an defective placentas and possibly causes maternal preeclampsia which increases fetal cells in mom by 4.5X. ABortion!!!

Good Microchimerism

Fetal pluripotent hematopoietic stem cells engraftment in mother's bone marrow and provide source of rejuvenating fetal stem cells that participate in maternal tissue repair. Evidence: some women had fetal cells in mothers bone marrow and some disease remission in associated with high concentration of fetal cells in affected tissue ex. HEP C. Alzheimer's disease associated with lower concentration of male cells in a women's brain.

Vinclozolin

Fungicide used by wine makers -Endocrine disruptor: metabolized into compounds with high binding affinity for androgen receptors. -this drug seemed to effect the male offspring of pregnant mice. It decreases number of sperm and reduced motility. It also increases apoptosis in rates.

Epigenome editing

Genetic engineering in which epigenome is modified at specific sites using engineered molecules targeted to those sites • Allows determination of causal role of an epigenetic modification at site in question - studying enhancer function and activity - determining function of specific methylation sites - determining role of histone modifications

E.W. Tobi et al. Dutch Hunger Winter revisited

Genome-wide analysis of differentially-methylated regions (DMRs) after periconceptual exposure to famine -greatest within-pain methylation differences occurred when conception coincided with lowest food availability. DMRs mapped to genes: -expressed during early development. -related to growth and metabolism.

histone deacetylase (HDAC) inhibitors

HDAC inhibitors include - short-chain fatty acids, hydroxamic acids, cyclic tetrapeptides and benzamides • HDAC inhibitors - prevent histone deacetylation and restore transcriptional activity of critical tumor suppressor genes such as p21 - also known to have anti-angiogenic effects and promote apoptosis • Two HDAC inhibitors recently approved by FDA for treatment of cutaneous T cell lymphoma

Epigenetic programming by maternal behavior (Weaver et al. 2004)

High LG-ABN mothers produce less fearful and stress reactive offspring • Maternal behavior affects DNA methylation and histone acetylation of glucocorticoid receptor gene in offspring • Cross fostering shows offspring epigenotype determined by maternal behavior not offspring genotype

Eutherian X-inactivation

Imprinted X-inactivation of paternal X established in all cells during preimplantation stage (blastocyst) • Paternal X-inactivation maintained in placenta but erased in embryo proper • Random X-inactivation then established in embryonic cells and maintained throughout adult life except in germline where X chromosomes are reactivated

Support for epigenetic model: I. Discordance for BWS in MZ twins

In 10 MZ twin pairs investigated, all affected twins exhibited loss of methylation at KCNQ1 locus • Failure of maintenance methylation during single cell cycle at (or just prior to) twinning event could result in complete discordance for imprinting defect

Reverse-transcription PCR (RT-PCR)

In RT-PCR, RNA strand is first reverse transcribed into its DNA complement (Complementary DNA or cDNA) using reverse transcriptase enzyme RT step carried out using -olido-de primer (for poly-A-3' tail) -sequence-specific primer -Random primers Resulting cDNA is amplified using traditional or realtime PCR with locus-specific primers Kits available for two step or one step or one step RT-PCR

Dideoxy (Sanger) sequencing

Key fact: incorporation of dideoxyribonucleoside triphosphates (ddNTPs) terminates chain elongation in DNA sequencing reaction • DNA sequencing reaction includes − many dNTPs − few ddNTPs − template DNA − sequencing primer − DNA polymerase Each ddNTP type (A, C, T, G) labeled with different fluorescent tag • Strand elongation occurs until incorporation of a ddNTP corresponding to the base in template strand • ddNTP incorporation is random, so reaction produces full set of sequences of length 2bp to 800 bp • Fragments separated according to length via electrophoresis in capillary tube • Dideoxymonomers on successive fragments reveal sequence of bases in template DNA

Air pollution disrupts global methylation

Male mice breathing polluted air for many weeks. Sperm DNA was hyper methylated in mice breathing ambient relative to HEPA-filtered air.

Sex determination in mammals

Mammals have XX/XY chromosomal system of sex determination • In humans - 155 Mb X contains 1,606 genes - 59 Mb Y contains only 397 genes including male-determining Sry gene • For autosomes, diploidy results in double-dose expression of most genes • For sex chromosomes, males are hemizygous for genes absent from Y • Mechanism therefore required to equalize level of X chromosome gene expression in males and females

MeDIP method for DNA methylation assessment

Methylated DNA immunoprecipitation • Utilizes chromatin immunoprecipitation (ChIP) followed by microaarray hybridization (ChIPchip) or next-generation sequencing (ChIP-seq) • Involves affinity enrichment of methylated regions using - antibodies specific for 5meC - methyl-binding proteins with affinity for methylated genomic DNA • In ChIP-chip method, input DNA and enriched DNA are labeled with different fluorescent dyes • Allows for rapid genome-wide assessment of methylation but not at the level of individual CpG nucleotides

Innocent bystander hypothesis

Microchimerism is incidental byproduct. Of pregnancy and fetal cells have no impact on maternal health. Evidence: Absence of fetal cell microchimerism in several autoimmune diseases that occurs primarily in women.

