EHS 660 Mid-Term

Characteristics of metastable epialleles

-- Epigenetic marks are stochastic -- Potential for transgenerational inheritance -- Targets for environmentally induced epigenetic modifications.

Why is this layer of epigenetic modification important (and essential for humans) (chromatin proteins)?

1) Humans are multicellular 2) structure = function (chromatin folding and remodeling) 3) when dysregulated leads to cancer and other disorders.

Major players in the epigenome project

1) International Human Epigenome Project 2) National Institutes of Health Epigenomic Roadmap.

Epigenetic mechanisms aiding in X-inactivation

1) Long non-coding RNA (Xist) 2) H3K27 methylation 3) Histone deacetylation 4) macroH2A histone variant 5) DNA methylation

What are Polycomb Group Proteins (PcG)?

1) Maintain silenced rate (off). 2) Two calsses PRC1 (vertebrates) and PRC2 (plants and animals). 3) Silent chromatin state inherited over many cell divisions. 4) Stem cell plasticity. 5) Dysregulation leads to cancer. (chromatin protein).

When was the field of genetics developed?

1871

CpG dinucleotides

70% of CpG sites methylated concentrated on repetitive sequences CpG dinucleotide is least frequent in human genome because of tendency for methylated cytosines to deaminate to thymines

Who proposed the term epigenetics and when?

C. Waddington in 1942. (trajectories and cell fates)

Major laboratory technique to investigate histone modifications.

Chromatin Immunoprecipitation (ChIP) 1) Not efficient for use in population studies bc proteins degrade very quickly. 2) Cross-link cells or embryos with formaldehyde to induce prtein DNA crosslinks. 3) Isolate chromatin and shear DNA along with bound proteins into small fragments. 4) Bind antibiotics specific to binding proteins of interest, precipitation, reverse cross-linking to release DNA and digest the proteins. 5) PCR to amplify specific DNA sequencies to see if they were precipitated by antibody of interest (gene specific) or do do tiling array or deep sequencing (epigenome-wide study).

What does DOHaD stand for?

Developmental Origins of Health and DIsease.

Histone Lysine Acetylation

Histones are mostly acetylated at lysine residues located in the amino termini of H3 and H4, with the exception of H3K56 localized in the globular domain.

What does RISC stand for?

RNA Induced Silencing Complex

mapping DNA methylation

restriction enzymes recognizing CpG containing sequences bisulfite modification of ssDNA PCR microarray

highly methylated sequences

satellite DNA's, repetitive elements, nonrepetitive intergenic and exons

MBD family proteins

signal to alter chromatin structure when DNA methylation detected largely non-overlapping in genome MBD binding shows DNA seq. specificity protect against spontaneous deamination mutations (unbound 5mc is prone to spontaneous mutation through deamination)

What is the law protecting citizens from discrimination based on genetic information

the Genetic Information Nondiscrimination Act (GINA).

What are transposons?

transposable elements or jumping genes that are mobile DNA sequences that can migrate to different regions of the genome. DNA methylation thought to suppress transposition. It is even suggested that DNA methylation evolved precisely for this purpose.

Natural proof of imprinting

uniparental disomy: inheritance of two copies of a chromosome of chromosomal region from one parent. Leads to negative phenotypes when occurs at imprinted loci.

Viable Yellow Agouti mouse model

1) in agouti mice, a range of coat colors is dependent on methylation status (mottled mice have agouti gene turned off -> are not obese later in life) 2) Epigenetic state determines the dominance of metastable epialleles. 3) Variable methylation, variable histone acetylation, and variable histone methylation (all work together in concert).

What is environmental mismatch?

A mother's diet, hormonal milieu, environmental exposures, etc, provide information about the environment the offspring will be born into but the conditions may change greatly between conception and adulthood.

What is a ncRNA?

