FANCL-/- knockout mutant in HCT116 background has been described previously (Huang et al.,. 2014). Construction of CRISPER/CAS9-mediated somatic ...
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UHRF1 Contributes to DNA Damage Repair as a Lesion Recognition Factor and Nuclease Scaffold Yanyan Tian, Manikandan Paramasivam, Gargi Ghosal, Ding Chen, Xi Shen, Yaling Huang, Shamima Akhter, Randy Legerski, Junjie Chen, Michael M. Seidman, Jun Qin,, and Lei Li
Supplementary Experimental Procedures
Construction of somatic cellular knockout mutants UHRF1 targeting vectors were constructed with the USER cloning system (NE-BioLabs). Briefly, two homology arms (1.3-1.5 kb) were PCR amplified from parental HCT116 genomic DNA, and inserted into the modified pAAV-USER vector where they flank a neo cassette. Packaging of rAAV and PCR screening of targeted clones were described previously (Wang et al., 2013). For complementation purposes, pLenti-UHRF1, pLenti-UHRF1C724A and pLenti-UHRF1H741A lentiviruses were generated and used to infect UHRF1 hypomorphic cells. Construction of the FANCL-/- knockout mutant in HCT116 background has been described previously (Huang et al., 2014). Construction of CRISPER/CAS9-mediated somatic mutants followed the procedure described by the Church group (Mali et al., 2013). DNA binding and competition assay For crosslinked DNA – UHRF1 competition assay, 50 ng
32
P-labelled 120 bp CL DNA were
added to 50µl of amylose beads pre-absorbed with -MBP-UHRF1 in the binding buffer (10 mM Tris-Cl, pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.05% NP-40, 5% glycerol) and incubated at room temperature before the addition of increasing amount of unlabeled crosslinked or unlabeled control 120 bp duplex DNA. The beads were washed three times with binding buffer and bound DNA was recovered by maltose elution and electrophoresed on 8% PAGE gel. Labeled DNA bands were visualized by phosphoimaging.
Figure S1
A Substrates A 5’ Biotin-GGAACCGATGACACTCAGCCACAAGGACACCGCAATCAAGTAGATGACGAACGACCCATGAAAGACCAAGGCACAGAAGACCACAGTACAAACAGGCGAGACGCGTGACCCAACAAGGGA CCTTGGCTACTGTGAGTCGGTGTTCCTGTGGCGTTAGTTCATCTACTGCTTGCTGGGTACTTTCTGGTTCCGTGTCTTCTGGTGTCATGTTTGTCCGCTCTGCGCACTGGGTTGTTCCCT-Biotin 5’
Substrates B
5’ Biotin-CGAACACAGTGGTGCAGCCGACACACAACCGCAACCAACTACAGACGACGACGACCCTGCCAAGAAAGAGCACACCAAGGTACAGCGAGAACAGACGCGTGCGAGTGACCAGTGACAACA GCTTGTGTCACCACGTCGGCTGTGTGTTGGCGTTGGTTGATGTCTGCTGCTGCTGGGACGGTTCTTTCTCGTGTGGTTCCATGTCGCTCTTGTCTGCGCACGCTCACTGGTCACTGTTGT-Biotin 5’
XL
MW (bp)
Ctrl
300 200 150 100 75
Figure S1A (related to Figure 1). ICL substrate sequences and preparation Upper panel: Nucleotide sequences of crosslinked substrates A and B used in the Affinity purification of ICL binding proteins. Nucleotide sequence of the 40oligonucleotide duplex used for the determination of disassociation constant UHRF1 ICL binding. Positions of the TA motifs where psoralen ICLs are introduced are marked red. Lower panel: Psoralen-crosslinked substrate were purified by denaturing agarose gel electrophoresis and resolved on denaturing PAGE to examine the purity of crosslinked substrate. Control substrate (Ctrl) migrates as denatured single strand 120-mer. Crosslinked substrate (XL) migrates slower as the two strands are joined by the psoralen crosslinks.
B EBNA -
EBNA+ EBV ori Fork progression
ICL
C
Mcm5
Relative enrichment (% of input)
5 4
Figure S1C (related to Figure 1). Enrichment of Mcm5 at ICL-blocked replication fork. eChIP substrate carrying a single defined psoralen ICL (XL) or control substrate (Ctrl) without the ICL were introduced into 293EBNA cells to enable episomal replication. Sixteen hours post transfection, cells were harvested and fixed. ChIP assay was performed with an anti-Mcm5 antibody. Significant enrichment of Mcm5 was observed at the site of the psoralen ICL, reflecting the presence of blocked replication forks.
3 2 1 0
Figure S1B (related to Figure 1). eChIP (episomal chromatin immunoprecipitation) assay for the in vivo identification of proteins recruited to a defined interstrand crosslink down stream of the unidirectional EBV viral replication origin. Schematics of the pORIP DNA substrate under replicative and nonreplicative states. Replication is enabled when the eChIP substrate is introduced to EBNA-expression cells. Cells transfected with the eChIP substrate are harvested and subjected to standard chromatin-IP procedure including formaldehyde fixation and sonication. An antibody against a protein recruited to the site of the ICL is expected to immunoprecipitate DNA fragments at the site of the ICL, which can be quantified via real-time PCR. Short vertical bars adjacent to the site of the ICL indicate the region of PCR amplification.
