Polycomb and Trithorax Group Genes

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  • Created by: Sarah
  • Created on: 23-05-19 18:49
how are hox complexes organised in drosophila?
organised along the AP axis in the order they're expressed
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how are the hox gene complex ANT-C and bithorax complex expressed?
they show colinearity in their organisation and domains of gene expression
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what happens in a loss of function mutation in sex combs reduced?
inhibit sex comb development on the first pair of legs
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who was one of the first people to use genetics to study developmental processes?
Eb Lewis- studied dosophila, many studies on homeotic genes
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what are homeotic genes?
genes that encode TFs with a homeodomain that organise the body plan of the fruit fly and define the AP identity acorss the animal
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what do hox genes give?
individual segmentation appendages like legs, halteres, winds and antennaes their specific characterstics
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what did Lewis find?
identified genes in the antenepedia and bithorax complex that make up the major hox clusters in flies
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what half of the animal is bithorax expressed in?
the anterior half of the animal
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where is antepedia expressed?
very restricted area in the middle
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what is colinearity of these hox genes?
the structural collinearity between the structural organisation of these complexes of genes and their expression domains show this collinear relationship
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what are sex combs used for? what happens in the sex comb mutant?
sex combs are small appendages on the forelegs of the male- used in mating, sex combs reduced = lose sex combs
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what do LOF mutations in enhancer of zeste cause?
extra sex combs on mesothroacic (2nd) and metathroacic (3rd) legs when normally only on 1st legs
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what happens in the absence of sex combs reduced?
no sex combs
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what did they find in producing extra sex combs on legs that don't normally have them?
several classes of genes produce this- when mapped these mutations were NOT in the hox gene cluster
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why is maternal loss of Enhancer of zest lethal to the larva?
the posterior transformation of the larval segment to the most posterior 8th abdominal segment and anterior expansion of hox gene expression
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why did it get called the polycomb gene family?
mutations producd more sex combs on the legs
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what did they start to make?
homozygous flies to look at he function of these genes as they wiped out most of the function
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what did they see when they made homozygous flies?
the segmental character of each segment was transformed- denticle belts distinct pattern each segment has a denticle belt used to crawl
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what happens to the denticle belts as you go more anteriorly?
the segmental denticle belts get thinner so each segment has a specific character of denticle pattern
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what happened to denticle belts in polycomb mutants?
they are the same in every segment = all resemble the 8th abdominal segment denticle pattern = like all segments become the most posterior one
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what is the explanation for this?
in all of the segments you now have the expression of many hox genes so posterior segmental identities are determined by the max combo of hox gene expression- most hox genes expr in posterior segment
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What happens to hox gene expression as you move towards the anterior of the larva?
you get fewer and fewer hox genes expressed in those anterior domains
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ISH for ultrabithorax show?
it is expressed in the posterir half of the embryo
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what happens to the domain of expression of ultrabithorax in enhancer of zeste mutant?
domain of expression of ultrabithorax is expanded anteriorly and this is true for other hox genes and polycomb mutants
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what other genes were identified to have this extra sex combs (polycombs) phenotype?
extra sex combs
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what is the nromal expression of Abd-B hox gene in the fly
very posterior restricted
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what happens in the extra sex combs mutant?
global derepression of this hox gene = spreads throughout the animal
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what happens to Ab-d expression in the polycomb mutant?
wider expression domain more anteriorly so these polycomb mutants that produce extra sex combs on M adult legs transform the larval segmental character to a more posterior one
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what happens to all segments in polycomb mutants?
all segments become posterior
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why do they all become posterior?
because expression of homeotic genes (Ab-d/a/b and ultrabithorax) is no longer narrowly defined to it's domain they're widely expressed throughout
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what did genetic analysis reveal from these studies?
there's about a dozen diff polycomb group genes- polycomb, polyhomeotic, posterior sex combs all discovered by the extra sex comb phenotype
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what do the polycomb genes encode?
proteins that operate in complexes that restrict the hox gene expression domain to a particular part in the embryo = silences genes as contain histone lysine methyltransferases
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what happens in the absence of polycomb genes?
domains of expression are broad and spread out
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what are the 3 polycomb complexes?
PCR1 + PRC2 and PcG DNA binding proteins (DNA binding complex)
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what do these 3 complexes work together to do?
