LS Lecture 17

How epigenetics is different from genetics.

changes BEYOND (ie, not) simply differences in DNA are inherited

• it is a form of genetic memory, because of the effect of past environmental stimuli on parent generations

• changes made in response to enviornmental stimuli that are passed down

• epigenetic changes made in a parent in response to environmental conditions,… results in the offspring being adapted to the conditions before actually experiencing them.

That DNA is packaged into chromatin, and how packaging of DNA can affect expression

packaging allows the genome to fit into tiny ass nucleus, they wrapped around a proteins called HISTONE

• packaging makes dna inaccessible, solution? we need loosening for transcription

• perhaps the epigenetic marks that are used to guide “the degree of condensation and thus the expression of dna“

EX: “To be transcribed, genes must be accessible to the transcriptional machinery, but when DNA is fully condensed, this machinery cannot engage with target regions like promoters.“

How chemical tags (methyl and acetyl groups) are deposited and removed and how they affect gene activity

• deposited via Writers (—transferases)

• removed by erasers (DE—)

tags specifiy the degree of condensation (and throuhg this the exression) of DNA

• methyl groups are deposited to the cytosines next to (immediately adjacent to) the guanines, they get removed by demethylases

How lncRNAs can inhibit gene expression.

these are long rnas, they lack an open reading fram

• these can work with proteins

• or themselves directly bind nucleosomes, can attract other proteins

x inactivation!

• x-ist gets activated, marks the chromosome to be inactivated

How epigenetic changes contribute to cellular differentiation (from totipotency)

totipotent - when an egg is fertilized, every cell in the embryo it has the potential to develop into ALL cell types

epigenetic modifications lead to a specific pattern of gene expression, this pattern of expression is what then leads to the formation of a cell’s identity

cell idenrtity is stable once defined!

Why epigenetic modifications are thought to be reversible

injections n sturf can reverse lol

• see the ability of bees to become a queen bee, Larvae can be turned into queens without royal jelly by using genetic tools that specifically repress Dnmt3 activity.

• Again, we see that these changes are reversible. A rat destined to be anxious – because of methylation of the GR gene – can be converted into a calm rat by injections of an inhibitor of methylation

The challenges of proving transgenerational epigenetic inheritance

reprogramming is a key issue

• gametes have their epigenetic marks “reset“. transgenerational marks must sturive these moments (happens twice)

Differentiate between transcriptional control by regulatory TFs and epigenetic modification

• TF control of transcription -

  • affects expression through directly binding to DNA via factors

• epigenetic modification -

  • controls expression of dna via environmental stumuli.. beyond the gene sequence!

    • determines how easy it is to transcribe in the first place “determining in the process how accessible the DNA is to transcriptional machinery”

    • WHERE and to whAt EXTENT?

Describe the primary mechanisms for epigenetic modification (histone acetylation, DNA methylation, RNA interactions) and predict their effects on gene expression

• acetylation increases expression

• methylation decreases. promotes coilin

• RNA…

Analyze bisulfite sequencing data to identify methylated bases, and predict the results of bisulfite sequencing reactions on methylated vs unmethylated sequences

converts:

UNmethylated (Cs) —> Methylated (Us, which get detected as Ts)

Explain how epigenetic modifications are involved in X-inactivation at a molecular level

• notably, x inactivation is stable. REmember how in x inactivation all cells express only the X chromosome that is NOT inactivated

Steps:

• the cell ‘counts’ the number of X-chromosomes present to determine whether inactivation is necessary

• Second, when two X chromosomes are present, one is randomly selected for inactivation, while the other remains active

Identify the “writers” and “erasers” involved in DNA methylation and histone acetylation, and predict the effects of activating or inhibiting them

writers - add modification

erasers -remove

readers__> enzymes that read and interpret to loosen or condense dna based on the epigenetic modifications

Explain how epigenetic modifications can be passed on from one cell generation to another

(maybe review for clearer answer to this)

• Sometimes, interactions between the environment and epigenome may have transgenerational consequences.

