Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: CCDC134 deficiency selectively impaired TLR-mediated immune responses. (A) Immunoprecipitates (IP) and whole-cell extracts (WCE) from HEK293T cells transfected as indicated. SH: Strep-HA tag. (B and C) Immunoblots of indicated knockout THP1 cells unstimulated (B) or stimulated with R848 (5 µg/ml, for 0–1 h) (C). (D) Indicated knockout THP1 DUAL reporter cells stimulated with R848 (5 μg/ml) or LPS (0.1 μg/ml) for 24 h. Supernatants were analyzed for ISRE and NF-κB reporter activity. Untr.: untreated. (E) Immunoblots of cell lysates from indicated knockout THP1 cells treated with EndoH (H) or PNGase F (F). (F) TNFα production of indicated knockout THP1 cells stimulated for 24 h with LPS (0.1 μg/ml), R848 (5 μg/ml), Pam3CSK4 (0.1 μg/ml) or Pam2CSK4 (0.01 μg/ml). Untr.: untreated. (G) Immunoblots of cell lysates from indicated knockout U937 cells. (H) IL-6 (left panel) and TNFα (right panel) production of indicated knockout U937 cells differentiated with 200 nM of PMA for 24 h before stimulation with LPS (0.1 μg/ml), Pam3CSK4 (Pam3.) (1 μg/ml), or Pam2CSK4 (Pam2.) (0.1 μg/ml) for 24 h. Untr.: untreated. (I and J) Immunoblots of cell lysates from indicated knockout U937 cells differentiated with 200 nM of PMA for 48 h, treated with EndoH (H) or PNGase F (F) (I) or from indicated knockout human primary dermal fibroblast cells (J). Mat.: Mature form; Imat.: Immature form. (K) IL-6 production of indicated knockout human primary dermal fibroblast cells stimulated with LPS (0.1 μg/ml) or Pam2CSK4 (Pam2.) (0.01 μg/ml) for 24 h. Untr.: untreated. In A–C, E, G, I, and J, data are representative of two independent experiments. In D, F, H, and K, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. Source data are available for this figure: .

Article Snippet: THP1 DUAL reporter cell lines were obtained from Invivogen (cat. dhpd-nfis).

Techniques: Transfection, Western Blot, Knock-Out, Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: Impact of CCDC134 loss on TLRs and integrins. (A and B) THP1 DUAL reporter cells stimulated with R848 (5 μg/ml), flagellin (0.1 μg/ml), Pam3CSK4 (Pam3.) (0.1 μg/ml), LPS (0.1 μg/ml), Poly(I:C) complexed with lipofectamine (1 μg/ml), cGAMP (3 μg/ml), C12-iE-DAP (5 μg/ml), L18-MDP (10 μg/ml), TNFα (10 ng/ml), or IL-1β (10 ng/ml) for 24 h. Supernatants were analyzed for ISRE and NF-κB reporter activity. (A, left panel, and B) Data show mean ± SD from one representative experiment performed in stimulation triplicates. (A, right panel) Reporter activity relative to sg Ren . Data show three independent experiments. Line represents the mean ± SD ISRE reporter activity, R848 sg CCDC134-1 P value <0.0001, sg CCDC134-4 P value <0.0001; Pam3CSK4 sg CCDC134-1 P value = ns, sg CCDC134-4 P value = ns; LPS sg CCDC134-1 P value <0.0027, sg CCDC134-4 P value <0.0030; flagellin sg CCDC134-1 P value = 0.0006, sg CCDC134-4 P value <0.0001; Poly(I:C) complexed with lipofectamine sg CCDC134-1 P value = ns, sg CCDC134-4 P value = ns; cGAMP sg CCDC134-1 P value = ns, sg CCDC134-4 P value = ns. NF-κB reporter activity, R848 sg CCDC134-1 P value <0.0001, sg CCDC134-4 P value <0.0001; Pam3CSK4 sg CCDC134-1 P value = ns, sg CCDC134-4 P value = 0.0315; LPS sg CCDC134-1 P value = 0.0012, sg CCDC134-4 P value = 0.0022; flagellin sg CCDC134-1 P value = 0.0053, sg CCDC134-4 P value = 0.0051; C12-iE-DAP sg CCDC134-1 P value = ns, sg CCDC134-4 P value = ns; L18-MDP sg CCDC134-1 P value = ns, sg CCDC134-4 P value = ns; TNFα sg CCDC134-1 P value = ns, sg CCDC134-4 P value = ns; IL-1β sg CCDC134-1 P value = 0.0329, sg CCDC134-4 P value = 0.0291. Two-tailed one sample t test. Untr.: untreated. (C) TNFα production of indicated knockout THP1 cells stimulated with flagellin (0.1 μg/ml) for 24 h. (D) IL-6 (Top panel) and TNFα (Bottom panel) production of indicated knockout THP1 cells differentiated with 10 nM of PMA for 24 h before stimulation with LPS (0.1 μg/ml), Pam3CSK4 (Pam3.) (0.1 μg/ml) or Pam2CSK4 (Pam2.) (0.1 μg/ml) for 24 h. Untr.: untreated. (E) Immunoblots of cell lysate treated with EndoH (H) or PNGase F (F) of indicated knockout THP1 cells differentiated with 10 nM of PMA for 48 h. (F and G) Immunoblots of indicated knockout Hoxb8-macrophages stimulated with R848 (0.1 μg/ml), CpG-B (ODN1668) (1 μM) or LPS (10 ng/ml) for 0–1 h (F) or knockout Raw 264.7 cells (G). Asterisks indicate a non-specific band. (H) TNFα production of indicated knockout Raw 264.7 cells stimulated with CpG-B (ODN1668) (150 nM), LPS (10 ng/ml), or poly(I:C) (500 ng/ml) for 24 h. (I) Representative histograms of surface (upper panel) or surface and intracellular (lower panel) expression of CD11a, CD18, CD49d, or CD44 in sg Ren (red) or sg CCDC134 (blue) Hoxb8-macrophages. Gray curves represent unstained controls. (J) Volcano plot of quantified proteins in whole proteome of sg CCDC134 versus sg Ren Hoxb8-macrophages (upregulated: red, fold change [FC] >2 and P value <0.01; downregulated: blue, FC less than −2 and P value <0.01); green: downregulated ISGs signature. In A–D and H, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. In E–G, data are representative of two independent experiments. In I, data are representative of three independent experiments. Source data are available for this figure: .