carry out bicohemical functions to establish transcriptionally repressed and silenced chromatin
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what happens when thee genes in these compelxes are mutated?
derepression of hox genes -> gives mutant phenotypes
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what regulates AP patterning of the vertebral column?
Hox genes
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how do we know core polycomb group proteins are highly conserved in other organisms?
almost all of those genes have vertebrate homologues that play roles in the patterning of the vertebral primary axis- set expression domains of hox genes in vertebral column and SC eg cervical or thoracic
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what does the conservation of hox complex colinear structure and expression pattern across AP of all metazoa imply?
conservation of regulatory mechanisms governing hox gene transcription
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how many hox clusters in mouse? what are they?
Hox D/C/A/B - 4 clusters
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the genes towards the end of each segment are expressed where?
in the posterior segments of the axial skeleton- set expression pattern exists in SC and brain
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so we know that hox genes are conserved what else is conserved?
polycomb genes
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what happens if you mutate vertebrate polycomb genes?
you see posterior transformations of vertebrate and skeletal elements
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what genes did they look at int he mouse tgat are 2 closely related homologues of a fly gene?
polyhomeotic 1 + 2 (phc1+phc2)
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what happens in an animal that is homozygous for a mutation in one of the 2 phc genes?
cervical vertebrae puts out ribs- remove phc2 or phc1 function then see lateral projections coming off which are similar to ribs- make it look like a thoracic vertebra
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how many ribs are missing in the animals that lack phc1 function?
one pair of ribs missing on the ribcage- unilateral rib 14
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what does it seem like this is doing?
seems like this final spinal segment of the vertebral column has been instructred to become a vertebrae within the lumbar region which is a ribless region
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what do polycomb group proteins do and how?
transcriptionally silence PcG target genes (hox genes) by forming distinct multisubunit protein complexes (PRC1 + PRC2)
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what do mutations in the vertebrae orthologues seem to be doing?
mutations in vertebrae orthologoues of the polycomb genes seem to confer posterior character to anterior segments
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what is enhancer of zeste part of?
polycomb repressive complex 2 (PRC2)
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what proteins are always seen together?
PCR1 + 2
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what contains the polycomb protein?
PRC1
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sec combs protein seems to do what?
make interactions with all or at least of these complexes
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whats the 3rd complex?
PhoRC- DNA binding bit
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enhancer of zest sequence has what?
extended region of sequence similarity at the end of its encoded protein, lst 20% of C terminus is highly conserved and related to a number of genes found in C elegans called MES2
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what is the vertebrate homologue of enhancer of zest?
Enx1
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what does Enx1/enhancer of zest have homolgy to?
trithorax
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what does enhancer of zeste have a domain very similar to?
a sequence in trithorax
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what has suvar3-9 got to do with this?
encodes a histone methyltransferase specific for Histone H3 lysine 9 methylation- domain was discovered thro this analys
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what is the SET domain common to?
Suvar 3-9, enhancer of zest and trithorax
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what lies within the SET domain?
the catalytic activity of histone lysine methyltransferases
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what is different about suvar3-9, enhancer of zest and trithorax?
they methylate a distinct lysine residue in a specific core histone subunit
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enhnacer of zest and its vertebrate homologues Enx1 and EZH2 methylate what?
histone H3 lysine 27
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what does suvar3-9 methylate?
histone H3 lysine 9
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what does trithorax methylate?
histone 3 lysine 4
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what do all these enzymes do?
carry out distinct methylation events on core histones
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how did find which each of the enzymes was binding to (biochemical experiment)
dros cells cultured in presence of radioactively labelled precursors of mehylation marks (s-adenosyl methionine) then cells were biochemically fractionated 100 diff fractions incubated with s-adenosyl methionine then WB run and autoradiographed
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what was this trying to identify
the fractions of the cell extract that had the triated methyl group from s-adenoysl methionine onto histone H3 (visualise histone H3 because its methylated with this triated methyl group)
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simplified experiment what they did?
dros cells cultured -> extract is made -> extract poured over a hydroxyapatite column to fractionate the proteins -> fraction creates 100 diff fractions that have more/less affinity for column - take each fraction and uncubate with S-adenosyl methion
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what was s-adenoysl methionine incubated with?
histone H3
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what do you do with those extracts with the s-adenosyl methionine and histone H3?
run a WB transfer contents of the gel to a membrane then expose to autoradiographed film and show you which fractions can transfer that traited methyl methyl groups onto histone H3
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when they took the fractions that could transfer S-adenosyl methionine onto Histone H3 and run them on another WB what did they they do?
