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Genomic Sequencing Technologies > Module 9.1 Epigenetics > Flashcards

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.
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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)
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3
Q

interphase

A
  • longest cell phase
  • cell actively expressing genes and synthesizing proteins
  • S phase: chromosomes duplicated before cell division
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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
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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
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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.
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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
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8
Q

heterochromatin

A
  • aka closed chromatin
  • tightly packed and less accessible for transcription
  • constitutive heterochromatin, facultative heterochromatin, and varieties in between
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9
Q

constitutive heterochromatin

A
  • usually repetitive regions and serves structural functions eg. centromeres or telomeres
  • always tightly condensed
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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
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11
Q

chromatin remodeling

2

A
  • rearrangement of chromatin from condensed state to transcriptionally accessible state
  • mechanisms: Histone modification or DNA methylation
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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
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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
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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
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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
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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
  1. covalent histone modifications through histone modifying enzymes
  2. nucleosome remodeling by ATP dependent chromatin remodeling complexes
  3. histone chaperones mediated nucleosome removal
  4. 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
  1. histone modifying enzyme marks certain H3 or H4 histones with specific modification
  2. heterochromatin proteins bind to modified H3/H4 histones
  3. histone modifying enzyme binds to heterochromatin region, ensuring modification is maintained
  4. When chromosome is replicated, marked histone H3/H4 of parent chromosome distributed randomly into two daughter strands = mixture of old and new nucleosomes
  5. In heterochromatin, histone modifying enzymes bound to old nucleosomes rapidly mark new nucleosomes = new binding sites for heterochromatin proteins
  6. heterochromatin proteins can bind to each other, further promoting assembly of protein polymer along chromosome
  7. 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
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
26
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
27
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
28
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
29
Q

DNA methylation

transcription repression

methods

A
  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
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

31
Q

Epigenetic mechanisms

Transcription activation

A

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
Q

Epigenetic mechanisms

DNA / Histone Methylation Interactions

A
  • 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

Genomic Sequencing Technologies (22 decks)

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