Lecture 3 Flashcards
What is the role of the Homeobox (HOX) genes?
Transcription factors that control segmental patterning during development. Only one of the HOX genes is turned on in each segment and lie on the chromosome in the same order as the body segments
What type of mutant arise due to issues with the HOX genes?
Homeotic mutants - alterations in cell fate due to Hox loss of function or deregulation og Hox gene expression
Give an 2 example Homeotic mutants
antennapedia mutant
Mis-expression of Antp; Antennae to legs transformation
polycomb mutant
Sex combs on males 2nd and 3rd legs, rather than just their 1st leg
What two groups of proteins maintain Hox gene expression?
Polycomb proteins; maintain repression of other Hox
Trithorax group; maintain activation of one Hox gene
Which organisms have Polycomb genes?
Found across species and kingdoms
What are the the two principal classes of polycomb protein?
Polycomb repressive complex 1 and 2 (PRC1 & PRC2)
What are the functions of the different proteins that make up PRC2?
1 methyltransferase
1 protein-protein interaction WD40 protein
1 histone binding protein
1 Zinc finger: DNA binding protein
What is the role of PRC2?
Adds methyl group to form H3K27me3 which is a repressive mark
Why does PCR1 require PRC2 activity?
Chromodomain of PRC1 binds to the repressive mark added by PRC2
What are the functions of the different proteins that make up PRC1?
1 chromodomain to bind to repressive mark
1 Zn finger domain to bind DNA
1 E3 ubiquitin ligase for the mono-ubiquination of H2AK119
1 Ring finger for the ubiquitination
How can PRCs inhibit transcription?
1) physically block RNA PolII
2) H2AK119 uniquitination
3) Chromatin compactors
4) Recruitment of DNMTs
How are PRCs targeted to the correct gene in Drosophila?
Polycomb response elements (PRE’s) found in the promoters of PRC regulated genes. No equivalent seen in vertebrates or plants
How are the PRCs targeted in vertebrates and plants?
Likely via DNA binding proteins and/or non-coding RNA
What role does HOTAIR play in vertebrate HOX gene expression?
Recruits PRC2 to add repressive H3K27me3 mark and KDM1A-coREST-REST to remove H3K4me3 active marks
Which Hox cluster transcribes HOTAIR? Which Hox cluster does it work on?
Transcribes from HoxC
Acts on HoxD
How was HOTAIR’s interaction with PRC2 (methyltransferase component -EZH2) and KDM1A shown?
1) Immunoprecipitate these proteins assess for HOTAIR presence using rtPCR (with U1 as the negative control- to ensure the proteins are not just non-specific RNA binders). Found that HOTAIR was pulled out with both proteins.
2) Biotinylate HOTAIR and use the biotin label to purify the RNA and its associated proteins. Perform western blot for proteins of interest. Showed interaction with EZH2, LSD1, CoREST and REST.
How was interaction between EZH2 and LSD1 shown?
Immunoprecipitate of one and check for association with other using western blotting (and vice versa), showed interaction that was lost if HOTAIR was knocked out using siRNAs
Give an example of when HOTAIR is highly upregulated
In Metastatic breast cancer
ChIP analysis showed that the over expression of HOTAIR lead to altered PRC2 occupancy - sometimes in genes that inhibit breast cancer progression
What are the biochemical functions of the Trithorax group (trxG) proteins?
Chromatin remodelling
Histone methyltransferations
Acetylations
How do TrxG proteins antagonize the action of the PcG complexes?
Acetylate H3K27 to stop its being methylated by PRC2
Methylated H3K4 and 36 to stop them being ubiquitinated by PRC1
How can TrxG complexes be recruited to target sites?
Interactions with unmethylated CpG-rich sequences via zinc-finger domains
Interactions with transcription factors or polymerase associated factors (PAFs)
non-coding RNAs and adaptor proteins
Outline an example of recruitment of TrxG complexes by non-coding RNAs
HOTTIP is expressed from the HOXA cluster and acts as a scaffold to coordinate activation of HOXA9-13. HOTTIP interacts with WDR5 to recruit MLL1 (a TrxG complex) to methylate H3K4me, an active mark, and loops the chromosome to bring all four HOX genes under its regulation together to allow MLL1 to access them .
How has the chromosome looping due to HOTTIP been shown?
Using 3C - shows that when HOTTIP removed chromatin no longer loops together
What do stem cells need to be capable of?
1) Self renewal
2) Differentiation down different pathways
Where do embryonic stem cells originate from?
Inner cell mass of pre-implantation blastocysts
What are the stem cell “master regulators”?
Oct4, SOX2 and Nanog
What does transcriptional profiling tell you? What can’t it tell you?
Can tell you which genes are on/off but not why they are - is it active repression or lack of activation?
How can you assess the global chromatin status of ES cells?
1) Immuno-labelling of chromatin proteins
2) FRAP
3) ChIP
What cells were used Meshorer?
Neural progenitor cells (NPCs)- created by depleting leukemia inhibitory factor of embryonic stem cells and the ES cells themselves
How did Meshorer stain the DNA?
What markers were used for ES cells and NPCs?
How was heterochromatin stained?
How were the repressive marks stained?
- DAPI to stain the DNA
- OCT4 for ES cells and Nestin for NPCs
- HP1 - a heterochromatin associated proteins
- H3-triMEK9
What was different about the heterochromatin between the ES cells and NPCs?
More heterochromatin in the NPCs
What was different about the repressive marks between the ES cells and NPCs?
Increase in the repressive marks in the NPCs
What histone modifications were seen more in the ES cells?
Acetylation - an active mark
Outline FRAP
Add fluorescence mark onto protein of interest. bleach, destroy proteins, a region of cell with a laser. Measure how quickly fluorescence is recovered
What did Meshorer’s FRAP show?
ES cells recover with fluorescence faster, as the chromatin proteins are more loosely bound and hence hyper-dynamic
Outline ChIP
Crosslink DNA and proteins and then shear the crosslinked product. Immunoprecipitate proteins of interest with associated DNA.
Reverse cross linking.
Then qPCR the DNA, use microarray on DNA or sequence the DNA
What did ChIP show about histone modifications of ES cells?
ES cells have bivalent chromatin in the promoters of some genes; are high acetylated at H3 and H4 and highly methylated at H3K4, active marks, but also highly methylated at H3K27, repressive mark
Outline the chromatin profiles in terms of developmental regulator genes in ES and lymphocyte cells
Early embryonic genes expressed in ES and off in lymphocyte
Haematopoietic genes poised in ES and expressed in lymphocyte
Neural-associated genes poised in ES and off in lymphocyte
Expressed = only activating marks
Off= Only repressive marks
Poised= Bivalent chromatin
What role does the PcG proteins play in ES cells?
PcG mutants can still self-renewal and express Oct4, Sox2 and Nanog.
Lack of EED, mammalian PRC2 component, reported to be associated with spontaneous ES differentiation.
More evidence that PcG proteins are required for correct differentiation and lineage commitment
What is relationship between TrxG proteins and ES cells?
Gene expression profiles by microarray show that as cells differentiate TrxG member WDR5 expression decreases
What role does WDR5 play in Es cells?
Loss of WDR5 causes loss of active mark and loss of stem cell self-renewal, as can no longer interact with Oct4 to keep Oct4/Nanog expression on
