Week 4.1: Epigenomics Flashcards
(41 cards)
1
Q
epigenetics
A
- study of heritable traits that happen without changes to DNA sequence
- usually involves changes that affect regulation of gene expression.
2
Q
histones
4
A
- histones form H2A/H2B and H3/H4 heterodimers
- DNA strands wrap around octamer anchor
- linker histone H1 binds and changes DNA exit path from nucleosome
- histone fold region and N-terminal tail that extends out from DNA-histone core.
3
Q
nucleosome
4
A
- nucleosome = core particle + linker DNA
- core particle = ~147 DNA bp wrapped in little less than two 2 turns around protein core (8 histone proteins)
- linker DNA = 10-80 bp depending on species and tissue types
- Most eukaryotic cells have characteristic average nucleosome spacing of ~190 bp = 45 bp linker
4
Q
heterochromatin
A
- aka closed chromatin
- tightly packed and less accessible for transcription
- constitutive heterochromatin, facultative heterochromatin, and varieties in between
5
Q
euchromatin
5
A
- aka open chromatin
- nucleosomes in euchromatin much more widely spaced
- lighter stain
- enriched in genes and often under active transcription.
- allows gene regulatory proteins and RNA polymerase complexes to bind to DNA sequence and initiate transcription
6
Q
chromatin
A
- unraveled, condensed structure of DNA, packaged by histones in nucleus
- structure tightly linked with gene expression regulation
- interphase: chromatin exists as long, thin, tangled threads in nucleus so that individual chromosomes cannot be easily distinguished
- interphase: 30nm fiber
- unfolded: beads on a string (nucleosome)
7
Q
chromatin remodeling
2
A
- rearrangement of chromatin from condensed state to transcriptionally accessible state
- mechanisms: Histone modification or DNA methylation
8
Q
histone modification
7
A
- post translational modification of histone proteins
- histone tails help pack nucleosomes together
- tails subject to covalent modifications (acetylation, methylation, phosphorylation, ubiquitylation, biotinylation, etc)
- constantly added and removed depending on chromosome location and cell history
- Some modifications can happen in histone globular core
- carefully controlled
- recruit specific proteins to modified chromatin, work together to control gene expression and other chromosome functions
9
Q
core histone proteins
3
A
- H2A, H2B, H3, H4
- among most highly conserved eukaryotic proteins
- synthesized primarily during S phase of cell cycle and assembled into nucleosomes on daughter DNA behind replication fork
10
Q
histone variants
7
A
- present in much smaller amount than major histones
- less well conserved during evolution
- synthesized throughout interphase
- often inserted into already formed chromatin
- requires histone exchange process via chromatn remodeling complex
- inserted in highly selective manner
- involved in specialized chromosome control functions
11
Q
histone variants
examples
A
- H2AX: DNA repair and recombination
- H2AZ: gene expression, chromosomal segregation
- macroH2A: transcriptional repression, X-chromosome inactivation
- H3.3: transcriptional activation
- CENP-A: centromere function and kinetochore assembly
12
Q
Eukaryotic Transcription Activators
altering chromatin structure of promoter
mechanisms (4)
A
- covalent histone modifications through histone modifying enzymes
- nucleosome remodeling by ATP dependent chromatin remodeling complexes
- histone chaperones mediated nucleosome removal
- histone replacement using histone variant proteins
13
Q
epigenetic inheritance
process (7)
A
- histone modifying enzyme marks certain H3 or H4 histones with specific modification
- heterochromatin proteins bind to modified H3/H4 histones
- histone modifying enzyme binds to heterochromatin region, ensuring modification is maintained
- When chromosome is replicated, marked histone H3/H4 of parent chromosome distributed randomly into two daughter strands = mixture of old and new nucleosomes
- In heterochromatin, histone modifying enzymes bound to old nucleosomes rapidly mark new nucleosomes = new binding sites for heterochromatin proteins
- heterochromatin proteins can bind to each other, further promoting assembly of protein polymer along chromosome
- cooperative action and recruitment of proteins propagates specific form of chromatin across cell generations
14
Q
epigenetic inheritance
features
A
- particular chromatin structure can be directly inherited to DNA following each round of replication
- enables cell to have both longer and short term memory of gene expression patterns
- plays central part in creating multicellular organisms
- differentiated cell types become established during development and persist through repeated cell division cycles (eg. daughters of liver cell persist as liver cells)
15
Q
DNA methylation
A
- biological process by which methyl groups are added to DNA molecule
- In mammalian cells, mainly at carbon 5 position of selected cytosine nucleotides located in sequence of CpG dinucleotides
- forms 5- methyl cytosine (5mC)
- DNA methyl transferases
- de novo and maintenance methylation
16
Q
de novo methylation
A
- establish new methylation pattern on unmodified DNA
- Dnmt3a and Dnmt3b
17
Q
maintenance methylation
A
- functions during DNA replication to copy DNA methylation pattern from parent DNA strand onto daughter strand
- Dnmt1
- no maintenance methylation = unmethylated daughter strand -> passive DNA demethylation
- acts preferentially on CpG sequences base paired with already-methylated CpG sequence
