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L3 Epigenetics > Polycomb and trithorax proteins > Flashcards

Polycomb and trithorax proteins Flashcards

1
Q

What are the Hox gene complexes? (6)

A
  • Homeobox genes
  • Antennapedia complex and bithorax complex
  • Specify positional identity along the anterior/posterior axis
  • Regulatory elements are linked to the genes to cause expression of each one in the right place
  • In the same order on the chromosome as they appear on the body (Hox gene complexes show collinearity in their organisation and domains of gene transcription)
  • Major loss of function mutations in these genes can be embryonic lethal
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2
Q

What are the genes within the drosophila Hox gene complexes? (8)

A
  • ANT-C: labial, proboscipedia, deformed, sex combs reduced, antennapedia
  • BX-C: ultrabithorax, abdominal A, abdominal B
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3
Q

What is the phenotype caused by antennapedia mutation?

A

Changes antennae into legs

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4
Q

What is the phenotype caused by ultrabithorax mutation?

A

Extra thorax region so there are 4 wings

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5
Q

What is the phenotype caused by sex combs reduced (Scr) loss of function (but viable) mutation?

A

Inhibits sex comb development on first pair of legs

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6
Q

What is Scr?

A

Sex combs reduced

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7
Q

What is the phenotype caused by enhancer of zeste (E(z)) mutation?

A

Sex comb development on all 3 pairs of legs

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8
Q

What group of genes does E(z) belong to?

A

The polycomb genes

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9
Q

What is the normal function of polycomb genes e.g. E(z)?

A

Meant to repress expression of hox genes in regions where they aren’t meant to be expressed e.g. E(z) represses Scr in the other legs

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10
Q

What is the hypothesis about the PcG phenotype?

A

PcG phenotype (extra sex combs) is caused by depression of Scr (hox gene causing normal sex comb development)

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11
Q

What are PcG genes?

A

Polycomb genes

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12
Q

What is the effect of E(z) deletion? (3)

A
  • Embryonic lethal
  • Each segment usually has characteristic shape and denticle pattern
  • E(z) deletion: loss of segment identity, each segment transformed into most posterior 8th abdominal segment because loss of ability to constrain Hox gene expression to correct segment so full expression of all Hox genes in every segment
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13
Q

What is the phenotype caused by extra sex combs and polycomb mutations?

A

Derepression of abdominal B so expression isn’t constrained to posterior embryo

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14
Q

What complexes are formed by polycomb group proteins? (3)

A
  • PRC1: recognises methylation mark made by PRC2 (‘read’)
  • PRC2: includes E(z), responsible for methylation of core histones (‘write’)
  • PHO-RC: recruitment of all elements to the polycomb response element (PRE)
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15
Q

What are polycomb response elements (PREs)?

A

Regulatory sites that mediate silencing of Hox genes

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16
Q

Which complex contains E(z)?

A

PRC2

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17
Q

What is encoded by E(z)?

A

Histone methyltransferase for H3K27 methylation

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18
Q

Which chromosome contains the Hox genes in drosophila?

A

Chromosome 3

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19
Q

What is collinearity?

A

The order of the Hox genes on the chromosome coincides with the order of domain of expression

20
Q

How is the homeotic complex conserved in humans? (3)

A
  • Humans have 4 hox gene clusters with the same collinear structure and expression across A/P axis
  • Also regulated by polycomb genes
  • Spine has different sections
20
Q

What are the human orthologues of the polycomb genes?

A

Phc1 and Phc2

20
Q

What is the impact of loss of function mutations in Phc1 and Phc2? (3)

A
  • Extra rib on the cervical vertebrae
  • Most posterior thoracic vertebrae has lost 1 or 2 ribs
  • Most posterior vertebra gained characteristics of the most anterior thoracic vertebra, the most posterior thoracic vertebra gained characteristics of the most anterior lumbar vertebra = posterior shift in the vertebral identity
21
Q

What are the critical components of PRC1?

A

Polycomb (Pb)

22
Q

What are the critical components of PRC2? (2)

A
  • Extra sex combs (Esc)
  • Enhancer of zeste (E(z))
23
Q

What does PHO-RC stand for?

A

Pleiohomeotic repressive complex

24
Q

What is the SET domain? (3)

A
  • Catalytic core for methylation which is conserved in Su(var)3-9, E(z) and Trx
  • Different SET domains catalyse methylation of different lysine residues within H3 and H4
  • Su(var)3-9 is H3K9, E(z) is H3K27, Trx is H3K4
25
Q

What is Trx?

