1. Volume 37, Issue 6, Pages 854-864 (March 2010)
Regulation of Chromatin Architecture by the PWWP Domain-Containing DNA Damage- Responsive Factor EXPAND1/MUM1 
Michael S.Y. Huen, Jun Huang, Justin W.C. Leung, Shirley M.-H. Sy, Ka Man Leung, Yick-Pang Ching, Sai Wah Tsao, Junjie Chen 
Molecular Cell 
Volume 37, Issue 6, Pages (March 2010)
DOI: /j.molcel
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Figure 1 Identification of EXPAND1 as a Damage-Response Element
(A) Untreated or irradiated (10 Gy) HeLa cells were lysed sequentially in NETN buffer containing increasing amounts of salt. Cell lysates were then separated by SDS-PAGE and immunoblotted using indicated antibodies.
(B) Tandem affinity purification identifies EXPAND1 as a 53BP1-associated protein. The table summarizes proteins identified by mass spectrometry analysis of 53BP1 protein complex. Sequence of the identified peptide corresponding to EXPAND1 was LDGSQRPPAVQLEPMAAGAAPSPGPGPGPR.
(C) Reciprocal immunoprecipitation of 53BP1 and EXPAND1. Nuclease-treated 293T cell lysates were incubated with protein A agarose beads conjugated with indicated antibodies, and western blotting was performed according to standard procedures.
(D and E) EXPAND1 colocalizes with 53BP1 (D) and pH2AX (E). HeLa cells grown on coverslips were transfected with plasmids encoding flag-tagged EXPAND1, irradiated (10 Gy), and processed as described in the Experimental Procedures. Indirect immunostaining was performed using indicated antibodies.
(F) EXPAND1 relocalizes in response to DNA damage. HeLa cells were either irradiated (10 Gy) or left untreated. Immunostaining was conducted using indicated antibodies.
(G) EXPAND1 is a chromatin-associated protein. HeLa cells were biochemically fractionated as described in the Experimental Procedures. Immunoblotting experiments were performed using indicated antibodies. WCE, whole-cell extract.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Genetic Requirements of Focal Accumulation of EXPAND1 to DNA Damage Sites
(A) EXPAND1 is a component of the DNA damage signaling pathway. MEFs and patient cells with deficiencies in various components of the DNA damage signaling pathway were retrovirally infected with constructs encoding flag-tagged EXPAND1. Cells were irradiated (10 Gy) and processed 6 hr later. Immunostaining experiments were performed using indicated antibodies.
(B) EXPAND1 does not affect 53BP1 loading to the chromatin. siRNA-treated HeLa cells were lysed on ice for 10 min in NETN (soluble fraction). Soluble fraction was clarified by centrifugation, and the pellet (chromatin fraction) was resuspended in sampling buffer and boiled. Western blotting was performed using indicated antibodies.
(C) EXPAND1 does not affect 53BP1 IRIF. siRNA-treated HeLa cells were irradiated and processed. Immunofluorescent studies were done using indicated antibodies.
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4. Figure 3 The Binding of EXPAND1 to 53BP1 Requires Its N Terminus
(A) Schematic illustration of EXPAND1 FL and deletion mutants.
(B) Coimmunoprecipitation experiments using streptavidin-binding peptide-flag-tagged 53BP1 and myc-tagged EXPAND1 or its mutants. 293T cells were transfected with constructs encoding tagged 53BP1 and EXPAND1. Precipitation was conducted using streptavidin beads, and immunoblotting was performed using indicated antibodies.
(C and D) EXPAND1 IRIF requires its 53BP1-binding domains. 293T cells were transfected with constructs encoding flag-tagged EXPAND1 or its mutants. Cells were irradiated 24 hr posttransfection, and immunostaining was performed to visualize EXPAND1 localization.
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5. Figure 4
53BP1 Recruits EXPAND1 to Sites of DNA Damage via Its BRCT Domain
(A) Schematic illustration of 53BP1 FL and deletion mutants.
(B) Coimmunoprecipitation experiments were performed using SBP-flag-tagged EXPAND1 and HA-tagged 53BP1 full length (FL) or its mutants (see A).
(C) 53BP1 interacts with EXPAND1 via its BRCT domain. Pull-down experiments were performed using bacterially expressed GST-53BP1 BRCT or GST-BRCA1 BRCT together with lysates prepared from 293T cells expressing myc-tagged EXPAND1.
(D) Pull-down experiment using 53BP1 BRCT domain together with lysates containing myc-tagged FL or D1 mutant of EXPAND1.
(E and F) 53BP1 BRCT-EXPAND1 interaction is phosphorylation independent. Lysates pretreated with lambda phosphatase were incubated with GST-53BP1 BRCT or control GST-BRCA1 BRCT domain. Immunoblotting was performed using indicated antibodies.