- methylated parent strand serve as template for methylation of daughter DNA strand = direct pattern inheritance
18
Q
Methylation during development
mammalian
A
- Early development: Genome-wide passive and active demethylation shortly after fertilization
- passive: suppression of maintenance DNa methyltransferase activity -> loss of methyl groups during each round of DNA replication
- active: series of enzymatic reactions converting 5- methylcytocine (5mC) to 5-hydroxymethylcytosine (5hmC) through TET enzymes ->replaced by cytosine via DNA repair or replication
- Later development: new methylation patterns established by several de novo DNA methyl transferases directed to DNA by sequence-specific DNA binding proteins
- Once new patterns established, can be propagated through replication by maintenance DNA methyltransferase
19
Q
cysotine deamination
A
- deamination: removal of amino group from molecule
- occurs naturally inside mammalian cell with high cytosine frequency
- unmethylated cytosine: accidental deamination ->uracil -> recognized and corrected by base excision repair
- methylated cytosine: 5mC - amino group = thymine
- TG mismatch could change to TA that’s not corrected
- C to T = most common single nucleotide mutation in mammalian cells
20
Q
cystosine deamination
base excision repair pathway
A
- cytosine deamination = uracil = UG mismatch
- U base removed by DNA repair enzyme Uracil DNA glycosylase (UDG) = abasic site
- abasic site recognized by enzymes AP endonuclease
- breaks phosphodiester bond to replace with cytosine
21
Q
CpG islands
A
- generally unmethylated -> spared accelerated mutation rate of bulk CpG sequences and retain expected CpG content
- regions >200 bp, GC >50%, ratio observed to expected CpG >0.6
- ~25,000 CPG islands in human genome
- major regulatory units
- ~50% in gene promoter regions, 25% in gene bodies as alternative promoters
- ~60-70% of human genes have CpG island in promoter region, especially housekeeping genes
- majority remain unmethylated in most somatic tissues whether or not associated gene is expressed.
- sequence-specific DNA binding proteins bind to cis-regulatory elements in CpG islands to shield from methyltransferase
- proteins recruit DNA demethylation enzymes to stay unmethylated
- enriched for permissive chromatin modifications and suitable for promoters.
- only 10% are methylated in somatic tissues, mostly in intergenic and intragenic regions
22
Q
DNA methylation
gene regulation
A
- methylated cytosines in CpG sites in promoter and enhancer regions = repressed gene
- methylated cytosines in CpG sites in gene body / coding region (excluding transcription start sites) = enhanced gene
23
Q
DNA methylation
transcription repression
methods
A
-
methylated cytosines in DNA major groove: interfere directly with binding of proteins like transcription regulators and general transcription factors
- Transcription factors usually bind to non methylated DNA motifs
- interaction disrupted by methylated CpG site in motifs - Methyl CpG binding protein (MBD): outcompete transcription factors via higher affinity to methylated CpG site in sequence independent fashion
- protein repertoire: the most well-known associate with histone modifying enzymes -> chromatin structure and DNA methylation act synergistically to repress chromatin state
24
Q
Epigenetic mechanisms
Transcription suppression
A
Methylation:
- DNA methyltransferase add methyl group to DNA
- DNA methyltransferase target CpG site and can be enhanced by association with histone tails
- methylation recognized by methyl binding proteins that recruit enzymes to modify histone tails (HDAC, etc)
Histone Modification Enzymes:
- histone d-acetylase (HDAC): remove acetylation
- histone methyl transferase: methylates histones
25
# Epigenetic mechanisms
Transcription activation
**Demethylation**:
- TET protein removes DNA methylation on histone tails
- TET proteins chemically modify DNA methylation to form hydroxy methylation
**Histone Modification**:
- histone tails in region often contain modifications that inhibit methyl transferase binding to unmethylated CpG sites = permissive transcription environment
26
# Epigenetic mechanisms
DNA / Histone Methylation Interactions
- reciprocal relationship between dna methylation and histone lysine methylation
- MBD can methylate DNA CpG site and methylate histone via histone methyl transferase
- DNA maintenance methylation by dnmt1 partly relies on recognition of histone methylation on nucleosome present at DNA site to carry out cytosine methylation on newly synthesized DNA
- further cross talk between DNA methylation by dnmt3A and 3B and histone methylation = correlation between genome wide distribution of DNA methylation and histone methylation
27
Epigenomic sequencing
| layers (4)
1. DNA methylation
2. Histone modifications
3. Chromatin accessibility
4. Long-range interactions
28
Chromatin accessibility
- degree to which nuclear micromolecules are able to physically contact chromatin DNA
- chromatin densely arranged within facultative and constitutive heterochromatin
- histones can be replaced by transcription factors at regulatory loci (enhancers, insulators, transcribed gene bodies)
- Transcription factors dynamically compete with histones and other chromatin binding proteins to modulate nucleosome occupancy and promote local access to DNA
- Nucleosome and linker histone occupancy create accessibility spectrum
- chromatin states: closed -> permissive -> open