A

Trithorax

26
Q

How could you visualise methylation?

A

Use a fluorescently labelled antibody which is specific for methylated H3K27/K9/K4 whichever you’re looking for

27
Q

What is the function of Esc? (2)

A
  • Esc accompanies E(z) in the PRC2 complex
  • Recognises the H3K27 methylation mark made by E(z), surrounds H3K27me3 creating an aromatic cage which binds H3K27me3
28
Q

What is the significance of Esc interaction with E(z)? (3)

A
  • Allows creation and propagation of H3K27me3 modification in chromatin
  • E(z) creates H3K27me3, recognised by Esc which repositions E(z) so it can methylate the adjacent nucleosome and so on
  • PRC2 creates and propagates H3K27me3, recognised by PRC1
29
Q

What is the impact of Su(var)3-9 mutation on wn4 white locus inversion mutation? (3)

A
  • wm4 causes repositioning of white locus into heterochromatin, no longer expressed, eye appears white instead of red
  • Su(var)3-9 mutation rescues the phenotype, eye appears red again
  • Su(var)3-9 is a H3K9 methyltransferase which allows HP1alpha dimer to bind to adjacent methylated nucleosomes, causes chromatin condensation into heterochromatin
30
Q

What is the action of PRC1 complex components? (2)

A
  • Polycomb (Pb) protein contains a chromodomain which is specific for H3K27me catalysed by E(z)
  • PRC1 and PCR2 complex components shown to all colocalise with H3K27me in drosophila
31
Q

What are the polycomb response elements (PREs)? (2)

A
  • Cis acting sequences mediating transcriptional repression of polycomb group proteins on target genes (Hox genes)
  • PHO-RC encodes DNA binding protein with zinc finger domain which is responsible for sequence specific interactions with PREs to recruit PRC1 and PRC2 complexes to DNA
32
Q

How are PREs defined experimentally? (5)

A
  • Put bithoraxoid PRE upstream of lacZ reporter under the control of Ubx promoter, insert into drosophila
  • Detect lacZ protein as brown signal with anti-beta-galactosidase antibody
  • Normal: expression repressed in anterior
  • Polycomb mutant: loss of anterior repression
  • PRC1 and PRC2 components bind H3K27 with strong localisation to PRE sequences
33
Q

How does PHO-RC recruit complexes to the PRE? (2)

A
  • Pho encodes DNA binding protein with zinc finger domain specific for PRE sequences
  • PHO-RC complex interacts with Scm which brings together PRC1 and PRC2
34
Q

What is encoded by Trx?

A

Histone methyltransferase for H3K4

35
Q

What is the phenotype caused by Trx mutations?

A

Loss of sex combs on first set of legs (opposite to polycomb mutant)

36
Q

What is the normal function of Trx?

A

Promotes homeotic gene regulation e.g. Scr, positively regulating sex comb development

37
Q

What is the phenotype of a double mutant for loss of function of polycomb and Trx? (2)

A
  • Fly looks wildtype
  • Effect of polycomb mutation (extra sex combs on second and third leg) is neutralised by Trx loss of function
38
Q

What is the normal effect of polycomb proteins on trithorax proteins? (2)

A
  • Polycomb proteins counteract Trx proteins, prevent ectopic Trx expression, so aren’t needed in Trx LoF mutant (i.e. double mutant looks wildtype)
  • Polycomb H3K27me restricts trithorax H3K4me
39
Q

What is the structure of Trx proteins? (4)

A
  • Highly conserved gene family
  • C-terminal SET domain (catalytic methylates H3K4)
  • Zinc finger domain specific for methylated H3K4 residues
  • Trx is a H3K4me2,3 write and reader
40
Q

How do PRC1 and PRC2 counteract Trx function?

A

Prevent Trx performing H3K4me and recruiting CBP in target gene promoters

41
Q

What is CBP? (2)

A
  • CREB binding protein
  • Histone acetyltransferase
42
Q

How is the anterior boundary of Hox gene expression determined?

A

Polycomb proteins recruited to the PRE repress Hox gene transcription anteriorly to determine normal position of each anterior boundary

43
Q

What is the function of Trx protein? (3)

A
  • H3K4 methyltransferase which binds to CBP which causes histone acetylation (e.g. H3K27ac)
  • Opposed by H3K27 methylation activity of PRC2
  • Polycomb proteins antagonise Trx functions to repress Hox gene expression, unclear how polycomb functions are normally overridden to enable anterior Hox gene expression

L3 Epigenetics (15 decks)

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