Module 9.1 Epigenetics Flashcards
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
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)
3
Q
interphase
A
- longest cell phase
- cell actively expressing genes and synthesizing proteins
- S phase: chromosomes duplicated before cell division
4
Q
M phase
7
A
- mitosis: nucleus divided into two daughter nuclei
- chromosomes condense
- nuclear envelopes break down
- mitotic spindles form
- Mitotic chromosomes captured by mitotic spindle, one complete set of chromosomes pulled to each end of cell
- nuclear envelope reforms around each nucleosome set
- cell divides into two daughter cells
5
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
6
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.
7
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
8
Q
heterochromatin
A
- aka closed chromatin
- tightly packed and less accessible for transcription
- constitutive heterochromatin, facultative heterochromatin, and varieties in between
9
Q
constitutive heterochromatin
A
- usually repetitive regions and serves structural functions eg. centromeres or telomeres
- always tightly condensed
10
Q
facultative heterochromatin
4
A
- not repetitive
- formed due to epigenetic regulation in response to developmental or environmental signals
- reversible formation
- regions of packaged DNA can differ between cell types
11
Q
chromatin remodeling
2
A
- rearrangement of chromatin from condensed state to transcriptionally accessible state
- mechanisms: Histone modification or DNA methylation
12
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
13
Q
histone modification
lysine acetylation
example
A
- acetyl group added to lysine removes positive charge
- reduces affinity of histone tail for adjacent nucleosomes = looser chromatin
14
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
15
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
16
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
17
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
18
Q
promoter chromatin alterations
features (5)
A
- provide greater access to DNA and facilitate assembly of RNA polymerase and general transcription factors at promoter
- can also allow binding of additional regulators
- repeated use of principle = large assemblies of proteins form on control regions of genes to regulate transcription
- activators and regulators are produced at different times and places in life of organism
- binds DNA in sequence specific manner = sequence determines how histones are modified
19
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)
20
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
21
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
22
Q
de novo methylation
A
- establish new methylation pattern on unmodified DNA
- Dnmt3a and Dnmt3b
23
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
24
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
25
cysotine deamination
- **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
26
# cystosine deamination
base excision repair pathway
* 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
27
CpG islands
- 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
28
# DNA methylation
gene regulation
- 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
29
# DNA methylation
transcription repression
| methods
1. **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
2. **Methyl CpG binding protein (MBD)**: outcompete transcription factors via higher affinity to methylated CpG site in sequence independent fashion
3. **protein repertoire**: the most well-known associate with histone modifying enzymes -> chromatin structure and DNA methylation act synergistically to repress chromatin state
30
# Epigenetic mechanisms
Transcription suppression
**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
31
# 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
32
# 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
