1. Volume 22, Issue 7, Pages 1849-1860 (February 2018)
Shwachman-Diamond Syndrome Protein SBDS Maintains Human Telomeres by Regulating Telomerase Recruitment 
Yi Liu, Feng Liu, Yizhao Cao, Huimin Xu, Yangxiu Wu, Su Wu, Dan Liu, Yong Zhao, Zhou Songyang, Wenbin Ma 
Cell Reports 
Volume 22, Issue 7, Pages (February 2018)
DOI: /j.celrep
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2. Cell Reports 2018 22, 1849-1860DOI: (10.1016/j.celrep.2018.01.057)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1 SBDS Knockdown Leads to Telomere Shortening
(A) Two different SBDS shRNA sequences (shSBDS-1 and shSBDS-2) were used for stable expression in HTC75 cells. An shRNA targeting GFP was used as a negative control. The cells were harvested for western blot analysis as indicated. GAPDH served as the loading control.
(B) Cells from (A) were cultured for the indicated population doublings and examined using the telomere restriction fragment (TRF) analysis.
(C) Data from (B) were quantified to derive the average telomere length of cells using TeloRun and plotted as shown.
(D) Cells from (A) were collected and lysed for TRAP assays. For each cell line, two reactions were carried out, with 40 and 20 cells each. IC, internal standard control.
(E) Data from (D) were quantified and plotted as shown. Three biological repeats were analyzed. Error bars indicate SEM (n = 3). N.S., not significant, with a p value of (one-way ANOVA).
See also Figure S1.
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4. Figure 2
SBDS Deficiency Disrupts Telomerase Co-localization with Telomeres during S Phase
(A) Control and SBDS knockdown HTC75 cells were synchronized at the S phase of cell cycle by double-thymidine block. The cell cycle profile of the treated cells was analyzed by flow cytometry.
(B) Cells from (A) were examined by IF-FISH using an hTERC probe and an antibody against TRF1. Arrowheads indicate overlapping signals of hTERC and TRF1.
(C) Data from (B) were quantified and plotted. Cells with ≥3 overlapping foci were scored as positive, with >100 cells examined for each cell line. Three biological repeats were performed. Error bars represent SD (n = 3). ∗∗p < 0.01, two-tailed Student’s t test.
(D) To generate inducible SBDS KO cells, two sgRNAs that target the first exon and 3′ UTR of SBDS were generated. Simultaneous cleavage by Cas9 at the two sgRNA target sites was predicted to delete the entire locus (∼7 kb).
(E) Two sgRNAs from (D) were co-transfected into HeLa cells expressing inducible Cas9 and cultured with (+) or without (−) doxycycline (Dox) (1 μg/mL) for 6 days before being collected for western blot analysis as indicated. GAPDH served as a loading control. Endogenous SBDS protein levels were quantified using the Odyssey Infrared Imaging System and normalized against the level of GAPDH.
(F) Cells from (E) were synchronized by double-thymidine block. The cells were then collected for IF-FISH assays using an hTERC probe and antibody against TRF1. Arrowheads indicate overlapping signals of hTERC and TRF1.
(G) Data from (F) were quantified and plotted for the percentage of cells with ≥2 overlapping foci; >100 cells were examined for each cell line in three biological replicates. Error bars represent SD (n = 3). ∗∗p < 0.01, two-tailed Student’s t test. Scale bar, 10 μm.
See also Figure S2.
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5. Figure 3 SBDS Associates with TPP1
(A) Schematic diagram of the BiFC assay. The N- and C-terminal fragments of YFP (YFPc and YFPn) were fused to two different proteins whose interaction brought the YFP fragments together to co-fold into a functional fluorophore.
(B) HeLa cell stably expressing YFPn-tagged SBDS were transfected with constructs encoding YFPc-tagged telosome subunits before being analyzed by FACS. YFPn-tagged POT1 served as a positive control.
(C) HEK293T cells co-transfected with SFB-tagged TPP1 and GST-tagged SBDS were harvested for streptavidin pull-down of TPP1. The precipitates were western blotted with the indicated antibodies.
(D) HEK293T cells were co-transfected with GST-TPP1 and FLAG-HA-tagged SBDS (FLHA-SBDS) before being treated with double-thymidine block. The cells were collected at the indicated time points after being released into cell cycle. The GST pull-down precipitates were western blotted with the indicated antibodies. FLAG-HA-tagged GFP and POT1 were used as negative and positive controls, respectively.