A non-coding RNA is an RNA molecule that is not translated into a protein. Until recently, focus was on protein coding RNAs. Initial draft of the GFP omitted ncRNAs. 1) strongly conserved = important function. 2) Plays central role in regulation of gene expression and genomic stability. 3) Defense against transposons and RNA viruses. 4) X-inactivation. 5) Acts with everything else in concert to shut things down.

Parent of origin vs. random mono-allelic expression

In parent of origin, the autosomes appeared to be dependent upon the parent of origin of a given chromosome. random mono-allelic expression is seen in things like X-inactivation in which the silencing of one gene is selected at random.

CpG Island and Shores

Island: a region around the promoter with more than the expected number of CpG's Shore: 1-2 kb upstream of the island -- may be responsible for epigenetic derived tissue specific gene expression. -- methylation goes awry in cancer.

Ubiquitylation

Large peptide modification - increases histone size by 2-3rds. Mono-ub. Activating and repressive. (depending on which histones are acted upon).

Methyl binding proteins (genetic and epigenetic reactions)

Methyl binding protein binds to methylated CpG sites, which prevents the promoter from turning on. This is associated with gene repression.

chromodomains

bind methylated lysine residues

Thrifty Hypothesis

malnourished individuals are programmed during their plastic states to expect energy-deficient environment and, in response, set their biochemical parameters to conserve energy and store fat.

When was the Human Genome Project?

1990's-2003

When was the Human Epigenome Project initiated?

2006-2008

Example of gene-specific tests

MS PCR, Bisulfite sequencing (cloning, pyrosequencing, sequenom)

TIP60 complex

incorporation of variant histones depends on the ability of some chromati remodeling complexes to loosen the structure of nucleosimes and promote histone exchange functions in replacing H2A by H2A.Z

bromodomains

interact with acetylated lysine residues

What is MeCP2

is a polycomb protein that binds to methylated DNA and recruits chromatin-remodeling complexes that contain histone deacetylases. This leads to chromatin condensation owing to histone deacetylation.

What is the Conflict Hypothesis

1) "not as beneficial adaptation of species" but as a reproductive battles between the sexes. 2_ difference in maternal and paternal investiment. Men want to promote prenatal growth while women want to suppress prenatal growth. 3) imprinting is not a beneficial adaptation, but a response to Conflict Hypothesis. 4) Evidence used to be thought that imprinting evolved with placentas but its been found that it evolved with marcupials and can be lose later in the evolutional tree too.

What is Rett Syndrome?

1) An x-linked dominant postnatal neurodevelopment disorder 2) mutation in RTT gene on X-chromosome that encodes meCP2 (PcG protein) 3) seen in females, males neonatal lethality (xxy males survive). 4) associated with unusual hand movements, teeth grinding, posture, clinically linked with small hands and feet. Neurodevelopment disoder.

What are the challenges in epigenetic epidemiology?

1) Association vs. causality (reverse causation) 2) heterogeneity rampant in samples and cohort. 3) critical windows of vulnerability 4) DNA methylation 'easy' to study - not so for histone modifications 5) validation of epigenome-wide methodologies 6) case/control cannot infer causality

Enzymes responsible for methylation (genetic and epigenetic interactions)

1) Catalyzed by cytosine methyltransferases using SAM as methyl donor 2) Dnmt 3 De novo methylation (from unmethylated to both strands being methylated) 3) Dnmt 1 Maintenance methylation (from hemi-methylated after replication back to both strands methylated) 4) Modification of the major groove (steric hindrance of transcription factors) 5) In order for a cytosine to be methylated it must sit next to a guanine (CpG)

Two Classes of PcG

1) PRC2 aids in the co-recruitment of repressive histone modifications (EZ dependent histone methylation H3K9 and or K27) 2) PRC1 recognizes 1 and binding leads to maintenance of PcG-dependent repression of reporter genes. - Repression is enhanced by the presence of multiple (Polycomb Response Elements) PRE copies.

Two major classes of transposons.

