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Bio 205 > Chapter 12 > Flashcards

Chapter 12 Flashcards

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1
Q

Regulatory proteins in eukaryotes

A

BINDING
1. Transcription factors
2. General transcription factors (GTF)

COREGULATORS
1. COACTIVATORS
2. COREPRESSORS

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2
Q

Enhancers

A

transcription factors that directly bind regulatory dna sequences

close to core promoter = part of proximal promoters = proximal enhancers

far from promoter = part of distal enhancers = distal enhancer

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3
Q

GTFs

A

Diretly bind dna reg sequences within core promoters surrounding transcription start sites

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4
Q

Coactivators

A

increase trasncription

binding or enxymatically modifying transcription factors

some = bridge trans factors and RNA pol II
otehrs = alter structure of chromatin

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5
Q

Proximal Enhancer (PE) importance

A

Point mutations in PE and core promoters (CP) reduce trans of B-globin gene

general features enhancers:
- short sequence elements
- mutiple elemnts clustered together
- frquently occur as inverted repeats of same dna (for binding 2 similar/identical trans factors)

  • randomly occur lots in genome
  • not all bound bt trans factors
    (binding often needs partnered trans factors nearby)
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6
Q

C/EBP

A

binds one of proximal enhancer elements in b-globin to CCAAT box

dna binding domain and dimerization domain

activation domain: interacts with other components to turn on trans

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7
Q

dna binding domain

A

site on dna binding protein that directly ionteracts with specific dna sequences

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8
Q

dimerization domain

A

protein region that permits binding between 2 identical or similar proteins

facilitates formation of homodimers
- binding between 2 C/EBP proteins

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9
Q

repression domains

A

turn off transcription

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10
Q

ligand binding domain

A

binds ligand (hormone, vitamin etc)
changes structure of trans factor
activates it

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11
Q

Estrogen example ligand binding domain

A

Estrogen binds Estrogen receptor in cytoplasm
Causes dimerization, nuclear localizatin, binding to enhancer elements (estrogen response elements)

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12
Q

What do all Trans factors contain

A

DNA binding domain and activation/repressor domain

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13
Q

What do only some trans factors contain

A

dimerization
ligand binding domains

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14
Q

yeast GAL system

A

Uses extracellular galactose (gal)
- imports and converts it into glucose

gal1,2,7,10 encode enzymes for this
gal3,4,80 encode regulatory proteins

Triggered in presence of galactose
Same as lac operon

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15
Q

Gal 4

A

Binds enhancers “Upstream activation sequences”

Sequence specific dna binding protein

PRESENCE OF GAL
mRNA levels of gal1,2,7,10 1000x higher

gal 4 mutants: mRNA levels dont change

each gal gene has 2+ gal-4 binding sites upstream promoter

if inding sites deleted (gal 4 mutation) genes can’t transcribe

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16
Q

Gal 4 in eukaryotes

A

activates trans of UAS genes in insect and human cells

used to manipulate gene expression

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17
Q

Gal 4 independent

A

Has activation domain

Trans factors = modular = independently function

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18
Q

Study of independent Trans factors - gal4

A
  1. fused gal4 activation domains to dna binding domain of Lex A (from e coli)
  2. trans measured via reporter genes with gal 4 sites or lexA sites upstream of promoter and coding region
  3. full length gal4 activated trans when bound to UAS
  4. gal4 dna domain lacking activation domain did not activate trans when bound to UAS
  5. lexA dna domain did not activate from lex A sites
  6. protein fusion of gal4 activation domain and gal 4 binding domains to other activation domain activated trans
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19
Q

modularity of TF

A

cause of some cancers
APL (leukemia)
- chromosome translocation creates gene fusion
- between activation domain PML and dna/ligand binding domains of RARA
- assembles with corepressor proteins INSTEAD OF COACTIVATOR PROTEINS
- blocks transcription of normal RARA gene targets
- they control differentiation of blood cells
- without control, leads to uncontrolled proliferation of the cells

