53BP1 and USP28 mediate p53-dependent cell cycle arrest in response to centrosome loss and prolonged mitosis

  1. Chii Shyang Fong
  2. Gregory Mazo
  3. Tuhin Das
  4. Joshua Goodman
  5. Minhee Kim
  6. Brian P O'Rourke
  7. Denisse Izquierdo
  8. Meng-Fu Bryan Tsou  Is a corresponding author
  1. Memorial Sloan Kettering Cancer Center, United States
  2. Oberlin College, United States
  3. Weill Cornell Medical School, United States
5 figures and 1 table

Figures

Figure 1 with 2 supplements
Genome-wide CRISPR-mediated loss-of-function screen for components required for centrosome loss-induced G1 arrest.

(A) Acentrosomal cells exhibits prolonged mitosis. Measurement of mitotic duration of wild type RPE1 and PLK4as cells dividing in the presence or absence of 3MBPP1 with live-cell imaging. With …

https://doi.org/10.7554/eLife.16270.002
Figure 1—figure supplement 1
Centrosome loss upon PLK4 inactivation.

Wild type RPE1 and PLK4as cell line treated with 3MBPP1 for seven days stained with antibodies against centrin-2 and γ-tub to mark centrosomes. Scale bar, 5 μm.

https://doi.org/10.7554/eLife.16270.003
Figure 1—figure supplement 2
Genotyping of p53 CRISPR cell line.

Positions of sgRNA target site within the ORF of the p53 gene is depicted in the map. Descriptions of mutant indels are depicted below. Green colored nucleotides are insertions. sgRNA target site is …

https://doi.org/10.7554/eLife.16270.004
Figure 2 with 2 supplements
53BP1 and USP28 are broad acting components acting upstream of p53 in response to mitotic stress.

(A) Schematic representation of the loss-of-function screen for components required for centrosome loss induced G1 arrest using the human genome-scale CRISPR knockout library (GeCKO). (B) Binary …

https://doi.org/10.7554/eLife.16270.005
Figure 2—figure supplement 1
Genotyping of CRISPR cell lines.

Positions of sgRNA target sites within the ORF of each gene are depicted on maps. Descriptions of mutant indels are depicted below. Red colored bases represent deleted nucleotides. Green colored …

https://doi.org/10.7554/eLife.16270.006
Figure 2—figure supplement 2
Schematic outlining the timeline of synchronization and drug treatments used.

BrdU incorporation assay for Figure 2H.

https://doi.org/10.7554/eLife.16270.007
Figure 3 with 3 supplements
53BP1 mediates centrosome loss-induced G1 arrest independently to its DNA repair activity.

(A) Domain organization of 53BP1. BRCT (BRCA1 carboxy-terminal), UDR (ubiquitylation-dependent recruitment). p53 and USP28 interact with 53BP1 through the tandem BRCT domain. (B) 53BP1ΔBRCT mutant …

https://doi.org/10.7554/eLife.16270.009
Figure 3—figure supplement 1
Wild type and mutant 53BP1 are exogenously expressed to similar levels in PLK4as; 53BP1-/- cells.

Western blot of PLK4as; 53BP1-/- cells mildly expressing (10 ng/ml doxycycline) wild type and mutant 53BP1.

https://doi.org/10.7554/eLife.16270.010
Figure 3—figure supplement 2
Schematic outlining the timeline of drug treatments used.

BrdU incorporation assay for Figure 3B.

https://doi.org/10.7554/eLife.16270.011
Figure 3—figure supplement 3
DDR function is intact in 53BP1WT, 53BP1ΔBRCT and 53BP1TASA, but not in 53BP1TESE.

53BP1TESE delocalizes from the DNA damage sites marked by γ-H2AX. Mutant proteins of 53BP1 were mildly expressed under the tetracycline inducible promoter in stable, clonal, centrosomal PLK4as; …

https://doi.org/10.7554/eLife.16270.012
Figure 4 with 2 supplements
USP28 is catalytically required for p53 stabilization during centrosome loss-induced G1 arrest.

(A) Organization of the conserved catalytic domains in USP28. UCH-1 (Cys box, amino acids 162–196) and UCH-2 (His box, amino acids 580–649). (B) Catalytic-inactive USP28CI cannot rescue the G1 …

https://doi.org/10.7554/eLife.16270.013
Figure 4—figure supplement 1
USP28WT and USP28CI are exogenously expressed to similar levels in PLK4as; USP28-/- cells.