1) Retrotransposons (cDNA copies of RNA transripts "copy and paste") 2) DNA Transposons ("cut and paste")

Opprotunities in epigenetic epidemiology

1) biomarker discovery 2) DNA methylation is stable and easily measured quantitatively 3) Technology is rapidly evolving 4) Reference epigenomes are in the works 5) Emerging view: two-way street (epid informs invitro/animal research and vice versa.

The Human Genome Project Goals

1) identify the approximately 20,000 genes in the human DNA. 2) determine the sequences of 3 billion chemical base pairs that make up human DNA. 3) Store this information in databases 4) Improve tools for data analysis 5) Transfer related technologies to the private sector 6) Address the ethical, legal, and social issues that may arise from the project

Why are metastable epialleles important?

1) potential explanation for phenotypic plasticity. 2) related to health outcomes (agouti mouse -> obesity) 3) Evident that: parental nutritional supplementation as a preventative intervention approach to counteract environmental influences on the fetal epigenome.

What are a few early "exposures" associated with later in life disease?

1) prenatal protein restricted data, postnatal high protein = twice as high risk in obesity and increased fasting insulin (in mice) 2) o Highest risk of heart disease is in those individuals born and low birth weight (thrifty phenotype) but then experience ample nutrition (in humans)

Name 1-2 ethical considerations epigenetics pose?

1) privacy and confidentiality issues; being able to tell paternity from methylation patterns. 2) environmental justice 3) inter-generational effects 4) personal responsibility (your actions affect the epigenetics of your children and grandchildren).

What are the types of transposons?

1) retrotransposons (cDNA copies of RNA transcripts) "copy and paste" viral and non-viral. 2) DNA transposons ("cut and paste")

Is Risk Assessment ready to include epigenetic data?

1) the growing feeling amoung scientists is that yes, risk assessment should include epigenetic data even though it's a new field. 2) What model systems should be employed? -- viable agouti mouse (in vivo) (+) closer to humans than cells (-) not humans and expensive. -- cell based systems (in vitro) (+) less ethnical issues than killing mice, cheaper (-) far from humans. what endpoints might be evaluated? -- no agreement on this so far. for risk assessment, end points should be 1) apical (epigenetic changes are not apical) 2) adverse effect (changes are not always adverse). 3) Non-reversible (epigenetic changes are reversible). 4) caused by the exposure 5) biologically plausible in humans. when is it appropriate to incorporate new science into the regulatory process? (yikes).

modes of repression by methylation

1. methyl group in major groove interferes with binding of txn favtors (a # of txn factors can recognize GC rich sequence motifs containing CpG sequence) 2. attraction of methyl-CpG binding proteins (DNA-protein complexes specific for methylated DNA) > MBD family: MDB1 & 2, Mecp2

2 classifications of methylation over time

1. stochastic/random (epigenetic drift) 2. programmed unidirectional (age-related methylation)

When was DNA as a double helix discovered?

1953

DNA methylation and histone modification are mutually reinforcing

> methylated DNA binds MECP2 and MBD1... these recruit HDACs and *histone methyltransferases* > histone deacetylation and methylation > methylated histones bind chromodomain proteins, which can recruit *DNA methyltransferases* > DNA methylation

Metastable epiallele

An allele that is variably expressed in genetically identical individuals due to its epigenetic state. Results in distribution of phenotypes from genetically identical cells. --agouti gene -- axin funsed metastable allele (kinky tail in mice... less methylation = more kinks) associated with repetitive transposable elements, epigenetic marks are stochastin, potential for transgenerational inheritance, targets for environmentally induced epigenetic modifications.

What is genomic imprinting?

An epigenetic chromosomal modification that results in the preferential expression of a gene in a mono-allelic parent-of-origin dependent manner.

What is a histone variant

Are non-allelic variants of histones representing one or a few amino acid differenes that are expressed at very low levels compared to their conventional counterparts. Histone variants have specific expression, localization, and species-distribution patterns , and conver novel structural functional properties on the nucleosome.