probed with an antibody for enhnacer of zete or other polycomb family members- see which fractions contain the protein of interest
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what did they dind?
enhancer of zeste was in the same fractions where there was histone H3 methylating ability
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what was there a correlation between?
the presence of histone H3 methylation marks and the presence of enhancer of zeste
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what does it tell you if these proteins are in the same fraction as histone methyltransferase?
likely that these proteins are playing a role in the methylation activity- biochemical evidence for histone methyltransferase activities of these proteins
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what complexes are important in C elegans?
MES complex EXH2 orhologue (enhnacer of zeste orthologue)
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what does loss of MES2 do in C elegans?
reduces H3K27 methylation (obvious because it's the enhancer of zest homologue)
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orthologue of enhancer of zest in C elegans? where?
MES2- in the worm gonads
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why is MES2 (enhancer of zest orthologue) found in worms what does it seem to be important for?
germ cell development- MES2 dfound in the gonads
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what did they see with DAPI (DNA staining)
DNA stained nuclei of worm gonads
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what did they use an antibody against?
antibody with green fluroform against trimethylated lysine on histone H3 = recognises that specific methylation mark, red fluroform antibody against DNA
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What did this show?
colocalisation of trimethyl-lysine with DNA nueclei in the WT worm gonads
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what did they see in the MES2 Mutant?
lots of DNA signal but no trimethyl-lysine signal so this is the mutant in the orthologue of enhancer of zest = proves its capable of methy transferase activity
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how else did they show enhancer of zeste could methylated H3?
biochemical experiment where they fractionated and run WBs with antibody for methylation mark
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what happens in the absence of enhancer of zest function?
the level of tri methylation on lysine 27 is almost completely abolished
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what conclusion did this give?
MES2/enhancer of zest is required for tri methylation on lysine 27
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what does enhancer of zest physically interact with?
extra sex combs/ Eed = the WD40 protein
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what does Esc/Eed create?
an aromatic cage that binds to trimethylated histone H3K27 lysine
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what goes everywhere with enhancer of zest?
extra sex combs- subunit of PRC2
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what kind of protein is extra sex combs?
a beta propeller protein- creates an aromatic cage in which that trimethylated H3 lysine 27 fits snugly
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so how does enhancer of zeste and extra sex combs work together?
enhancer of zest makes the lysine 27 methylation marks and extra sex combs recognises them marks
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Ez and Esc do what?
allow a region of chromatin to be progressively methylated- once you've you've recruited this complex to a doamin Ez methylates it and Esc this beta propeller protein can bind to it + positions Ez ready to methylate the next histone H3 in next nucleu
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its a way of generating what?
a localised focus of trimethyl lysine through the paired activity of these 2 proteins
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so what does PRC2 do?
has Esc and Ez- makes marks and propagates it in chromatin
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what does PRC1 do?
recognises that trimethyl mark
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how does PRC1 recognise that trimethyl mark on lysine 27?
has a chromodomain
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what did we talk about the chromodomain before with suvar 3-9?
suvar 3-9 methylates lysine 9 and that creates a binding site for HP1 alpha which sits in between nucleosomes and constatinos chromatin down
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what does the PRC1 do?
recognises the methylation marks using its chromodomain, sits in between nucleosomes and condenses chromatin so its silenced through constantining it down = creates condensed chromatin
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what did the chromatin immunoprecipitation data show?
across dros chromosome 3 distribution of methyl lysine- enhancer of zest has a distribution that perfectly matches lysine 27 methylation as does PRC1 which recognises H3K27me - writers+readers of lysine 27 marks colocalise across genome
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what colocalisation did posterior sex combs (part of the PRC1 complex show)?
perfect colocalisation acorss a region of condensed chromatin
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how are the polycomb proteins specifically recruited to their target genes?
cis regulatory elements- POLYCOMB RESPONSE ELEMENT -
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what is the polcyomb response element?
a cis acting element that recruits the polycomb repressive complexes to establish a domain of silent chromatin around the hox cluster
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where do you see particuarly high levels of accumulation of methylated lysine 27, polycomb, enhancer of zest and posterior sex combs?
on the polycomb response elements the bxd and ultrabithorax response elements- peak of Ez + post se combs + polycomb activity all colocalising to these discrete cis regulatory elements that have functional activity
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what happens when you hook the polycomb response element up to a promoter and make a transgenic construct?
these transgenic constructs are expressed in a domain with a very discrete boundary of anterior expression that is lost in animals that lack polycomb function
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what is polycomb protein function essential for?
to define the anterior limit of Ultrabithroax expression