• People in this cohort are more likely to develop metabolic diseases like obesity-related diabetes, and so too are their children. Is this a transgenerational epigenetic effect, involving epigenetic alteration of the mother’s gametes in response to her own nutritional status (and, implicitly, that of her child when it is born)? Possibly. However, it’s difficult to exclude direct in-utero effects on the fetus: if the mother is starving, then there is less nutrition available to the fetus across the placenta and the fetus itself might respond with a view to coping with a resource-limited environment.

Describe experimental evidence for the role of postnatal environment on offspring phenotype (e.g., the rat licking experiment)

EX: This means that rats that were little-licked as pups are, throughout life, anxious and jumpy, whereas those that were much-licked are more relaxed and less anxious. While this might seem a raw deal for the anxious rats, it’s likely that this is in fact adaptive.

• we see that these changes are reversible. A rat destined to be anxious – because of methylation of the GR gene – can be converted into a calm rat by injections of an inhibitor of methylation

Interpret data from experiments of model systems in which epigenetic modifications have an effect on gene expression to predict the types of epigenetic modifications that would be found on the genes being studied and explain why.

sure, give me prac prob

Predict the effect of different drug treatments (inhibitors of the enzymes that add and remove methyl and acetyl groups) on epigenetic marks and gene expression and explain why.

*practice prob ex*

Design an experiment to test the effect of a particular drug treatment on epigenetic regulation of genes in a model organism

• the one that directly affects the problem is typically the best answer

  • if methylation is a problem, yes you can acetylate, but the best option is DEmethyltranserase, such that you are directly targeting the problem

Epigenetics

is the phenomenon by which changes above or beyond differences in the DNA sequence are inherited, either from one generation to the next or from one cell generation to the next. Epigenetic changes affect the expression of DNA, but not the identity of the constituent DNA sequences – the sequence of the DNA bases is unaffected.

DNA Packaging

. How the genome fit inside such a tiny compartment. DNA does not float freely inside the nucleus, but instead is wrapped around histone

histone

proteins which DNA wraps around

• have positively charged tails

• The best understood histone modification is the addition of acetyl groups to the amino acids on the histone tails.

nucleosome

repeating units of DNA, that are wrapped (packaged into) around histones

chromatin

collective term for (many) nucleosomes

DNA Accessibility

directly involves the ability of transcription machinery to access DNA transcription in order to express genes

Epigenetic tag/mark

• these have a chemical composition

• some of which are on the DNA itself while others are present instead on the histone proteins, all in all, they work to specify degree of condensin and relaxin of dna, and thereby the expression

DNA Methylation/methylated DNA

• DNA methylation refers to the addition of methyl (CH3) groups to the nucleotide bases. The predominant form of DNA methylation is the methylation of the DNA base cytosine

• DNA methylation promotes coiling and condensation of chromatin so methylation of CpG dinucleotides in the promoter region of a gene decreases transcription and reduces expression of the gene

Hemi-methylated

• basically refers to the different strand of dna where c is methylated and not

After semiconservative replication, any cytosine that was methylated prior to replication will now only be methylated on one strand – the template strand. The cytosine on the newly replicated strand will be unmethylated. We refer to this state as hemimethylated

Special methyltransferases recognize hemimethylated CpG dinucleotides and add methyl groups to the unmethylated cytosine bases on the new strand (remember the CpG on one strand is paired with a complementary GC on the other strand), thus restoring the methylation pattern to the same state as that of the parent cell.