What are histones?

Are proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes. Acts as spools around which DNA winds and playing a role in gene regulation. n-terminal tails are important regulators of transcription. A change in chemistry of the tails changes the structure and function. Most modification is through the tails.

Cis vs. trans histone modification consequences

Cis: A covalent modification of a histone tail residue results in an altered structure or charge that manifests as a change in chromatin organization Trans: the enzymatic modification of a histone tail residu results in an affinity for chromatin associated protein. -- inhibits binding of a negative-acting factor (blocking) -- recruits positive-acting factor

What is bisulfite treatment?

Creates sequence dependent differences, yet you lose a lot of DNA because it is a harsh treatment. To convert unmethylated cytosines to uracil, leaving methylated cytosine unchanged. This allows one to distinguish methylated from unmethylated DNA via PCR amplification.

What are the ramifications of DNA methylation?

DNA methylation is often associated with gene repression. Methylation is heritable (stable but reversible). Sensitive to the environment. Blocks assess of transcription factors to CG-rich response elements, attracted methyl CpG binding proteins, which also inhibit transcription and may stimulate additional chromatin modification.

DNA vs RNA nucleic acids

DNA: double stranded, introme and exome, can be methylated on the 5th position cytosine, thymine, major/minor groove (two are pyrimdine hexagonal rings: cytosine and thymine) (two are purines adenine and guanine) uracil in place of thymine RNA: single stranded, only exomic sequence, uracil, comes from transcription from antisense strand of DNA

What is Developmental Plasiticity and its assumptions?

Developmental plasticity results in adaptive changes allowing an emerging phenotype appropriate for the environment it is expected to encounter. Assumptions 1) cues are accruate 2) early life is a good predictor of adult environment. if either assumption is not met, than the survival and/or health of the resulting individual may be compromised.

Euchromatin versus heterochromatin

Euchromatin - loosely packed, enriched in genes, active transcription, hyperacetylated histones. "poised" for gene expression. Other euchromatin: 1) activating histone modifications 2) lack of DNA methylation. 3) 30nm fibers and looped domains 4) binding of oncogenic viral dna 5) binding of chemical carcinogens. 6) increase during cell neoplasia Heterochromatin - tightly packed, cannot be transcribed, hypoacetylated histones Other heterochromatin 1) must play an important role in genome stability and organization. Constitutive (permenantly silence) Facultative (repressed during a specific cell cycle or developmental stage). Genome stability (middle and ends of chromosomes). histone variants and repressive histone marks, specialized proteins like polycomb, DNA methylation.

Compare and contrast genetic and epigenetic changes

Genetic changes: are referred as mutations (actual changes in the DNA), This change in the sequence results in an altered gene product. Genotoxic. Epigenetic changes: are heritable changes in DNA modification that alters the transcription and no actual changes to the DNA. Non-genotoxic and potentially reversible.

Examples of histone variants

H3.3: Marks actively transcribed regions. Replacement with this variant occurs at active genes, a dynamic process with potential epigenetic consequences. H2A.Z: Less known, probably reverses heterochromatin. Studies in yeast have implicated H2AZ in establishing transcriptional competience and in counteracting heterochromatin silencing. HeA.X: DNA Repair: Is defined as a variant whose serine residue is the site for phosphorylation at sites of DNA double-strand breaks. Poshphorylation of H2AX is an early event in double strand break repair where it is thought to concentrate components of the repair machinery.

Achievements in the Epigenome Project

HEP: published two studies that identified inter-tissue variation and that methylation is correlated with gene expression patterns. Transcription start site = less methylation Nice databases set up. NIH: Epigenome Roadmap, cumulation of what we contributed. 8 different threads interactive covering different aspects of the epigenome. TaRGET II: Phase II - so difficult that we are starting with mice. How the environment affects the epigenome over time. Toxicants and different exposures. Beginning with inbred mice. Humans are quite complex. Can't always use the target genes.