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20
Q

regulation of gal 4

A

regulated by gal80 and gal 3 proteins

gal80 mutants: trans occurs without galactose therefore always one

gal80 inhibits trans

gal3 mutants: trans not active even with galactose

gal3 promotes trans of gal genes

gal 80 = corepressor of gal 4
gal 80 binds gal4 activation domain with high affinity = blocls promotin of gal 4

gal3 releases genes from repression by gal80 when galactose present
- binds gal and atp
- changes structure
- promotes binding gal 80

gal 80,3,4 all part of switch

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21
Q

cell type specific regulation - mating in yeast

A

yeast can exist in 3 cell types
a, alpha, a/alpha
a and alpha are haploid - one of each chromosome
a/alpha is diploid
differentiate cells by mating type

alpha mates only with a
a mates only with alpha

both secrete phermones to arrest the other in cell cycle

both must be arrested for mating

a/alpha does not mate and does not repsond to the hormones

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22
Q

alpha factor

A

sex hormone of alpha cells

arrests a cells in cell cycle

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23
Q

a factor

A

sex hormone of a cells

arrests alpha cells in cell cycle

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24
Q

MAT locus

A

mating type locus
single genetic locus
controlls mating

2 alleles of MAT

alpha cells have MATalpha allele
a cells have MATa allele

a/a cells have both

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25
Q

How yeast switches mating types

A

Homologous recombination event
Replaces one MAT allele with the other

alleles activate differnet sets of genes
(encode diff trans factors)

MCMC1 - trans factor encoded by MAT - regulates cell type

MATalpha: encodes a1 trans factor
a1 no effect in haploid cells
MCM1 turns on expression of structural genes by binding enhancers

Alpha cells: alpha structural genes need MCM1 prevented from activating a specific genes

MATa allele encodes a1 and a2:
a1= activator of alpha transcription
a2= repress transcription of a genes

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26
Q

Diploid yeast cell

A

Both a1 and a2 expressed
Leads to repression of all mating genes

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27
Q

Chromatin Structure General

A

DNA + Proteins = chromatin

  • Histones
  • Nucleosomes
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28
Q

Histones

A

Major protein components of chromatin

H1,2A,2B,3,4

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29
Q

Core histones

A

H2A,H2B,H3,H4

form a complex - DNA wrapped around core histones to form nucleosomes

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30
Q

Linker histone

A

H1

binds dna that links adjacent nucleosomes

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31
Q

Nucleosome

A

basic structural unit of chromatin

Core histones + DNA

Nucleosomes bind together with H1 (linker histones)

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32
Q

Canonical histones

A

package newly replicated genomes (the basic histones)

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33
Q

Variant Histones

A

Variants of canonical histones
Incorporated into nucleosomes in DNA-replication independent manner

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34
Q

Example of variant histone

A

H2A-Z

60% identical to H2A

Replaces H2A in nucleosome at promoters of active and silent genes

H2A-X
Incorporated into nucelsomes at sites of DNA damage

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35
Q

Features of histone proteins

A

Abundant
- 70% protein in chromatin in euks

Core histones = small, basic, positively charged @ neutral pH

36
Q

Determining structure of chromatin

A

Electrostatic interaction between (+) aminos and (-) charged phosphate backbone of DNA

Core histone sequences highly conserved through evolution

37
Q

Importance of conservation of histone in evolution

A

Studies of histones = genetically controllable

  • Yeast and drosophillia –> insights into function of histones in higher euks (humasn)
38
Q

Structural domains of core histones

A
  1. Histone Folds
  2. Histone fold-extensions
  3. Flexible Tails
39
Q

Histone folds

A

Located at central region of histone proteins
3 alpha helices separated by loops
hydrophobic contacts between loops CRUCIAL for pairing of H2A with H2B, H3 with H4

40
Q

Histone fold extensions

A

Contribut e to specificity of histone pairing

41
Q

Flexible tails

A

At ends of histone proteins

Interact with non-histone proteins and nearby nucleosomes

42
Q

Structure of H1 in humans

A

Larger
Greater sequence/structural diversity

3 domains in humans
1. Central
2. Unstructured N terminal tail
3. Unstructures C terminal tail

43
Q

Structure H1 in yeast

A

1 unstructured domain

44
Q

Nucleosomes General

A

DNA wraps 1.7 times around histone octamer

Stability due to…
- interaction with octamer
- electrostatic and hydrogen bonds between histones and dna

Flexible tails: involved in interactions with adjacent nucleosomes and nuclear factors

45
Q

Histone octamer

A

8 histone proteins
2 copies of each core histone

46
Q

Nucleosome formation

A
  1. Assembly of H3/H4 tetramer
  2. Tetramer binds to DNA
  3. Add 2 H2A/H2B dimers
  4. H1 binds
47
Q

Tetramer

A

2 dimers joined together

48
Q

Removal of histones

A
  1. Remove H2 dimers
  2. Remove tetramer
  3. linkers detach nucleosomes from eachother
49
Q

