Chromatin & DNA Methylation Flashcards
Describe embryonic development in terms of epigenetics
Embryonic dev is a complicated epigenetic program
Describe embryonic dev - single cell, epigenetics
Starts as a single cells that gives rise to all the cell types in an embryo
Arise during dev
All of these cells are genetically identical
What is the fundamental question
What mechanisms allow epigenetic diversification in the right cells at the right time
What are the major questions we are going to address
How do epigenetic mechanisms (dna meth and histone mods) lead to heritable changes in gene expression
What impact do these have on dev, how they apply in developmental context
Describe epigenetic inheritance
Stable or heritable changes
What is epigenetics - broadest sense
Environment —> genes —> phenotype
Connection between environment —> impacts gens expression = lead to diff phenotypes at cellular level
If put cells in diff environment= diff gene expresison
What is epigenetics for developmental biologists
Stable propagation of phenotype without a change in genotype
Induce phenotypes (due to environment) = stable, can be propagated
What does stable mean - epigenetics
Stable means change is maintained over time, cell Disions or generations, in absence of initiating event
Long in duration, daughter cells hold changes
Even if take out of environment =still have change
What does epigenetics help maintain
Cellular identity during development = memory
Describe cell differentiation pathway
Start off with multipotent stem cells —> diff triggers lead to differentiation = 2 branches of pathway = primitive progenitor cell now —> continue to differentiate = lineage committed cells —> specialized blood cells = final differentiation state, more cOMPLICATED
What genes are on early during differentiation pathway
Genes associated with multi potency on = high expression
Linage specific genes = low expression
What genes are on at end of differentiation pathway
As move along pathway = turn off multipotency = restricts
Genes associated with multipotency off and higher expression of lineage specific genes
Describe what epigenetics does in differentiation pathway
As move along = turn off multipotency genes = become restricted
Mechanisms needed so multipotent genes stay off
If come on in differentiated cells - bad = defects, not function well
How do epigenetics help maintain cellular identity
Epigenetic mechanisms help cells remember those genes must be off —>initiation event may no longer be present, but can remember = epigenetic mechanisms
Describe when epigenetic and cellular memory are important
In other biological contexts
Describe young —> old neuron
Epigenetics = confer cellular memory
Implicated in changes that occur in young neuron so old neuron cell can remember
Describe person —> baby
Occurs n germ cells =passed on to next generation
What do epigenetic effects typically involve
Chromatin = vehicle for mechanisms
What does chromatin do
Complex of genomic dna with histones
Nucleosome = fundamental unit of chromatin structure, composed of dna and histones octamer, 150bp dna wrapped around
Packages dna
Describe euchromatin gen
On
Transcription high
Accessible loos conformation
Describe heterochromatin gen
Off
Transcription low
Heterochromatin
Compact and less accessible conformation
What is main type of chromatin
Can switch between these 2 states = close have ways of doing it, on vs off, on and off states have distinct chromatic features
Focus mostly on heterochromatin = off state, clearer pic of its formation and transmission during development (epigenetics and heritability)
Describe chromatin regulatory targets - 3
Developmental regulated genes
Repetitive dna elements - like transposons
Large chromosomal domains or even whole chromosomes - like XCI
Actually are important for embryo dive
What does heterochromatin do
Regulates developmental genes and other aspects of chromosomal structure
Describe the 2 types of heterochromatin
Constitutive heterochromatin
Facultative heterochromatin
Mechanisms are distinct but overlapping
Both types epigenetic = stable from developmental perspective
Describe constitutive heterochromatin
In ever cell, throughout dvelopment
Centromeres, telomeres, retrotransposons
Always present = genome regions organiziewd to be heterochromatin all the time, mostly structural elements, repetitive dna elements
Describe facultative heterochromatin
Varies with cell type and stage of development
Developmental genes, imprinting, inactive x chrom
For development
More plastic = varies dependent on type and developmental stage
Name 4 cellular mechanisms for altering chromatin structure
Nucleosomal movement
Nucleosome assembly (turn thing off) and disassembly (thing turn on)
Large scale movement of chromatin in the nucleus
Covalent modification of chromatin components (dna and/or histones)= important mechanisms, contribute to off/on state