Article Snippet: THP1 DUAL reporter cell lines were obtained from Invivogen (cat. dhpd-nfis).

Techniques: Activity Assay, Two Tailed Test, Knock-Out, Western Blot, Expressing

Journal: The Journal of Cell Biology

Article Title: Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis

doi: 10.1083/jcb.202405169

Figure Lengend Snippet: Quantification of loop extruders throughout the cell cycle and during mitotic exit. (A) Effective confocal detection volume (V eff ) determined using AF488-NHS dye and Atto488 (this study) at low concentrations (10 nM). 5 30-s-long FCS measurements are performed for V eff determination. Autocorrelation analysis of these measurements and fitting of the diffusion time parameter τ D is performed to calculate a 3D-gaussian volume. Differences between AF488-NHS and Atto488 are significant as determined by Student’s t test. The updated V eff determination routine resulted in a systematic drop in protein numbers measured in living cells. Protein numbers bound to chromatin (with cytosolic background correction performed as in ) are displayed for SMC4-mEGFP, NCAPH-mEGFP, and NCAPH2-mEGFP, measured in the same HeLa Kyoto cell lines under similar culture conditions at anaphase onset in and this study ( n SMC4 = 13, n NCAPH = 11, n NCAPH2 = 16). Error bars represent standard deviation of the mean. (B) Exemplary segmentation of a full cell cycle track. Segmentations correspond to the top right cell in . (C) Illustration of full cell cycle data processing based on cell volume information. (D) Average cell and nuclear volume for all single-cell trajectories combined. Error bands represent standard deviation. (E–G) Mean nuclear (E), cellular (F), and cytosolic (G) concentrations of HeLa Kyoto homozygous knock-in cell lines. Error bands represent a 95% confidence interval. (H) The nuclear NCAPH pool of cells endogenously expressing NCAPH-EGFP was photobleached and monitored for up to 12 h. Full bleaching could even be achieved with bleach ROIs that do not target the entire nuclear volume, indicating fast and freely moving protein. The bleached nuclear pool was not recovered by unbleached cytosolic pool throughout the measurement period. (I–L) Comparison of cell volume and absolute protein numbers in S-phase synchronized versus asynchronous cells. Bar plots compare cell volume or cellular protein content in the first mitosis after release from S-phase arrest. S-phase arrest resulted in notably increased cell size and protein abundance, influencing protein abundance, production and duration of the next cell cycle. See L and M for the influence of synchronization on Cohesin-STAG2 protein import. (I and J) n sync = 21, n async = 12; K and L n sync = 13, n async = 4. Boxes indicate the quartiles of the dataset and the whiskers show the rest of the distribution. (M and N) Comparison of Cohesin-STAG2 protein import kinetics in S-phase synchronized versus asynchronous cells. In contrast to S-phase synchronized cells, we found that WT cells required only about 2 h for full Cohesin-STAG2 import into the newly formed nuclei. Error bands represent standard deviation. (O) FCS-calibrated imaging of genome-edited HK cells with homozygously EGFP-tagged Cohesin-STAG1 throughout mitotic exit. (P) Live cell imaging of the nuclear envelope marker LBR-GFP to assess the timing of nuclear envelope reformation after mitosis. While LBR-GFP accumulated on chromatin (stained via 5-SiR-Hoechst) as early as +4 min after anaphase onset (AO), chromatin was almost fully engulfed at +6 min past AO and fully covered at +10 min past AO. (Q) Absolute protein numbers co-localizing with chromatin/the two daughter nuclei displayed for genome-edited HK cells with homozygously (m)EGFP-tagged proteins (SMC4: n = 21 cells, NCAPH: n = 14 cells, NCAPH2: n = 19 cells, CTCF: n = 15 cells, RAD21: n = 18 cells, STAG1: n = 25 cells, STAG2: n = 11 cells). Reformation and full establishment of the nuclear envelope as determined by Lamin B receptor is indicated through a grey background. Error bands represent 95% confidence interval.

Article Snippet: We found that this correlated with a leaky degradation of RAD21 in the RAD21-EGFP-AID CTCF-Halo cell line, reducing RAD21 levels about 40% relative to our CTCF-EGFP line (using Simple Western of asynchronous cell lysates, RAD21 detected via anti-RAD21 antibody (05-908, 1:50; Merck Millipore, ).

