Journal: Cell

Article Title: Mechanism of orphan subunit recognition during assembly quality control

doi: 10.1016/j.cell.2023.06.016

Figure Lengend Snippet: Interactors of unassembled chaperonin subunits destined for degradation (A) HEK293T cells were transfected with a GFP-RFP dual-color reporter of orphan CCT4 degradation (red; see Figure S1 A) or a control GFP-RFP reporter lacking the CCT4 insert (black), then analyzed by flow cytometry. CCT4 is fused to GFP, with RFP serving as a translation control. Shown are overlaid scatter plots of individual transfected cells (top) and the corresponding histograms of the GFP:RFP ratio (bottom). (B) Cells were treated with control or CCT2-targeting small interfering RNAs (siRNAs) for 72 h, and total cell lysates were analyzed by immunoblotting for the proteins indicated on the left. (C) CCT subunits containing a C-terminal TwinStrep tag (TST) or 3xFLAG tag were translated in rabbit reticulocyte lysate (RRL) containing 35 S-methionine and His-tagged ubiquitin (His-Ub). Samples were analyzed directly (total IVT, in vitro translation) or after ubiquitin pull-down under denaturing conditions via the His-tag (His-Ub PD). (D) CCT4-TST was translated in RRL, affinity-purified under native conditions, and analyzed by label-free quantitative mass spectrometry. Proteins in the upper right quadrant are significantly enriched with CCT4. (E) Cells transiently expressing 3xFLAG-tagged CCT subunits were subjected to anti-FLAG immunoprecipitation (IP) under non-denaturing conditions. Input and IP samples were analyzed by immunoblotting for the indicated proteins. See also Figures S1 and .

Article Snippet: Each aliquot was diluted ∼5-fold in native IP buffer and incubated at 4°C with 4 μg of either rabbit control IgG or anti-CCT4 antibody (Bethyl, Cat# A304-725A) for 1.5 h and subsequently with 10 μl of protein A resin (Repligen) for another 1.5 h with end-over-end rotation.

Techniques: Transfection, Control, Flow Cytometry, Western Blot, Ubiquitin Proteomics, In Vitro, Affinity Purification, Mass Spectrometry, Expressing, Immunoprecipitation

Journal: Cell

Article Title: Mechanism of orphan subunit recognition during assembly quality control

doi: 10.1016/j.cell.2023.06.016

Figure Lengend Snippet: Recognition of partially assembled CCT subunits by the ZNRD2-HERC2 complex (A) The CCT4-ZNRD2-RLD3 model was docked into the complete CCT complex structure (PDB: 7lum ) by aligning the CCT4 chains. The CCT4-ZNRD2-RLD3 model is colored, as in Figure 5 A. The non-CCT4 subunits within the top ring of the CCT complex are shown as cartoons in khaki. The CCT subunits of the bottom ring are shown in transparent surface representation. (B) Cartoon depicting a hypothetical partial CCT assembly comprising three subunits (colored in light coral, yellow, and cyan). One of the end subunits is compatible with binding the ZNRD2-HERC2 complex. The interior chamber is indicated for reference. (C) HEK293T cells transiently overexpressing the indicated 3xFLAG-tagged proteins were subjected to non-denaturing anti-FLAG IP. The bound proteins were eluted with 3xFLAG peptide and subsequently analyzed by immunoblotting for the indicated proteins relative to serial 2-fold dilutions of the cytosolic fraction (cytosol) prepared from control transfected cells. 20-fold less of the 3xFLAG-PhLP1 sample was loaded on the gel relative to the control and ZNRD2-3xFLAG (WT and L42E) samples. Two exposures of the blots are shown for all CCT subunits. (D) ZNRD2-KO cells stably overexpressing ZNRD2-3xFLAG (or untagged ZNRD2 as a negative control) were subjected to non-denaturing anti-FLAG IP. The bound proteins were eluted with 3xFLAG peptide and subjected to a second round of non-denaturing IP with anti-CCT4 antibody or control IgG. Samples were analyzed by immunoblotting as indicated. See also Figure S7 .