Chromatin folding

A

DNA compacts when wrapped around octamer

Nucleosomes fold on themselves

50
Q

Cell cycle and compaction of nucleosomes

A

Differs in stages of cycle

Mitosis: highly compacted
Interphase: less compacted

Varries within interphase too

51
Q

Heterochromatin

A

More compacted regions of chromatin

52
Q

Euchromatin

A

Less compacted regions of chromatin

53
Q

Constitutive heterochromatin

A

Remains heterochromatin throughout cell cycle

Concentrated at centromeres and telomeres

Rich in repetitive sequences - poor in genes

54
Q

Facultative heterochromatin

A

Does not remain as heterochromatin throughout entire cell cycle

Chromosome arms
Gene rich
Can lose compact structure - can become euchromatin

55
Q

Chromatin Immunoprecipitation (ChIP)

A

technology

Determine genome-wide distribution of nucleosomes

56
Q

Nucleosome free region (NFR)

A

Transcription start side - within 150 bp
Contains promoter
Flanked by nucleosome (-1 and +1 nucleosomes)

Positioning of nucleosome reduced upsteam and downstream of promoter

Enhancers flanked by pair of nucleosomes ar binding sites of TF (these nucs eliminated when trans starts)

57
Q

3d structure chromatin

A

Individual chromosomes
- distinct territories
- gene dense chromosomes near centre of nucleus

  • active chromatin associate with one another
  • Inactive associate with eachother
58
Q

Topologically associating domains (TADs)

A

Genomic regions the contact each other and are bound by proteins

Smaller regions of chromatin organized

Gene enhangers and promoters = TADs

Anchor points for looping out of chromatin have specialized regulatory requences (insulators/boundaries)

59
Q

Insulators/boundaries

A

Anchor points for looping out of chromatin in TADS

Interact with eachother/nuclear envelope through association proteins

Mammals: most bound by zinx-finger DNA binding protein called CTCF

DIVIDE CHROMOSOME SINTO DEFINED LOOPS TO DETERMINE WHICH ENHANCER-PROMOTER INTERACTIONS ARE ALLOWED/PREVENTED

60
Q

Chromatin modification

A

Enzymes alter chemical structure of aminos in histones/nucleotides in dna

Affects recruitment of transcription factors, corregulators, general TF to chromatin

PURPOSE: enable dynamic access of transcription machinery to DNA

61
Q

Chromatin remodelling

A

accessibility of DNA to TF, corregulatos, general TF altered by enzymes

Uses ATP hydrolysis to remodel nucleosomes: reposition octamers, remove octamers, or replace canonical histones in octamers with variants

62
Q

Histone modification

A

Acetylation: post-translational modification
- add acetyl group

Hyopthesized acetylation affected transcription
- acetrylation neutralizes positive charge = decreases affinity = reduce chromatin compaction, increase accessibility of transcription, promote transciption activation

Deacetylation = increase chromatin compaction, promote transcription repression

63
Q

Histone Acetyltransferase (HAT)

A

Enzyme transfers acetyl group from acetyl choline to histones

Provides mechanistsic link between acetylation and trans activation

64
Q

Histone deacetylases (HDACs)

A

repress transcription via removal of acetyl groups

65
Q

Acetylation residues - effects on transcription

A
  1. Less compact chromatin
  2. Creates binding site for protein motif - increase affinity of factor for genes
66
Q

Methylation of lysine and arg

A

1-3 times: lysine
1-2 times: arg

Controlled by writers, erasers, readers

66
Q

Post-translation mods of histone

A
  • Concentrated in tails
  • detected experimentally in vivo and in vitro
  • mass spectrometry
  • can’t detect extent of coexistance on individual histone
  • histone code hypothesis
66
Q

Histone code hypothesis

A

multiple histone mods specify unique transcription outcomes

  • Lysine (K)
  • Arginine (R)
  • Serine (S)
  • Threonine (T)
  • KRST at N terminal flexible tail of H3 are post-translationally acetylated, methylated, or phosphorylated

Trans active genes: usually trimethylated on K4

Promoters of trans repressed genes: trimethylated on K9

67
Q

DNA methylatyion

A
  • reversible methylation of 5th carbon of cytosine (5mC)
68
Q

Detecting dna methylation

A

5mC detectable via sodium bisulfite reaction

  • dna treated w sodium bisulfite
  • converts cytosine to uracil
  • 5mC not converted
  • those changed read as thymine
  • those unchanged (5mC) read as cytosine
69
Q

CpGs in mammals

A

Unmethylated clusters of CpG islands at promoters typically correlated with active trasncription