Techniques: Diffusion-based Assay, Standard Deviation, Knock-In, Expressing, Comparison, Imaging, Live Cell Imaging, Marker, Staining

Journal: The Journal of Cell Biology

Article Title: Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis

doi: 10.1083/jcb.202405169

Figure Lengend Snippet: Functional nuclear pores are required for nuclear import of Cohesin and CTCF. (A) Experiment scheme for mitotic synchronization of genome-edited HK cells with homozygously mEGFP-FKBP12 F36V tagged Nup153, followed by targeted protein degradation of Nup153, release into mitotic exit, and timed fixation during early G1. Cells are immunestained for RAD21 or CTCF and for diffraction-limited imaging. (B and C) Validation data for the generation of the homozygous Nup153-mEGFP-FKBP12 F36V knock-in line, clone #10. (B) Digital PCR was performed to assess the copy number of GFP inserted into the HK genome and to assess the number of successful homology-directed repair (HDR) events at the Nup153 endogenous gene locus. While one copy of mEGFP-FKBP12 F36V was inserted elsewhere in the genome, this copy is not expressed (C). Error bars represent minimum and maximum values of the three measured replicates. (C) Simple Western analysis of HK WT cells and the Nup153-mEGFP-FKBP12 F36V #C10 cell line created and used within this study. The anti-GFP western blot shows no expression of free GFP. The anti-Nup153 western blot shows a clear shift of the Nup153 band, indicating successful and homozygous gene editing. (D) Fluorescence micrographs of early G1 cells (∼45 min past mitosis) stained with DAPI in WT or ΔNup153 condition. (E) Average fluorescence intensity plots per 3D-segmented nucleus in grey (WT) or colored (ΔNup153). ΔNup153 nuclei do not expand in size, show no residual Nup153 intensity, and show a clear reduction in RAD21 intensity inside the nuclear lumen. (F) Average fluorescence intensity of early G1 cells in WT ( n = 539) or ΔNup153 ( n = 463) condition stained for RAD21. 33–48% reduction in average fluorescence intensity after 45 min release time. Changes above/below 20% are considered a significant change. (G) Average CTCF fluorescence intensity upon immunostaining in early G1 cells in WT condition ( n = 465) or after mitotic depletion of Nup153 ( n = 400). ΔNup153 cells show a 25–40% reduction in average CTCF fluorescent intensity after 45 min release time. Changes above/below 20% are considered a significant change. Source data are available for this figure: .

Article Snippet: We found that this correlated with a leaky degradation of RAD21 in the RAD21-EGFP-AID CTCF-Halo cell line, reducing RAD21 levels about 40% relative to our CTCF-EGFP line (using Simple Western of asynchronous cell lysates, RAD21 detected via anti-RAD21 antibody (05-908, 1:50; Merck Millipore, ).

Techniques: Functional Assay, Imaging, Knock-In, Digital PCR, Simple Western, Western Blot, Expressing, Fluorescence, Staining, Immunostaining

Journal: The Journal of Cell Biology

Article Title: Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis

doi: 10.1083/jcb.202405169

Figure Lengend Snippet: The absolute amount of Condensins, Cohesins, and CTCF inside the nucleus and bound to DNA, as well as their dynamic residence time during early G1

Article Snippet: We found that this correlated with a leaky degradation of RAD21 in the RAD21-EGFP-AID CTCF-Halo cell line, reducing RAD21 levels about 40% relative to our CTCF-EGFP line (using Simple Western of asynchronous cell lysates, RAD21 detected via anti-RAD21 antibody (05-908, 1:50; Merck Millipore, ).

Techniques:

Journal: The Journal of Cell Biology

Article Title: Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis

doi: 10.1083/jcb.202405169

Figure Lengend Snippet: Fluorescence photobleaching reveals the dynamic transition of chromatin-bound loop extruders from mitosis to interphase. (A) Scheme of FRAP experiments. Half of the metaphase plate/nucleus was bleached and fluorescent recovery was monitored in bleached and unbleached regions. (B) Exemplary FRAP data of SMC4-mEGFP in metaphase and early G1 cells. While one bleach step (150 repetitions 100% laser power) is performed in metaphase cells, three bleach steps (50 repetitions, 100% laser power) are performed in early G1 cells to bleach the entire soluble pool and allow for the determination of the total chromatin-bound fraction. (C–F) Metaphase and early G1 FRAP measurements of Condensin I (C, NCAPH-mEGFP, n meta = 16, n eG1 = 10), Condensin II (D, NCAPH2-mEGFP, n meta = 12, n eG1 = 9), CTCF-EGFP (E, n meta = 15, n eG1 = 9) and Cohesin (F, RAD21-EGFP, n meta = 11, n eG1 = 10). Error bands represent a 95% confidence interval. (G) Representative example of a spot-bleach measurement of HK WT cells exogenously expressing low concentration of stably chromatin-bound H2B-EGFP. H2B-EGFP chromatin bound fraction was used to calibrate spot-bleach measurements. (H) Representative example of a spot-bleach measurement of HK WT cells exogenously expressing the low concentration of monomeric EGFP. mEGFP chromatin-bound fraction was used to calibrate spot-bleach measurements. (I) Chromatin-bound fractions of H2B-EGFP (used as a calibration reference for 100% chromatin-bound, n = 27) and mEGFP (used as a calibration reference for 0% chromatin-bound pool, n = 23). Chromatin-bound fractions of all other proteins of interest were scaled accordingly. (J) Mean cellular fluorescence intensity of endogenous NIPBL tagged with AID-EGFP in WT condition or upon addition of auxin measured in Nocodazole-arrested mitotic cells. Depletion of the protein pool happened within 20 min. Low signal/background ratio required high light-doses leading to bleaching of NIPBL-EGFP and autofluorescence. (K) The fraction of chromatin-bound Condensin and Cohesin isoforms as well as CTCF determined using the spot-bleach assay at different time points during mitotic exit. Every bar plot represents at least 10 individual datapoints measured in 10 separate cells. Boxes indicate quartiles and error bars show the rest of the sample distribution. (L) Absolute number of proteins bound to chromatin was determined by multiplication of chromatin-bound fractions shown in K with absolute protein numbers colocalizing with chromatin as determined in ; and and displayed as per-megabase-count assuming an equal distribution of the proteins on the entire 7.9 Mb HeLa genome . Grey background indicates the reformation of the nuclear envelope. Error bands represent 95% confidence interval.

