USP1 Deubiquitinase maintains Phosphorylated CHK1 by limiting its DDB1-dependent Degradation...
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Transcript of USP1 Deubiquitinase maintains Phosphorylated CHK1 by limiting its DDB1-dependent Degradation...
![Page 1: USP1 Deubiquitinase maintains Phosphorylated CHK1 by limiting its DDB1-dependent Degradation Jean-Hugues Guervilly, Emilie Renaud, Minoru Takata, and Filippo.](https://reader030.fdocuments.us/reader030/viewer/2022032702/56649cc05503460f94986550/html5/thumbnails/1.jpg)
USP1 Deubiquitinase maintains Phosphorylated CHK1 by limiting its
DDB1-dependent Degradation
Jean-Hugues Guervilly, Emilie Renaud, Minoru Takata, and Filippo Rosselli.
Presented by Stella Snyder
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OutlineIntroduction
◦Checkpoint kinase 1 (CHK1)◦Fanconi anemia, complementation group D2
(FANCD2)◦Ubiquitin specific peptidase 1 (USP1)
Experiments◦USP1 depletion impact on CHK1◦USP1/CHK1 relationship independent of cell
cycle and DNA repair◦USP1 limits DDB1 degradation of CHK1
ConclusionFuture research
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Checkpoint Kinase 1 (CHK1)Controls DNA repair and cell cycle
regulationInvolved in monoubiquitinylation of
FANCD2 and PCNAActivates RAD51Phosphorylation activates CHK1 and
promotes degradation◦S345 phosphorylated form
Associated with resisting anticancer therapy
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FANC pathway15 FANC proteins required for
normal function◦Eight in FANCcore complex that
catalyzes mono-Ub-FANCD2 and –FANCI
◦Mono-Ub-FANCD2 regulates CHK1Fanconi Anemia- Caused by
mutations in DNA damage response genes
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Ubiquitin-specific Peptidase 1 (USP1) DeubiquitinaseRestrains monoubiquitinylation of
FANCD2/FANCIWill be shown to affect CHK1,
steady state and phosphorylated◦With or without FANCD2
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USP1 depletion effect on CHK1
FANCD2 monoubiquitinylation can restrain checkpoint signaling ◦Higher amount of mono-Ub-FANCD2
should mean lower p-CHK1 levelResearchers depleted USP1 to
increase mono-Ub-FANCD2◦Used siRNA in HeLa and U2OS cells◦Higher amounts of mono-Ub-FANCD2
and –PCNA without genotoxic stress
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U2OS cellsUSP1 depletion
shows reduction in CHK1
Co-depletion of USP1 and FANC proteins rescued CHK1
USP1 depletion reduces pCHK1 and steady-state CHK1 after MMC
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HeLa CellsUSP1 depletion
reduces pCHK1USP1 depletion
more variable effect on CHK1
Needed to do quantitative analysis…
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Quantitative Analysis of HeLa Cells7 individual experimentsShow CHK1 levels to be
significantly lower in USP1 deficient cells
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CHK1-FLAG constructCHK1-FLAG expressed
in MRC5 fibroblastsDeprivation of USP1 or
FANCD2USP1 depletion shows
increased mono-Ub-FANCD2, decreased CHK1
FANCD2 depletion shows increased CHK1-FLAG and pCHK1
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FANCD2 Knock-out DT40 cellsEctopically corrected
with WT FANCD2 and non-mono-Ub-FANCD2 (KR)
FANCD2-deficient shows hyper-phosphorylated CHK1
Rescued by WT but not FANCD2-KR form
Shows negative role of activated FANCD2 in CHK1 phosphorylation
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USP1 depletion in FA cells
Depleted USP1 in FA-D2, FA-G, FA-C fibroblasts◦FANCD2-independent
Still decreased CHK1 levels and phosphorylationOther pathways are available…
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Does USP1 Depletion Impact CHK1 Independent of Cell Cycle and DNA Repair?
Lower level of CHK1 due to◦Changes in cell cycle?◦Increased DNA repair?
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Cell Cycle DistributionUSP1 depletion shows no effect on
unchallenged cellsSo, lower level CHK1 not due to cell
accumulation in G1
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Cell Cycle Recovery with MMC
◦USP1-deficient U2OS re-enter cell cycle more rapidly than USP1 proficient
◦USP1-deficient HeLa show prolonged G2 checkpoint
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Difference between Cell Lines?Differ in p53 status
◦ Repeated experiments with p53 inhibitor, no changeWestern blot analysis
◦ HeLa cells more p-CHK1 and p-CHK2 than U2OS◦ MEFs more p-CHK2, less p-CHK1
CHK1 decrease not factor in checkpoint differences
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CHK1 Degradation due to Increased DNA Repair?Dot blot analysis
◦Repair of 6-4 pyrimidine-pyrimidone lesions
◦6-4 PP eliminated equally, USP1 present or not
◦DNA repair pathways are normal without USP1
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CHK1 Degradation due to Increased DNA Repair by FANCD2?
USP1 depletion showed defective relocalization◦Not
consequence of ϒH2AX
◦No enhanced DNA repair by mono-Ub-FANCD2
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USP1 Limits DDB1-specific Degradation of CHK1USP1 depletion
shows lowered pCHK1
DDB-1 depletion shows rescued pCHK1
Co-depletion of USP1 and DDB1 rescued pCHK1 levels
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CHK1 and DDB1 Interaction◦Interaction between
CHK1 and DDB1 enhanced without USP1
◦USP1 depletion did not affect CDT1 No steady-state
reduction Did not promote
degradation with MMC
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ConclusionUSP1 controls a
feedback loop that limits CHK1 activity
CHK1 needed for mono-Ub-FANCD2
DDB1-dependent degradation of p-CHK1
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Future ResearchDDB1 needs CUL4-associated
factor◦One specific for CHK1?
PCNA/FANCD2 interaction found◦Required for proficient mono-Ub-
FANCD2?Bi-allelic FANC mutations in FA
patients◦Ex. Patient with FANCA and FANCM
mutations◦FANCM mutation compensated for
FANCA mutation?
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