Polycomb group genes and cellular memory

Question 1

What are polycomb group genes (Pc-G)?

Answer 1

Genes encoding transcriptional repressors of homeotic genes.

Question 2

How were Pc-G discovered?

Answer 2

Screening of developmental Drosophila mutants that had extra sex combs.
Phenotypes caused by mis-expression of homeotic genes.

Question 3

Where are sex combs usually found?

Answer 3

Only on the T1 leg.

Question 4

Where are sex combs found in Polycomb mutants?

Answer 4

In the T1, T2 and T3 legs. Due to expression of a homeotic gene usually only on in T1.

Question 5

Are Pc-G mutants homozygous or heterozygous?

Answer 5

Heterozygous; only slightly reduced function.

Question 6

What happens in homozygous Pc-G mutants?

Answer 6

The organism dies very early in development. The embryo exhibits extreme homeotic transformations - all segments have posterior segment identity (homeotic genes expressed more anteriorly than usual).

Question 7

Why may sex comb mutants struggle in drosophila courtship?

Answer 7

• May be rejected by females who detect abnormal sex combs with their mechanosensory organs.
• Can’t grasp the females’ genitalia properly.

Question 8

Are Pc-Gs needed for initiation of homeotic gene expression patterns?

Answer 8

No - they only maintain the patterns initially set up by transient factors. Homeotic gene expression patterning in mutants is initially normal then is lost.

Question 9

Which gene groups provide an epigenetic memory of homeotic gene patterning?

Answer 9

• Pc-G genes maintain repression.
• Trx-G genes maintain activation.

Question 10

What is the function of Pc-G genes?

Answer 10

Encode a variety of different proteins that form complexes together that repress expression. Knocking out any gene inhibits complex’s function.

Question 11

Which polycomb repressive complex is best conserved?

Answer 11

PRC2 (Polycomb repressive 2).

Question 12

How many core proteins are in PRC2?

Answer 12

4.

Question 13

Which protein is the engine of the PRC2 complex?

Answer 13

E(Z) - enhancer of zest.

Question 14

Which domain confers the E(Z)’s histone methyl transferase activity?

Answer 14

The SET domain.

Question 15

Under what condition does E(Z) have histone methyl transferase activity?

Answer 15

When in complex with the other PRC2 proteins. They stimulate proper folding.

Question 16

Where is the methylation by E(Z) carried out?

Answer 16

H3K27. Repressive and stable.

Question 17

How does H3K27 repress transcription?

Answer 17

Bound by readers e.g. polycomb (PRC1). Has a chromodomain for methylated histone tail binding. Readers recruit chromatin remodellers.

Question 18

How was polycomb methylation of H3K27 proven in vitro?

Answer 18

When K27 was mutated to R, no methylation of the histone tail occured. It could still be methylated when other residues were mutated.

Question 19

How was polycomb methylation of H3K27 proven in vivo?

Answer 19

Antibodies against H3K27 showed that this methylation was present in WT flies but not in E(Z) ts mutants.
ChIP-seq further proved the methylation at this site.

Question 20

What is an example of a plant’s epigenetic memory of a transient environmental signal?

Answer 20

Vernalization.

Question 21

What is vernalization?

Answer 21

The exposure of a plant to cold during germination to accelerate flowering when the plant is returned to warmth.

Question 22

What makes vernalization an epigenetic switch?

Answer 22

It causes a mitotically inherited change in gene activity without changing the DNA sequence. This change can be reversed. The fact that there is separation between the signal and the effect (doesn’t flower in winter) shows cellular ‘memory’.

Question 23

Which plant requires vernalization AND long days (16 hours) in order to flower?

Answer 23

Henbane.

Question 24

How was memory of vernalization proven in Henbane?

Answer 24

Plants that were vernalized still flowered when transferred to long days, even after all the vernalized tissue had been replaced by new tissue. Plants in the same conditions that weren’t vernalized did not flower.

Question 25

How does vernalization induce flowering in Arabidopsis?

Answer 25

Cold reduces the expression of FLC. FLC is an inhibitor of flowering. Therefore cold induces flowering.

Question 26

Why do vrn mutants not respond to vernalization?

Answer 26

There are issues with the memory - FLC does not remain repressed when plant is transferred to warmth.

Question 27

What are the VRN2 proteins required for?

Answer 27

Memory - stable repression of FLC.

Question 28

What do VRN2 genes encode?

Answer 28

Elements of the PRC2. Pc-G mediated changes in chromatin structure may provide the mechanism for the cellular memory of vernalization.

Question 29

How is FLC repressed in the cold?

Answer 29

H3K27me3 by VRN2. Much less methylation in vrn mutants.

Question 30

Is vernalization quantitative?

Answer 30

Yes - longer vernalization means faster flowering (inverse relationship).

Question 31

How is vernalization quantitative if methylation is an on off switch?

Answer 31

FLC expression is proportional to the length of cold treatment. The longer the cold treatment lasts, the more cells in the plant have FLC switched off (‘digital model’).

Question 32

What is an incorrect theory on why vernalization is quantitative?

Answer 32

Cells in different plants are able to stably inherit different FLC expression levels.

Question 33

How are epigenetic modifications removed when gametes are produced (FLC switched back on)?

Answer 33

• ELF6 protein (related to jumonji proteins) erases the methylation marks at H3K27. Doesn’t happen in elf6 mutants, and marks are inherited between generations. Ectopic expression of ELF6 reduces H3K27 methylation.
• Histone H3 is replaced by HTR10 protein during pollen development; is not methylated by PRC2.