Bisulfite Sequencing

How it works: 1) converts cytosine to uracil 2) creates sequence-dependent differences (+) 3) increases redundancy (-) 4) Low yield of DNA post treatment because it damages the DNA

What are some epigenome wide tests?

Illumina Infinium,. Methylation27, and BeadArray (biased: subset of the genome) (unbiased: whole genome).

How is imprinting a violation of Mendel's law of allelic behavior?

Imprinting is a violation because he contended for independent assortment.

What are Trithorax (trxG)?

Maintain active state of gene expression (on). 1) place covalent modifications on nucleosomes 2) part of transcriptional machinery. Maintaining the "on" switch is very complex. 3) ATP-dependent chromatin remodeling 4) histone methyltransferase activity 5) transcription factors 6) growth factor receptors.

Histone Lysine Methylation

Methylation of histones occurs at lysine residies in histones H3 and H4. Certain methylated lysine residues are associated with activating transcription whereas others are involved with repressive processes.

Responsibilities of histone modifications

Plays a role in gene regulation and transcription. Compartmentalize the genome into domains of different transcriptional potentials

Two major classes of chromatin proteins

Polycomb (PcG) and Trithroax (trxG) Both protein complexes promote cellular memory. Large multi-globular complexes that act on their target genes via chromatin structure modulation and form the molecular basis of cellular memory. -- Conserved throughout evoltion. In humans, they maintain the fates of differentiated cells but are also required for cell proliferation and maintence of several types of stem cells. Regulate X-inactivation.

What is pyrosequencing?

Pro is that it is site specific and quantitative, high throughput (-) short reads and assay design can be tricky in CG dense reads. Instead of gels, you get a number through a system.

What does RITS stand for?

RNA Induced Transcriptional Silencing

Example of global testing methods.

Restricting enzyme digest for methylation detection: methylation specific enzymes (endonucleases HpaII cuts unmethylated CCGG and MspI cuts any CCGG (compare the ratio between these to see how much methylation is occurring). Can also look at repeated elements. Luminometric Methylation Assay (Assay): pro: don't need to know the underlying sequence. (con) don't know anything about what genes are being affected.

Human diseases associated with dysregulated chromatin

Rett Syndrome and Cancer

Experimental proof of imprinting

Surani (1984) nuclear transplant experiment in mammals that showed that a fertilized embryo will only develop correctly with both maternal and paternal genomes (but genes are only expressed from one parent and silenced in the other)

What do we need to know about the concept of a histone code?

The concept implies that particular combinations of histone modifications defined conformation of chromatin and hence the activity of the DNA contained therein. Although there is undoubtedly a strong general correlation, no single histone modification is completely predictive of chromatin state or DNA activity.

How would you look at these chromatin proteins

Use ChIP except you would use an antibody that would be attracted to the proteins that you're interested in.

Special Considerations/ Obstacles for the Epigenome Project

VARIATION!! A single person has multiple epigenomes: Tissue type Age Circadian Disease status Environment Which kind of epigenetic mark should be used?? 1) nutritional, chemical, physical and social factors modify the epigenome 2) particularly vulnerable during development. 3) tissue and cell type specificity 4) non-traditional dose-response and/or complex manifestations 5) does an epigenetic change necessarily reflect and adverse effect? 6) epigenetic plasticity may allow for nutritional/pharmacological intervention and prevention approaches.

What is the non-coding RNA responsible for X-inactivation?

Xist (long ncRNA) is expressed in one X. Coats that X. Reverse complement Tsix expression from the other (the active) X and represses Xist to induce further compacting of the X chromosome.

Macro H2A

a characteristic of nucleosomes on the inactivated X chromosome

blastocyte stage

after demethylation concurrently with initial differentiation events ,the genome undergoes a wave of de novo DNA methylation level of methylation varies in different cell lineages primordial germ cells of embryo undergo a second wave of genome wide reprogramming

Where does DNA methylation occur primarily in humans (mammals)?

at 5th position cytosine in a CpG.