Article Snippet: We found that this correlated with a leaky degradation of RAD21 in the RAD21-EGFP-AID CTCF-Halo cell line, reducing RAD21 levels about 40% relative to our CTCF-EGFP line (using Simple Western of asynchronous cell lysates, RAD21 detected via anti-RAD21 antibody (05-908, 1:50; Merck Millipore, ).

Techniques: Fluorescence, Expressing, Concentration Assay, Stable Transfection

Journal: The Journal of Cell Biology

Article Title: Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis

doi: 10.1083/jcb.202405169

Figure Lengend Snippet: Condensins and Cohesins co-occupy chromatin during telophase and early G1, as revealed by time-resolved bleaching. (A) Illustration of the spot-bleach assay. Genome-edited HK cells homozygously expressing (m) EGFP-tagged Condensin and Cohesin subunits are illuminated at a single spot on chromatin for a total duration of 30 s and the resulting fluorescence intensity is continuously measured. The chromatin-bound fraction of a given protein of interest is calculated based on the mean fluorescence intensity of the first and last 500 ms. Exemplary image and bleach data are shown for the common Condensin subunit SMC4. (B) The fraction of chromatin-bound Condensins (SMC4) and Cohesins (RAD21) determined using the spot-bleach assay at different timepoints during mitotic exit. Every bar plot represents at least 10 individual datapoints measured in 10 separate cells. (C) Absolute number of proteins bound to chromatin was determined by multiplication of chromatin-bound fractions shown in B with absolute protein numbers colocalizing with chromatin ( n [SMC4] = 21 cells, n [RAD21] = 18 cells) as determined in and displayed as per-megabase-count assuming an equal distribution of the proteins on the entire 7.9 Mb HeLa genome . Grey background indicates the reformation of the nuclear envelope. Error bands represent 95% confidence interval. (D) Fluorescence micrographs and quantification of early G1 cells in WT condition ( n = 496) or after degradation of the isoform-shared Condensin subunit SMC4 ( n = 278). Cells were pre-extracted for 1 min prior to fixation and were stained for RAD21. SMC4 depletion caused a delay in cell division as well as major cell division errors (see merged daughter nuclei in fluorescence micrograph indicated by arrow). Time of release from Nocodazole block had to be increased to 60–70 min to fix cells in early G1 stage. Difference in mean fluorescence intensity: 8–12.5%. Changes above 20% are considered a significant change. (E) Fluorescence micrographs and quantification of early G1 cells in WT condition ( n = 307) or after degradation of the Cohesin loader NIPBL ( n = 272). Cells were pre-extracted for 1 min before fixation and were stained for SMC2. The difference in mean fluorescence intensity: ∼15%. Changes above 20% are considered a significant change.

Article Snippet: We found that this correlated with a leaky degradation of RAD21 in the RAD21-EGFP-AID CTCF-Halo cell line, reducing RAD21 levels about 40% relative to our CTCF-EGFP line (using Simple Western of asynchronous cell lysates, RAD21 detected via anti-RAD21 antibody (05-908, 1:50; Merck Millipore, ).

Techniques: Expressing, Fluorescence, Staining, Blocking Assay

Journal: The Journal of Cell Biology

Article Title: Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis

doi: 10.1083/jcb.202405169

Figure Lengend Snippet: The absolute amount of Condensins, Cohesins, and CTCF inside the nucleus and bound to DNA, as well as their dynamic residence time during G1

Article Snippet: We found that this correlated with a leaky degradation of RAD21 in the RAD21-EGFP-AID CTCF-Halo cell line, reducing RAD21 levels about 40% relative to our CTCF-EGFP line (using Simple Western of asynchronous cell lysates, RAD21 detected via anti-RAD21 antibody (05-908, 1:50; Merck Millipore, ).