X inactivation and mosaicism

Mosaicism most apparent when one X carries distinctive phenotypic marker - Blaschko' s lines • specific cause unknown but linked to X inactivation-based mosaicism - anhidrotic ectodermal dysplasia • deficiency in sweat glands in which defective skin occurs in patches

Epigenetic therapy reverses effect of negligent mothers

Mothering style effects reversed in adult offspring by treatment with HDAC inhibitor trichostatin A (TSA) or methyl-donor methionine • TSA and methionine treatment reversed the effect of maternal care on open-field behavior - TSA decreased anxiety in low LG-ABN offspring - Methionine increased anxiety in high LG-ABN offspring

RNAi-based delivery: non viral methods

Nonselective delivery: -Chemically unmodified siRNAs & miRNAs: unstable and do not easily penetrate cell membranes. -Chemically modified siRNAs: increases stability, increases transport across cell membrane Cell-specific delivery: -Target small RNAs to cell-surface receptors: achieved with aptamers, antibody fragments or nanoparticle-borne ligands, lowers dosage requirements and reduces off-target effects.

Marsupials

Placenta poorly developed and gestation very short with fetus completing development while nursing, usually in pouch • In marsupials, paternal X is imprinted (silenced) in all tissues

Clinical trial phases

Pre-clinical studies: in vitro and/or animal studies • Phase 0: exploratory, first in-human trials • Phase I: small groups (20-50) healthy volunteers or terminal cancer or HIV patients; dose-ranging (escalation) testing • Phase II: intermediate-sized groups (20-300) of volunteers and patients; dosing, efficacy and toxicity testing • Phase III: randomized, controlled multicenter trial on large patient groups (300 - 3,000) • Phase IV: post-marketing, surveillance trial to test for rare or long-term adverse effects

GWAS - preliminary conclusions

Quantitative traits influenced by many loci with small effects • Only small proportion of genetic variation can be explained, even for traits with high heritability • Heritability of human height is ~ 0.8 (very high) - 40 loci linked to height variation by GWAS account for only 20% of heritability - Locus with greatest effect is HMGA2 - individuals with two copies of HMGA2 "tall allele" are, on average, 1 cm taller than those with two copies of short allele

Skewed X inactivation

Random X inactivation should produce on average 50:50 ratio of active maternal to active paternal X chromosomes Skewed X inactivation can result from - chance - genetic factors involved in X inactivation process, e.g., strong versus weak X chromosome controlling element (XCE) alleles - selection process involving cell competition between two cell lines differing in their proliferative capacity

Afeiche M.C. Processed meat reduces fertility

Red meat=significantly higher percentage of morphologically abnormal sperm. White meat fish= higher percentage of normal sperm. Dark meat fish= increased total sperm count by 34%

Hypermethylation & cancer

Selective cytosine hypermethylation within CpG islands of 5´ regulatory regions of genes common in cancer • Likely tumor suppressor genes become aberrantly hypermethylated and silenced in nearly every tumor type - genes involved in cell-cycle regulation, DNA repair, chromatin remodelling, cell signalling and apoptosis • Provides tumor cells with growth advantage and allows them to metastasize

PacBio - transformative NGS for whole genome assembly and epigenetics?

Single Molecule Sequencing (SMRT) - No PCR • Enormous read length - up to 60 kb, with a mean read length of 18 kb • Can detect DNA base modifications - based on polymerization reaction kinetics

Epigenotype

Specific set of DNA methylation marks and histone modifications that determines a functional properties of a cell.

Jensen, S.A.

Spherical nucleic acid nanoparticles conjugates as RNAi-based therapy for glioblastoma. -these particles consist of siRNAs surrounding gold core used to treat human glioblastome brain tumors in mouse model. -these spherical nucleic acid nanoparticles (SNAs): crossed blood-brain barrier, entered tumor, targeted known oncogene, induced apoptosis of gloom a cells. -resulted in impaired tumor growth and increased survival.

RNA-Seq

Steps in RNA sequencing 1. Long RNAs converted to cDNA via RNA or DNA fragmentation 2. Sequencing adapters added to each cDNA fragment 3. Short sequence obtained through NGS technology 4. Sequence reads aligned to reference genome or transcriptome Unlike traditional transcriptome analyses, can detect all categories of RNA • mRNA • miRNAs • piRNAs • long ncRNAs In contrast to microarray methods, cDNA or EST libraries are directly sequenced

Environmental sex determination (ESD)

Temperature - warm temperatures produce males / cool temperatures produce females (alligators & some lizards) - vice versa (turtles) • Social context and body size - female to male (bluehead wrasse) - male to female (some damselfish) • Population density (parasitic nematodes) Aromatase (cyp19a1) in non-mammalian vertebrates - steroid that converts androgens to estrogens - essential for ovarian development • In European sea bass, both genetics and temperature contribute to sex determination - promoter cyp19a1 methylation higher in males than in females • High temperature - increases level of promoter cyp19a1 methylation in gonads • Above threshold level of methylation - genetic females converted into phenotypic males

