p53 GOF paper lectures Flashcards
what is the p53 paradox?
- p53 looks like a tumor supressor (cancer when deleted)
- p53 looks like an oncogene (mutated p53 leads to gain-of-function and cancer)
- p53 could be either a TSG or an oncogene
what explains the mutation spectrum of p53 (i.e. the fact that we see lots of missense mutations, which is uncommon in cancer-causing genes)?
Dominant/negative mutations – p53 is a tetramer and only a full WT complex can have proper function (each subunit must be able to seperate bind DNA)
deletion that leave the protein “null” will have no significant effect since will not be able to oligermize.
most mutations seen in p53 map to which regions?
DNA-binding domain (and are missense mutations)
are the same mutations found in all types of cancer?
no, different mutations are found in different cancers (if each mutation site had equal chance/same chnace between different cancer types, the prevalance should be the same, but it is not what we see)
most common p53 mutation in colon cancer?
R175H/C
contact mutant
most common p53 mutation in pancreatic cancer?
R175H
contact mutant
most common p53 mutation in CNS cancer?
R273C
conformation mutant
most common p53 mutation in NSCLC?
R273L
conformation mutant
what happens in this situation:
Addition of mutant p53 in mouse cell line lacking WT p53.
and why is this weird?
Cause transformation (i.e. cancer) but only in those lacking WT p53.
Weird since if these mutantions were in fact “contact mutants” they should not be able to bind to DNA and cause an abnormal function, but they do.
what happens in this situation:
Addition of mutant p53 in human cell line lacking WT p53.
and why is this weird?
It does not cause transformation.
weird since these same mutants caused transformation in mouse cell lines lacking WT p53 BUT they did score positive in the soft agar assay (so hints towards carcinogenesis potential)
what method could be used to generate mouse models of R270H and R270H p53 mutants to mimic physiological levels?
- make a construct where you make a single mutated p53 allele (“knock-in”) in ES cells (other allele is null)
- insert LoxP sites inside this construct where it would not be expressed in “normal” conditions
- Then, use Cre recombinase to delete the “stop” in between the LoxP sites, leading to expression of mutant allele
what was seen in -/- , 172/- , and 270/- p53 mice?
why do we see this?
-/- : null so no p53
172/- and 270/- : see p53 expression
see this because p53 is expressed and cannot get degraded since it doesn’t bind mdm2
what do we see in mice with these p53 genotypes: +/- , 270H/+ , 172H/+ ?
the mutant ones devlop tumors, as expected since it is dominant negative against the WT, but they develop different types of tumors :
270H/+ : b-lymphoma, carcinoma, adenoma, hemangio
172H/+ : osteosarcoma
Does heterozygous mutant/WT and mutant/null p53 mutants induce expression of its downstream negative regulators?
mutant/null: does not induce p21 and mdm2 in response to DNA damage (mutant p53 not regulated)
mutant/WT: does induce p21 and mdm2 in reponse to DNA damage (still a WT p53 present)
which tumors do 270H/- and 172H/- cause when compared to -/-?
more endothelial tumors in 172H
null p53 does not induce carcinoma and brain but mutant/null does
what evidence is there that p32 is a tumor supressor?
- Loss of heterozygosity at p53 locus prevalent in human tumours
- Expression of wild-type p53 causes cell cycle arrest or apoptosis
- p53 null alleles are recessive in mice
what evidence is there that p53 is an oncogene?
- mutation acts in a dominant fashion (in cells)
- mutant forms transform cells in concert with oncogene
- protein is over expressed in human tumors
- different alleles cause different types of cancers (mice)
- different alleles are enriched in specific cancers (humans)
what is the function or product of point mutations in p53?
- mutations function as dominant negatives
- mutant forms have gain-of-function properties
- mutant expression in p53 null cells confers new properties
- mutant p53 does not activate p53-target genes
How does chromatin immunoprecipitation work?
- Crosslink proteins to DNA (formaldehyde -5min)
- Lyse cells and break up DNA (sonication -3x15sec)
- Immunoprecipitate protein with antibody (or use some other affinity method)
- Purify DNA protein with antibody (decouple protein)
- Analyze DNA (PCR arrow, massove parallel sequencing)
- Map the sequences to the genome
What p53 ChIP results were seen in these cell lines:
- WT
- R273H
- R248Q
- R249S
- WT: p53 boudn to normal, WT sites
- R273H: did not bind to most WT sites, but do bind DNA to new sites, which WT p53 does not bind to
- R248Q: same thing here
- R249S: same thing here
Which TF binding motifs were found in WT and mutant cell lines?
WT: MEME/TomTom, which is expected
Mutant: also MEME/TomTom, but also new motifs (ETS family proteins, SeqPos)
do R273H mutant p53 cells bind ETS?
yes, Flag tage and HA antibody
how did GO analysis differ between WT and mutant p53 cell lines?
WT: as expected, high apoptosis/DNA damage stuff
Mutant: genes regulating translation and histone methylation (specifically saw MLL1 and MLL2 that were interesting)
what does MLL1 do?
it is a methylatrasnferase that does H3K4me3
promoter gene activation