Techniques:

Journal: The Journal of Cell Biology

Article Title: Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis

doi: 10.1083/jcb.202405169

Figure Lengend Snippet: Cohesin-STAG1 and CTCF cooperate to form interphase TAD structures after mitosis. (A) FCS-calibrated protein numbers colocalizing with chromatin displayed for genome-edited HK cells with homozygously EGFP-tagged Cohesin-STAG1 ( n = 25 cells), Cohesin-STAG2 ( n = 11 cells), and CTCF ( n = 15 cells) relative to the measurement 2 h after anaphase onset. Error bands represent 95% confidence interval. (B) Scheme explaining LoopTrace chromatin tracing workflow. Fixed cells were subjected to single-strand resection via exonuclease treatment (RASER) for maximal structure-preservation and subsequent hybridization with a tiled FISH library. Every FISH probe contains a non-genome-complementary docking handle that can be hybridized with a fluorescently labeled imager strand to read out the 3D location of a genomic locus ( , Preprint ). (C) Overview of the traced 1.2 megabase locus on chromosome 14 with genes as well as ChIP-seq binding sites for RAD21 and CTCF (from the ENCODE portal [ , https://www.encodeproject.org/ ] with the following identifiers: ENCFF239FBO [RAD21], ENCFF111RWV [CTCF]; CTCF directionality annotations from ). (D and E) Exemplary chromatin traces of WT (D) or ΔSTAG2 (E) early G1 cells. (F) Distance and contact matrices of a 1.2 megabase region on chromosome 14 locus traced at a genomic resolution of 12 kb in early G1 cells with and without Cohesin-STAG2. Differences between WT and ΔSTAG2 are highlighted for distance and contact probability maps.

Article Snippet: We found that this correlated with a leaky degradation of RAD21 in the RAD21-EGFP-AID CTCF-Halo cell line, reducing RAD21 levels about 40% relative to our CTCF-EGFP line (using Simple Western of asynchronous cell lysates, RAD21 detected via anti-RAD21 antibody (05-908, 1:50; Merck Millipore, ).

Techniques: Preserving, Hybridization, Labeling, ChIP-sequencing, Binding Assay

Journal: The Journal of Cell Biology

Article Title: Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis

doi: 10.1083/jcb.202405169

Figure Lengend Snippet: Cohesin-STAG1 and CTCF co-bind chromatin early after mitosis and cooperate in TAD reformation. (A) FCS-calibrated protein numbers colocalizing with chromatin are displayed for genome-edited HK cells with homozygously EGFP-tagged Cohesin-STAG1 ( n = 25 cells), Cohesin-STAG2 ( n = 11 cells), and CTCF ( n = 15 cells). Error bands represent 95% confidence interval. (B) Experimental scheme for the synchronization of cells in early G1 and G1, with subsequent pre-extraction of soluble protein and immunofluorescence to visualize chromatin-bound proteins using specific antibodies. (C) Exemplary microscopy images of non-extracted and pre-extracted G1 cells in endogenous STAG1-EGFP knock-in cell lines, STAG1-EGFP was detected via GFP nanobody. (D) Validation of pre-extraction of the soluble Cohesin-STAG1 ( n non-extracted = 80, n extracted = 230), Cohesin-STAG2 ( n non-extracted = 16, n extracted = 92), and CTCF ( n non-extracted = 16, n extracted = 92) pools. Pre-extraction results are compared with bound-fraction estimates derived from half-nuclear photobleaching FRAP experiments . Error bars represent the standard deviation of the mean. (E) Exemplary microscopy images of pre-extracted early G1 and G1 cells, stained for Cohesin-STAG1, Cohesin-STAG2, or CTCF. (F) Integrated fluorescent intensity of pre-extracted cells in mitosis, early G1 and G1 stained for Cohesin-STAG1, Cohesin-STAG2 or CTCF represent the total chromatin-bound pool. >150 cells were analyzed for early G1 and G1, respectively. Total chromatin-bound pool measured by pre-extraction & IF is compared to expected chromatin-bound protein numbers estimated by FCS-calibrated imaging and spot-bleach . Error bars represent standard deviation of the mean. (G) Experimental scheme for mitotic degradation of Cohesin-STAG2 using genome-edited HK cells with homozygously AID-EGFP tagged Cohesin-STAG2, followed by release into mitotic exit and chromatin tracing using LoopTrace ( , Preprint ). (H) Mean nuclear intensity of immune-stained STAG2 in WT condition or after depletion of endogenously tagged STAG2. Nuclei and the corresponding traces could be clearly classified into WT or ΔSTAG2. (I) Scaling plot showing the genomic versus Euclidian distance relationship of the three 1.2 Mb regions sampled at 12 kb resolution in WT or ΔSTAG2 cells. Traces from ΔSTAG2 are slightly less compact compared to WT. (J and K) Overview of the traced 1.2 megabase locus on chromosome 5 (J) and chromosome 2 (K) with genes as well as ChIP-seq binding sites for RAD21 and CTCF (from the ENCODE portal , https://www.encodeproject.org/ ) with the following identifiers: ENCFF239FBO (RAD21), ENCFF111RWV (CTCF); CTCF directionality annotations from . (L and M) Distance and contact matrices of a 1.2 megabase region on chromosome 5 (L) and chromosome 2 (M) traced at a genomic resolution of 12 kb in early G1 cells with and without Cohesin-STAG2. Differences between WT and ΔSTAG2 are highlighted for distance and contact probability maps.