Article Snippet: Each aliquot was diluted ∼5-fold in native IP buffer and incubated at 4°C with 4 μg of either rabbit control IgG or anti-CCT4 antibody (Bethyl, Cat# A304-725A) for 1.5 h and subsequently with 10 μl of protein A resin (Repligen) for another 1.5 h with end-over-end rotation.

Techniques: Binding Assay, Western Blot, Control, Transfection, Stable Transfection, Negative Control

Journal: Cell

Article Title: Mechanism of orphan subunit recognition during assembly quality control

doi: 10.1016/j.cell.2023.06.016

Figure Lengend Snippet:

Article Snippet: Each aliquot was diluted ∼5-fold in native IP buffer and incubated at 4°C with 4 μg of either rabbit control IgG or anti-CCT4 antibody (Bethyl, Cat# A304-725A) for 1.5 h and subsequently with 10 μl of protein A resin (Repligen) for another 1.5 h with end-over-end rotation.

Techniques: Recombinant, Transfection, Protease Inhibitor, Mutagenesis, In Vitro, Plasmid Preparation, Negative Control, Sequencing, Expressing, Software, Western Blot, Clear Native PAGE

Journal: bioRxiv

Article Title: ESCO1 and CTCF enable formation of long chromatin loops by protecting cohesin STAG1 from WAPL

doi: 10.1101/779058

Figure Lengend Snippet: A. Immunoblot analysis of SMC3, STAG1 and STAG2 immuno-precipitates obtained from chromatin extracts of cells synchro­nized in G1 and G2. Immunoprecipitated material was normal­ized to SMC3 levels and immunoblotting was performed using the indicated antibodies. Flow cytometry profiles are shown on the left. B. Flow cytometry and chromatin extract immunoblot analysis of cells synchronized in G1 and depleted of the proteins indicated. Chromatin extracts were normalized relative to SMC3. Note that SMC3ac levels are decreased after ESCO1 and STAG1 deple­tion and increased after STAG2 depletion. C. SDS-PAGE analysis of EGFP-STAG1 and STAG2-EGFP immunoprecipitates obtained from chromatin extracts of cells synchronized in G1 and G2. Immunoprecipitations were performed using anti-GFP antibodies and were analysed with silver staining. Flow cytometry profiles are shown on the left. D. Percentage of non-acetylated peptides (0 Ac), peptides acetylated at position K105 (K105) and peptides acetylated peptide at positions K105 and K106 (K105/106) was determined by quantitative mass spectrometry from material immunoprecip­itated using anti-GFP antibodies from EGFP-STAG1 and STAG2-EGFP cells synchronized in G1 and G2. Peptide percentage was calculated relative to the total number of SMC3 peptides. Note that part of this experiment has been previously published in Ivanov at al., 2018; .

Article Snippet: The following commercial antibodies were used: SMC1 (Bethyl Laboratories, A300-055A), SMC3 (Bethyl Laboratories, A300-060A), SCC1 (EMD Millipore Corporation, 53A303), PDS5A (Bethyl Laboratories, A300-089A), α-tubulin (Sigma-Aldrich, T5168), histone H3 (Santa Cruz Biotechnology, sc-8654), phospho-histone H3 (Ser10) (Cell Signaling, #9701), PCNA (Santa Cruz, PC10), GFP (Roche, clones 7.1 and 13.1; Abcam, ab13970).

Techniques: Western Blot, Immunoprecipitation, Flow Cytometry, SDS Page, Silver Staining, Mass Spectrometry

Journal: bioRxiv

Article Title: ESCO1 and CTCF enable formation of long chromatin loops by protecting cohesin STAG1 from WAPL

doi: 10.1101/779058

Figure Lengend Snippet: A. Immunoblot analysis of HeLa cell lines. Immunoblotting of whole cell, chromatin and soluble extracts was performed using the indicated antibodies, u-tubulin and H3 were used as loading controls. B. Immunoblot analysis of whole cell extract from cells depleted of the proteins listed. Immunoblotting was performed using the indicated antibodies, α-tubulin and H3 were used as loading controls. C. Curve fitting to experimental iFRAP curves obtained from SMC3-LAP in G1 phase by a single exponential and bi-exponential functions is shown on the left. Residuals representing the difference between the experimental- and simulated-value, of the fits from the left panel are plotted on the right. D. Curve fitting to experimental iFRAP curves obtained from EGFP-STAG1 and STAG2-EGFP in G1 phase by a single exponential and bi-exponential functions is shown on the left. Residuals representing the difference between the experimental- and simulat­ed-value, of the fits from the left panel are plotted on the right, and bi-exponential functions is shown on the left. Residuals represent­ing the difference between the experimental- and simulated-value, of the fits from the left panel are plotted on the right.