Ctrl
XL
Figure S2
A 3
UHRF1 knockout allele UHRF1 hypomorphic allele
5
P1
3
Alleles Wild type
P2
4
5 NeoR
P1
P3
UHRF1
Hypomorphic
P2
Tubulin
Knockout Cre
+
-
-
+
+
1.00 0.73 0.15 0.18
Figure S2A (related to Figure 2). Left panel: Schematics of UHRF1 hypomorphic mutant genotypes. Solid squares and numbers indicate UHRF1 exons. Triangles depict Cre-LoxP sites. NeoR: Neomycin resistant cassette. Blue arrows indicate PCR primers used in genotyping. Middle panel: PCR genotyping of UHRF1-/Neo cells. UHRF1-/Neo-1 and UHRF1/Neo-2 cells were mock-infected or infected with AdCre, resulting in the incision of the NeoR cassette and exon 4 from the hypomorphic allele. Inverted Agarose-EB gel imagines are shown. Right panel: Immunoblot detecting UHRF1 protein in wild type (UHRF1+/+), heterozygous (UHRF1+/-), and hypomorphic (UHRF1-/Neo-1 and UHRF1-/Neo-2) mutant cells. UHRF1 protein levels relative to the parental HCT116 cells (UHRF1+/+) were derived after normalization against the loading control (Tubulin).
B Clonogenicity (% relative to UHRF1+/+)
120 100
C
80 60 40
UHRF1+/+
20
UHRF1-/Neo-1
0
UHRF1-/Neo-2
Cre(-)
Cre(+)
Figure S2B (related to Figure 3). Clonogenicity of UHRF1-/Neo cells upon inactivation of the conditional allele. UHRF1-/Neo and UHRF1+/+ cells were infected with AdCre (MOI = 50) and plated for clonogenic counting. Removal of exon 4 from the hypomorphic allele lead to severe reduction in colony formation in UHRF1-/Neo cells, compared to UHRF1+/+ cells. This result indicates that UHRF is essential for sustained cell proliferation in the HCT116 background. Error bars depict SDs derived from four independent experiments.
7
Cell proliferation (% relative to initial seeding)
6 5
UHRF1+/+
4
UHRF1-/Neo-1
3
UHRF1-/Neo-2
2
UHRF1+/+ +Cre
0
D
UHRF1-/Neo-1 + Cre
1
UHRF1-/Neo-2 + Cre Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Figure S2C (related to Figure 3). Cell proliferation of UHRF1-/Neo cells upon inactivation of the conditional allele. UHRF1-/Neo and UHRF1+/+ cells were infected with AdCre (MOI = 50) as indicated and plated for MTTbased cell proliferation assay. Cell numbers were measured for six consecutive days and plotted against the initial number of seeded cells. Error bars represent SDs derived from three independent experiments.
Cell proliferation (% relative to initial seeding)
6 5 4
UHRF1+/+ + pCtrl
3
UHRF1-/Neo-1 + pCtrl
2
UHRF1-/Neo-2 + pCtrl
1
UHRF1-/Neo-1 + pUHRF1
0
UHRF1-/Neo-2 + pUHRF1 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Figure S2D (related to Figure 3). Cell proliferation of UHRF1-/Neo and wild type UHRF1 complemented derivative cells upon inactivation of the conditional allele. UHRF1-/Neo cells with the control expression vector pCtrl) or WT UHRF1 expression vector (pUHRF1) were subjected to AdCre (MOI = 50) treatment and plated for MTT-based cell proliferation assay. Cell numbers were measured for six consecutive days and plotted against the initial number of seeded cells. Error bars represent SDs derived from three independent experiments.