Article Snippet: We found that this correlated with a leaky degradation of RAD21 in the RAD21-EGFP-AID CTCF-Halo cell line, reducing RAD21 levels about 40% relative to our CTCF-EGFP line (using Simple Western of asynchronous cell lysates, RAD21 detected via anti-RAD21 antibody (05-908, 1:50; Merck Millipore, ).

Techniques: Extraction, Immunofluorescence, Microscopy, Knock-In, Derivative Assay, Standard Deviation, Staining, Imaging, ChIP-sequencing, Binding Assay

Journal: The Journal of Cell Biology

Article Title: Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis

doi: 10.1083/jcb.202405169

Figure Lengend Snippet: Fluorescence recovery of Cohesin isoforms and CTCF. (A) Fluorescence recovery after photobleaching (FRAP) was performed by bleaching half of a nucleus in early G1 cells (20–40 min after anaphase onset) or later G1 cells selected by nuclear volume. (B) FRAP shown for genome-edited HK cells with homozygously EGFP-tagged CTCF. The difference between the bleached and unbleached region is normalized by the maximal difference at time t = 0 after bleaching. Black line indicates the data fit by a single-exponential function with an immobile fraction. Single exponential functions with immobile fraction also fit the FRAP recovery of RAD21, STAG1/2 well. (C–E) FRAP measurements using homozygous EGFP-knock-in HK cell lines in early G1 and G1 cells, respectively. Bar plots display the mean fraction of protein that is stably bound to chromatin. Two-sample t test was used to calculate significance levels. Error bars show standard deviation. (C) Cohesin-STAG1 (early G1: n = 10 cells, G1: n = 9 cells). (D) Cohesin-STAG2. (early G1: n = 10 cells, G1: n = 13 cells). (E) CTCF. (early G1: n = 9 cells, G1: n = 10 cells). (F) FRAP measurements of endogenous CTCF with WT levels of RAD21, after degradation of endogenous RAD21, and after rescue of RAD21 degradation by exogenous RAD21 expression for at least 24 h. Bar plots display the mean fraction of protein that is stably bound to chromatin. Error bars indicate standard deviation (CTCF WT: n = 10 cells, CTCF dRAD21: n = 9 cells, CTCF dRAD21 rescue: n = 10 cells). Two-sample t test was used to calculate significance levels. Data from CTCF-EGFP knock-in line is used as WT reference as it displays WT expression levels of RAD21. The double-knock-in line Rad21-EGFP-AID CTCF-Halo-3xALFA #C7 displayed leaky degradation of RAD21, reducing CTCF-chromatin binding already in -IAA cells (see and Materials and methods).

Article Snippet: We found that this correlated with a leaky degradation of RAD21 in the RAD21-EGFP-AID CTCF-Halo cell line, reducing RAD21 levels about 40% relative to our CTCF-EGFP line (using Simple Western of asynchronous cell lysates, RAD21 detected via anti-RAD21 antibody (05-908, 1:50; Merck Millipore, ).

Techniques: Fluorescence, Knock-In, Stable Transfection, Standard Deviation, Expressing, Binding Assay

Journal: The Journal of Cell Biology

Article Title: Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis

doi: 10.1083/jcb.202405169

Figure Lengend Snippet: FRAP reveals increased chromatin association of Cohesins and CTCF from early to late G1. (A–C) Chromatin-association metrics derived from FRAP measurements in HK cells in which CTCF, RAD21, STAG1, and STAG2 were endogenously tagged with EGFP. Comparison between early G1 and later G1 measurement timepoint. ( A) Chromatin bound fractions were calculated based on the remaining fluorescent intensity in the unbleached region after bleaching. After the third bleach iteration the entire soluble nuclear protein pool was bleached. Differences between early G1 and G1 were significant for all proteins tested given a significance level of P = 5% (Kolmogorov-Smirnov test, CTCF: P = 0.0037, RAD21: P = 3.09 × 10 −6 , STAG1: P = 0.0002, STAG2: P = 1.75 × 10 −6 ). (B) Chromatin residence times were derived by fitting FRAP recovery with a single exponential function with an immobile fraction component. Differences between early G1 and G1 were non-significant for all proteins tested given a significance level of P = 5% (Kolmogorov-Smirnov test, CTCF: P = 0.39, RAD21: P = 0.28, STAG1: P = 0.54, STAG2: P = 0.22). (C) Immobile fractions were derived by fitting FRAP recovery with a single exponential function with an immobile fraction component. Differences between early G1 and G1 were significant for CTCF and STAG1 given a significance level of P = 5% (Kolmogorov-Smirnov test, CTCF: P = 0.0004, RAD21: P = 0.5577, STAG1: P = 0.0037, STAG2: P = 0.1497). (D–F) Validation data for the correct tagging of CTCF in the HK RAD21-EGFP-AID CTCF-Halo-3xALFA (#C7) cell line generated in this study. (D) The copy number of Halo-3xALFA tags integrated at the target locus (HDR assay) and within the whole recipient genome (all-Halo assay) was determined in HK WT and edited (clone #7) cell lines by digital PCR. The complete tagging of all three endogenous CTCF copies was confirmed by PCR-amplification of the target locus and sequencing analysis. Digital PCR results indicate that no extra off-target copies of the tag are present at the genome of edited cells. (E and F) Simple western analysis of protein extracts from HK WT cells and the RAD21-EGFP-AID CTCF-Halo-3xALFA #C7 line created and used in this study. For each condition, 3 μl of total protein lysate at 0.4 µg/µl was loaded into the assay’s microplate. (E) Immunolabeling of CTCF shows a clear shift of the CTCF band to higher molecular weight in the edited cell line, indicating successful and homozygous gene tagging. Anti-CTCF antibody (07-729; EMD Millipore) was used at 1:40 dilution. (F) Immunolabeling of Halo shows correct tagging of a protein of the expected MW for CTCF-Halo-3xALFA, and no expression of free Halo tag. Anti-Halo Antibody (G9211; Promega) was used at 1:50 dilution. (G) Simple western analysis of protein extracts from HK WT cells, HK CTCF-EGFP cells and the RAD21-EGFP-AID CTCF-Halo-3xALFA #C7 line created and used in this study, the later grown in the absence (−IAA) and presence (+IAA) of auxin in the last 3 h of culture. For each condition, 3 μl of total protein lysate at 0.4 µg/µl was loaded into the assay’s microplate. Anti-RAD21 antibody (05-908; Sigma-Aldrich) was used at 1:50 dilution. Complete depletion of RAD21 in the genome-edited cell line was achieved by the addition of auxin. In the absence of auxin, this cell line showed a reduced expression of RAD21 compared to HK WT or the HK CTCF-EGFP cell line due to leaky degradation of RAD21. Nonetheless, the effect of the loss of the remaining RAD21 in the double-knock-in cell line still led to a more dynamic interaction of CTCF with chromatin (assessed by FRAP, data not shown). Source data are available for this figure: .