Article Snippet: The following commercial antibodies were used: SMC1 (Bethyl Laboratories, A300-055A), SMC3 (Bethyl Laboratories, A300-060A), SCC1 (EMD Millipore Corporation, 53A303), PDS5A (Bethyl Laboratories, A300-089A), α-tubulin (Sigma-Aldrich, T5168), histone H3 (Santa Cruz Biotechnology, sc-8654), phospho-histone H3 (Ser10) (Cell Signaling, #9701), PCNA (Santa Cruz, PC10), GFP (Roche, clones 7.1 and 13.1; Abcam, ab13970).

Techniques: Western Blot

Journal: bioRxiv

Article Title: ESCO1 and CTCF enable formation of long chromatin loops by protecting cohesin STAG1 from WAPL

doi: 10.1101/779058

Figure Lengend Snippet: A. Images of inverse fluorescence recovery after photobleaching (iFRAP) experiments in SMC3-LAP cells synchro­nized in G1 and depleted of the indicated proteins by RNAi. Scale bar, 10 µm. Half of the nuclear SMC3-LAP fluores­cent signal was photobleached and the mean fluorescence in the unbleached and bleached regions was monitored by time-lapse microscopy. B. Graph depicting the mean normalized difference in fluorescence intensity between the bleached and unbleached regions from cells treated as described in A. Error bars denote standard error of the mean (s.e.m.), n >= 15 cells per condition. C. Quantification of the fraction of nuclear SMC3-LAP that was stably chromatin-bound in cells synchronized in G1 and depleted of the indicated proteins by RNAi. D. Quantification of the residence time of stably chromatin-bound SMC3-LAP in cells synchronized in G1 and depleted of the indicated proteins by RNAi. E. Quantification of residence time of dynamically chromatin-bound SMC3-LAP in cells synchronized in G1 and depleted of STAG1 by RNAi. The numbers are derived from the single exponential fit. F. Quantification of the residence time of dynamically chromatin-bound SMC3-LAP in cells synchronized in G1 and depleted of STAG2 and WAPL by RNAi. The numbers are derived from the bi-exponential fit. G. iFRAP images in EGFP-STAG1 and STAG2-EGFP cells synchronized in G1 Scale bar, 10 µm. H. Graph depicting the mean normalized difference in fluorescence intensity between the bleached and unbleached regions from cells treated as in G. Error bars denote standard error of the mean (s.e.m.), n = 10 cells per condition. I. Quantification of the fractions of nuclear EGFP-STAG1 that was stably chromatin-bound in G1 cells. J. Quantification of the residence time of stably chromatin-bound EGFP-STAG1 in G1 cells. K. Quantification of the residence time of dynamically chromatin-bound EGFP-STAG1 in G1 cells. The numbers are derived from the biexponential fit. L. Quantification of the residence time of dynamically chromatin-bound STAG2-EGFP in G1 cells. The numbers are derived from the single exponential fit.

Article Snippet: The following commercial antibodies were used: SMC1 (Bethyl Laboratories, A300-055A), SMC3 (Bethyl Laboratories, A300-060A), SCC1 (EMD Millipore Corporation, 53A303), PDS5A (Bethyl Laboratories, A300-089A), α-tubulin (Sigma-Aldrich, T5168), histone H3 (Santa Cruz Biotechnology, sc-8654), phospho-histone H3 (Ser10) (Cell Signaling, #9701), PCNA (Santa Cruz, PC10), GFP (Roche, clones 7.1 and 13.1; Abcam, ab13970).