70
Q

Chromatin Remodelling mutants

A

2 genetic screens in yeast

1) mutants can’t grow on sucrose = sugar non-fermenting mutants (snf)
2) mutants defective for mating type switching (swi)

1 mutant gene caused both phenotypes
- ATPase subunit of SWI/SNF remodelling complex at swi2/snf2 locus

71
Q

Chromatin mediated control of IFN-B transcription

A
  • regulated changes in chromatin structure affect trans in IFN-B gene over 24 hour period
  • in repsonse to virus infection of human cells
72
Q

IFN-B gene in humasn

A

Encodes antiviral protein

Upon infection, it is highly activated

73
Q

IFN-B 24 hours

A

Hour 0 - infection
Hour 3: Histone mods detected (H4K8ac, H3S10P, H3K9ac)

Recruits SWI/SNF, promotes secondary recruiter of TFIID, recruits TFIID

Hour 6:
IFN-B mRNA detected
TFIID detected

Hour 8: H4K8ac and H3S10P detected for last time

Hour 24: Only secondary recruiter of TFIID left

74
Q

Chromatin-mediated trasncription regulation - General

A
  1. Binding sites in chromatin
  • regulatory sequences, histone code, dna mods
  1. Association of proteins with binding site

-Reader proteins bind DNA sequences, histone mods, and dna mods
- Proteins read information

  1. Recruitment of chromatin modifiers
  • Writers, erasers, remodelers in complex with readers OR temporarily associate with readers
  • assemble enzymes to change chromatin structure
  1. Modification of chromatin structure
  • Histone and DNa mods are added and removed
  • Enzymes edit/change info at gene
  1. Altered activity of transcription machinery
  • assemble and function of INIT and ELON factors (includes rna pol ii) are increased or decreased
75
Q

Epigenetic regulation

A
  • cellular memory
  • position effect variegation
  • genomic imprinting
  • x chromosome inactivation
76
Q

Epigenetic inheritence

A

inheritance of information stored within the structure of chromatin through cell divisions

Affects traits of daughter cells without altering dna

77
Q

Cell memory

A

2 groups proteins
- Polycomb: maintain genes in repressed state
- Trithorax: maintain genes in active state

post-trasnlational histone mods

Maintain states of parent cells in daughter cells

78
Q

Position effect variegation

A
  • expression of genes can be silenced when moved
  • chromosomal rearrangement
  • Spreading of heterochromatin to euchromatic regions and vice versa
79
Q

Example of Position effect variegation in drosophillia

A
  • Flies had patches of red and white on eyes
  • region of x-chromosome with white gene was inverted
  • white gene normally in euchromatic region
  • was moved to heterochromatic centromere
  • spread of heterochromatin to white gene = silencing of white transcription in only some cells

-

80
Q

Using PEV for research

A
  • Exploit PEV to identify proteins needed to form heterochromatin
  • isolate mutations
    a) enhanced
    b) supressed variegated pattern

Supressor of variegation: Su(var) = gene when mutated reduces spread of heterochromatin

Therefore, WT of SuVAR needed for spreading

Enhancer of Variegation: E(var) = increases spread of heterochromatin when mutated

Therefore, WT of Evar blocks spreading

81
Q

Genomic Imprinting

A
  • autosomal genes expressed in parent of origin manner
  • Maternal or Paternal copy of chromosome is silenced

Maternal imprinting: maternal is silenced, all transcripts come form paternal allele

Controlled by imprinting control regions (ICRs)

82
Q

Imprinting Example: Mice Insulin

A

Igf2 and H19

Methylated DNA between the 2 genes in male germ cell

Unmethylated in female germ cell

Methylation = Igf2 active, H19 inactive

Only unmethylated can be bound by CTCF

CTCF acts as enhancer-blocking insulator: prvents enhancer activation of Igf2 transcription

83
Q

X chromosome inactivation

A

Silencing of one of 2 x chromosomes in a female to prevent imbalance of transcription of genes on x chromosome

Inactivated = bar body
- dark stained heterochromatic structure in nucleus

Decision of which chromosome is silenced is random but permanent for life

84
Q

Xist

A

Non coding RNA
Initiated silencing of one of two x chromosomes

Tsix = antisense

Tsix expressed from both alleles during embryonic development (both chromosomes active)

Transient pairing of x chromosome represses transcription of Tsix from one, that will be the inactive one

Transcription from “good” allele blocks xist = active chromsome

Xist spreads along future inactive x and induces silencing

Uses methylation, deacetylation, CpG island methylation etc. to keep in a heterochromatin state and prevent transcription

Bio 205 (3 decks)

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