Article Snippet: We found that this correlated with a leaky degradation of RAD21 in the RAD21-EGFP-AID CTCF-Halo cell line, reducing RAD21 levels about 40% relative to our CTCF-EGFP line (using Simple Western of asynchronous cell lysates, RAD21 detected via anti-RAD21 antibody (05-908, 1:50; Merck Millipore, ).

Techniques: Derivative Assay, Comparison, Generated, Halo Assay, Digital PCR, Amplification, Sequencing, Simple Western, Immunolabeling, Molecular Weight, Expressing, Knock-In

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: Validation of MLKL-IRF5(122–498) reporter cell line and genome-wide CRISPR/Cas9 screening. (A) Schematic of MLKL-IRF5(122–498) construct. DBD: DNA-binding domain; LK: linker region; IAD: IRF association domain; AR: auto-inhibitory region; NBB: N-terminal bundle and brace; PKD: pseudo kinase domain. (B) Representative dot-plot of FSC versus SSC gating used to assess cell viability (upper panel) with histogram for FSC (left lower panel), quantification of cell viability relative to the respective unstimulated (unst.) condition (middle lower panel) and ratio of mCherry/GFP gMFI relative to sg Ren unstimulated (unst.) cells (right lower panel). CAL-1 cells stably expressing MLKL-IRF5(122–498)-T2A-mCherry construct (population) and indicated knockout were induced with doxycycline (0.5 µg/ml) for 17 h before being stimulated or not with R848 (5 µg/ml) for 6 h. (C) Representative histogram of mCherry/GFP gMFI ratio (relative to sg Ren uninduced unstimulated [unind. unst.] condition) of CAL-1 reporter cells (clone) and indicated knockout. Cells were induced or not with doxycycline (Dox.) (0.5 µg/ml) for 17 h before being stimulated or not with R848 (2 µg/ml), CL307 (2 µg/ml) or CpG-B (ODN2006, 2 µM) for 6 h. (D) Immunoblots of CAL-1 reporter cells (clone) induced or not with doxycycline (Dox.) (0.5 µg/ml) for 17 h before being stimulated with R848 (5 µg/ml, for 0–1 h). Unlike the anti-phospho-IRF5 antibody, the IRF5 antibody used detects endogenous IRF5 but not the MLKL-IRF5(122–498)-T2A-mCherry construct. Red arrow indicates MLKL-IRF5(122–498) construct, black arrow endogenous IRF5 or MLKL and asterisk a non-specific band. (E) Representative dot-plot gating used to assess cell viability (left panel) and quantification of cell viability relative to uninduced untreated (unind. untr.) condition (right panel). CAL-1 cells stably expressing MLKL-IRF5(122–498)-T2A-mCherry construct (clone) were induced with doxycycline (Dox.) (0.5 µg/ml) and simultaneously treated or not with Z-VAD-FMK (Z-VAD) (20 µM) or NSA (5 µM) for 17 h before being stimulated or not with R848 (2 µg/ml) for 6 h. Small debris and cell aggregates were neglected using FSC and SSC gating while dead cells were excluded by gating on the negative/low population (Near-IR Live/Dead [L/D]). na: condition not assessed. (F) Schematic of genome-wide loss-of-function screen. (G) Venn diagram showing the overlap of hits with a fold change >1.55 for each of the indicated comparisons (left panel), and a list of the 29 overlapping hits in the different comparisons including the induced CL307 condition (right panel). The ranking, indicated in bracket, is based on the induced CL307 versus induced untreated comparison. (H) Cell viability quantification of an extended panel of CAL-1 reporter (clone) knockout cell lines of one independent experiment previously illustrated in . Cells were induced or not by doxycycline (Dox.) (0.5 µg/ml) for 17 h before being stimulated or not with CL307 (2 µg/ml) for 6 h. Data are representative of two (B–D) or one (H) independent experiments. In E (right panel), data show mean ± SD from three independent experiments. Source data are available for this figure: .