Methyl-CpG Binding Proteins (MBD)

bind DNA containing methylated CpG dinucleotides recruit other proteins associated with repressive structures such as HDACs contribute to txn silencing humans have 5

Genome organization - layers of compaction

chromatin: DNA uses complex scaffolding of proteins to wind the DNA into tightly ordered structures. Histones: specialized proteins in which DNA wounds around in the development of chromatin. Nucleosomes: 20-50 base pairs of DNA (the beads on a string).

methylation is mutagenic

cytosine deaminates spontaneously to uracil, which is then mispaired w/ guanine > this is recognized by uracil DNA glycosylases and REPAIRED HOWEVER, when 5mc deaminates, thymines are formed > thymines are a normal base so they are not recognized as needing repair and can persist through DNA replication > passed to progeny as C-T mutation ** CpG usually unstable over evolutionary time

PcG

establish a silenced chromatin state

MBD2

exclusive affinity for mCpG > methyl-CpG binding proteins recruit co-repressors > MBD2 mediates methylation dependent txn repression > cells lacking MBD2 are unable to effectively repress methylated reporter constructs

Types of epigenetic study designs

global, candidate gene, and epigenome/genome wide.

trxG

in general propogate gene activity

paternally imprinted genes

maternally expressed

bisulfite identification

method for IDing methylation status of cytosines in genomic DNA > cytosine is deaminated to uracil > methylated cytosine remains unchanged! (stays a cytosine)

DNMT's

methyl transferases cytosine methylation 3 functional DNMT's in humans

miRNA vs siRNA

miRNA: hairpin precursor, cleaved by Dicer into miRNA, forms mRNA-protein complex, miRNA recognizes a target site on mRNA siRNA: Does not have the hairpin loop, yet the processing is similiar. What do small nc RNAs do? A natural form of gene regulation that protects cells from harmful propagation of viruses and transposons. 1) RNA-inducing silencing complex (RISC) - Recognize and degrade mRNA (genetic) 2) RNA- induced transcriptional silencing (RITS) -- target the complex of chromosome regions and induce chromatin modifications.

What is one imprinting disorder?

o Angelman and Prader-Willi --Loss of region 15q11-13. This region contains the paternally expressed genes (SNRPN and NDN) and the maternally expressed gene CBE3A) 1) Loss of maternal Angelmann (very very skinny) 2) Loss of paternal Prader-Willi (overweight, non-stop growth)

Study designs/considerations in evalulating epigenetics as a mechanism of DOHad

o Study Question Choice --If really and truly interested in very early exposures, then perhaps choice of tissue type to analyze for epigenetics is not as tricky! However, story doesn't end with birth --Early life environment/nutrition --Adolescence? --Drift with aging (programmed, stochastic, environmental components?) -- Gut bacteria colonization

Who is David Barker?

one of the most influential epidemiologists of our time. Barker hypothesis: transformed thinking about the causes of common disorders (diabetes and cancer). He challenged the idea that they are explained by a combination of bad genes and unhealthy adult lifestyle and proposed that their roots lie in early life. Critical periods of development which insults the stimuli cause specified changes in the body's physiology. "fetal programming." changes in nutrition produce permanent changes in the pattern of metabolic activity.

DNA methylation in development

paternal = active demethylation maternal = passive

Maternally imprinted genes

paternally expressed

cell memory

patterns of DNA methylation replicate semi-conservitively DNA methylation sensitive restriction endonucleases do not cleave if methylation

CpG islands

protected from methylation a fraction of vertebrate DNA cleaved unusually frequently by DNA methylation-sensitive restriction enzymes most mark the promoters and 5' domains of genes CpG islands on one X chromosome become de novo methylated during x chromosome inactivation in cancer cells, many normally non-methylated CpG islands become methylated