(E) TPP1 and SBDS co-expressing cells from (D) were examined by FACS for DNA content analysis.
See also Figures S3–S5.
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6. Figure 4 SBDS Facilitates TPP1 Association with the Telomerase
(A) The inducible SBDS-knockout HeLa cells were harvested 6 days after doxycycline treatment (1 μg/mL) for western blot analysis. Endogenous SBDS protein levels were quantified using the Odyssey Infrared Imaging System and normalized against the GAPDH loading control.
(B) FLAG- and HA-tagged TPP1 (FLHA-TPP1) was transfected into the inducible SBDS-knockout HeLa cells 3 days after doxycycline induction. The cells were then collected for anti-FLAG IP and western blotting with the indicated antibodies. Parental HeLa cells were used as a negative control for IP. GAPDH served as a loading control.
(C) The anti-FLAG immunoprecipitants from (B) were analyzed for the amount of telomerase activities that co-precipitated with TPP1 in TRAP assays. IC represents internal standard control.
(D) Data from (C) were quantified and plotted as shown. The signals of telomerase products in each sample were normalized to the internal control and the amount of immunoprecipitated TPP1 from (B), and they were compared against signals from HeLa cells not induced with doxycycline (wild-type [WT]). Results from three independent experiments were combined. Error bars represent SD (n = 3). ∗∗p < 0.01, two-tailed Student’s t test.
See also Figure S6.
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7. Figure 5
The RRM Domain of SBDS Interacts with the N-terminal OB Fold of TPP1
(A) Schematic diagram of the domains of TPP1. OB, oligosaccharide or oligonucleotide-binding fold; RD, POT1-binding domain; S/T, the serine-rich region; TID, TIN2-interacting domain.
(B) Schematic diagram of the domains of SBDS. FYSH, Fungal, Yhr087w, and Shwachman domains; HTH, helix-turn-helix domain; RRM, RNA recognition motif.
(C) HeLa cells stably expressing YFPc-tagged full-length TPP1 were transfected with vectors encoding YFPn-tagged full-length or truncation mutants of SBDS (left). Similarly, HeLa cells stably expressing YFPn-tagged full-length SBDS were transfected with vectors encoding truncation mutants of TPP1 (right). The cell lysates were immunoblotted using an anti-GFP antibody. Asterisk indicates YFPn-POT1. YFPn alone and YFPn-POT1 served as negative and positive controls for TPP1-YFPc.
(D) Cells from (C) were also analyzed by flow cytometry to obtain the percentage of YFP+ cells.
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8. Figure 6
Expression of SBDS Disease-Associated Mutants Leads to Telomere Shortening
(A) Two SBDS truncation mutants were generated, which correspond to the protein products resulting from the two most common SBDS mutations in patients (K62X and 84Cfs3).
(B) HTC75 cells stably expressing SFB-tagged full-length and disease mutants of SBDS were western blotted with the indicated antibodies. Cells with an empty vector were used as a negative control. GAPDH served as a loading control.
(C) Cells from (B) were cultured and collected at the indicated time points for TRF analysis.
(D) Data from (C) were quantified using TeloRun and plotted as shown.
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9. Figure 7
Disease-Associated Mutants of SBDS Impede TPP1 Binding to Telomerase
(A) FLAG- and HA-tagged (FLHA) TPP1 was co-transfected into HEK293T cells with GST-tagged full-length and disease truncation mutants of SBDS. The cells were then collected for anti-FLAG IP and western blot using the indicated antibodies. GST-tagged GFP was used as a negative control. GAPDH served as a loading control.
(B) The anti-FLAG immunoprecipitates were examined using TRAP assays to determine the amount of telomerase activities brought down by TPP1. IC represents internal standard control.
(C) Data from (B) were quantified and plotted as shown. The signals of telomerase products in each sample were normalized to internal control and the amount of immunoprecipitated TPP1 from (A), and they were compared against signals from GST-GFP cells. Results from three independent experiments were combined. Error bars represent SD (n = 3). N.S., not significant with a p value of ∗∗p < 0.01, one-way ANOVA with Bonferroni test.
(D) Model of SBDS-mediated telomere regulation. Wild-type SBDS, through the interaction between its RRM domain and TPP1 N terminus, can facilitate TPP1-mediated recruitment of telomerase during the S phase of cell cycle. In SDS patients, C-terminally truncated SBDS can no longer interact with TPP1, resulting in decreased telomerase recruitment and ultimately telomere shortening.
See also Figure S7.
Cell Reports  , DOI: ( /j.celrep )
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