Western blot of PLK4as; USP28-/- cells mildly expressing (10 ng/ml doxycycline) USP28WT and USP28CI. USP28 was probed with anti-HA antibody.

https://doi.org/10.7554/eLife.16270.014
Figure 4—figure supplement 2
Schematic outlining the timeline of drug treatments used.

BrdU incorporation assay for Figure 4B.

https://doi.org/10.7554/eLife.16270.015
Figure 5 with 2 supplements
SAC activation is not essential for mitotic stress-induced G1 arrest.

(A) 53BP1 normally disassociates from the kinetochores during anaphase, but can do so prematurely in prometaphase upon mitotic stress/delay. Immunofluorescence images of centrosomal (left) or …

https://doi.org/10.7554/eLife.16270.016
Figure 5—figure supplement 1
53BP1 delocalizes from kinetochores in prometaphase in response to mitotic stress.

(A and B) Cells arrested or delayed in prometaphase due to Eg5 inhibitor treatment or acentrosomal cell division stained with the antibodies indicated. Bright BubR1 or Mad2 signals indicate SAC …

https://doi.org/10.7554/eLife.16270.017
Figure 5—figure supplement 2
Schematic outlining the timeline of synchronization and drug treatments used.

(A) Assay for 53BP1 mitotic kinetochore localization for Figure 5B. (B) SAC reactivation assay for Figure 5C. (C) BrdU incorporation assay for Figure 5F.

https://doi.org/10.7554/eLife.16270.018

Tables

Table 1

Candidate genes enriched in the eight independent screens for genes involved in centrosome loss-induced G1 arrest.

https://doi.org/10.7554/eLife.16270.008
Enrichment compared to p53Gene symbolGene name or descriptionNumber of sgRNAs enriched out of total 6
1.0000TP53Tumor Protein P535/6
0.9386TP53BP1Tumor Protein P53 Binding Protein 14/6
0.9039TRIM37Tripartite Motif Containing 373/6
0.8895USP28Ubiquitin Specific Peptidase 284/6
0.8858P21Cyclin-Dependent Kinase Inhibitor 1A (P21, Cip1)6/6
0.7559CHD8Chromodomain Helicase DNA Binding Protein 84/6
0.6945FBXO42F-Box Protein 423/6
0.6846RNF20Ring Finger Protein 20, E3 Ubiquitin Protein Ligase3/6
0.6665PCDHGC5Protocadherin Gamma Subfamily C, 52/6
0.6604TSPY8Testis Specific Protein, Y-Linked 83/6
0.6395RWDD3RWD Domain Containing 32/6
0.6364UGT1A9UDP Glucuronosyltransferase 1 Family, Polypeptide A92/6
0.6285PDSS1Prenyl (Decaprenyl) Diphosphate Synthase, Subunit 12/6
0.6275BPIFB4BPI Fold Containing Family B, Member 42/6
0.6240PCDHAC2Protocadherin Alpha Subfamily C, 23/6
0.6218CSNK2A1Casein Kinase 2, Alpha 1 Polypeptide2/6
0.6201GAGE7G Antigen 73/6
0.6119WTAPWilms Tumor 1 Associated Protein2/6
0.6093PRDM11PR Domain Containing 112/6
0.6086OR56A1Olfactory Receptor, Family 56, Subfamily A, Member 12/6
0.6059TBCELTubulin Folding Cofactor E-Like2/6
0.5997DNAJC9DnaJ (Hsp40) Homolog, Subfamily C, Member 92/6
0.5991NPIPL3Nuclear Pore Complex Interacting Protein Family, Member B33/6
0.5987SYDE1Synapse Defective 1, Rho GTPase, Homolog 12/6
0.5959TECPR2Tectonin Beta-Propeller Repeat Containing 22/6
0.5683STOML3Stomatin (EPB72)-Like 32/6
0.5507DVL3Dishevelled Segment Polarity Protein 32/6
  1. Log2 scaling of sgRNAs enrichment normalized to the top scoring hit p53. The sgRNAs for genes highlighted in red were validated in this study. For a gene to be scored as a candidate, at least two of its six sgRNAs were repeatedly enriched in independent screens, with the HiSeq reads at least 3 times higher than the average of the baseline read.

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