All of these mechanisms operate in many/all cell types so are generally important in development
Name a kind of covalent modification
Dna methylation
1st chromatin modification associate with developmental process and regulate transcription
Describe dna meth generally
Cytosine - pyrimidine ring
Dnmt = fam of enzyme, catalyses reaction =
Methyl put on 5th position C = 5-methylcytosine
Where is 5meC present
In CpD dinucleotides (G right 3’ of C, can be methylated)
Present in dna of all vertebrates and flowering plants, some invertebrates, protists, bacteria
What is the original epigenetic mark
Dna methylation
Holiday and Pugh proposed the heritability of dna methylation pattern in 1975
Not genetic changes, passes info but not change in sequence dna
What is a critical components of the OFF state
5-mC
Critical component of heterochromatin
Important epigenetic mark
Where is dna methylation in the genome
Many places in genome , not random
Sines, lines, Ltrs, dna transposing fossils, unannotated regions too
Measuring CpG
Only a small fraction unmtheylated, really thin
What are usually methylated in mammalian genome
MOST CpGs in mammalian genome are methylated
General pattern applies to most mammalian cell types
Describe where a large portion of methylated CpGs arise
~40% reside in retrotransposons
What is methylation apart of
A silencing Mechanism that contributes to genome defense
Want to keep transposes in transcrptionally silent state
What happens when retrotransposons transcribed
Transposons = mobile dna elements
Retrotransposons = transcribed by rna pol 2, = mobilized now = insertional mutagenesis and aberrant recombination, antisense transcription - random places genome, strange transcriptional events
Where are retrotransposons typically found
Constiuitive heterochromatin = always kept in this state
Describe sines and lines and ltrs
Short/long interspersed nuclear elements
Long terminal repeats
Diff kinds of repetitive dna elements, retrotransposons remnants
Describe CpG islands
~70% of human genes contain CpG islands = CGIs
Length= 0.5-3kb, >55% C or G, enriched in CpG dinucleotides
Promoter 5’ of gene = transcriptionally on or off
Describe CpGs in CGIs
Almost always in unmethylated at both activate and inactive promoters (does not mater if gene transcriptionally on or off)
+ first exons
What is correlated with a strong repression of transcription
Rare CGI methylation - very strong signal
What is one type of CGI that is commonly methylated
ICRS THAT CONTROL imprinted genes
Name 2 examples of cell type specific variation on the general pattern of dna meth
When looking more closely = see patterns during development
Methylation at shores
Methylation at enhancers
Describe dna methylation at shores
Experiment that looks at dna methylation around CpG islands
Cell type specific methylation found at shores = often in first intron of the genes, changes depends on gene and cell types
Methylation status at shores is more strongly associated with whether gene on or off than at CGIs
Describe dna methylation of enhancers
Mammalian genes - developmental genes on enhancers = found further away from genes they regulate
Important for regulation of developmental genes
Describe enhancers
A regulatory dna elements that can act at large genomic distances and that are the binding sites for tfs
What is mechanism of repression by dna methylation
Either by methyl dna binding proteins
Blocking factors
If methylate c = create binding site for class of proteins= help dna chromatin into off state
Describe methyl dna binding proteins - mechanism of repression
Methyl dna binding proteins = MeCP1, MeCP2, MBD family, UHRF1- function in combo with histone modifications to repress transcription
Describe blocking factors - mechanisms of repression
Blocking factor access to dna - CTCF, YY1, SP1, EGR1 = blocks factors from binding
Dna binding tfs, binds to target site in dna, but if CpG methylated = cannot bind
Describe method detecting CpG methylation - gen
Bisulfite treatment = sequencing
Treatment of dna with sodium bisulfite = converts unmethylated cytosines to uracil through deamination
Methyl cytosine is unaffected = resistant, reaction does not work
Normally transition turns cytosine to uracil
Describe method detecting CpG methylation - results
C’s not methylated = converted to uracil
Do sodium bisulfite treatment then pcr and sequencing
= all of the T are unmethylated c, remaining c = methyl c
Can be adapted to sequence entire genome, many diff research methods
Describe dna methyl transferases generally
Catalytic domain = contains enzymatic parts, Conserved, through species, bacteria to humans, fundamental catalytic mechanisms
Targeting domain = to right place in genome, much more different from each other
Name and briefly describe the 3 types of dna methyl transferase - families