Cytosine

a typa nucleotide BASE

CpG

a CpG dinucleotide (‘di’ meaning two and ‘p’ representing the phosphate group that connects the C and G nucleotides)

CpG Island

where CpGs cluster, typically in the promoter regions

Uracil

a typa nucleotide BASE

Bisulfite sequencing

Here, genomic DNA is treated with the chemical sodium bisulfite, which chemically converts unmethylated cytosines to uracils, which are then detected as thymines during sequencing.

converts:

UNmethylated (Cs) —> Methylated (Us, which get detected as Ts)

• if a cytosine is methylated, it is said to be protected, andd as such will not turn to a uracil, or thymine once detected lol

ATAC-Seq

one of “techniques for detecting the ‘openness’ of chromatin“

Assay for Transposable Accessible chromatin via Sequencing

• uses an enzyme, tn5 transposase, facilates movement of DNA segments all about da genome

  • which attacks anyyy accesible DNA

    • it appears, at random

    • the enzyme, tn5 transposase only affects those unsafe dna bros that are in an open configuration, cannot access the tight bros

  • *fragments generated by Tn5 transposase therefore correspond to the parts of the genome with open chromatin.

DNA Demethylase

removes methyl groups

Writers, Readers, Eraser?

Writers are responsible for the addition of the 5 modification

whereas erasers are responsible for the removal of the modification (e.g., DNA demethylases)

Readers —‘readers’, which interpret the suite of epigenetic modifications to loosen or condense the DNA accordingly.

lncRNA

• a type of chemical tag!

• long non-coding RNAs

• a special type of RNA molecule that is transcribed but not translated. These RNAs are typically greater than 100 nucleotides in length (hence, the name “long”) and lack an open reading frame.

• interact with transcription factors and chromatin

  • can also directly bind to nucleosomes, “often in the process attracting additional proteins involved in epigenetic modification.“

Chromatin binding protein

type of proteins that regulate transcription

• interacts with lncRNA

Xist

(X-inactive specific transcript)

There are a number of lncRNAs involved in mediating X-inactivation; Xist (X-inactive specific transcript) is the best characterized

• gets UPregulated when inactivation is needed for an x, marks the chromosome for inactivation (The coated chromosome is then silenced via high levels of DNA methylation and low levels of histone acetylation.)

Totipotent

when a celll has the potential to become any type

• when? —> fertilization

Cellular (tissue) differentiation

when a cell goes away from totipotency to specialize into a specific cell

• once cells assume unique identities (e.g., the cells of the kidney are distinct in form and function from neurons and red blood cells)

***even THO all cells have the same genome

Epigenetic reprogramming

However, upon fertilization most or all of these epigenetic marks are removed to generate a totipotent zygote that can divide and give rise to all tissues of the body. The removal of epigenetic marks is triggered by the cellular environment in the zygote; this process is referred to as reprogramming.

• some genes escape reprogramming via imprinting (the gene’s expression in the individual is mediated by the epigenetic markers set by the parent during the gamete’s formation. Typically, the promoter of the silenced copy (whether maternal or paternal) is methylated)

Thrifty phenotype hypothesis

Whether environmentally induced epigenetic changes can be transmitted from parent to offspring is highly controversial, as they would have to escape both steps of reprogramming.

• For example, if a pregnant woman experiences a poor nutritional environment, this could be signaled to the fetus via specific epigenetic modifications. This, in turn, prompts a metabolic response in favor of being metabolically ‘thrifty’ to minimize energy expenditure, in addition to eating whenever possible and hoarding calories.

Somatic cell nuclear transfer

This involved removing the nucleus from an egg cell and replacing it with the nucleus of a somatic cell (in Dolly’s case, a mammary cell) and, finally, inserting the cell into a surrogate mother ewe. Presumably, the DNA from the mammary cell nucleus has a whole set of epigenetic markers specifying its (highly specific) mammary cell characteristics. That the genetic material from the mammary cell became effectively totipotent (via reprogramming) in Dolly shows how readily these marks can be eliminated and the cell’s differentiation status reversed.

Epigenome

the totality of epigentic modifications on a genome

• relevant because each cell exists with different EPIGENOMES

primordial germ cells (PGCs)

a window of reprogramming

• Reprogramming of DNA methylation also occurs at a second time point: in primordial germ cells (PGCs), which are the direct progenitors of sperm or oocyte. Epigenetic marks are then laid down, based on the cellular context, to direct these PGC progenitors to develop into a sperm or egg.