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ANAT 381 > Chromatin & DNA Methylation > Flashcards

Chromatin & DNA Methylation Flashcards

1
Q

Describe embryonic development in terms of epigenetics

A

Embryonic dev is a complicated epigenetic program

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2
Q

Describe embryonic dev - single cell, epigenetics

A

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

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3
Q

What is the fundamental question

A

What mechanisms allow epigenetic diversification in the right cells at the right time

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4
Q

What are the major questions we are going to address

A

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

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5
Q

Describe epigenetic inheritance

A

Stable or heritable changes

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6
Q

What is epigenetics - broadest sense

A

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

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7
Q

What is epigenetics for developmental biologists

A

Stable propagation of phenotype without a change in genotype
Induce phenotypes (due to environment) = stable, can be propagated

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8
Q

What does stable mean - epigenetics

A

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

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9
Q

What does epigenetics help maintain

A

Cellular identity during development = memory

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10
Q

Describe cell differentiation pathway

A

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

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11
Q

What genes are on early during differentiation pathway

A

Genes associated with multi potency on = high expression
Linage specific genes = low expression

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12
Q

What genes are on at end of differentiation pathway

A

As move along pathway = turn off multipotency = restricts
Genes associated with multipotency off and higher expression of lineage specific genes

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13
Q

Describe what epigenetics does in differentiation pathway

A

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

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14
Q

How do epigenetics help maintain cellular identity

A

Epigenetic mechanisms help cells remember those genes must be off —>initiation event may no longer be present, but can remember = epigenetic mechanisms

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15
Q

Describe when epigenetic and cellular memory are important

A

In other biological contexts

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16
Q

Describe young —> old neuron

A

Epigenetics = confer cellular memory
Implicated in changes that occur in young neuron so old neuron cell can remember

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17
Q

Describe person —> baby

A

Occurs n germ cells =passed on to next generation

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18
Q

What do epigenetic effects typically involve

A

Chromatin = vehicle for mechanisms

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19
Q

What does chromatin do

A

Complex of genomic dna with histones
Nucleosome = fundamental unit of chromatin structure, composed of dna and histones octamer, 150bp dna wrapped around
Packages dna

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20
Q

Describe euchromatin gen

A

On
Transcription high
Accessible loos conformation

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21
Q

Describe heterochromatin gen

A

Off
Transcription low
Heterochromatin
Compact and less accessible conformation

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22
Q

What is main type of chromatin

A

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)

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23
Q

Describe chromatin regulatory targets - 3

A

Developmental regulated genes
Repetitive dna elements - like transposons
Large chromosomal domains or even whole chromosomes - like XCI
Actually are important for embryo dive

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24
Q

What does heterochromatin do

A

Regulates developmental genes and other aspects of chromosomal structure

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25
Q

Describe the 2 types of heterochromatin

A

Constitutive heterochromatin
Facultative heterochromatin
Mechanisms are distinct but overlapping
Both types epigenetic = stable from developmental perspective

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26
Q

Describe constitutive heterochromatin

A

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

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27
Q

Describe facultative heterochromatin

A

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

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28
Q

Name 4 cellular mechanisms for altering chromatin structure

A

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

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29
Q

Name a kind of covalent modification

A

Dna methylation
1st chromatin modification associate with developmental process and regulate transcription

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30
Q

Describe dna meth generally

A

Cytosine - pyrimidine ring
Dnmt = fam of enzyme, catalyses reaction =
Methyl put on 5th position C = 5-methylcytosine

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31
Q

Where is 5meC present

A

In CpD dinucleotides (G right 3’ of C, can be methylated)
Present in dna of all vertebrates and flowering plants, some invertebrates, protists, bacteria

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32
Q

What is the original epigenetic mark

A

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

33
Q

What is a critical components of the OFF state

A

5-mC
Critical component of heterochromatin
Important epigenetic mark

34
Q

Where is dna methylation in the genome

A

Many places in genome , not random
Sines, lines, Ltrs, dna transposing fossils, unannotated regions too
Measuring CpG
Only a small fraction unmtheylated, really thin

35
Q

What are usually methylated in mammalian genome

A

MOST CpGs in mammalian genome are methylated
General pattern applies to most mammalian cell types

36
Q

Describe where a large portion of methylated CpGs arise

A

~40% reside in retrotransposons

37
Q

What is methylation apart of

A

A silencing Mechanism that contributes to genome defense
Want to keep transposes in transcrptionally silent state