- Tracing accessibility changes critical to understand epigenetic regulation of gene expression and cellular status
29
Active demethylation process
- TET family proteins convert 5-mC -> 5-hmC ->5-fC -> 5-caC
- 5-mC: most abundant
- 5-hmC: highly abundant in neurons
- 5-fC & 5-caC: lowest abundance, converted to cytosine via BER enzymes
30
Cytosine converted forms
1. Cytosine (C)
2. 5-methylcytosine (5-mC)
3. 5-hydroxymethylcytosine (5-hmC)
4. 5-formylcytosine (5-fC)
5. 5-carboxylcytosine (5-caC)
31
5-hmC
| features
- highly abundant in neurons
- related to activating genes
- plays important role in embryonic development and cell differentiation
- disregulation been shown in tumorogenesis
32
# DNA methylation analysis
bisulfite conversion
| features
- unmethylated cytosine converted to uracil via deamination
- methyl group at carbon 5-position (5-mC & 5-hmC) protected against deamination
- denaturation required: sodium bisulfite can only react with cytosine in ssDNA
- PCR: uracils converted to thymines, 5-mC and 5-hmC unchanged
- compared to reference genome
- single nucleotide resolution info about methylation status of DNA segment
33
post-bisulfite adapter tagging
| features
- harsh chemical treatment that damages and nicks DNA
- low library conversion rate, significant loss
- utilize single strand library prep to add sequencing adapters to bisulfite-treated ssDNA
- can salvage fragmented DNA caused by bisulfite treatment to mitigate bisulfite induced loss of sequencing templates
- greatly improved library conversion efficiency
- enabled sequencing with very low sample input eg. single cell
34
# methylation detection
bisulfite sequencing
| benefits and drawbacks
**Benefits**:
- >99% conversion efficiency of unmodified cytosine to uracil
- original gold standard for mapping 5-mC and 5-hmC
**Drawbacks**:
- limited read length
- 95% of cytosine in genome converted to T
- 60% AT / 40% GC before treatment -> 75% AT, 20% G & <5% C
- lower mapping efficiency and biased genomic coverage
- makes targeted sequencing more difficult
- unable to distinguish 5mc from 5hmc
35
Nanopore methylation detection
- Methylated bases have different ionic current signature fluctuation than unmethylated base that can be interpreted by base-calling algorithms
- methylation status can be picked up directly from sequencing reads in real time without requiring pre-processing of DNA template with conversion or selective pull down
- accuracy dependent upon nanopore, processive enzyme, and bioinformatics methodology
- Improved sensitivity of channels allows for increasingly subtle modifications to be detected
36
SMRT Technology methylation detection
| features
- Data generated from pulse width and interpulse duration
- unique kinetic signatures can theoretically be used to identify epigenetic modifications
- RCA allows measurement of both strands of initial dsDNA molecule, enabling identification of hemi- and symmetrically methylated positions
- sequencing of longer sequences at higher depths requires robust polymerase
37
# histone modification detection
Chromatin immunoprecipitation followed by sequencing
(ChIP-Seq)
| features
- sequence histone modifications in a genome wide manner
* Analyze chromatin-associated protein interactions with DNA
- can be used to map global binding sites precisely for any protein of interest
* Antibodies target specific histone modifications / TFs/ other chromatin binding proteins
* sensitive and specific antibody gives high level of enrichment = easier to detect binding events
* poor specificity antibody = high background noise
38
Chromatin immunoprecipitation followed by sequencing
(ChIP-Seq)
| process
1. DNA binding protein crosslinked to DNA in vivo by treating cells with fixation reagents (eg. formaldehyde)
2. chromatin fragmented and incubated with specific antibodies to enrich for DNA-protein complex containing target histone modifications or specific transcription factors
3. cross links reversed and purified DNA sequenced on any NGS platform to detect genome wide histone modifications or protein binding distributions
39
Chromatin Accessibility quantification
| methods (4) and feature
1. DNase-seq
2. ATAC-seq
3. MNase-seq
4. NOMe-seq
quantify susceptibility of chromatin to either enzymatic modifications or cleavages of its constituent DNA
40
Assay for Transposable Accessible Chromatin
(ATAC-seq)
- uses hyperreactive transposase Tn5 to simultaneously cleave and ligate sequencing adapters into accessible chromatin regions
- selectively amplifies double cleavage events in accessible chromatin
- widely adopted partly because robustly identifies accessible chromatin
- straightforward and rapidly implemented
- amenable to materially limited clinical and primary tissue samples
- libraries routinely generated in < 2 hours, with 10,000 to 20,000 cells
- capture similar regulatory information to DNase-Seq
41
Cancer epigenetics
- global DNA hypomethylation often accompanied by hypermethylation in specific regions containing CpG islands
- hypomethylation induces expression of oncogenes
- hypermethylation suppress expression of tumor suppressor genes
- occurs early in cancer development
- disturbance of other epigenetic mechanisms such as histone modification, DNA binding proteins, non-coding regulatory RNAs observed in different types of cancers
- can occur before cancer genetic mutations are detectable
- epigenetic changes in cancer cells >> genetic mutations
- epigenetic signal contains tissue specific information
- promising biomarkers for cancer early screen
- potential therapeutic targets