Monotremes

The only egg-laying mammals • Possess mammary glands but lack nipples - milk secreted from glands on mother's belly - baby sucks milk from mother's skin • Extended period of nursing - platypus females produce 1-3 offspring per year - echidna young suckle to age 6 months • Platypus has set of 10 sex chromosomes - XXXXXXXXXX are females - XYXYXYXYXY are males • Genes involved in fetal and placental growth in eutherians and marsupials (Igf2 and Igf2r) NOT imprinted in platypus

Rijnink

Tissue microchimerism during pregnancy. -autopsy samples from women who died during or just after pregnancy. -male fetal cells present in: lungs, spleen, liver, kidneys, heart, and brain.

Bisulfite sequencing for determining DNA methylation

Treatment of DNA with bisulfite - converts unmethylated cytosines to thymine (by uracil) - methylated cytosines unchanged • PCR then performed on bisulfitetreated DNA - Non-methylation specific PCR amplifies both methylated and unmethylated sequences - Methylation-specific PCR uses primers that only anneal to unaltered sequences, thus amplifying only sequences that are methylated at 5' ends on opposite DNA strands • Gold standard for DNA methylation analysis involves non-methylation specific PCR followed by sequencing of both untreated and bisulfitetreated DNA • Comparison of two sequences allows determination of methylation status for every cytosine within amplicon

Agouti Avy mice and IAP retrotransposon

Viable yellow agouti (Avy) mouse -Avy mutataion results from insertion of LTR retrotransposon IAP upstream of Agouti gene. -agouti gene normally expressed transiently to produce brown (agouti) coat color -when active, IAP promotor enhances Avy allele expression, resulting in yellow coat, obesity, type 2 diabetes, and tumor susceptibility. -Offspring coat color directly correlated with % CpG sites methylated in IAP promoter. -Low percent methylated--> promoter active--> yellow coat -High percent methylated--> promoter inactive--> brown (pseudoagouti) coat -offspring given supplements which are able to reverse the effects of the agouti overexpression.

Klinefelter syndrome and genes that escape X inactivation

XXY males (and even XXXY males) viable because counting mechanism of X inactivation "inactivates" all but one X in cell • X-linked genes escaping inactivation may explain partial feminization (enlarged breasts, small testicles, lack of facial and body hair) of Klinefelter males • Individual variation in X inactivation may explain high variation in level of feminization

Illumina

currently most popular NGS platform − cheap, accurate but short reads and long run times − generates paired-end reads: major advantage for de novo genome sequencing

X-chromosome inactivation

known only in female marsupial and eutherian mammals • in marsupials, paternal X is imprinted (silenced) in all tissues • in eutherian mammals - paternal X is inactivated in placenta - random X inactivation in fetal/adult tissues

Bad Microchimerism:

maternal immune system response to Foreign fetal cells explains higher frequency of certain autoimmune disease in women than in men. SOme apparent autoimmune disease may actually be alloimmune diseases. Evidence: More fetal cells more women who have autoimmune diseases.

Non-nucleoside analogues

not incorporated into DNA - bind directly to catalytic region of DNMT or target sequence Potentially more effective than nucleoside analogues - bind directly to DNMTs to induce hypomethylation

Tsankova

poor mice experiment

Ion Torrent

simpler technology, long reads and rapid run times − measures changes in pH

Dosage compensation

up- or down-regulation of sex-chromosome gene expression in one sex • believed to occur in all egg laying animals with chromosomal sex determination • homogametic sex downregulates gene expression (C. elegans) • heterogametic sex upregulates gene expression (Drosophila) • incomplete in birds - partial upregulation in heterogametic WZ females

Eutherian mammals

• Commonly called placental mammals because placentas more complex than those of marsupials Eutherians complete fetal development within uterus joined to mother by placenta - transports nutrients, O2 and wastes between fetus and mother • Paternal X inactivated in placenta • Random X inactivation in fetal/adult tissues

Roche 454 GS FLX

− one of first next generation machines - used for pioneering studies such as Neanderthal genome sequencing project Sample input and fragmentation - fragment library prepared by randomly shearing genomic DNA into small fragments (also can process PCR products, BACs, etc.) 2. Library preparation - adaptors containing universal priming sites ligated to target ends, allowing complex genomes to be amplified with common PCR primers 3. One fragment = one bead - each single-stranded DNA fragment immobilized on a single DNA capture bead and emulsified in water-oil mixture 4. Emulsion PCR (emPCR) - each fragment amplified within own microreactor and amplification of entire fragment library performed in parallel 5. One bead = one read - amplified fragments loaded onto PicoTiterPlate for sequencing with only one bead per well - fluidics subsystem flows individual nucleotides in fixed order across hundreds of thousands of wells each containing one bead - addition of one or more nucleotides complementary to template results in chemiluminescent signal recorded by CCD camera