38
Q

What happens when retrotransposons transcribed

A

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

39
Q

Where are retrotransposons typically found

A

Constiuitive heterochromatin = always kept in this state

40
Q

Describe sines and lines and ltrs

A

Short/long interspersed nuclear elements
Long terminal repeats
Diff kinds of repetitive dna elements, retrotransposons remnants

41
Q

Describe CpG islands

A

~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

42
Q

Describe CpGs in CGIs

A

Almost always in methylated at both activate and inactive promoters (does not mater if gene transcriptionally on or off)
+ first exons

43
Q

What is correlated with a strong repression of transcription

A

Rare CGI methylation - very strong signal

44
Q

What is one type of CGI that is commonly methylated

A

ICRS THAT CONTROL imprinted genes

45
Q

Name 2 examples of cell type specific variation on the general pattern of dna meth

A

When looking more closely = see patterns during development
Methylation at shores
Methylation at enhancers

46
Q

Describe dna methylation at shores

A

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

47
Q

Describe dna methylation of enhancers

A

Mammalian genes - developmental genes on enhancers = found further away from genes they regulate
Important for regulation of developmental genes

48
Q

Describe enhancers

A

A regulatory dna elements that can act at large genomic distances and that are the binding sites for tfs

49
Q

What is mechanism of repression by dna methylation

A

Either by methyl dna binding proteins
Blocking factors
If methylate c = create binding site for class of proteins= help dna chromatin into off state

50
Q

Describe methyl dna binding proteins - mechanism of repression

A

Methyl dna binding proteins = MeCP1, MeCP2, MBD family, UHRF1- function in combo with histone modifications to repress transcription

51
Q

Describe blocking factors - mechanisms of repression

A

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

52
Q

Describe method detecting CpG methylation - gen

A

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

53
Q

Describe method detecting CpG methylation - results

A

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

54
Q

Describe dna methyl transferases generally

A

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

55
Q

Name and briefly describe the 3 types of dna methyl transferase - families

A

Dnmt1 = maintenance methyl transferase
Dnmt3A/B= de novo methyl transferases
Dnmt3L = not catalytically active, Germline specific, targeting function- unusual variant

56
Q

What is dnmt1 involved in

A

Maintenance, inheritance of dna methylation

57
Q

Describe what would happen if no dnmt1

A

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

58
Q

Describe role of dnmt1

A

Keeps methyl group there are rep, methylated during rep when have hemimethylated duplexes, specifically

59
Q

What does dna methylation do = S PHASE

A

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

60
Q

DESCRIBE DNMT3A/B

A

De novo methyltransferases
Introduce dna methylation at a place it did not exist
Targeting = binds to chromatin

61
Q

Describe role of DNMT3 enzymes

A

Dnmt3A and Dnmt3B are de novo methyl transferases - establish methylation

62
Q

Describe role of DNMT3 enzymes - in embryo

A

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

63
Q

Describe role of DNMT3 enzymes - domains

A

ADD, PWWP domains bind to chromatin

64
Q

Key dnmt mutant phenotypes

A

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

65
Q

What is dna methylation very important for - explains

A

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

66
Q

What is DNMT1 required for - also explain exp

A

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

67
Q

What is DNMT1 required for - exp Control results

A

Mature keratinocytes on top of skin
Need pool of progenitors below = keeps regenerating

68
Q

What is DNMT1 required for - exp mutaNT results

A

Grady didn’t work as well = never gets regeneration of progenitor cells

69
Q

What is DNMT1 required for - RESULSTS

A

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

70
Q

DESRIbe skin cell differentiation - before differentiation

A

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

71
Q

DESRIbe skin cell differentiation - after differentiation

A

Most lose dnna methylation for differentiation to occur properly

72
Q

DESRIbe skin cell differentiation - how is methylation removed

A

Has to be a way to demethylate for genes
Must occur frequently = for gens to differentiate properly

73
Q

WHEN DOES DNA METH NEED TO BE REMOVED

A

During development
Period of dev = dna meth reloaded rapidly at zygote stage or 2 cell transition
And then again = germ cell development, global removal

74
Q

HOW is dna demethylated

A

No dnmt1 = passive demethylation = will get diluted out and lose after a few dna reps
Active demethylation = need someway to get of methyl

75
Q

Describe active demethylaton - gen

A

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

76
Q

Describe active demethylaton - the 2 ways how it works - oxidized 5mC derivatives

A

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

77
Q

Describe meanings of methylation patterns of shores

A

High methylation means low expression, low methylation means high expression

78
Q

Describe meanings of enhancer methylation

A

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)

ANAT 381 (24 decks)

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