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how does polycomb define the anterior limit of Ubx express what's it mediated through?
interactions with polycomb repressive element
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what experiment did they do to show that polycomb is needed to give the anterior limit of Ubx expression mediated by PRE?
had a lacZ transgenic reporter that gives beta galactosidase expression (brown) hooked up to Ubx promoter- with polycomb response elements next to it when you put that construct into WT = get back Ubx expression domain in polycomb mutant
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in a polycomb mutant what happens to the Ubx expression domain
polycomb complex comes into the Bx-d polycomb response element and defining this domain as repressed that anterior domain is repressed but in polycomb mutant there's widespread derepression so Ubx spreads throughout
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what is the molecular basis for targeting polycomb complexes to the polycomb response element?
recuritment of PRC2 and PRC2 requires Pho-Rc (3rd PRC complex)
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what is Pho-RC?
a DNA binding protein (pleiohomeotic)
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what does Pho-RC do?
Pleo homeotic protein interacts with SFMBT which is a connecting partner to recruit PRC1 + PRC2 to polycomb response elements
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what are polycomb response elements?
cis regulatory elements that bind a DNA binding protein component of the polycomb family- ple homeotic thro a series of secondary interactions and it can make these interactions with the enzyme complex that marks the chromatin for condensation
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how were trithorax genes identified?
by a sex comb mutant phenotype
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what happens in a trithorax mutant?
they have less or no sex combs but these mutations not linked to the mex complex
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what must trithorax be?
must be in another set of genes that are interacting with hox genes perhaps regulating their exprssion in an opposite way to polycomb genes
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how is trithorax opposite to polycomb?
polycomb mutant gives extra sex combs because it de-represses hox genes, trithorax genes give extra sex combs because they promote hox genes to generate extra sex combs
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how do you know in the absence of trithorax that you no longer need polycomb activity?
the double polycom/trithorax mutant looks like the WT is normal
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what do you have in the polycomb mutant?
multiple sex combs as psoterior identity as hox genes aren't repressed
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what do you get in the polycomb/trithorax mutant?
those extra sex combs have now gone- normal
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whats the WT expression of sex combs reduced?
imaginal disc of legs lots of expression in 1st leg but little in 2nd and 3rd legs
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what happens to sex combs reduced expression in the polycomb mutant?
ectopic expression of sex comb reduced in imaginal discs in the 2nd and 3rd leg
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what happens to expression in double mutant?
goes back to WT- in the absence of trithorax you no longer need polycomb
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what did this tell us?
that the polycomb proteins are blcoking the activity of trithorax in regions where sex comb reduced are prevented
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what do the function of polycomb proteins counteract the function of
trithorax proteins
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what is another expeirment that drove it home that you dont need polycomb proteins in the absence of trithorax?
enhancer of zest in hoozygous larvae- every larval segment is transformed to the 8th abdominal segment (posteriosed) but in enhacer of zest + trithorax double mutant thw pattern is normal no need for Ez function in the absence of trithorax- acitvitie
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what is trithorax proteins doing?
trying to activate these target genes so is antagonistic to PRC1 and PRC2- to define the domains of hox genes and other genes
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is trithorax a member of a highly conserved t=gene family?
yes
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what does the trithorax family members usually possess?
a PHD zinc finger domain and a C terminal SET domain
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what does trithorax contain?
trithorax is a SET domain containing protein with methyltransferase activity
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what does trithorax methylate?
specific for H3 lysine 4
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What domain is the histone 3 lysine 3 recognised by?
PHD zinc fingers
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how does poycombs work?
Ez catalytic core for lysine 27 methylation and polycomb protein contains a chromodomain that recognises mod (PRC2)
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How does trithorax work?
has its own methytransferase activity and counteracts polycomb (unknown), recognsies through it's PHD zinc finger domain that methylation makrs so both writers and reader in 1 protein
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what is the outcome of trithorax?
transcriptionally activate genes
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what do trithorax and polycomb specify?
the domains of expression of hox genes
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what is the recruitment process for polycomb triggered by?
binding of pleiohomeotic zinc finger containing protein t these polycomb response elements that sit within the hx clusters
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how are the hox gene complex ANT-C and bithorax complex expressed?

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they show colinearity in their organisation and domains of gene expression

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what happens in a loss of function mutation in sex combs reduced?

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Card 4

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who was one of the first people to use genetics to study developmental processes?

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Card 5

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what are homeotic genes?

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