(G) Direct interaction between 53BP1 BRCT and EXPAND1. Bacterially purified MBP-EXPAND1 and GST-53BP1 BRCT proteins were coincubated, and pull-down experiment was performed.
(H) EXPAND1 IRIF requires 53BP1 BRCT domain. 53BP1-deficient cells expressing flag-tagged EXPAND1 were reconstituted with 53BP1 FL or mutants. Cells were irradiated (10 Gy) and processed to visualize EXPAND1 localization.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Roles of EXPAND1 in Chromatin Dynamics
(A and B) EXPAND1 is dispensable for checkpoint activation. siRNA-treated HeLa cells were irradiated or left untreated. Cells were lysed 1 hr post-IR (10 Gy), and immunoblotting was performed using the indicated antibodies (A). EXPAND1-depleted cells properly arrest at the G2/M checkpoint (B). Experiments were repeated three times, and similar results were obtained. Representative figure shows mitotic cell population in siRNA-treated cells following DNA damage.
(C and D) Micrococcal nuclease (MNase) sensitivity assay. siRNA-treated cells were incubated with 200 ng/ml NCS or DMSO for 1 hr. Nuclei were extracted as described in the Experimental Procedures and digested using MNase (C). Bar chart showing average size (±SD, n = 3) of nucleosomes (D).
(E) EXPAND1 depletion does not affect KAP1 expression or its phosphorylation. WCE, whole-cell extract; Chr, chromatin-enriched fraction.
(F and G) EXPAND1 is required for efficient DNA repair and cell survival in response to genotoxic stress. siRNA-treated cells were irradiated (3 Gy), and pH2AX foci were monitored (F). Clonogenic assay showing reduced survival rates in EXPAND1-depleted cells (G). siRNA-treated cells were irradiated with the indicated dose of IR. The number of colonies was determined 14 days postirradiation. Results represent mean ± SEM. Experiments were performed twice each in triplicate.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
EXPAND1 Associates with the Chromatin via Its PWWP-Mediated Nucleosome Interaction
(A) Biochemical fractionation of EXPAND1 FL, D1, and dPWWP mutants. Immunoblotting was done using the indicated antibodies.
(B and C) Nucleosome-binding experiments using MBP-fused EXPAND1 and its mutants. Interaction was assessed by immunoblotting for histone H3.
(D) EXPAND1 interacts with components of the chromatin in vivo. Nuclease-treated lysates derived from cells expressing SBP-flag-tagged EXPAND1 were immunoprecipitated using streptavidin beads. Immunoblotting was performed using indicated antibodies.
(E and F) EXPAND1 PWWP domain is sufficient for nucleosome interactions. Purified GST proteins were incubated with nucleosomes. (E) Protein-nucleosome complex was separated by agarose gel electrophoresis, and nucleosomes were stained with ethidium bromide. (F) Shown are GST pull-down experiments using GST-EXPAND1 PWWP or GST alone. Interaction with nucleosomes was assessed by immunoblotting with histone H3 antibodies.
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8. Figure 7 The PWWP Domain Is Indispensable for EXPAND1 Functions
(A) Generation of siRNA-resistant EXPAND1 constructs. 293T cells were cotransfected with indicated constructs and siRNAs. Twenty-four hours posttransfection, cells were lysed and immunoblotting experiment was done using the indicated antibodies.
(B–D) PWWP is required for EXPAND1-dependent chromatin condensation. Stable cells expressing siRNA#1-resistant EXPAND1 FL, its D1, or dPWWP mutants were treated with siRNA#1 twice at 24 hr intervals. Cells were incubated with 200 ng/ml NCS for 1 hr before nuclei were extracted. Micrococcal nuclease sensitivity was assessed as described in the Experimental Procedures. Expression of siRNA-resistant EXPAND1-expressing cells are shown (B). Micrococcal nuclease sensitivity assay was displayed in (C), and average size (±SD, n = 3) of nucleosomes is shown in (D).
(E) Stable cells as described in (B) were treated with EXPAND siRNA#1 twice at 24 hr intervals. Twenty-four hours after the second transfection, cells were seeded onto coverslips and irradiated (3 Gy) 24 hr later. At indicated time points, cells were fixed and processed for immunostaining using pH2AX antibodies.
(F) Clonogenic assay of EXPAND1-expressing cells following radiation. EXPAND1 siRNA-transfected cells were seeded onto a 60 mm dish and were irradiated with the indicated dose. Cells were incubated for 14 days before being fixed and counted. Results represent mean ± SEM from two independent experiments each done in triplicate.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions