Journal: The Journal of Biological Chemistry

Article Title: Interaction of Sororin Protein with Polo-like Kinase 1 Mediates Resolution of Chromosomal Arm Cohesion *

doi: 10.1074/jbc.M111.305888

Figure Lengend Snippet: Interaction of Plk1 and Sororin. A, HeLa cells were transfected with pFLAG-Sororin or pFLAG empty vector (EV). Twenty-four hours after transfection, the cells were synchronized with double thymidine (DT). Non-synchronized (NS) cells were used as a control. Cells were harvested 6–11.5 h after release from double thymidine. Endogenous Plk1 was co-immunoprecipitated by FLAG-Sororin and visualized with immunoblotting. ** indicates a nonspecific band. B, WT, but not mutant (AA) PBD, interacts with Sororin. FLAG-Sororin was expressed in HeLa S3 Myc-PBD WT and Myc-PBD(AA) Tet-On cells for 40 h before Myc-PBD WT and mutant Myc-PBD(AA) were induced with doxycycline for 16 h. Co-IP of FLAG-Sororin and Myc-PBD was performed. C, Sororin T159A mutation significantly reduces its interaction with PBD. HeLa S3 Tet-On cells stably expressing Myc-tagged PBD were transfected with FLAG-tagged Sororin WT and four putative PBD binding motif mutants (T115A, S125A, S157A, and T159A). Empty vector (EV)-transfected cells were used as a control. Reciprocal IP and immunoblotting were performed. D, sequence comparison for the five putative PBD binding motifs at ST115P, SS125S, SS126K, ST157S, and ST159P of human Sororin with other vertebrate species. The numbers on the top line show the site of human Sororin. Invariant, conserved, and semiconserved residues are indicated by an asterisk (*), colon (:), and period (.), respectively. pAb, polyclonal antibody.

Article Snippet: The sources of the antibodies used in this study were as follows: rabbit polyclonal antibodies FLAG (Sigma), human Rad21 ( 29 ), and GFP (Abcam, Cambridge, MA); goat polyclonal antibody SA2 (Novus Biologicals, Littleton, CO); and mouse monoclonal antibodies: FLAG (Sigma), Myc (Calbiochem), Cdk1 (Millipore, Billerica, MA), and Plk1 (Zymed Laboratories Inc.).

Techniques: Transfection, Plasmid Preparation, Control, Immunoprecipitation, Western Blot, Mutagenesis, Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Binding Assay, Sequencing, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Interaction of Sororin Protein with Polo-like Kinase 1 Mediates Resolution of Chromosomal Arm Cohesion *

doi: 10.1074/jbc.M111.305888

Figure Lengend Snippet: Sororin T159A mutant inhibits resolution of chromosomal arm cohesion. A, fluorescence microscopy showing the expression of GFP, GFP-Sororin WT, and GFP-Sororin T159A mutant in HeLa stable cell lines. B, co-IP of GFP-Sororin (Sor) and cohesin. GFP and GFP-Sororin were immunoprecipitated with rabbit anti-GFP polyclonal antibody (pAb) (lanes 4–6). Normal rabbit IgG (NIgG) was used as a control (lane 7). 2.5% of the total input protein used for co-IP was loaded in lanes 1–3. Co-IP of cohesin by GFP-Sororin was analyzed with immunoblotting using SA2 polyclonal antibody and Rad21 mAb. The middle panel shows both SA2 and Rad21 bands, which were probed with Rad21 antibody after SA2 was blotted. * indicates non specific bands. C–E, efficiency of endogenous Sororin knockdown by siRNA. C, exponentially growing HeLa cells were transfected with Sororin coding region siRNA (Sor CDR) or Sororin 3′-UTR (Sor 3UTR). Silencer Negative Control 1 siRNA was used as a control. Forty-eight hours after transfection, total RNA in HeLa cells was extracted, and RT-PCR was performed. The mRNA level in cells transfected with Sororin 3′-UTR or coding region siRNA was significantly reduced compared with that in cells transfected with control siRNA. Data are the means ± S.E. from three samples. D, Myc-Sororin in HeLa Tet-On Sororin stably transfected cells induced with 2 μg/ml doxycycline. The cells were transfected with control or Sororin coding region siRNA for 48 h. Whole cell lysates were used for immunoblotting with anti-Myc mAb, Sororin polyclonal antibody (Sor pAb), and Actin mAb. E, HeLa cells were transfected with control siRNA or Sororin 3′-UTR siRNA for 24 h. The cells were fixed and stained with DAPI. Cells treated with Sororin 3′-UTR siRNA arrested at prometaphase (arrow). F–I, HeLa stable cell lines shown in A were treated with control siRNA (G) or Sororin 3′-UTR siRNA (H) before metaphase spread of chromosomes was performed. More than 100 mitotic cells were randomly examined and categorized according to the resolution of sister chromatid cohesion in F (panels a–d). The frequency of each category was plotted in G and H. I, immunoblotting shows the GFP-Sororin level in the cells used for metaphase spread in G and H. * indicates nonspecific bands. Bar size, 25 μm.

Article Snippet: The sources of the antibodies used in this study were as follows: rabbit polyclonal antibodies FLAG (Sigma), human Rad21 ( 29 ), and GFP (Abcam, Cambridge, MA); goat polyclonal antibody SA2 (Novus Biologicals, Littleton, CO); and mouse monoclonal antibodies: FLAG (Sigma), Myc (Calbiochem), Cdk1 (Millipore, Billerica, MA), and Plk1 (Zymed Laboratories Inc.).

Techniques: Mutagenesis, Fluorescence, Microscopy, Expressing, Stable Transfection, Co-Immunoprecipitation Assay, Immunoprecipitation, Control, Western Blot, Knockdown, Transfection, Negative Control, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: The Journal of Biological Chemistry

Article Title: Interaction of Sororin Protein with Polo-like Kinase 1 Mediates Resolution of Chromosomal Arm Cohesion *

doi: 10.1074/jbc.M111.305888

Figure Lengend Snippet: Model for Sororin-mediated resolution of arm cohesion. During generation of cohesion at S phase, Sororin is incorporated into the cohesin complex by interacting with cohesin and is required for the maintenance of sister chromatid cohesion both on chromosome arms and centromeres (Step 1). Protection of centromeric cohesion by Shugoshin is not shown. To resolve chromosomal arm cohesion during prophase, Sororin is phosphorylated by Cdk1/cyclin B at the PBD binding site ST159P (Step 2), which functions as a docking protein to recruit Plk1 to the vicinity of the cohesin complex and results in SA2 phosphorylation by Plk1 (Step 3). Once SA2 is phosphorylated, Sororin is further phosphorylated and dissociates from cohesin (Step 4). Wapl displaces phosphorylated Sororin and binds with Pds5 to facilitate the removal of cohesin complexes from sister chromatid arms (Step 5).

Article Snippet: The sources of the antibodies used in this study were as follows: rabbit polyclonal antibodies FLAG (Sigma), human Rad21 ( 29 ), and GFP (Abcam, Cambridge, MA); goat polyclonal antibody SA2 (Novus Biologicals, Littleton, CO); and mouse monoclonal antibodies: FLAG (Sigma), Myc (Calbiochem), Cdk1 (Millipore, Billerica, MA), and Plk1 (Zymed Laboratories Inc.).

Techniques: Binding Assay, Phospho-proteomics

Journal: Nature Communications

Article Title: STAG2 regulates interferon signaling in melanoma via enhancer loop reprogramming

doi: 10.1038/s41467-022-29541-9

Figure Lengend Snippet: a Circos plot for differential peaks of CTCF, SMC1A, STAG1, and STAG2 binding upon STAG2 knockdown in M14 cells. Red bar represents gain of binding and blue bar represents loss of binding. b Distinct profiles of changes in STAG1 binding at the loss of STAG2 binding sites upon STAG2 knockdown in M14 cells. The STAG switch and non-switch groups are identified by k-means clustering and show significant gain or nearly no gain in STAG1 binding at the loss of STAG2 binding sites, respectively. c Heatmap of normalized ChIP-seq signals for STAG1, STAG2, SMC1A, and CTCF in M14 cells with (KD) and without (WT) STAG2 shRNA knockdown and their changes (Δ), as defined by log 2 (KD/WT), in both STAG switch (cluster 1) and non-switch groups (cluster 2). d Average contact probability at a different genomic distance for KD and WT, using 100 kb as window size. e Average TAD length is significantly different between STAG2 WT ( n = 3789 TADs) and KD ( n = 3496 TADs). The box plot is defined by bounds at the 25th percentile and 75th percentile, center at 50th percentile, the minima and maxima are at the 10th percentile and 90th percentile. P value is 3.488e-13 and is based on a two-sided Wilcox test. f Difference in TAD size for expanded TADs ( n = 1125 TADs) is significantly longer than those of shrinked TADs ( n = 1007 TADs). P value is 2.312e-17 and is based on a two-sided Wilcox test. g CTCF, SMC1A, STAG1, and STAG2 profiles at stable and variable TAD boundaries in M14 STAG2 WT cells. RPM reads per million. h Remarkable STAG2 to STAG1 switch occurs at boundaries of expanded TADs but not shrinked TAD upon STAG2 knockdown. The schematic diagram depicts that the variable boundary (VB) start site is defined as the site in WT, and the related end site as the site in KD. Statistical significance is determined as: ns P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The primary antibodies against STAG2 (Santa Cruz, SC-81852), IRF9 (Cell Signaling, #76684), IRF7 (Cell Signaling, #4920), USP18 (Cell Signaling, #4813), ISG15 (Santa Cruz, SC-166755), IRF1 (Cell Signaling, #8478), IRF3 (Cell Signaling, #11904), GAPDH (Cell Signaling, #2118), GAPDH (Cell Signaling, #51332), STAG1 (Novus Biologicals, NB100-298), PD-L1 (R&D Systems, AF156), CTCF (Cell Signaling, #2899), and Flag M2 (Sigma, F3165) were used.

Techniques: Binding Assay, Knockdown, ChIP-sequencing, shRNA

Journal: Nature Communications

Article Title: STAG2 regulates interferon signaling in melanoma via enhancer loop reprogramming

doi: 10.1038/s41467-022-29541-9

Figure Lengend Snippet: a Heatmap representation of differential H3K27Ac-associated loops between M14 cells with (KD) and without (WT) STAG2 shRNA knockdown as indicated by black arrows. b Majority of loop changes upon STAG2 knockdown occurs within TADs. c Motifs of transcription factors enriched at anchors of enhanced and impaired loops. d Numbers of gained, lost, and maintained super-enhancers upon STAG2 knockdown in M14 cells. e Gain of H3K27ac signal at the STAG2 to STAG1 switch sites, but not non-switch sites, upon STAG2 knockdown. f Differential CTCF, SMC1A, STAG1, STAG2, and H3K27ac profiles at anchors of enhanced and impaired loops upon STAG2 knockdown. g The relative gain of STAG1 in compensation to the loss of STAG2 is significantly higher at anchors of enhanced loops than those of impaired loops. The degree of the switch is calculated as the sum of log 2 (KD/WT) for STAG1 and STAG2 (method).

Article Snippet: The primary antibodies against STAG2 (Santa Cruz, SC-81852), IRF9 (Cell Signaling, #76684), IRF7 (Cell Signaling, #4920), USP18 (Cell Signaling, #4813), ISG15 (Santa Cruz, SC-166755), IRF1 (Cell Signaling, #8478), IRF3 (Cell Signaling, #11904), GAPDH (Cell Signaling, #2118), GAPDH (Cell Signaling, #51332), STAG1 (Novus Biologicals, NB100-298), PD-L1 (R&D Systems, AF156), CTCF (Cell Signaling, #2899), and Flag M2 (Sigma, F3165) were used.

Techniques: shRNA, Knockdown