Article Snippet: CRISPR–Cas9-based knockout cell line generation was performed using pLentiCRISPRv2 (ID: 52961; Addgene). sgRNA sequences targeting SLC15A4 or TASL as well as a non-targeting, control sgRNA sequence designed against Renilla (sg Ren ) has been previously described ( ).

Techniques: Genome Wide, CRISPR, Construct, Binding Assay, Stable Transfection, Expressing, Knock-Out, Western Blot, Comparison

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: Impact of CCDC134 loss on TLRs and integrins. (A and B) THP1 DUAL reporter cells stimulated with R848 (5 μg/ml), flagellin (0.1 μg/ml), Pam3CSK4 (Pam3.) (0.1 μg/ml), LPS (0.1 μg/ml), Poly(I:C) complexed with lipofectamine (1 μg/ml), cGAMP (3 μg/ml), C12-iE-DAP (5 μg/ml), L18-MDP (10 μg/ml), TNFα (10 ng/ml), or IL-1β (10 ng/ml) for 24 h. Supernatants were analyzed for ISRE and NF-κB reporter activity. (A, left panel, and B) Data show mean ± SD from one representative experiment performed in stimulation triplicates. (A, right panel) Reporter activity relative to sg Ren . Data show three independent experiments. Line represents the mean ± SD ISRE reporter activity, R848 sg CCDC134-1 P value <0.0001, sg CCDC134-4 P value <0.0001; Pam3CSK4 sg CCDC134-1 P value = ns, sg CCDC134-4 P value = ns; LPS sg CCDC134-1 P value <0.0027, sg CCDC134-4 P value <0.0030; flagellin sg CCDC134-1 P value = 0.0006, sg CCDC134-4 P value <0.0001; Poly(I:C) complexed with lipofectamine sg CCDC134-1 P value = ns, sg CCDC134-4 P value = ns; cGAMP sg CCDC134-1 P value = ns, sg CCDC134-4 P value = ns. NF-κB reporter activity, R848 sg CCDC134-1 P value <0.0001, sg CCDC134-4 P value <0.0001; Pam3CSK4 sg CCDC134-1 P value = ns, sg CCDC134-4 P value = 0.0315; LPS sg CCDC134-1 P value = 0.0012, sg CCDC134-4 P value = 0.0022; flagellin sg CCDC134-1 P value = 0.0053, sg CCDC134-4 P value = 0.0051; C12-iE-DAP sg CCDC134-1 P value = ns, sg CCDC134-4 P value = ns; L18-MDP sg CCDC134-1 P value = ns, sg CCDC134-4 P value = ns; TNFα sg CCDC134-1 P value = ns, sg CCDC134-4 P value = ns; IL-1β sg CCDC134-1 P value = 0.0329, sg CCDC134-4 P value = 0.0291. Two-tailed one sample t test. Untr.: untreated. (C) TNFα production of indicated knockout THP1 cells stimulated with flagellin (0.1 μg/ml) for 24 h. (D) IL-6 (Top panel) and TNFα (Bottom panel) production of indicated knockout THP1 cells differentiated with 10 nM of PMA for 24 h before stimulation with LPS (0.1 μg/ml), Pam3CSK4 (Pam3.) (0.1 μg/ml) or Pam2CSK4 (Pam2.) (0.1 μg/ml) for 24 h. Untr.: untreated. (E) Immunoblots of cell lysate treated with EndoH (H) or PNGase F (F) of indicated knockout THP1 cells differentiated with 10 nM of PMA for 48 h. (F and G) Immunoblots of indicated knockout Hoxb8-macrophages stimulated with R848 (0.1 μg/ml), CpG-B (ODN1668) (1 μM) or LPS (10 ng/ml) for 0–1 h (F) or knockout Raw 264.7 cells (G). Asterisks indicate a non-specific band. (H) TNFα production of indicated knockout Raw 264.7 cells stimulated with CpG-B (ODN1668) (150 nM), LPS (10 ng/ml), or poly(I:C) (500 ng/ml) for 24 h. (I) Representative histograms of surface (upper panel) or surface and intracellular (lower panel) expression of CD11a, CD18, CD49d, or CD44 in sg Ren (red) or sg CCDC134 (blue) Hoxb8-macrophages. Gray curves represent unstained controls. (J) Volcano plot of quantified proteins in whole proteome of sg CCDC134 versus sg Ren Hoxb8-macrophages (upregulated: red, fold change [FC] >2 and P value <0.01; downregulated: blue, FC less than −2 and P value <0.01); green: downregulated ISGs signature. In A–D and H, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. In E–G, data are representative of two independent experiments. In I, data are representative of three independent experiments. Source data are available for this figure: .

Article Snippet: CRISPR–Cas9-based knockout cell line generation was performed using pLentiCRISPRv2 (ID: 52961; Addgene). sgRNA sequences targeting SLC15A4 or TASL as well as a non-targeting, control sgRNA sequence designed against Renilla (sg Ren ) has been previously described ( ).

Techniques: Activity Assay, Two Tailed Test, Knock-Out, Western Blot, Expressing