Techniques: Fluorescence, Time-lapse Microscopy, Stable Transfection, Derivative Assay

Journal: bioRxiv

Article Title: ESCO1 and CTCF enable formation of long chromatin loops by protecting cohesin STAG1 from WAPL

doi: 10.1101/779058

Figure Lengend Snippet: A. Immunoblot analysis of whole cell extract from cells depleted of sororin or ESCO1. α-tubulin was used as a loding control. B. Graph depicting the mean normalized difference in EGFP-STAG1 fluorescence intensity between the unbleached and bleached regions following iFRAP in G1 cells and depleted of the indicated proteins by RNAi. Error bars denote standard error of the mean (s.e.m.), n = 10 cells per condition. C. Quantification of the fraction of nuclear EGFP-STAG1 that was stably chromatin-bound in cells synchronized in G1 and depleted of the indicated proteins by RNAi. Quantification of dynamic and stable residence time of EGFP-STAG1 upon RNAi treatment for indicated proteins is shown on the right. D. Immunoblot analysis of STAG1 and STAG2 chromatin immunoprecipitates obtained from cells synchronized in G1. Immunoprecipitations were performed using control-IgG, anti-STAG1 and anti-STAG2 antibodies (shown on the left). Immu-noprecipitated material was normalized to SMC3 levels and immunoblotting was performed using the indicated antibodies. E. Immunobiot analysis of chromatin extracts from control-, ESCO1- and CTCF-depleted HeLa cells synchronized in G1. Immunoblotting was performed using the indicated antibodies. Note that acetylation levels were decreased following deple­tion of CTCF. H3 was used as a loading control. F. Graph depicting the mean normalized difference in EGFP-STAG1 fluorescence intensity between the bleached and unbleached regions following iFRAP in cells synchronized in G1 and depleted of the indicated proteins by RNAi. Error bars denote standard error of the mean (s.e.m.), π = 10 cells per condition. G. Quantification of the fraction of nuclear EGFP-STAG1 that was stably chromatin-bound in cells synchronized in G1 and depleted of the indicated proteins by RNAi. Quantification of dynamic and stable residence time of EGFP-STAG1 in control and CTCF depleted cells by RNAi is shown on the right. H. Coverage-corrected Hi-C contact matrix of the 95.2 - 95.7 Mb region of HeLa chromosome 1. ChIP-seq signals for EGFP-STAG1, STAG2-EGFP, SMC3, SMC3ac and CTCF are shown below the Hi-C contact matrix. STAG1 and STAG2 immunoprecipitation was performed using anti-GFP antibodies. Note that the SMC3ac signal is decreased following ESCO1 depletion, confirming the specificity of the anti-SMC3ac antibody. I. Venn diagram illustrating the genome-wide co-localisation between STAG1 and STAG2 ChIP-seq signals. STAG1/2 immunoprecipitations from EGFP-STAG1 and STAG2-EGFP cells were performed using anti-GFP antibodies. Average read density plots for EGFP-STAG1 and STAG2-EGFP at SMC3ac and SMC3 sites is shown on the right. J. Venn diagrams illustrating the genome-wide overlap between SMC3ac and STAG1/2 common sites, SMC3 and CTCF ChIP-seq sites respectively. K. Venn diagrams illustrating the genome-wide overlap between the loop anchors determined using the Hi-C map shown in (H) and SMC3 (left panel) and SMC3Ac (right panel) ChIPseq binding sites.

Article Snippet: The following commercial antibodies were used: SMC1 (Bethyl Laboratories, A300-055A), SMC3 (Bethyl Laboratories, A300-060A), SCC1 (EMD Millipore Corporation, 53A303), PDS5A (Bethyl Laboratories, A300-089A), α-tubulin (Sigma-Aldrich, T5168), histone H3 (Santa Cruz Biotechnology, sc-8654), phospho-histone H3 (Ser10) (Cell Signaling, #9701), PCNA (Santa Cruz, PC10), GFP (Roche, clones 7.1 and 13.1; Abcam, ab13970).

Techniques: Western Blot, Control, Fluorescence, Stable Transfection, Hi-C, ChIP-sequencing, Immunoprecipitation, Genome Wide, Binding Assay