Dnmt1 = maintenance methyl transferase
Dnmt3A/B= de novo methyl transferases
Dnmt3L = not catalytically active, Germline specific, targeting function- unusual variant
What is dnmt1 involved in
Maintenance, inheritance of dna methylation
Describe what would happen if no dnmt1
CpG - methylated,
Dna rep happens and end up with 2 products = each has one strand methyl cytosine
Would end up losing methyl - diluted out, with each dna rep, so dnmt1 prevents this
Describe role of dnmt1
Keeps methyl group there are rep, methylated during rep when have hemimethylated duplexes, specifically
What does dna methylation do = S PHASE
Localizes to site of dna rep in s phase
See it = tagged dnmt1, with fluorescencent tag,
Same loci as replication factor = where rep actually happening
DESCRIBE DNMT3A/B
De novo methyltransferases
Introduce dna methylation at a place it did not exist
Targeting = binds to chromatin
Describe role of DNMT3 enzymes
Dnmt3A and Dnmt3B are de novo methyl transferases - establish methylation
Describe role of DNMT3 enzymes - in embryo
Dnmt3B = major de novo methyltransferase in embryo
Dnmt3A ESSENTAIL FOR GERmline development and certain somatic tissues = blood cells- place during dev where much de novo methylation occurs, - new for patterning
Complex with dnmt3L in germ cells
Describe role of DNMT3 enzymes - domains
ADD, PWWP domains bind to chromatin
Key dnmt mutant phenotypes
Impair dna methylation
Reamination of retrotransposons = transcribed and jump around
Chromosome instability = aneuploidy, compromised centromere function, could be related to jumping retrotransposons
Loss of imprinting
Defective expression of developmentally regulated genes
What is dna methylation very important for - explains
Self renewing progenitor cells
Ex = has to do with transition from progenitor cells to differentiated cells - progenitor has 2 paths
Not embryonic - adult, differentiation, into skin, but applies to development
If lose dnmt1 = progenitors cannot self renew
What is DNMT1 required for - also explain exp
Renewal of skin cell progenitors
Exp= graph piece of skin on wound on mice and see how well graft takes and heals wound in mice, how could dnmt1 function affect graph = use pharmacological agent of dnmt 1
What is DNMT1 required for - exp Control results
Mature keratinocytes on top of skin
Need pool of progenitors below = keeps regenerating
What is DNMT1 required for - exp mutaNT results
Grady didn’t work as well = never gets regeneration of progenitor cells
What is DNMT1 required for - RESULSTS
Wild type keratinocytes = progenitors or ones with dnmt1 ko = introduced into an in vivo skin regeneration away
Wild tape progenitors can restore the graft, dnmt1 ko cells cannot
Dmnt1 ko progenitors show increases expression of differentiation genes
DESRIbe skin cell differentiation - before differentiation
Genes associated with skin cell differentiation are methylated in progenitors but not in differentiated cells = measured transcription and genome expression in genome wide wall, and measures dna methylation,
Differential methylation found to occur in CGI shore regions
In absence dnmt1 = cannot maintain methylation, so think they are supposed to start differentiating
DESRIbe skin cell differentiation - after differentiation
Most lose dnna methylation for differentiation to occur properly
DESRIbe skin cell differentiation - how is methylation removed
Has to be a way to demethylate for genes
Must occur frequently = for gens to differentiate properly
WHEN DOES DNA METH NEED TO BE REMOVED
During development
Period of dev = dna meth reloaded rapidly at zygote stage or 2 cell transition
And then again = germ cell development, global removal
HOW is dna demethylated
No dnmt1 = passive demethylation = will get diluted out and lose after a few dna reps
Active demethylation = need someway to get of methyl
Describe active demethylaton - gen
Oxidation of 5mC by TET enzymes
Through oxidation reaction
Chemical mod = 5hmC-hydroxymethylcytosine —> 5fC-formylcytosine —> 5caC-carboxylcytosine
TETs have a variety of important roles in development
Describe active demethylaton - the 2 ways how it works - oxidized 5mC derivatives
Oxidation can help get of dna meth in 2 wards =
1 - oxidized forms are no longer substrate for dnmt1, cannot be efficiently maintained (oxidized forms) = PASSIVELY
2 - becomes substrates for BER = USUALLY after mistakes or mutation, cuts out and replaces with normal cytosine, see oxidixed form of methyl cytosine as mutation = ACTIVELY
Describe meanings of methylation patterns of shores
High methylation means low expression, low methylation means high expression
Describe meanings of enhancer methylation
High methylation is often anti correlated with transcription factor binding and enhancer activity
More productive if target genes on (no meth) or off (high meth)