Figure S2
E
F HeLa
293T
50
Clonogenic survival (%)
Clonogenic Survival (%)
100
UHRF1 Tubulin
XPA
10
β-Actin
Ctrl shRNA
1
UHRF1 shRNA-3
293T + shCtrl
UHRF1 shRNA-4
293T + UHRF1 shRNA3 5
0
500
1000
1500
XPA shRNA1
293T + UHRF1 shRNA4
0.1
0
10 UV (J/m2)
Mitomycin C (nM)
G
20
XPA shRNA2
H HCT116
HCT116 1
UHRF1+/+ UHRF1-/Neo-1 UHRF1-/Neo-2
Clonogenic survival (100%)
Clonogenic survival (%)
100
0.1
UHRF1 Tubulin
UHRF1+/+ + vector
0.01
UHRF1-/Neo-1 + vector UHRF1-/Neo-1 + wtUHRF1
FAAP24-/10
0
250 Mitomycin C (nM)
500
FANCM-/-
UHRF1-/Neo-1 + UHRF1H724A 0.001
0
500
1000
1500
UHRF1-/Neo-1 + UHRF1C741A
Mitomycin C (nM)
Figure S2 E-D (related to Figure 3). Survival analyses of UHRF1-deficient cells (E) Clonogenic survival of 293 cells and stable cell lines with UHRF1 knockout by two independent shRNAs (shRNA3 and shRNA4) against mitomycin C. Error-bars were derived from three independent technical repeats. (F) Clonogenic survival of HeLa cells with UHRF1 or XPA knockdown against UV-C treatment. Error-bars were derived from three independent technical repeats (G) Clonogenic survival of HCT116 wt (UHRF1+/+) and isogenic derivatives, UHRF1-/Neo-1, UHRF1-/Neo-1, FANCM-/-, FAAP24-/-, against mitomycin C. Error-bars were derived from three independent technical repeats. (H) Clonogenic survival of UHRF1-/Neo-1 complemented with two E3 ligase mutants, UHRF1H724A and UHRF1C741A. Error bars represent SDs from three independent experiments with triplicated plates. independent experiments.
Figure S3
A
B DAPI
53BP1
DAPI
UHRF1+/+
53BP1
UHRF1-/Neo-2
UHRF1-/Neo-1
53BP1 foci % (>5/cell)
1 0.8 0.6 0.4
UHRF1+/+
0.2
UHRF1-/Neo-1
0
Mock treated
UHRF1-/Neo-2 0 hr
6 hr
18 hr
MMC 50ng/ml 18 hr
Figure S3 (related to Figure 3). UHRF1 loss attenuates the onset of 53BP1 foci. (A) Formation of MMC-induced 53BP1 nuclear foci in wild type (UHRF1+/+) and hypomorphic mutant (UHRF1-/Neo-1 and UHRF1-/Neo-2) cells 18 hours after treatment. (B) Percentage of 53BP1 foci-positive nuclei from MMC-treated UHRF1+/+ and UHRF1-/Neo-1 and UHRF1-/Neo-2 cells at indicated time points after MMC exposure.
C
IP
IB UHRF1 MUS81 ERCC1
Figure S3C (related to Figure 4). Immunoblot detecting endogenous MUS81 and ERCC11 from control (IgG) or UHRF1 antibody coimmunoprecipitation of 293T cells
Figure S4
A SLX4 1
3 2
5 4
7 6
9
11
8 10
13
15
12
14 SLX4 β-Actin
gRNA sequence 3: GGAGCGACAGTCACGGCTGTCGG
SLX4 WT
ACAGCAGTGCCAAGTCCCTCCAAACCCCGCACAGCACAATTGGTCCTACAGCGAATGCAGCAGTTCAAGAGAGCAGAC T A V P S P S K P R T A Q L V L Q R M Q Q F K R A D Deletion
Allele-1
ACAGCAGTGCCAAGTCCCTCCAAACCCCGCCTAAAACGAAGAGGTGACTAG T A V P S P S K P R L K R R G D STOP
Allele-2
ACAGCAGTGCCAAGTCCCAGTAGAGGTCTGAAACGGAGAGACAACAGCAGTGCCAAGTCCCAGTAG T A V P S P S R G L K R R D N S S A K S Q STOP
insertion
B
WT Positive Clones
FANCL 1
3 2
5 4
7 6
9 8
10
11
4
13 12
6
21
31
FANCL
Β-actin gRNA sequence 1: GGGAAGAGACTTCCACCTT
FANCL WT
Allele-1
GGAAGAGACTTCCACCTTAGGATAGTGTTGCCTGAAGATTTACAACTGAAGAATGCAAG G R D F H L R I V L P E D L Q L K N A Deletion GGAAGAGACTTCCACTAGGATAGTGTTGCCTGAAGATTTACAACTGAAGAATGCAAG G R D F H STOP Deletion
Allele-2
GGAAGAGACTTAGGATAGTGTTGCCTGAAGATTTACAACTGAAGAATGCAAG G R D L G STOP
Figure S4 (related to Figure 5). CRISPR/CAS9-mediated construction of SLX4-/- and FANCL-/- knockout mutants in HeLa cells. (A) HeLa SLX4-/- cells were obtained by using gRNA3 (shaded sequence) which targets exon 7 of the SLX4 locus. Western blot shows the protein null status of the SLX4 mutant used in this study. Sequencing verification of the mutant show bi-allelic nonsense deletion/insertions. (B) HeLa FANCL-/- cells were obtained by using gRNA1 (shaded sequence) which targets exon 2 of the FANCL locus. Western blot shows the protein null status of four FANCL-/- mutants. Mutant 4 was used in this study. Sequencing verification of the mutant show biallelic nonsense deletions. Note: The SLX4-/- and FANCL-/- designations may be arbitrary since HeLa cells does not possess strict diploidy. However, sequencing of multiple PCR clones from each mutant recovered only biallelic mutations, suggesting that both the SLX4 and FANCL loci in the parental HeLa cells are diploid.
